Posts Tagged ‘metastasis’

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI

Author: Marzan Khan, B.Sc


A team of mechanical engineers at Worcester Polytechnic Institute (WPI) have developed a fascinating technology – a liquid biopsy chip that captures and detects metastatic cancer cells, just from a small blood sample of cancer patients(1). This device is a recent development in the scientific field and holds tremendous potential that will allow doctors to spot signs of metastasis for a variety of cancers at an early stage and initiate an appropriate course of treatment(1).

Metastasis occurs when cancer cells break away from their site of origin and spread to other parts of the body via the lymph or the bloodstream, where they give rise to secondary tumors(2). By this time, the cancer is at an advanced stage and it becomes increasingly difficult to fight the disease. The cells that are shed by primary and metastatic cancers are called circulating tumor cells (CTCs) and their numbers lie in the range of 1–77,200/m(3). The basis of the liquid biopsy chip test is to capture these circulating tumor cells in the patient’s blood and identify the cell type through specific interaction with antibodies(4).

The chip is comprised of individual test units or small elements, about 3 millimeters wide(4). Each small element contains a network of carbon nanotube sensors in a well which are functionalized with antibodies(4). These antibodies will bind cell-surface antigens or protein markers unique for each type of cancer cell. Specific interaction between a cell surface protein and its corresponding antibody is a thermodynamic event that causes a change in free energy which is transduced into electricity(3). This electrical signature is picked up by the semi-conducting carbon nanotubes and can be seen as electrical spikes(4). Specific interactions create an increase in electrical signal, whereas non-specific interactions cause a decrease in signal or no change at all(4). Capture efficiency of cancer cells with the chip has been reported to range between 62-100%(4).

The liquid biopsy chip is also more advanced than microfluidics for several reasons. Firstly, the nanotube-chip arrays can capture as well as detect cancer cells, while microfluidics can only capture(4). Samples do not need to be processed for labeling or fixation, so the cell structures are preserved(4). Unlike microfluidics, these nanotubes will also capture tiny structures called exosomes spanning the nanometer range that are produced from cancer cells and carry the same biomarkers(4).

Pancreatic cancer is the fourth leading cause of cancer-associated deaths in the United states, with a survival window of 5 years in only 6% of the cases with treatment(5). In most patients, the disease has already metastasized at the time of diagnosis due to the lack of early-diagnostic markers, affecting some of the major organs such as liver, lungs and the peritoneum(5,6). Despite surgical resection of the primary tumor, the recurrence of local and metastatic tumors is rampant(5). Metastasis is the major cause of mortality in cancers(5). The liquid biopsy chip, that identifies CTCs can thus become an effective diagnostic tool in early detection of cancer as well as provide information into the efficacy of treatment(3). At present, ongoing experiments with this device involve testing for breast cancers but Dr. Balaji Panchapakesan and his team of engineers at WPI are optimistic about incorporating pancreatic and lung cancers into their research.


1.Nanophenotype. Researchers build liquid biopsy chip that detects metastatic cancer cells in blood: One blood sample can be tested for a comprehensive array of cancer cell biomarkers. 27 Dec 2016. Genesis Nanotechnology,Inc


2.Martin TA, Ye L, Sanders AJ, et al. Cancer Invasion and Metastasis: Molecular and Cellular Perspective. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.


3.F Khosravi, B King, S Rai, G Kloecker, E Wickstrom, B Panchapakesan. Nanotube devices for digital profiling of cancer biomarkers and circulating tumor cells. 23 Dec 2013. IEEE Nanotechnology Magazine 7 (4), 20-26

Nanotube devices for digital profiling of cancer biomarkers and circulating tumor cells

4.Farhad Khosravi, Patrick J Trainor, Christopher Lambert, Goetz Kloecker, Eric Wickstrom, Shesh N Rai and Balaji Panchapakesan. Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube–CTC chip. 29 Sept 2016. Nanotechnology. Vol 27, No.44. IOP Publishing Ltd


5.Seyfried, T. N., & Huysentruyt, L. C. (2013). On the Origin of Cancer Metastasis. Critical Reviews in Oncogenesis18(1-2), 43–73.


6.Deeb, A., Haque, S.-U., & Olowoure, O. (2015). Pulmonary metastases in pancreatic cancer, is there a survival influence? Journal of Gastrointestinal Oncology6(3), E48–E51. http://doi.org/10.3978/j.issn.2078-6891.2014.114


Other related articles published in this Open Access Online Scientific Journal include the following:


Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN



Trovagene’s ctDNA Liquid Biopsy urine and blood tests to be used in Monitoring and Early Detection of Pancreatic Cancer

Reporters: David Orchard-Webb, PhD and Aviva Lev-Ari, PhD, RN



Liquid Biopsy Assay May Predict Drug Resistance

Curator: Larry H. Bernstein, MD, FCAP


New insights in cancer, cancer immunogenesis and circulating cancer cells

Larry H. Bernstein, MD, FCAP, Curator



Prognostic biomarker for NSCLC and Cancer Metastasis

Larry H. Bernstein, MD, FCAP, Curato



Monitoring AML with “cell specific” blood test

Larry H. Bernstein, MD, FCAP, Curator



Diagnostic Revelations

Larry H. Bernstein, MD, FCAP, Curator



Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation

Reporter: Aviva Lev-Ari, PhD, RN





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A Mechanism of Cancer Metastasis

Larry H. Bernstein, MD, FCAP, Curator



A Protein that Spreads Cancer

Nils Halberg at the University of Bergen has identified a protein that makes it possible for cancer cells to spread


The cells inside a tumour differ a lot. While some remains “good” and do not cause trouble, others become aggressive and starts to spread to other organ sites. It is very hard to predict which cells become aggressive or not.

Nevertheless, by isolating these aggressive cancer cells in in vivotests on animals, Nils Halberg at the Department of Biomedicinet the University of Bergen (UiB) and the researchers Dr. Sohail Tavazoie and Dr. Caitlin Sengelaub at The Rockefeller University have discovered a certain protein (PITPNC1) that characterise aggressive cancer cells.

“We discovered that the aggressive cancer cells that are spreading in colon, breast, and skin cancer contained a much higher portion of the protein PITPNC1, than the non-aggressive cancer cells,” says researcher Nils Halberg of the CELLNET Group at the Department of Biomedicine at UiB.

“This means we can predict which of the cancer cells are getting aggressive and spread, at a much earlier stage than today.”

How cells penetrate tissue

The researcher also discovered that this protein, that characterizes the aggressive cancer cells, has got a very specific function in the process of spreading cancer.

The cancer cells spread from one place in the body to another, through the blood vessel. To get into the blood vessels, the cell needs to penetrate tissue, both when it leaves the tumour and when it is attaching to a new organ.

“The protein PITPNC1 regulates a process whereby the cancer cells are secreting molecules, which cut through a network of proteins outside the cells, like scissors. The cancer cell is then able to penetrate the tissue and set up a colonies at new organ sites,” Halberg explains.

Custom-made therapy

A tumour that is not spreading, is usually not dangerous for the patient if it is removed. The hard part in cancer therapy is when the tumour starts to spread. Guided by the new discoveries, supported by the Bergen Research Foundation´s (BFS) Recruitment Programme, Halberg hopes to contribute to a better treatment of cancer patients.

“If we get to the point where we can offer a custom-made therapy that targets the function of this protein, we might be able to stop it spreading,” says Nils Halberg.



This gene encodes a member of the phosphatidylinositol transfer protein family. The encoded cytoplasmic protein plays a role in multiple processes including cell signaling and lipid metabolism by facilitating the transfer of phosphatidylinositol between membrane compartments. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene, and a pseudogene of this gene is located on the long arm of chromosome 1. [provided by RefSeq, May 2012]


Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid*

Kathryn Garner‡ , Alan N. Hunt§ , Grielof Koster§ , Pentti Somerharju¶ , Emily Groves‡1, Michelle Li‡ , Padinjat Raghu , Roman Holic‡ , and Shamshad Cockcroft‡2
JBC Papers in Press, July 21, 2012,      http://dx.doi.org:/10.1074/jbc.M112.375840

Background: Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgB) promotes metastatic colonization and angiogenesis in humans.

Results: We demonstrate that RdgB is a phosphatidic acid (PA)- and phosphatidylinositol-binding protein and binds PA derived from the phospholipase D pathway.

Conclusion: RdgB is the first lipid-binding protein identified that can bind and transfer PA.

Significance: PA bound to RdgB is a likely effector downstream of phospholipase D


PITPNC1 Recruits RAB1B to the Golgi Network to Drive Malignant Secretion

Nils Halberg3,4,, Caitlin A. Sengelaub3, Kristina Navrazhina, Henrik Molina, Kunihiro Uryu, Sohail F. Tavazoie
Cancer Cell 14 Mar 2016; Volume 29, Issue 3:339–353   http://dx.doi.org/10.1016/j.ccell.2016.02.013
  • PITPNC1 promotes metastasis by melanoma, breast cancer, and colon cancer cells
  • PITPNC1 recruits RAB1B to the Golgi compartment of the cell
  • Golgi localization of RAB1B enhances vesicular secretion via GOLPH3 recruitment


Enhanced secretion of tumorigenic effector proteins is a feature of malignant cells. The molecular mechanisms underlying this feature are poorly defined. We identify PITPNC1 as a gene amplified in a large fraction of human breast cancer and overexpressed in metastatic breast, melanoma, and colon cancers. Biochemical, molecular, and cell-biological studies reveal that PITPNC1 promotes malignant secretion by binding Golgi-resident PI4P and localizing RAB1B to the Golgi. RAB1B localization to the Golgi allows for the recruitment of GOLPH3, which facilitates Golgi extension and enhanced vesicular release. PITPNC1-mediated vesicular release drives metastasis by increasing the secretion of pro-invasive and pro-angiogenic mediators HTRA1, MMP1, FAM3C, PDGFA, and ADAM10. We establish PITPNC1 as a PI4P-binding protein that enhances vesicular secretion capacity in malignancy.



Cancerous Conduits

Metastatic cancer cells use nanotubes to manipulate blood vessels.

By Amanda B. Keener | April 1, 2016


MAKING CONTACT: Breast cancer cells (white arrows) in culture deliver microRNAs to endothelial cells through filamentous nanotubes (yellow arrow).



The paper
Y. Conner et al., “Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype,” Nat Commun, 6:8671, 2015.

Branching Out
Harvard bioengineer Shiladitya Sengupta and his team were establishing a culture system to model the matrix and blood vessel networks that surround tumors when they found that human breast cancer cells spread out along blood vessel endothelial cells rather than form spheroid tumors as expected. Taking a closer look using scanning electron microscopy, they spied nanoscale filaments consisting of membrane and cytoskeletal components linking the two cell types.

Manipulative Metastases
These cancer cell–spawned nanotubes, the team discovered, could transfer a dye from cancer cells to endothelial cells both in culture and in a mouse model of breast cancer metastasis to the lungs.The cells also transferred microRNAs known to regulate endothelial cell adhesion and disassociation of tight junctions, which Sengupta speculates may help cancer cells slip in and out of blood vessels. This study is the first to suggest a role for nanotubes in metastasis.

Breaking the Chain
Sengupta’s team then used low doses of cytoskeleton-disrupting drugs to block nanotube formation. Emil Lou, an oncologist at the University of Minnesota who studies nanotubes in cancer and was not involved in the study, says this approach is a “good start,” though such drugs would not be used in human patients because they are not specific to nanotubes.

In the Details
Lou says the study emphasizes the importance of understanding interactions between tumors and their surrounding tissues on a molecular level. Going forward, Sengupta plans to study how the tubes are formed in melanoma as well as breast and ovarian cancers to try to identify other drug targets.


Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype

Yamicia ConnorSarah TekleabShyama NandakumarCherelle Walls,….., Bruce ZetterElazer R. Edelman & Shiladitya Sengupta
Nature Communications6,Article number:8671

Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of ‘metastatic hijack’: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.


Metastasis is the culmination of a cascade of events, including invasion and intravasation of tumour cells, survival in circulation, extravasation and metastatic colonization4. Multiple studies have reported a dynamic interaction between the metastatic tumour cell and the target organ, mediated by cytokines4, 12 or by exosomes that can prime metastasis by creating a pre-metastatic niche13. Interestingly, the interactions between cancer cells and endothelium in the context of metastasis, which occurs during intravasation, circulation and extravasation, remains less studied. Cancer cell-secreted soluble factors can induce retraction of endothelial cells and the subsequent attachment and transmigration of tumour cells through the endothelial monolayers14, 15. Recently, studies indicate a more intricate communication between cancer cells and the endothelium. For example, a miRNA regulon was found to mediate endothelial recruitment and metastasis by cancer cells16. Similarly, exosome-mediated transfer of cancer-secreted miR-105 was recently reported to disrupt the endothelial barrier and promote metastasis17. We rationalized that a better understanding of cancer–endothelial intercellular communication, primarily during extravasation, could lead to novel strategies for inhibiting metastasis18.

Recently, nanoscale membrane bridges, such as tunnelling nanotubes (TNTs) and filopodias, have emerged as a novel mechanism of intercellular communication19. For example, specialized signalling filopodia or cytonemes were recently shown to transport morphogens during development20. Similarly, TNTs, which unlike filopodia have no contact with the substratum21, were shown to facilitate HIV-1 transmission between T cells, enable the spread of calcium-mediated signal between cells and transfer p-glycoproteins conferring multi-drug resistance between cancer cells22, 23, 24, 25. TNTs were also recently implicated in trafficking of mitochondria from endothelial to cancer cells and transfer miRNA between osteosarcoma cells and stromal murine osteoblast cells, and between smooth muscle cells and the endothelium26, 27, 28. However, whether similar intercellular nanostructure-mediated communication can be harnessed by cancer cells to modulate the endothelium is not known.

Here we report that metastatic cancer cells preferentially form nanoscale intercellular membrane bridges with endothelial cells. These nanoscale bridges act as physical conduits through which the cancer cells can horizontally transfer miRNA to the endothelium. We observe that the recipient endothelial cells present an miRNA profile that is distinct from non-recipient endothelial cells isolated from the same microenvironment. Furthermore, the co-cultures of cancer and endothelial cells upregulate markers associated with pathological endothelium, which is inhibited by pharmacological disruption of the nanoscale conduits. Additionally, the pharmacological inhibitors of these nanoscale conduits can decrease metastatic foci in vivo, which suggests that these nanoscale conduits may potentially emerge as new targets in the management of metastatic cancer.


Figure 1: Nanoscale structures physically connect metastatic cells and the endothelium

Nanoscale structures physically connect metastatic cells and the endothelium.

(a) Representative image of MDA-MB-231 cancer cells exhibiting an invasive phenotype in the presence of preformed endothelial tubes in co-culture. (b) Representative image of a mammosphere typically formed by MDA-MB-231 cells when cultured on 3D tumour matrix in the absence of endothelial cells. MDA-MB-231 cells were loaded with CFSE. Actin was labelled with rhodamine phalloidin and nuclei were counterstained with DAPI. (c) A representative SEM of epithelial (EPI) MDA-MB-231 cells aligning on HUVEC (ENDO) tubules in the co-culture. Lower panel shows higher magnification. (d) SEM image reveals nanoscale membrane bridges connecting (nCs) metastatic breast cancer (EPI) cells and endothelial vessels (arrows). (e) A representative transmission electron micrograph shows intercellular connectivity through the nanoscale membrane bridge between MDA-MB-231 and an endothelial cell. (f) A cartoon represents the types of homotypic and heterotypic intercellular nanoscale connections that an epithelial cell may form in the presence of endothelial tubules. Highly metastatic (MDA-MB-468, MDA-MB-231 or MDA-MB-435) or low metastatic (MCF7 and SkBr3) cancer cells were co-cultured with the endothelial tubes. Normal HMECs were used as control. Graphs show percentage of total population of epithelial cells that exhibit either homotyptic (Epi–Epi) or heterotypic (Epi–Endo) nanoscale connections and (g) average number of nanoscale connections formed per cell. Quantification analysis was done on >300 cells of each cell type. Data shown are mean±s.e.m. (n=6 replicates per study, with 2–3 independent experiments). **P<0.01, ***P<0.001 (analysis of variance followed by Bonferroni’s post-hoctest).

The nanoscale bridges are composed of cytoskeletal elements

Figure 3: Structure and function of the heterotypic intercellular nanoscale membrane bridges.

a) Representative images show the heterotypic nanoscale membrane bridges are composed of both F-actin and α/β-tubulin cytoskeletal components. Co-cultures were stained with α/β-tubulin antibody (green) and phalloidin (purple) to label actin, and counterstained with DAPI (nuclear)+WGA (plasma membrane) (blue). Endothelial cells were labelled with DiL-Ac-LDL (red). (b) Mathematical modelling of the structure of the nanoscale connections. The physical properties of actin filaments necessitate microtubules for projections of certain length scales. The maximum projection length for a given minimum diameter at the buckling limit is plotted for actin-only nanoscale structures (purple line). This curve is overlaid with the experimental length and diameter measurements (red dots) from the observed thin projections measured in these studies. Projections containing only actin or projections containing both actin and tubulin can exist to the right of the curve (purple line). However, actin-only projections cannot exist to the left of the curve (green region). (c) The effect of incorporating tubulin in these projections. The maximum length is plotted against the minimum diameter for varying fractions of tubulin incorporated in the nanoscale projection. Addition of microtubules to the projections increases the overall flexural rigidity, shifting the curves left of the actin-only limit (purple line), thus allowing for longer and thinner nanoscale connections. However, owing to the larger radius of microtubules (4 × radius of actin filaments), there is an optimal fraction of tubulin (green line) that can be incorporated into the projection before the effect is reversed. (d) The optimal fraction of microtubules is about 6.6% (red dashed line) to maximize nanostructure flexural strength, while minimizing thickness. (e) Representative confocal image shows the presence of myosin V motor proteins within the intercellular nanostructure (inset shows higher magnification). Scale bar 10μm.

Nanoscale bridges act as conduits for communication

Figure 4: The nanoscale membrane bridges act as conduits for intercellular communication between cancer and endothelial cells.

(a) Confocal image of nanoscale membrane bridge-mediated transfer of cytoplasmic contents. CFSE (green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. Transfer of the CFSE dye was observed after 24-h co-culture. CFSE dye can be seen within HUVEC cells (yellow arrow). Tumour cells can form a nanobridge with a distal endothelial cell (EC1) than an endothelial cell (EC2) in close proximity. (b,c) Cartoon shows the experimental design, where dual cultures control for vesicle-mediated intercellular transfer. FACS plot show gating for sorting endothelial cells from the co-cultures using dual staining for DiI-Ac-LDL and PECAM-1, and then quantification for CFSE transfer in the isolated endothelial cells. (d) Graph shows quantification of FACS analysis, highlighting increased transfer of CFSE to endothelial cells in the co-culture. (N>100,000 events, n=36 replicates, 3 replicates per study). (e) Graph shows the temporal kinetics of nanoscale connection-mediated intercellular transfer of CFSE from MDA-MB-231 cells to the endothelium (n=2 studies, 3 replicates per study). (f) Effect of small molecule inhibitors of cytoskeletal components on membrane nanobridges. (g) Graphs show treatment with vehicle (control) or a low-dose combination of docetaxel and cytochalasin do not affect the exosome shedding (n=2 independent studies). (h,i) Graphs show the effect of pharmacological inhibitors on the formation of heterotypic and homotypic nanoscale bridges (arrows). (n=2 studies, 6 replicates per study). (j) Graph shows the effect of pharmacological inhibitors on intercellular transfer of CFSE to endothelial cells from cancer cells (n=10 studies, 3 replicates per study). Data shown are mean±s.e.m. (*P<0.05,**P<0.01, ****P<0.001, analysis of variance followed by Bonferroni’s post-hoc test).

Effect of pharmacological inhibition of nanoscale bridges

Nanobridges transfer miRNA from cancer cells to endothelium

Figure 5: The nanoscale membrane bridges act as conduits for intercellular transfer of miRNA between cancer and endothelial cells.

Representative confocal images show the transfer of Cy3-labelled miRNA from MDA-MB-231 cells (EPI) to endothelial cells (ENDO) at (a) 24h and (b) 36h of co-culture. Alexa Fluor 488-Ac-LDL (green)-labelled endothelial cells were co-cultured with Cy3-labelled miRNA-transfected MDA-MB-231. Co-cultures were counterstained with phalloidin (purple) and DAPI+WGA (blue). A 3D visualization shows the localization of miRNA within the nanoscale connections (white arrows), which act as conduits for horizontal transfer of miRNAs to endothelial cells. (c) Schema shows quantification of Cy3-labelled control miRNA and Cy3-labelled miR132 transfer between cancer cell and endothelium using flow cytometry. Endothelial cell populations were isolated from the co-cultures and percentage of miRNA+ve cells was determined. Dual cultures in Boyden chambers were included as controls. (d) Graph shows the effect of pharmacological disruption of nanoscale conduits on miRNA transfer. (e) Schema shows experimental design for reverse transcriptase–PCR-based detection of transferred miR-132 in endothelial cells under different experimental conditions. MDA-MB-231 cells transfected with miR-132 and α-miR-132 were co-cultured with endothelial tubes. FACS-isolated endothelial cell populations were analysed for the expression of miR-132. (f) Graph shows miR-132+ve cell populations (solid red) show 5 × increase compared with miR-132−ve populations (solid blue) (P<0.0001), whereas anti-miR-132+ve cells (striped red) show 26 × decrease in miR-132 expression (P<0.0001) compared with α-miR-132−ve cells (striped blue). Direct transfection of miR-132 (black) and α-miR-132 (light blue) in endothelial cells is used as positive and negative controls, respectively. Upregulation of miR-132 from baseline was observed in dual culture (solid green), which could be inhibited with anti-miR-132 (striped green). MiR-132 levels are increased compared with dual only in those cells that are positive for intercellular transfer. Fold change was determined compared with endothelial cell transfection with control miRNA (grey). (g) FACS analysis shows nanoscale bridges-mediated transfer of miRNAs leads to changes in p120RasGAP and pAkt (S473) expression downstream of the miR-132 pathway in endothelial cell populations isolated from co-cultures. (h) Graphs show p120RasGAP expression is decreased in the miR-132+ve cell populations and increased in the α-miR-132+ve cell populations, while further downstream miR-132 positively regulates pAkt expression. Data shown are mean±s.e.m. (N=2–5 independent studies, with 3 replicates per study,*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analysis of variance followed by Bonferroni’s post-hoctest).

Nanobridge-mediated transfer alters endogenous miRNA profile

Figure 6: Cancer cell–endothelial intercellular transfer alters the endogenous miRNA profile and phenotype of recipient endothelial cells.


The complexity of regulatory tumour parenchyma–endothelial communication is increasingly being unravelled7, 50. The altered phenotypic behaviour of the metastatic cancer cells in the presence of endothelial cells observed in this study, instead of forming classical mammospheres, is consistent with the emerging paradigm of modulatory tumour parenchyma–stroma communication and the creation of a pre-metastatic niche. Indeed, a recent study proposed the concept of the formation of a pre-metastatic niche mediated via metastatic cell-secreted exosomes, leading to vascular leakiness at the pre-metastatic sites13. Here we demonstrate that cancer cells form nanoscale membrane bridges, which can act as conduits for horizontal transfer of miRNA from the cancer cells to the endothelium, switching the latter to a pathological phenotype. Our findings reveal that the ability to form the nanoscale conduits with endothelial cells correlates with the metastatic potential of the cancer cell, and that the pharmacological perturbation of these nanoscale connections can lead to a reduction in the metastatic burden in experimental metastasis models. Together, our studies shed new insights into the tumour parenchyma–endothelial communication, adding depth to the emerging paradigm of the ability of a cancer cell to ‘hijack’ a physiological stromal cell for self-gain13.

Indeed, exosomes have emerged as an extensively studied mechanism of horizontal intercellular transfer of information51. However, a key distinction exists between the exosome-mediated versus the nanoscale membrane bridge-mediated intercellular communication. Although the former is stochastic, that is, it is unlikely the cancer cell has control over which cell will be targeted by a secreted exosome, the communication via nanoscale membrane bridges is deterministic, that is, the cancer cell can connect to a specific endothelial cell, which could be further away than the most proximal endothelial cell.

Although the aim of this study was to study the nanoscale membrane bridges as a mode of horizontal transfer of miRNAs from the metastatic cancer cells to the endothelium, and not to characterize a specific miRNA that are implicated in metastasis, many of the miRNAs, which were differentially regulated in the recipient endothelial cells, have previously been shown to regulate metastasis (Supplementary Discussion).

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New insights in cancer, cancer immunogenesis and circulating cancer cells

Larry H. Bernstein, MD, FCAP, Curator


Revised 4/20/2016


Circulating Tumor Cells Traverse Tiny Vasculature

Clusters of tumor-derived cells can pass through narrow channels that mimic human capillaries, scientists show in vitro and in zebrafish.

By Tanya Lewis | April 18, 2016



A stained cluster of cancer cells passes through a 7-μm channel in vitro. PNAS, AU ET AL

Clusters of circulating tumor cells (CTCs) may play a larger role in cancer metastasis than previously thought. Researchers at Massachusetts General Hospital have now shown that these clusters can squeeze through microfluidic channels just 7 microns (μm) in diameter. The team’s findings were published today (April 18) in PNAS.

“There’s a common belief in the field that even single CTCs traversing through capillary beds would destroy the majority of them through physical shearing,” Edward Cho of Spectrum Genomics wrote in an email toThe Scientist. This study “demonstrates new evidence that clusters of CTCs may have a mechanism to prevent shearing as they traverse through small capillaries, and thus may have greater metastatic potential than previously thought,” added Cho, who was not involved in the work.

Most cancer deaths are caused by tumors metastasizing to different organs. Traditionally, clusters of these cells were thought to be too large to pass through capillaries, instead getting stuck and forming blood clots. Yet, more recently, these clusters have been detected in blood drawn from cancer patients. “If they’re so big, how can we find them in blood collection in the arm?” study coauthor Mehmet Toner of Massachusetts General Hospital told The Scientist.

To investigate, Toner and his colleagues created 7-μm-wide microfluidic channels designed to mimic the mechanical properties of human capillaries, and filmed clusters of CTCs—derived from patient blood samples and from cell lines—as they moved through the channels.

As the videos revealed, clusters of 10 to 20 cells loosely disassembled as they entered the channels and moved through the tiny passageways, single-file—like a crowd of people holding hands as they squeeze through a narrow alleyway in a line formation. The cells were “squeezed beyond belief,” said Toner. Upon exiting the channel, the CTCs reassembled into nonlinear clusters.

Next, the researchers sought to validate their model in vivo. They injected human CTC clusters into the bloodstreams of 3-day-old transgenic zebrafish embryos. Zebrafish are a good model for humans because their capillaries are almost identical to those of humans in size and pressure, Toner explained. Again the researchers found that the CTC clusters could traverse the fish capillaries.


A cluster of four tumor cells elongates and compresses as it travels through a 7-μm microfluidic capillary.PNAS, AU ET AL.


Clusters of tumor cells (green) moving through a transgenic zebrafish blood vessel (arrows indicate direction of flow through dorsal aorta, caudal vein, and pivot point)PNAS, AU ET AL.

Finally, Toner’s team showed that these CTC clusters could be broken up with certain drugs. The researchers treated clusters with either FAK inhibitor 14, a molecule overproduced by many tumors that inhibits a protein involved in cell-cell adhesion, or the chemotherapy drug paclitaxel, which also weakens cell junctions. When the researchers injected the treated clusters into the microfluidic channels, the clusters broke up into smaller clumps or single CTCs, suggesting a possible avenue for treatment.

“It’s a very interesting paper,” Sanjiv Sam Gambhir of Stanford, a professor of radiology and nuclear medicine who did not take part in the study, told TheScientist. “It’s not known how these clusters of cells end up being the bad [guys] in terms of metastasis. This work very nicely—both through computational modeling, as well as microfluidic devices and zebrafish models—attempts to elucidate this finding.”

However, Gambhir added, the results are still based on models. “Unless you’re doing this in actual human capillaries, you still can’t prove this is what really goes on in humans,” he said.

However, Gambhir added, the results are still based on models. “Unless you’re doing this in actual human capillaries, you still can’t prove this is what really goes on in humans,” he said.

Missing from both the in vitro and zebrafish models was information on how CTC clusters behave in branching capillary beds like those seen in human capillaries, Cho noted. Cho’s team and others have previously shown that CTC clusters may also become stuck in veins, creating clots that can be fatal.

Further, developing treatments based on breaking up these clusters relies on the assumption that CTC clusters have greater metastatic potential than single CTCs, which is still up for debate.

“This study is a good first effort to help us understand how cells might transit through a simplified model of the circulatory system,” Cho wrote in an email, “but until we can model the true complexity of CTCs and CTC clusters traveling through the human circulatory system, we should be cautious not to extrapolate too much in terms of the potential therapeutic potential from the conclusions of studies like these.”

S.H. Au et al., “Clusters of circulating tumor cells traverse capillary-sized vessels,” PNAS,doi:10.1073/pnas.1524448113, 2016.

Clusters of circulating tumor cells traverse capillary-sized vessels

Sam H. Aua,bBrian D. StoreycJohn C. Moored,e,fQin Tangd,e,f, et al.    Sam H. Au,  http://dx.doi.org:/10.1073/pnas.1524448113

Metastasis is responsible for 90% of cancer-related deaths and is driven by tumor cells circulating in blood. However, it is believed that only individual tumor cells can reach distant organs because multicellular clusters are too large to pass through narrow capillaries. Here, we collected evidence by examining clusters in microscale devices, computational simulations, and animals, which suggest that this assumption is incorrect, and that clusters may transit through capillaries by unfolding into single-file chains. This previously unidentified cell behavior may explain why previous experiments reported that clusters were more efficient at seeding metastases than equal numbers of single tumor cells, and has led to a strategy that, if applied clinically, may reduce the incidence of metastasis in patients.


Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.
Signal Loop Pulls Healthy Cells into Cancer’s Echo Chamber


In the cellular media environment, some of the most pernicious messaging occurs within tumors, which form a kind of echo chamber that amplifies molecular interactions. These interactions, which support the growth and spread of cancer, occur not only between genetically diverse cancer cells, but also between cancer cells and healthy cells.

That healthy cells should participate in such distorted discourse is disappointing but undeniable, say scientists based at the Institute of Cancer Research (ICR). These scientists report that stromal cells are all too receptive to KRAS signals secreted by cancer cells. Under the influence of oncogenic KRAS, stromal cells secrete a message of their own, one that cancer cells cannot produce themselves, and the stromal cells’ messaging ends up reinforcing the cancer cells’ malignant behavior.

These findings appeared April 14 in the journal Cell, in an article entitled, “Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.” The article describes how the ICR researchers studied communication networks in cells from a type of pancreatic cancer called pancreatic ductal adenocarcinoma, one of the most deadly forms of cancer.

“By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells,” wrote the authors of the Cell article. “Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D.”

Normal KRAS makes occasional signals that tell a cell to divide; but when the gene is mutated, it becomes hyperactive and helps drive cancer cells’ rapid and uncontrolled growth. KRAS is mutated in more than 90% of pancreatic cancer, and in nearly 20% of all cancers.

The authors determined that, “…reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis.”

In other words, by monitoring reciprocal signaling, the ICR scientists discovered that healthy cells were responding with a totally new message, one propagated via IGF1R/AXL-AKT. This message doubled the capacity for KRAS to drive malignant behavior in cancer cells.

“We now know that tumors are a complex mix of genetically diverse cancer cells and multiple types of healthy cells, all communicating with each other via an intricate web of interactions,” noted Claus Jørgensen, Ph.D., the ICR scientist who led the study and is currently a junior group leader at the Cancer Research UK Manchester Institute. “Untangling this web, and decoding individual signals, is vital to identify which of the multitude of communications are most important for controlling tumor growth and spread.”

“We have identified a key role played by the most commonly mutated gene in cancer in communicating with healthy cells. Blocking its effects could be an effective cancer treatment.”


Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation

Christopher J. Tape, Stephanie Ling, Maria Dimitriadi4,…., Douglas A. Lauffenburger, Claus Jørgensen
Cell Apr 2016    http://dx.doi.org/10.1016/j.cell.2016.03.029

In Brief – Cell-specific proteome labeling reveals that oncogenic KRAS stimulates stromal cells to initiate reciprocal signaling back to pancreatic tumor cells, thereby enabling signaling capacity beyond the traditionally studied cell-autonomous pathways.


  1.  KRASG12D establishes a reciprocal signaling axis via heterotypic stromal cells
  2.  Reciprocal signaling further regulates tumor cell signaling downstream of KRASG12D
  3.  Reciprocal signaling regulates tumor cell behavior via AXL/ IGF1R-AKT
  4.  Heterocellularity expands tumor cell signaling beyond cellautonomous pathways

Figure thumbnail fx1


Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRASG12D) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12Dproduces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer.

Solid cancers are heterocellular systems containing both tumor cells and stromal cells. Coercion of stromal cells by tumor cell oncogenes profoundly impacts cancer biology (Friedl and Alexander, 2011, Quail and Joyce, 2013) and aberrant tumor-stroma signaling regulates many hallmarks of cancer (Hanahan and Weinberg, 2011). While individual oncogene-driven regulators of tumor-stroma signaling have been identified, the propagation of oncogene-dependent signals throughout a heterocellular system is poorly understood. Consequently, our perspective of oncogenic signaling is biased toward how oncogenes regulate tumor cells in isolation (Kolch et al., 2015).

In a heterocellular cancer, tumor cell oncogenes drive aberrant signaling both within tumor cells (cell-autonomous signaling) and adjacent stromal cells (non-cell-autonomous signaling) (Croce, 2008, Egeblad et al., 2010). As different cell types process signals via distinct pathways (Miller-Jensen et al., 2007), heterocellular systems (containing different cell types) theoretically provide increased signal processing capacity over homocellular systems (containing a single cell type). By extension, oncogene-dependent signaling can theoretically engage additional signaling pathways in a heterocellular system when compared to a homocellular system. However, to what extent activated stromal cells reciprocally regulate tumor cells beyond cell-autonomous signaling is not well understood.

We hypothesized that the expanded signaling capacity provided by stromal heterocellularity allows oncogenes to establish a differential reciprocal signaling state in tumor cells. To test this hypothesis, we studied oncogenic KRAS (KRASG12D) signaling in pancreatic ductal adenocarcinoma (PDA). KRAS is one of the most frequently activated oncogenic drivers in cancer (Pylayeva-Gupta et al., 2011) and is mutated in >90% of PDA tumor cells (Almoguera et al., 1988). PDA is an extremely heterocellular malignancy—composed of mutated tumor cells, stromal fibroblasts, endothelial cells, and immune cells (Neesse et al., 2011). Crucially, the gross stromal pancreatic stellate cell (PSC) expansion observed in the PDA microenvironment is non-cell-autonomously controlled by tumor cell KRASG12D in vivo (Collins et al., 2012, Ying et al., 2012). As a result, understanding the heterocellular signaling consequences of KRASG12D is essential to comprehend PDA tumor biology.

Comprehensive analysis of tumor-stroma signaling requires concurrent measurement of cell-specific phosphorylation events. Recent advances in proteome labeling now permit cell-specific phosphoproteome analysis in heterocellular systems (Gauthier et al., 2013, Tape et al., 2014a). Furthermore, advances in proteomic multiplexing enable deep multivariate phospho-signaling analysis (McAlister et al., 2012, Tape et al., 2014b).

Here, we combine cell-specific proteome labeling, multivariate phosphoproteomics, and inducible oncogenic mutations to describe KRASG12Dcell-autonomous, non-cell-autonomous, and reciprocal signaling across a heterocellular system. This study reveals KRASG12D uniquely regulates tumor cells via heterotypic stromal cells. By exploiting heterocellularity, reciprocal signaling enables KRASG12D to engage oncogenic signaling pathways beyond those regulated in a cell-autonomous manner. Expansion of KRASG12D signaling via stromal reciprocation suggests oncogenic communication should be viewed as a heterocellular process.


Whether oncogenes regulate tumor cell signaling via stromal cells is a fundamental question in tumor biology. Using heterocellular multivariate phosphoproteomics, we demonstrate how oncogenic KRAS signals through local non-tumor cells to achieve a differential reciprocal signaling state in the inceptive tumor cells. In PDA, this reciprocal axis supplements oncogenic cell-autonomous signaling to control protein abundance, transcription, mitochondrial activity, proliferation, apoptosis, and colony formation. Reciprocal signaling is the exclusive product of heterocellularity and cannot be achieved by tumor cells alone. These observations imply oncogenes expand their capacity to deregulate cellular signaling via stromal heterocellularity (Figure 7).

Despite the well-established heterocellularity of cancer, our understanding of oncogenic signaling within tumor cells has largely excluded non-tumor cells. We observe that stromal cells approximately double the number of tumor cell signaling nodes regulated by oncogenic KRAS, suggesting both cell-autonomous (internal) and reciprocal (external) stimuli should be considered when defining aberrant oncogenic signaling states. For example, although KRAS is thought to cell-autonomously regulate AKT in PDA (Eser et al., 2014), we show that KRASG12D activates AKT, not cell-autonomously, but reciprocally. As PI3K signaling is essential for PDA formation in vivo (Baer et al., 2014, Eser et al., 2013, Wu et al., 2014) reciprocal signaling may control oncogene-dependent tumorigenesis. Our findings suggest future genetic studies should consider the heterocellular signaling consequences of oncogene/tumor-suppressor deregulation.

The observation that many oncogene-dependent tumor cell signaling nodes require reciprocal activation has important implications for identifying pharmacological inhibitors of oncogene signaling. For example, if PDA tumor cells were screened alone, one would expect MEK, MAPK, and CDK inhibitors to perturb KRASG12D signaling. However, when screened in conjunction with heterotypic stromal cells, our study additionally identified SHH, AKT, and IGF1R/AXL inhibitors as KRASG12D-dependent targets in tumor cells. Inhibitors of signaling specific to reciprocally engaged tumor cells, such as or AKT or IGF1R/AXL, block heterocellular phenotypes (e.g., protein expression, proliferation, mitochondrial performance, and anti-apoptosis), but have little effect on KRASG12D tumor cells alone. An appreciation of reciprocal nodes increases our molecular understanding of drug targets downstream of oncogenic drivers and highlights focal points where reciprocal signals converge (e.g., AKT). These trans-cellular observations reinforce the importance of understanding cancer as a heterocellular disease.

Previous work in PDA tumor cells under homocellular conditions demonstrated cell-autonomous KRASG12D shifts metabolism toward the non-oxidative pentose phosphate pathway (Ying et al., 2012), whereas KRASG12D-ablated cells depend on mitochondrial activity (Viale et al., 2014). Here, we show that heterocellular reciprocal signaling can restore the expression of mitochondrial proteins and subsequently re-establish both mitochondrial polarity and superoxide levels. This suggests KRASG12D regulates non-oxidative flux through cell-autonomous signaling and mitochondrial oxidative phosphorylation through reciprocal signaling. These results provide a unique example of context-dependent metabolic control by oncogenes and reinforce the emerging role of tumor-stroma communication in regulating cancer metabolism (Ghesquière et al., 2014).

In PDA, the stroma has dichotomous pro-tumor (Kraman et al., 2010, Sherman et al., 2014) and anti-tumor (Lee et al., 2014, Rhim et al., 2014) properties. It is becoming increasingly evident that non-cell-autonomously activated stromal cells vary within a tumor and can influence tumors in a non-obvious manner. For example, while vitamin D receptor normalization of stromal fibroblasts improves PDA therapeutic response (Sherman et al., 2014), total stromal ablation increases malignant behavior (Lee et al., 2014, Rhim et al., 2014). Thus, while stromal purging is unlikely to provide therapeutic benefit in PDA, “stromal reprogramming” toward an anti-tumor stroma is now desirable (Brock et al., 2015). Although we describe a largely pro-tumor reciprocal axis, both pro- and anti-tumor stromal phenotypes likely transduce across reciprocal signaling networks. Our work suggests future efforts to therapeutically reprogram the PDA stroma toward anti-tumor phenotypes will require an understanding of reciprocal signaling. In describing the first oncogenic reciprocal axis, this study provides a foundation to measure the cell-cell communication required for anti-tumor stromal reprogramming.

We demonstrate heterocellular multivariate phosphoproteomics can be used to observe reciprocal signaling in vitro. Unfortunately, cell-specific isotopic phosphoproteomics is not currently possible in vivo. To delineate reciprocal signaling in vivo, experimental systems must support manipulation of multiple cell-specific variables and provide cell-specific signaling readouts. Simple pharmacological perturbation of reciprocal nodes (e.g., IGF1R, AXL, AKT, etc.) in existing PDA GEMMs will in principle affect all cell types (e.g., tumor cells, PSCs, immune cells) and cannot provide axis-specific information in vivo. Future in vivo studies of reciprocal signaling will require parallel inducible genetic manipulation (e.g., oncogene activation in cancer cell and/or inhibition of reciprocal node in stromal cell), combined with cell-specific signaling data (e.g., using epithelial tissue mass-cytometry) (Simmons et al., 2015).

We describe KRASG12D reciprocal signaling between PDA tumor cells and PSCs. However, it is likely oncogenic reciprocal signaling occurs across multiple different cell types in the tumor microenvironment. For example, in PDA, FAP+stromal fibroblasts secrete SDF1 that binds tumor cells to suppress T cells (Feig et al., 2013). Our model predicts oncogene signaling expands across several cell types in the tumor microenvironment—including immune cells. Moreover, as oncogenes non-cell-autonomously regulate the stroma in many other tumor types (Croce, 2008), our model predicts oncogenic reciprocal signaling to be a broad phenomenon across all heterocellular cancers. The presented heterocellular multivariate phosphoproteomic workflow now enables future characterization of oncogenic reciprocal signaling in alternative cancer types.

As differentiated cells process signals in unique ways, heterocellularity provides increased signal processing space over homocellularity. We provide evidence that KRASG12D exploits heterocellularity via reciprocal signaling to expand tumor cell signaling space beyond cell-autonomous pathways. Given the frequent heterocellularity of solid tumors, we suspect reciprocal signaling to be a common—albeit under-studied—axis in oncogene-dependent signal transduction.

New Genomic Analysis of Immune Cell Infiltration in Colorectal Cancer


Through whole-exome sequencing of colorectal tumors, researchers were able to identify additional driver genes that correlate high neoantigen load with increased lymphocytic infiltration and improved survival. [Giannakis et al., 2016, Cell Reports 15, 1–9]     http://www.genengnews.com/Media/images/GENHighlight/thumb_fx11248133146.jpg

The past several years have seen some exciting results for cancer immunotherapy. However, there remains a fundamental lack of understanding of immune system recognition in various cancers. Many large-scale sequencing efforts have added to our collective knowledge base, but too many of these studies have been deficient in comprehensive epidemiological and demographic information.

Now, researchers at the Dana-Farber Cancer Institute and the Broad Institute of MIT and Harvard report on their findings from a new study, which found that colorectal cancers festooned with tumor-related proteins called neoantigens were likely to be saturated with disease-fighting white blood cells, mainly lymphocytes.

Using several data sets from patients in two large health-tracking studies, the Nurses’ Health Study and the Health Professionals Follow-up Study, investigators performed whole-exome sequencing on colorectal tumor samples from 619 patients—itemizing each DNA base that specifies how cell proteins are to be constructed. This information was merged with data from tests of the immune system’s response to the tumors and with patient clinical data, including length of survival.

“We were looking for genetic features that predict how extensively a tumor is infiltrated by lymphocytes and which types of lymphocytes are present,” explained co-lead study author, Marios Giannakis, M.D., Ph.D., medical oncologist and clinical investigator at the Dana-Farber Gastrointestinal Cancer Treatment Center, and researcher at the Broad Institute of MIT and Harvard. “We found that tumors with a high ‘neoantigen load’—which carry large quantities of neoantigens—tended to be infiltrated by a large number of lymphocytes, including memory T cells, which provide protection against previously encountered infections and diseases. Patients whose tumors had high numbers of neoantigens also survived longer than those with lower neoantigen loads.”

The findings from this study were published recently in Cell Reports in an article entitled “Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma.”

Neoantigens are mutated forms of protein antigens, which are found on normal cells. Genetic mutations often cause cancer cells to produce abnormal proteins, some of which get trafficked to the cell surface, where they serve as a red flag to the immune system that something has gone awry with the cell.

“There can be hundreds or thousands of neoantigens on tumor cells,” noted Dr. Giannakis explained. “Only a few of these may actually provoke T cells to infiltrate a tumor. However, the more neoantigens on display, the greater the chance that some of them will spark an immune system response.”

Physicians often take advantage of therapies known as immune checkpoint inhibitors, which work by removing some of the barriers to an immune system attack on cancer. Although these agents have produced astonishing results in some cases, they’re effective only in patients whose immune system has already launched an immune response to cancer. This new study may help investigators identify which patients are most likely to benefit in new clinical trials of immune checkpoint inhibitors by showing that tumors with high antigen loads are apt to be laced with T cells—and therefore able to provoke an immune response.

Interestingly, this new analysis found several often-mutated genes that had not previously been strongly associated with the disease, including BCL9L, RBM10, CTCF, and KLF5, suggesting that they may be valuable targets for new therapies.

“Our study helps shed light on the overall development of colorectal cancer,” Dr. Giannakis remarked. “It also shows the insights that can be gained by integrating molecular research with findings from other areas such as epidemiology and immunology.”

“Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma”


Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival
Scott D. Brown,1,2 Rene L. Warren,1 Ewan A. Gibb,1,3 Spencer D. Martin,1,3,4 John J. Spinelli,5,6 Brad H. Nelson,3,4,7 and Robert A. Holt1,3,8,9
Genome Res. 2014 May; 24(5): 743–750.    doi:  10.1101/gr.165985.113

Somatic missense mutations can initiate tumorogenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. Tumor genome analysis has revealed extreme heterogeneity among tumor missense mutation profiles, but their relevance to tumor immunology and patient outcomes has awaited comprehensive evaluation. Here, for 515 patients from six tumor sites, we used RNA-seq data from The Cancer Genome Atlas to identify mutations that are predicted to be immunogenic in that they yielded mutational epitopes presented by the MHC proteins encoded by each patient’s autologous HLA-A alleles. Mutational epitopes were associated with increased patient survival. Moreover, the corresponding tumors had higher CTL content, inferred from CD8A gene expression, and elevated expression of the CTL exhaustion markers PDCD1 and CTLA4. Mutational epitopes were very scarce in tumors without evidence of CTL infiltration. These findings suggest that the abundance of predicted immunogenic mutations may be useful for identifying patients likely to benefit from checkpoint blockade and related immunotherapies.

The accumulation of somatic mutations underlies the initiation and progression of most cancers by conferring upon tumor cells unrestricted proliferative capacity (Hanahan and Weinberg 2011). The analysis of cancer genomes has revealed that tumor mutational landscapes (Vogelstein et al. 2013) are extremely variable among patients, among different tumors from the same patient, and even among the different regions of a single tumor (Gerlinger et al. 2012). There is a need for personalized strategies for cancer therapy that are compatible with mutational heterogeneity, and in this regard, immune interventions that aim to initiate or enhance anti-tumor immune responses hold much promise. Therapeutic antibodies and chimeric antigen receptor (CAR) technologies have shown anti-cancer efficacy (Fox et al. 2011), but such antibody-based approaches are limited to cell surface target antigens (Slamon et al. 2001; Coiffier et al. 2002; Yang et al. 2003;Cunningham et al. 2004; Kalos et al. 2011). In contrast, most tumor mutations are point mutations in genes encoding intracellular proteins. Short peptide fragments of these proteins, after intracellular processing and presentation at the cell surface as MHC ligands, can elicit T cell immunoreactivity. Further, the presence of tumor infiltrating lymphocytes (TIL), in particular, CD8+ T cells, has been associated with increased survival (Sato et al. 2005; Nelson 2008; Oble et al. 2009; Yamada et al. 2010; Gooden et al. 2011; Hwang et al. 2012), suggesting that the adaptive immune system can mount protective anti-tumor responses in many cancer patients (Kim et al. 2007; Fox et al. 2011). The antigen specificities of tumor-infiltrating T cells remain almost completely undefined (Andersen et al. 2012), but there are numerous examples of cytotoxic T cells recognizing single amino acid coding changes originating from somatic tumor mutations (Lennerz et al. 2005;Matsushita et al. 2012; Heemskerk et al. 2013; Lu et al. 2013; Robbins et al. 2013;van Rooij et al. 2013; Wick et al. 2014). Thus, the notion that tumor mutations are reservoirs of exploitable neo-antigens remains compelling (Heemskerk et al. 2013). For a mutation to be recognized by CD8+ T cells, the mutant peptide must be presented by MHC I molecules on the surface of the tumor cell. The ability of a peptide to bind a given MHC I molecule with sufficient affinity for the peptide-MHC complex to be stabilized at the cell surface is the single most limiting step in antigen presentation and T cell activation (Yewdell and Bennink 1999). Recently, several algorithms have been developed that can predict which peptides will bind to given MHC molecules (Nielsen et al. 2003; Bui et al. 2005; Peters and Sette 2005; Vita et al. 2010; Lundegaard et al. 2011), thereby providing guidance into which mutations are immunogenic.

The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/) is an initiative of the National Institutes of Health that has created a comprehensive catalog of somatic tumor mutations identified using deep sequencing. As a member of The Cancer Genome Atlas Research Network, our center has generated extensive tumor RNA-seq data. Here, we have used public TCGA RNA-seq data to explore the T cell immunoreactivity of somatic missense mutations across six tumor sites. This type of analysis is challenged not only by large numbers of mutations unique to individual patients, but also by the complexity of personalized antigen presentation by MHC arising from the extreme HLA allelic diversity in the outbred human population. Previous studies have explored the potential immunogenicity of tumor mutations (Segal et al. 2008; Warren and Holt 2010; Khalili et al. 2012), but these have been hampered by small sample size and the inability to specify autologous HLA restriction. Recently, we described a method of HLA calling from RNA-seq data that shows high sensitivity and specificity (Warren et al. 2012). Here, we have obtained matched tumor mutational profiles and HLA-A genotypes from TCGA subjects and used these data to predict patient-specific mutational epitope profiles. The evaluation of these data together with RNA-seq-derived markers of T cell infiltration and overall patient survival provides the first comprehensive view of the landscape of potentially immunogenic mutations in solid tumors.    …..

The results of the present study have clinical implications. We have shown that patients with tumors bearing missense mutations predicted to be immunogenic have a survival advantage (Fig. 3D). These tumors also show evidence of higher CD8+ TIL, which suggests that a number of these mutations might be immunoreactive. The existence of these mutations is encouraging because, in principle, they could be leveraged by personalized therapeutic vaccination strategies or adoptive transfer protocols to enhance anti-tumor immunoreactivity. Likewise, patients with tumors showing naturally immunogenic mutations and associated TIL are potential candidates for treatment with immune modulators such as CTLA4- or PDCD1-targeted antibodies. There is evidence that such therapies are most effective against tumors infiltrated by T cells (Moschos et al. 2006; Hamid et al. 2009). Our results indicate that tumors bearing predicted immunogenic mutations have not only elevated CD8A expression (Fig. 3C) but also elevated expression of CTLA4 and PDCD1 (Fig. 4), reinforcing the notion that these patients may be optimal candidates for immune modulation. Importantly, we observed that tumors with low levels of CD8+ TIL invariably have far fewer immunogenic mutations. Such patients would be better suited to conventional therapy or to immunotherapies (e.g., chimeric antigen receptor modified T cells) that target nonmutated antigens.

  1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229261/Apr 12, 2011 Keywords: tumor immune infiltrate, T-cells, cancer prognosis, colon … By conducting genomic and in situ immunostaining on resected tumors from ….. of tumor-infiltrating immune cells correlates with better overall survival.

  2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234325/May 27, 2011 Keywords: Colorectal cancer, Immune response, T lymphocytes, Microsatellite instability … In CRC, at least 3 distinct pathways of genomic instability have been …. The potential influence of these immunecell infiltrates in CRC on the … in controlling cytolytic activity of CD8+ T cells, inversely correlates to the …   


Cancer-Associated Immune Resistance and Evasion of Immune Surveillance in Colorectal Cancer

Gastroenterology Research and Practice
Volume 2016 (2016), Article ID 6261721, 8 pages

Data from molecular profiles of tumors and tumor associated cells provide a model in which cancer cells can acquire the capability of avoiding immune surveillance by expressing an immune-like phenotype. Recent works reveal that expression of immune antigens (PDL1, CD47, CD73, CD14, CD68, MAC387, CD163, DAP12, and CD15) by tumor cells “immune resistance,” combined with prometastatic function of nonmalignant infiltrating cells, may represent a strategy to overcome the rate-limiting steps of metastatic cascade through (a) enhanced interactions with protumorigenic myeloid cells and escape from T-dependent immune response mediated by CD8+ and natural killer (NK) cells; (b) production of immune mediators that establish a local and systemic tumor-supportive environment (premetastatic niche); (c) ability to survive either in the peripheral blood as circulating tumor cells (CTCs) or at the metastatic site forming a cooperative prometastatic loop with foreign “myeloid” cells, macrophages, and neutrophils, respectively. The development of cancer-specific “immune resistance” can be orchestrated either by cooperation with tumor microenvironment or by successive rounds of genetic/epigenetic changes. Recognition of the applicability of this model may provide effective therapeutic avenues for complete elimination of immune-resistant metastatic cells and for enhanced antitumor immunity as part of a combinatorial strategy.

1. Introduction

Metastasis remains the most significant cause of cancer-associated morbidity and mortality and specific targeting molecules have had limited success in reversing metastatic progression in the clinical setting [13]. Understanding the exact molecular and cellular basis of the events that facilitate cancer metastasis has been difficult so far. Over the past years, a well-accepted theory suggests that genomic alterations of the malignant cells accompanied by the so-called tumor microenvironment “nonmalignant cells” contribute to the metastatic cascade [4, 5]. As such, metastasis is frequently described as the sequential execution of multiple steps. To establish the metastatic tumor, cancer cells have to acquire the traits that enable them to efficiently cooperate with the host stroma and simultaneously avoid antitumor immune response [49]. At early stage of carcinogenesis, tumors appear to be vulnerable because mutant and thus potentially immunogenic tumor cells are being exposed to the immune system, which can recognize them and restrict their growth [10, 11]. This is the case of tumor-infiltrating immune cells particularly CD8+ T cells and NK cells which have the potential to restrict the tumor outgrowth or reject metastatic tumor cells [12, 13]. According to this notion, in most primary tumors, a strong Th1/cytotoxic T cells infiltration correlates with a longer patient’s survival [1214]. Unfortunately, tumor develops multiple mechanisms of evading immune responses, by forming a compromised microenvironment that allows the dissemination of malignant cells in a foreign microenvironment through molecular mechanisms still poorly characterized. A variety of stromal cells, particularly M2-phenotype macrophages and myeloid-derived suppressor cells (MDSCs), are recruited to primary tumors; these not only enhance growth of the primary cancer but also facilitate its metastatic dissemination to distant organs [13, 14]. Notably, cooperative “dialogue” between malignant cells and their microenvironment will go on in the systemic circulation and subsequently in the future metastatic site [1317]. In fact, recent studies have demonstrated that a high systemic inflammatory response, that is, blood neutrophil-lymphocyte ratio (NLR), predicts lower overall survival, higher tumor stage, and a greater incidence of metastasis in multiple tumor types [18, 19]. Therefore, a substantial amount of data suggests a novel dimension of the tumor biology and offers the opportunity to revisit the mechanisms describing evasion of cancer immunosurveillance during the metastatic process. The present review analyses recent studies that elucidate and reinforce the theory by which immune-phenotypic features or “immune resistance” by cancer cells may need to sustain the metastatic cascade and avoid antitumor immune response.

2. Tumor Antigens and Antitumor Immune Response by Effectors of Adaptive Immunity

A decade of studies has emphasized the nature of cancer as a systemic disease remarking a key role of host microenvironment as a critical hallmark. As a result, a new picture of cancer is emerging in particular due to unexpected cross-talk between malignant cells and the immune system [35]. Recent data have expanded the mechanisms of cancer-immune system interactions revealing that every known innate and adaptive immune effector component participates in tumor recognition and control [9, 10]. It is now recognized that in different individuals and with different cancers, at early stage of tumorigenesis, the few cancer cells are detected by NK cells through their encounter with specific ligands on tumor cells [5]. In turn, activation of macrophages and dendritic cells and particularly T and B cells expands production of additional cytokines and further promotes activation of tumor-specific T cells “CD8+ cytotoxic T cells” leading to the generation of immune memory to specific tumor components [1416]. However, in cases where the immune system is not able to eliminate the cancer, a state of equilibrium develops or eventually cancer cells can resist, avoid, or suppress the antitumor immune response, leading to the immune escape and a fully developed tumor (Figure 1) [915]. For example, investigations into the nature of cancer as a genetic disease have suggested two paradigmatic subtypes of colorectal cancer (CRC): chromosomal instability (CIN) and microsatellite instability (MSI), in which the expression of immune-checkpoint proteins can be differentially dysregulated to unleash the potential of the antitumor immune response [11]. In particular, tumors with mismatch-repair deficiency (dMMR) (10–20%) of advanced colorectal cancer tend to have 10 to 100 times more somatic mutations and higher amount of lymphocyte infiltrates than mismatch-repair-proficient colorectal cancers (pMMR), a finding consistent with a stronger antitumor immune response (Figure 1) [11, 20]. According to this notion, recent studies suggest that certain cancer subtypes dMMR CRC with high numbers of somatic mutations are more responsive to PD-1 blockade, a well-known immune-checkpoint inhibitor [20]. In particular, CD8-positive lymphoid infiltrate and membranous PDL1 expression on either tumor cells or tumor-infiltrating lymphocytes at the invasive fronts of the tumor are associated with an improved response to anti-PD-1 therapy in patients with mismatch-repair-deficient cancer [11, 20]. In addition, cancer subtypes with stronger antitumor immune responses (immunogenic) are characterized by surface-exposed calreticulin or heat shock protein 90 (HSP90), which serve as a powerful mobilizing signal to the immune system in the context of damage-associated molecular patterns (DAMPs) [17]. As danger signals, DAMPs accompanied by subversion MHC Class I and II antigens on the plasma membrane of cancer cells appear to be characteristic of stressed or injured cells and can act as adjuvant signals to enhance antitumor immunity mediated by the innate immune system [17]. As described in this review, unfortunately, the large majority of human tumors can suppress the immune system to enhance their survival, rendering them invisible to cytotoxic T lymphocytes through a variety of mechanisms. Furthermore, in most cases, tumor-infiltrating immune cells differentiate into cells that promote each step of the tumor progression supporting ability of cancer cells to invade and survive in foreign organs. In addition, the intricate network of malignant and immune components represents a prominent obstacle to the effects of therapeutic agents.


High-resolution genomic analysis: the tumor-immune interface comes into focus

Jonathan J Havel1 and Timothy A Chan1,2*
Havel and Chan     Genome Biology (2015) 16:65      http://dx.doi.org:/10.1186/s13059-015-0631-3

A genomic analysis of heterogeneous colorectal tumor samples has uncovered interactions between immunophenotype and various aspects of tumor biology, with implications for informing the choice of immunotherapies for specific patients and guiding the design of personalized neoantigen-based vaccines.

Please see related article: http://dx.doi.org/10.1186/s13059-015-0620-6

Immunotherapy is a promising new approach for treating human malignancies. Approximately 20% of melanoma and lung cancer patients receiving immune checkpoint inhibitors show responses [1,2]. Current major challenges include identification of patients most likely to respond to specific therapies and elucidation of novel targets to treat those who do not. To address these problems, a detailed understanding of the dynamic interactions between tumors and the immune system is required. In a new study, Zlatko Trajanoski and colleagues [3] describe a powerful approach to dissecting these issues through high-resolution analysis of patient genomic data. This study represents a significant advance over previous work from this group, which defined 28 immune-cell-type gene expression signatures and identified specific cell types as prognostic indicators in colorectal cancer (CRC) patients [4]. Here, the authors [3] integrate genomic analyses of CRC tumor molecular phenotypes, predicted antigenicity (called the ‘antigenome’), and immune-cell infiltration derived from multiple independent cohorts to gain refined insights into tumor-immune system interactions.

Not all tumor-infiltrating lymphocytes are created equal

Past studies have used immune-staining techniques to determine associations between a limited set of infiltrating immune cells and patient survival [5] or tumor molecular phenotype [6]. Here, the authors [3] use gene set enrichment analysis (GSEA) of immune cell expression signatures to ascertain associations of 28 immune-cell populations with patient survival and tumor molecular phenotypes. Effector memory CD8+ and CD4+ T cells, natural killer cells, and activated dendritic cells are significantly associated with improved overall survival. Interestingly, although the authors’ previous work found no significant prognostic value of regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs) [4], negative associations of these cell types with overall survival are among the strongest relationships observed in the current study. It is possible that variations in sample collection and preparation may have contributed to this discrepancy. The conclusions, supported by the numerous animal studies demonstrating the importance of cell-mediated immunosuppression, are substantially strengthened by a much larger cohort size used in this study.

Another important observation is the association of specific immune cell subsets with CRC tumor stage and molecular phenotypes as classified by mutation rate, microsatellite instability, and methylation status. This knowledge will be crucial in determining which types of immunotherapy are most likely to benefit individual patients. Interestingly, although hypermutated microsatellite-unstable tumors show strong enrichment of adaptive immune cells, similar enrichment is notably lacking in the small population of hypermutated microsatellite-stable tumors. This raises an intriguing question of whether and how microsatellite instability/mismatch repair may independently shape immune responses. Furthermore, Trajanoski and colleagues [3] find that tumor-infiltrating lymphocytes transition from an adaptive to an innate immunophenotype with increasing tumor stage. This raises an interesting issue of whether immunotherapies that depend on the adaptive immune response can be effective in later stage CRC tumors.

Diversity of tumor antigens

In addition to characterizing immune components involved in tumor immune responses, it is equally important to identify and understand the tumor-associated antigens that elicit these responses, called the ‘antigenome’. The authors [3] analyze RNA-seq and genomic data to identify two types of tumor antigens in CRC – non-mutated cancer germline antigens that are aberrantly overexpressed, and neoantigens, which are generated from non-synonymous somatic mutations. Importantly, the authors [3] find that cancer-germline antigens are highly shared among patients and are independent of molecular and immune phenotype. In contrast, neoantigens are enriched in the hypermutated microsatellite-unstable phenotype tumors and rarely shared among patients. These results imply a heightened importance of neoantigens in comparison to cancer-germline antigens [7]. In addition, similar analytical methods have recently been applied to identify functional neoantigens in human melanoma and cholangiocarcinoma [810]. An emerging theme of these studies is that the in vitro validation rate for predicted neoantigens is relatively low; however, it is unclear whether this is due to limited sensitivity of functional assays or epigenetic silencing to circumvent immunoediting, or whether the number of immunogenic neoantigens is in fact small. Interestingly, Trajanoski and colleagues [3] find a modest yet significant decrease in neoantigen frequency with increasing tumor stage. Considering the concomitant decrease in adaptive immune cell infiltration, it is tempting to speculate that this phenomenon reflects immunoediting of critical neoepitopes during tumor progression. Furthermore, the authors find an association, albeit not statistically significant, between increased neoantigen burden and improved patient survival. This finding complements a recent report [9] showing that whereas neoantigen burden roughly predicts survival of anti-CTLA-4-treated melanoma patients, a collection of consensus neoepitope motifs is strongly associated with patient survival. It will be interesting to see if future studies can determine the effect of CRC neoantigen burden in the setting of immunotherapy, and answer the questions of whether an analogous signature of prognostic neoepitope motifs exists for CRC, and whether there are any similarities between substring signatures of different tumor types.


Not Your Average Circulating Tumor Cells   

Translational Scientists Profile Cancer Cells That Have Gone on the Lam

GEN Apr 15, 2016 (Vol. 36, No. 8)    http://www.genengnews.com/gen-articles/not-your-average-circulating-tumor-cells/5737/


  • For most malignant tumors, morbidity and mortality are, to a great extent, the result of metastatic dissemination, as opposed to the presence of the primary tumor.

    The existence of circulating tumor cells, which can be shed into the circulation by primary or metastatic malignancies, was first recognized almost 150 years ago, and their diagnostic and therapeutic values have been increasingly appreciated during the last few decades.

    One of the unique characteristics of circulating tumor cells is that they are in a fundamentally different environment from that established in either the primary tumor or the metastatic one. Although circulating tumor cells can be kept in place so that they can be assessed, the usual technique—immobilization to a solid surface—tends to yield distorted results. Free-floating cells are molecularly and functionally distinct from immobilized cells. For example, nonadhering breast cancer cells were shown to have tubulin-based microtentacles that shape their dynamic behavior, including their aggregation, retention in organs, or interaction with the endothelium.

    “These microtentacles are very hard to study because they depolymerize when cells bind either an endothelial cell or another tumor cell,” says Christopher M. Jewell, Ph.D., assistant professor of bioengineering  at the University of Maryland College Park. “Cells that form microtumors undergo massive mechanochemical and phenotypic changes as compared to when they are floating or circulating.”

    Characterizing circulating tumor cells, then, seems to amount to capturing the substance of freedom, a task that sounds self-defeatingly paradoxical—or at least fraught with difficulties. Overcoming difficulties, however, would likely be worth the effort. Two areas that immediately benefit from the characterization of circulating tumor cells are diagnostics and therapeutics.

    Capturing and analyzing circulating tumor cells opens not only the possibility of diagnosing patients earlier and more accurately, but also the potential for identifying new approaches to targeting malignancies. “Many groups are working on important technologies to capture circulating tumor cells,” informs Dr. Jewell. “We’re working on new technologies to analyze these populations.”

  • Floating in Place

    To address the existing gap in characterizing the biology of free-floating cancer cells, Dr. Jewell and collaborators in the University of Maryland laboratory of physiologist Stuart Martin, Ph.D., have designed an unusual  microfluidic device. It can spatially immobilize free-floating tumor cells while maintaining their free-floating characteristics.

    In this microfluidic device, polyelectrolyte multilayers inhibit the attachment of cells to multiwall plates, allowing their free-floating functional and morphological characteristics to be visualized and studied. Lipid tethers incorporated into the device interact with the cell membrane and allow cells to remain loosely attached and spatially localized, offering the possibility to perform applications such as real-time imaging and drug screening.

    “We are trying to understand what the signaling changes are in individual circulating tumors cells that are not nucleating into a tumor,” explains Dr. Jewell, “as compared to cells that contact enough cells and nucleate to form a tumor.”

    Surface tethering of circulating tumor cells also provides the opportunity to capture arrays of tumor cells; to introduce a perturbation such as a drug or a change in flow rate or mechanical properties; and then to collect the same individual cells that had already been imaged. In these cells, morphological changes can be correlated with genomic or proteomic information, providing an opportunity to dynamically understand how the mechanochemical properties of the cells change in response to external perturbations.

    “Our collaborators,” notes Dr. Jewell, “are also developing algorithms to quantify some of the features of microtentacles and convert visual information into quantitative metrics.”

  • Filterless Filters

    At the University of California, Los Angeles, Dino Di Carlo, Ph.D., and colleagues have developed High-Throughput Vortex Chip (Vortex-HT) technology, which uses parallel microfluidic vortex chambers to accumulate the larger circulating tumor cells from flowing blood. Vortex-HT reportedly generates less contamination with white blood cells than other technologies and isolates cells in a smaller output volume.

    Early techniques to capture circulating tumor cells have taken advantage of cell size differences, leading to the development of filtration-based approaches. This was followed, more recently, by the emergence of inertial microfluidic-based approaches, of which vortex technology is one example.

    “We think of vortex technology as a filterless filter,” says Dino Di Carlo, Ph.D., professor of bioengineering and director of the Cancer Nanotechnology Program at the Jonsson Comprehensive Cancer Center of the University of California, Los Angeles. “There aren’t any structures that are smaller than the cell types, but cells are still isolated based on size.”

    Dr. Di Carlo and colleagues recently developed the High-Throughput Vortex Chip (Vortex HT), an improved microfluidic technology that allows the label-free, size-based enrichment and concentration of rare cells. The strategy involves minimal pretreatment steps, reducing cell damage, and allows an approximately 8 mL vial of blood to be processed within 15–20 minutes.

    “With this approach,” asserts Dr. Di Carlo, “we can concentrate cells from any volume to about 100 µL.”

    Circulating tumor cells can then be used for subsequent steps, such as real-time imaging or immunostaining. The capture efficiency, up to 83%, is slightly lower than with Dean flow fractionation and CTC-iChip, but Vortex HT generates much less contamination with white blood cells than other technologies and isolates cells in a smaller output volume.

    Along with circulating tumor cells, another promising noninvasive biomarker is provided by circulating tumor DNA. Such DNA can be detected in the plasma or serum of many cancer patients as a result of the active or passive release of nucleic acid from apoptotic or necrotic tumor cells.

    While circulating tumor DNA can be used to dynamically collect information about specific mutations, and provides advantages for some applications, it is not powered to offer certain types of information that can be captured only from circulating tumor cells. For example, it cannot provide details about cellular morphology or protein expression and localization. Also, it cannot enable investigators to perform proteomic profiling in parallel with genomic profiling.

    These are not the only situations in which circulating DNA serves as a poor substitute for circulating tumor cells. “Another example,” notes Dr. Di Carlo, occurs with “applications that involve a drug screen that seeks to determine whether cells are sensitive or resistant to a particular compound.” Additionally, for certain cancers that have no dominant mutations, or for which mutations are not well known, circulating tumor DNA cannot provide the information that can be interrogated from profiling circulating tumor cells.

    • Insights beyond Counting

      “The field started off by enumerating circulating tumor cells as a potential biomarker,” says David T. Miyamoto, M.D., Ph.D., assistant professor of radiation oncology at Harvard Medical School and the Massachusetts General Hospital (MGH). “It is currently moving toward performing detailed molecular analyses of these circulating tumor cells and using them as a form of liquid biopsy that allows us to gain insights into the molecular biology of the tumor itself.”

      The Circulating Tumor Cell Center at MGH, led by Daniel Haber, M.D., Ph.D., and Mehmet Toner, Ph.D., has developed three generations of microfluidic technology. The technology of the first two generations captured circulating tumor cells on microfluidic surfaces. The technology of the third generation, known as CTC-iChip technology, introduces the unique capability—isolating cells in solution. Once the circulating tumor cells are captured or isolated, notes Dr. Miyamoto, they can be subjected to “a variety of sophisticated molecular analyses.”

      In a recent study using the CTC-iChip technology, Dr. Miyamoto and colleagues performed single-cell RNA sequencing. The investigators used 77 circulating tumor cells isolated by microfluidic enrichment from 13 patients.

      “The goal of this work was to use the circulating tumor cell technology to identify potential resistance mechanisms in metastatic castration-resistant prostate cancer,” explains Dr. Miyamoto. In patients who were undergoing therapy with an androgen receptor inhibitor, the retrospective analysis of their circulating tumor cells revealed that the noncanonical Wnt signaling pathway may play a role in resistance to therapy.

      “We need to validate the findings in larger patient cohorts,” concludes Dr. Miyamoto. “But this proof-of-concept study shows that detailed molecular analyses of liquid biopsy samples can be used to identify potentially clinically relevant mechanisms of resistance that can then be exploited to guide patient care.”

    • Variable to the Last

      Morphotek, a biopharmaceutical company that specializes in the development of protein and antibody products through the use of gene-evolution technology, has developed the ApoStream device, which uses continuous field-flow-assist and dielectrophoresis technology to isolate and recover circulating tumor cells from the blood of cancer patients. In a recent study in Biomarker Insights, Morphotek scientists described how they interrogated ApoStream-isolated circulating tumor cells by employing laser-scanning cytometry using highly selective antibodies. The scientists detected folate receptor alpha (FRα) expression in CK+/CD45 cells isolated from lung cancer, as indicated in these representative images.

      “Most of the work on circulating tumor cells has been done in late-stage cancers to direct therapeutic interventions,” says Daniel J. O’Shannessy, Ph.D., head of translational medicine and diagnostics at Morphotek. “Even in late-stage cancers, there is a great deal of variability with respect to the numbers of cells shed for a cancer type but especially between cancer types.”

      Many studies correlated the presence of circulating tumor cells with prognosis in several cancers, including breast, lung, and colorectal malignancies. However, one of the challenges associated with analyzing circulating tumor cells is that not every cancer releases them into the circulation. Also, even among cancers that do, not every cancer generates a lot of these cells.

      For example, as estimated using current techniques, ovarian cancers do not appear to shed as many circulating tumor cells as several other malignancies. “Another challenge is that existing technologies are often limited by sensitivity much more than by specificity,” cautions Dr. O’Shannessy. This limitation has the potential to make interpatient comparisons, and even the longitudinal follow-up of patients, particularly difficult.

      Previously, investigators at Morphotek described ApoStream®, a device that uses continuous field-flow-assist and dielectrophoresis technology to isolate and recover circulating tumor cells from the blood of cancer patients. In a recent study, Dr. O’Shannessy and colleagues used laser-scanning cytometry and highly selective antibodies to identify folate receptor alpha-positive cells from circulating tumor cells that had been isolated using the ApoStream technology.

      This proof-of-principle study was able to detect folate receptor alpha-positive cells in patients with breast cancer, ovarian cancer, and non-small cell lung adenocarcinoma, but not in patients with squamous cell lung cancer. These findings supporting previous findings that were made using the respective primary or metastatic tumors.

      The study demonstrated the utility of following the enrichment and identification of circulating tumor cells with immunofluorescence staining for a specific tumor marker. This combination of approaches emerges as a valuable noninvasive strategy for differentiating among tumor types. It can also be used to examine heterogeneous cell populations within tumors, particularly when tissue samples are not available.

    • Outliers among Outliers

      “We know that circulating tumor cells are present in cancer patients, but we have a limited understanding of the prognostic significance of their presence, or how to identify the ones that have more metastatic potential,” says Shana O. Kelley, Ph.D., professor of biochemistry at the University of Toronto. “These are questions we are trying to address to obtain functional information.”

      Recently, Dr. Kelley and colleagues described a new molecular approach based on a fluidic chip that captures circulating tumor cells using two-dimensional sorting. At a first stage, DNA aptamers specific to cell-surface markers bound to magnetic nanoparticles are used to capture circulating tumor cells. Subsequently, at a second stage, the corresponding antisense oligonucleotides are used to release the cells, enabling two-dimensional cell sorting.

      In a proof-of-concept experiment, Dr. Kelley and colleagues illustrated the strength of this approach in isolating cellular subpopulations that exhibit different phenotypes. Also, the investigators validated their results using an invasion assay.

      “Progress in working on the biology of circulating tumor cells motivates us to make devices to collect information about markers much more readily,” declares Dr. Kelley. “We hope this will provide information about outcomes and prognosis.”



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Metabolic insight into cancer cell survival

Larry H. Bernstein, MD, FCAP, Curator



Revised 4/20/2016

AACR 2016: Novel Epigenetic Drug Therapeutics Revealed


As the 2016 American Association for Cancer Research meeting begins to downshift toward a close, the presentation sessions certainly did not suffer from a lack of enthusiasm from attendees or high-quality research from presenters. Of particular note was a major symposium that discussed next-generation epigenetic therapeutics.

In the past several years, there have been a variety of epigenetic targets exploited by newly developed drug compounds, many of which have already progressed into clinical trials. Often these compounds will target specific classes of epigenetic regulators like acetylases and histone demethylases, for instance, the small-molecule inhibitors of protein interacting bromodomains—implicated in a diverse range of cancers—and methyltransferase inhibitors, such as lysyl demethylases (KDM).

However, for all of their recently achieved success, researchers are continually searching for increasingly rapid methods to validate epigenetic drug targets. Session Chairperson Udo Oppermann, Ph.D., principal investigator at the University of Oxford, stressed that open access research and continued investigator cooperation were key factors for driving rapid development of novel therapeutics in the field. Anecdotally, Dr. Oppermann noted that if biologists were a bit more like the international cooperative teams of physicists that discovered the Higgs boson or gravitational waves, many biological endeavors would advance rather quickly.

After providing the audience with a brief introduction to the symposium’s topic, Dr. Oppermann described his current research on histone demethylase inhibitors in multiple myeloma and the connection to metabolic pathways. He surmised that tricarboxylic acid (TCA) cycle-derived metabolites can link cellular metabolism to cancer—impacting epigenetic landscapes. Specifically, the TCA intermediates are inhibitors of KDMs, ultimately controlling epigenetic and metabolic regulation.

Furthermore, Dr. Oppermann’s group was able to show that treatment of myeloma cell lines with the potent and specific histone demethylase inhibitor GSK-J4 was able to reverse the Myc-driven metabolic dependency, forcing a selected amino acid depletion. This deficiency led to the integrated stress response and the activation of proapoptotic genes. This work helps to solidify further the potent nature of GSK-J4 in cancer while simultaneously uncovering the metabolic mechanisms that cancer cells employ to keep their overproliferative phenotypes progressing forward.

Next, Tomasz Cierpicki, Ph.D., assistant professor at the University of Michigan, described his work on targeting leukemic stem cells with small-molecule inhibitors of the protein regulator of cytokinesis 1 (PRC1). Dr. Cierpicki took the audience through his research design, which was to target BMI1, an oncogene that determines the proliferative capacity and self-renewal potential of normal and leukemic stem cells. BMI1 has been implicated in a variety of tumors and is essential for the Polycomb Repressive Complex 1 (PRC1). Moreover, BMI1 interacts with the RING1B protein to form an active E3 ubiquitin ligase that targets histone H2A, modifying epigenetic regulation mechanisms.

Dr. Cierpicki’s laboratory looked at inhibitors of the RING1B–BMI1 E3 ligase complex as potential therapeutic agents targeting cancer stem cells. Using an array of techniques from fragment screening to medicinal chemistry, the researchers were able to identify potent compounds that bound to RING1B–BMI1 and inhibit its E3 ubiquitin ligase activity with low micromolar affinities. When testing in vitro, the inhibitors revealed robust downregulation of H2A ubiquitination. Dr. Cierpicki and his colleagues found that the RING1–BMI1 inhibitor blocked the self-renewal capacity of the stem cells and induced cellular differentiation—validating RING ligases as a novel epigenetic drug target.

Finishing up the session was William Sellers, M.D., vice president and global head of oncology for the Novartis Institutes for BioMedical Research. Dr. Sellers’ research is focused on what genes are necessary for epigenetic regulation of cancer and how they are linked to essential metabolic processes. He and his colleagues accomplished their studies through the use of large-scale shRNA screening across a diverse set of 390 cancer cell lines.

Utilizing deep small hairpin RNA (shRNA) screening libraries, at 20 shRNAs per genome, provided the investigators with highly robust gene-level data, which resulted in the emergence of several distinct classes of cancer-dependent genes. For example, Dr. Sellers’ group found that several known oncogenes fell into the genetic dependence class, whereas other genes were sorted into lineage, paralog, and collateral synthetic lethality dependent classes.

An interesting example from Dr. Sellers’ work was the link his laboratory discovered between polyamine metabolism and salvage and the protein arginine methyltransferase 5 (PRMT5). In particular, the loss of methylthioadenosine phosphorylase (MTAP)—which has been observed in many solid tumors and hematologic malignancies—resulted in the accumulation of S-methyl-5′-thioadenosine (MTA), which specifically inhibited the epigenetic mechanisms of PRMT5. The culmination Dr. Sellers’ analysis led to the finding that PRMT5 is a novel target for therapeutic development in MTAP deleted cancers.

These three presentations represented some of the excellent, cutting-edge research that is not only looking to develop novel drug therapeutics but also trying to uncover the underlying molecular mechanisms of epigenetic regulation and cancer.




A Metabolic Twist that Drives Cancer Survival


A novel metabolic pathway that helps cancer cells thrive in conditions that are lethal to normal cells has been identified.


“It’s long been thought that if we could target tumor-specific metabolic pathways, it could lead to effective ways to treat cancer,” said senior author Dr. Ralph DeBerardinis, Associate Professor of CRI and Pediatrics, Director of CRI’s Genetic and Metabolic Disease Program, and Chief of the Division of Pediatric Genetics and Metabolism at UT Southwestern. “This study finds that two very different metabolic processes are linked in a way that is specifically required for cells to adapt to the stress associated with cancer progression.”

The study, available online today in the journal Nature, reveals that cancer cells use an alternate version of two well-known metabolic pathways called the pentose phosphate pathway (PPP) and the Krebs cycle to defend against toxins. The toxins are reactive oxygen species (ROS) that kill cells via oxidative stress.

This work builds on earlier studies by Dr. DeBerardinis’ laboratory that found the Krebs cycle, a series of chemical reactions that cells use to generate energy, could reverse itself under certain conditions to nourish cancer cells.

Dr. DeBerardinis said most normal cells and tumor cells grow by attaching to nutrient-rich tissue called a matrix. “They are dependent on matrix attachment to receive growth-promoting signals and to regulate their metabolism in a way that supports cell growth, proliferation, and survival,” he said.

Detachment from the matrix results in a sudden increase in ROS that is lethal to normal cells, he added. Cancer cells seem to have a workaround.

The destruction of healthy cells when detached from the matrix was reported in a landmark 2009 Nature study by Harvard Medical School cell biologist Dr. Joan Brugge. Intriguingly, that same study found that inserting an oncogene – a gene with the potential to cause cancer – into a normal cell caused it to behave like a cancer cell and survive detachment, said Dr. DeBerardinis, who also is affiliated with the Eugene McDermott Center for Human Growth & Development, holds the Joel B. Steinberg, M.D. Chair in Pediatrics, and is a Sowell Family Scholar in Medical Research at UT Southwestern.

“Another Nature study, this one from CRI Director Dr. Sean Morrison’s laboratory in November 2015, found that the rare skin cancer cells that were able to detach from the primary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis said. Dr. Morrison, also a CPRIT Scholar in Cancer Research and a Howard Hughes Medical Institute Investigator, holds the Mary McDermott Cook Chair in Pediatrics Genetics at UT Southwestern.

Working under the premise that the two findings were pieces of the same puzzle, a crucial part of the picture seemed to be missing, he said.

It was known for decades that the PPP was a major source of NADPH, which provides a source of reducing equivalents (that is, electrons) to scavenge ROS; however, the PPP produces NADPH in a part of the cell called the cytosol, whereas the reactive oxygen species are generated primarily in another subcellular structure called the mitochondria.

“If you think of ROS as fire, then NADPH is like the water used by cancer cells to douse the flames,” Dr. DeBerardinis said. But how could NADPH from the PPP help deal with the stress of ROS produced in a completely different part of the cell? “What we did was to discover how this happens,” Dr. DeBerardinis said.

The current study in Nature demonstrates that cancer cells use a “piggybacking” system to carry reducing equivalents from the PPP into the mitochondria. This movement involves an unusual reaction in the cytosol that transfers reducing equivalents from NADPH to a molecule called citrate, similar to a reversed reaction of the Krebs cycle, he said. The citrate then enters the mitochondria and stimulates another pathway that results in the release of reducing equivalents to produce NADPH right at the location of ROS creation, allowing the cancer cells to survive and grow without the benefit of matrix attachment.

“We knew that both the PPP and Krebs cycle provide metabolic benefits to cancer cells.  But we had no idea that they were linked in this unusual fashion,” he said. “Strikingly, normal cells were unable to transport NADPH by this mechanism, and died as a result of the high ROS levels.”

Dr. DeBerardinis stressed that the findings were based on cultured cell models, and more research will be necessary to test the role of the pathway in living organisms. “We are particularly excited to test whether this pathway is required for metastasis, because cancer cells need to survive in a matrix-detached state in the circulation in order to metastasize,” he said.

CRI scientists find novel metabolic twist that drives cancer survival

https://pharmaceuticalintelligence.com/2016/04/09/programmed-cell-death-and-cancer-therapy/5 days ago Cancer Cell Survival Driven by Novel Metabolic Pathway … This new study describes an alternate version of two wellknown metabolic pathways, the pentose phosphate pathway (PPP) and the Krebs cycle,…

http://www.nature.com/nature/journal/v481/n7381/abs/nature10642.html Jan 19, 2012 Nature | Letter …. DeBerardinis, R. J. et al. Beyond aerobic …. Andrew R. Mullen,; Pei-Hsuan Chen,; Tzuling Cheng &; Ralph J. DeBerardinis ..   

Haematopoietic stem cells require a highly regulated protein – Nature
http://www.nature.com/nature/journal/v509/n7498/abs/nature13035.html May 1, 2014 Nature | Article. Print; Share/ ….. synthesis. Nature Methods 6, 275–277 (2009) …. Robert A. J. Signer,; Jeffrey A. Magee &; Sean J. Morrison …       
http://www.nature.com/nature/journal/v527/n7577/abs/nature15726.html Nov 12, 2015 Nature | Article ….. Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and ….. Sean J. Morrison …     
Deep imaging of bone marrow shows non-dividing stem Nature
http://www.nature.com/nature/journal/v526/n7571/abs/nature15250.html   Oct 1, 2015 Nature | Letter. Print; Share/ …… Morrison, S. J. & Scadden, D. T. The bone marrow niche for ….. Kiranmai S. Kocherlakota &; Sean J. Morrison …
https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/Glutamine and cancer: cell biology, physiology, and clinical opportunities …. Metabolism of glutamine-derived α-ketoglutarate in the TCA cycle serves … known as hexosamines, that are used to glycosylate growth factor receptors and …… of two wellknown metabolic pathways, the pentose phosphate pathway (PPP )
The Mitochondrial Warburg Effect: A Cancer Enigma – IBC Journal
http://www.ibc7.org/article/file_down.php?pid=48&mode=article This feature of cancer cells is known as the Warburg effect, named … new paradigm of collaboration and a well-designed systemic approach will supply … Krebs cycle. … The pentose phosphate pathway uses glucose to produce ribose, which is used … glucose is taken up into cells, it is used in two main metabolic pathways


New paper offers intriguing insights into tumor metabolism     William G. Gilroy    August 19, 2009

Posted In: Research

A paper appearing in this week’s edition of the journal Nature by a team of researchers that includes University of Notre Dame biologist Zachary T. Schafer has important new implications for understanding the metabolism of tumors.

Schafer, an assistant professor of biological sciences and Coleman Junior Chair of Cancer Biology, points out that in the early stages of tumor formation some cells become detached from their normal cellular matrix. These “homeless” cells tend to develop certain defects that stop them from becoming cancerous. In a process known as apoptosis, these precancerous cells essentially kill themselves, allowing them to be destroyed by immune system cells.

The prevailing wisdom among researchers has been that apoptosis was the only way that cells could die.

In studies conducted prior to the research described in the Nature paper, it was found that even when apoptosis was inhibited in detached, precancerous cells, they still eventually died. Intrigued by these results, a team of researchers led by Joan S. Brugge, Louise Foote Pfieffer Professor of Cell Biology at Harvard Medical School, and Schafer decided to take a closer look.

They report in this week’s Nature paper that they found that even when apoptosis was inhibited in detached cells endowed with a cancer-causing gene, they still were incapable of absorbing glucose, their primary energy source. Additionally, the cells displayed signs of oxidative stress, which is a harmful accumulation of oxygen-derived molecules called reactive oxygen species (ROS). The research also revealed decreased ATP production, a key factor in energy transport in the cells.

Schafer notes that this combination of loss of glucose transport, decreased ATP production and heightened oxidative stress reveal a manner of cell death that hadn’t been previously demonstrated to play a role in this context.

In the next phase of the study, Schafer engineered the cells to express a high level of HER2, a gene known to be hyperactive in many breast cancer tumors. He also treated the cells with antioxidants to relieve oxidative stress.

Both approaches helped the cells survive. The HER2-treated cells regained glucose transport, avoided oxidative stress and recovered ATP levels.
Most surprisingly, the antioxidants restored metabolic activity in the cells by allowing fatty acids to be effectively used instead of glucose as an energy source, providing them with a chance to survive.

“Our results raise the possibility that antioxidant activity might allow early stage tumor cells to survive where they would otherwise die from these metabolic defects,” Schafer said.

He also cautions that while the antioxidant findings were surprising, their research was done solely in cell cultures and more research needs to be done before there are clear implications for individuals and their diets.

The paper does, however, offer important new clues about the metabolism of tumor cells and important information that may lead to drugs that can developed to target them.


Antioxidant and Oncogene Rescue of Metabolic Defects Caused by
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931797/Aug 19, 2009
Nature. Author manuscript; available in PMC 2010 Sep 2. Published in … Y. Irie,1 Sizhen Gao,1 Pere Puigserver,1,2 and Joan S. Brugge1,*.

Proteasome inhibitors have been shown to be effective in cancer treatment, an ability … a specific inhibitor of 26S proteasome, also reduced cell viability ( 80% with 10 mu …
be a consequence of the increased generation of ROS caused by MG132. …. vectors endowed with the wild type forms of RB or p53 genes (Figure 1f).


The Metastasis-Promoting Roles of Tumor-Associated Immune Cells

Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors and proteases that remodel the tumor microenvironment. Invasion and metastasis requires neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment, and examines the factors allowing these cells to promote each stage of metastasis.


Once established, tumors are quite adept at preventing anti-tumor immune responses, and several defense mechanisms to circumvent immune detection have been described including antigen loss, down-regulation of major histocompatibility molecules (MHC), deregulation or loss of components of the endogenous antigen presentation pathway, and tumor-induced immune suppression mediated through cytokine secretion or direct interactions between tumor ligands and immune cell receptors [2]. These mechanisms contribute to the process of immunoediting in which tumor cell subpopulations susceptible to immune recognition are lysed and eliminated, while resistant tumor cells proliferate and increase their frequency in the developing neoplasia [3]. However, tumors not only effectively escape immune recognition, they also actively subvert the normal anti-tumor activity of immune cells to promote further tumor growth and metastasis.

During early stages of cancer development, infiltrating immune cell populations are primarily tumor suppressive, but depending on the presence of accessory stromal cells, the local cytokine milieu, and tumor-specific interactions, these immune cells can undergo phenotypic changes to enhance tumor cell dissemination and metastasis. For instance, CD4+ T cells, macrophages, and neutrophils have all been shown to possess opposing properties depending on the inflammatory state of the tumor environment, the tissue context, and other cellular stimuli intrinsic to the altered tumor cells [4, 5]. These features are dependent upon the inherent plasticity of immune cells in response to stimulatory or suppressive cytokines [6]. Notably, the switch from a Th1 tumor-suppressive phenotype such as CD4+ “helper” T cells, which aid cytotoxic CD8+ T cells in tumor rejection, to a Th2 tumor-promoting “regulatory” phenotype, which blocks CD8+ T-cell activity, is a characteristic outcome in the inflammatory, immune-suppressive tumor microenvironment [5, 7]. Likewise, M1 macrophages and N1 neutrophils are known to have pronounced anti-tumor activity; however, these immune cells are often subverted to a tumor-promoting M2 and N2 phenotype, respectively, in response to immune-suppressive cytokines secreted by tumor tissue [8].


The crosstalk that occurs between tumor and immune cells within the tumor microenvironment, the circulation, or at distant metastatic sites has been clearly shown to foster metastatic dissemination. Immune cells as well as the suppressive factors that they secrete represent potential targets for therapeutic intervention. Regardless of their source, cytokines, chemokines, proteases, and growth factors are some of the main factors contributing to immunosuppression and immune-mediated tumor progression. These molecules can be produced by immune, stromal, or malignant cells and can act in paracrine and autocrine fashion to promote each stage of tumor cell invasion and metastasis by enhancing inflammation, angiogenesis, tumor proliferation, and recruitment of additional immunosuppressive and tumor-promoting immune cells. These secreted factors provide the malignant cells with an abundant source of growth and survival signals that perpetuate a supportive microenvironment for tumor metastasis and represent some of the most attractive targets for directed anti-tumor therapy. Immune pathways provide numerous soluble targets for cancer treatment, and indeed, many drugs to target immune-suppressive molecules are moving forward in clinical trials. For instance, the anti-RANKL (Denosumab) antibody has been shown to effectively inhibit bone metastasis in prostate cancer patients [201], while a variety of neutralizing antibodies to IL-1β and IL-1 receptor have been shown to have efficacy in treating metastasis in pre-clinical animal models [202]. Several agents that target IL-1 or other immune-suppressive cytokines are already approved for the treatment of some inflammatory diseases and are prime candidates for human trails [202]. Additionally, other proteins involved in tumor progression that are induced directly or indirectly by immune cell populations, such as EMT-associated transcription factors, adhesion molecules, and tumor receptors and ligands which mediate immune suppression, could also be targeted with small molecules or blocking antibodies. Antibodies against two surface molecules expressed by suppressive lymphoid cells, anti-CTLA-4 (ipiliumimab) [203, 204] and anti-PD-1 have been recently gaining increasing support from clinical trials for their effective treatment for many forms of cancer including advanced melanoma and prostate cancer [205, 206]. Specifically, anti-CTLA-4 has been shown to be particularly efficacious in metastatic melanoma, while anti-PD-1 has only just begun a comprehensive evaluation in clinical trials [204, 207]. Likewise, non-steroidal anti-inflammatory drugs (NSAIDS) to prevent or treat chronic inflammation and lymphangiogenesis [208210], and anti-coagulants to prevent platelet aggregation on circulating tumor cells [211] are just two examples of a multitude of therapeutic agents that could be utilized to prevent immune-mediated tumor progression at unique stages of metastasis. Of course, new methods or biomarkers for the detection of patients at risk of tumor progression or metastasis are also desperately needed to tailor personalized therapy for patients to obtain the best possible clinical outcome.


  1. https://pharmaceuticalintelligence.com/category/cancer-and-therapeutics/Mar 26, 2016 This turns your immune systems ability to attack and kill cancer cells back on” …. the rare skin cancer cells that were able to detach from theprimary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis remarked.

  2. https://pharmaceuticalintelligence.com/tag/histone-deacetylase-inhibitors-hdac/The HDAC-inhibiting agent romidepsin significantly increased T-cell tumor … skin cancer cells that were able to detach from the primary tumor and successfully … of the body had the ability to keep ROS levels from getting dangerously high,” Dr. …. Sensitivity for EGFR or KRAS was higher in patients with multiplemetastatic …

  3. https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/In a study involving 320 patients, the researchers were able to infer cell death in …. Glutamine and cancer: cell biology, physiology, and clinical opportunities … On the other hand, GLS2 expression is enhanced in some neuroblastomas, …… of the body had the ability to keep ROS levels from getting dangerously high,” Dr.


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Lipids link to breast cancer

Larry H. Bernstein, MD, FCAP, Curator



Lipids Found Critical to Breast Cancer Cell Proliferation




Scientists in Spain report finding that breast cancer cells need to take up lipids from the extracellular environment so that they can continue to proliferate. The main protein involved in this process is LIPG, an enzyme found in the cell membrane and without which tumor cell growth is arrested. Analyses of more than 500 clinical samples from patients with various kinds of breast tumors reveal that 85% have high levels of LIPG expression.

The research (“FoxA and LIPG Endothelial Lipase Control the Uptake of Extracellular Lipids for Breast Cancer Growth”) is published in Nature Communications.

In Spain, breast cancer is the most common tumor in women and the fourth most common type in both sexes (data from the Spanish Society of Medical Oncology, 2012), registering more than 25,000 new diagnoses each year. According to figures from the World Health Organization, every year 1.38 million new cases of breast cancer are diagnosed and 458,000 people die from this disease (International Agency for Research on Cancer Globocan, 2008).

It was already known that cancer cells require extracellular glucose to grow and that they reprogram their internal machinery to produce greater amounts of lipids. The relevance of this study is that it reveals for the first time that tumor cells must import extracellular lipids to grow.

“This new knowledge related to metabolism could be the Achilles heel of breast cancer,” explains ICREA researcher and Institute for Research in Biomedicine–Barcelona group leader Roger Gomis, Ph.D., co-leader of the study together with Joan J. Guinovart, Ph.D., director of IRB Barcelona and professor at the University of Barcelona. Using animal models and cancer cell cultures, the scientists have demonstrated that blocking of LIPG activity arrests tumor growth.

“What is promising about this new therapeutic target is that LIPG function does not appear to be indispensable for life, so its inhibition may have fewer side effects than other treatments,” explains the first author of the study, Felipe Slebe, a Ph.D. Fellow at IRB Barcelona.

According to Dr. Guinovart, “because LIPG is a membrane protein, it is potentially easier to design a pharmacological agent to block its activity.”

“If a drug were found to block its activity, it could be used to develop more efficient chemotherapy treatments that are less toxic than those currently available,” adds Dr. Gomis.

The scientists are now looking into international collaborations for developing LIPG inhibitors.

FoxA and LIPG endothelial lipase control the uptake of extracellular lipids for breast cancer growth

Felipe SlebeFederico RojoMaria Vinaixa,…Joan AlbanellJoan J. Guinovart & Roger R. Gomis

Nature Communications7,Article number:11199      http://dx.doi.org:/10.1038/ncomms11199

The mechanisms that allow breast cancer (BCa) cells to metabolically sustain rapid growth are poorly understood. Here we report that BCa cells are dependent on a mechanism to supply precursors for intracellular lipid production derived from extracellular sources and that the endothelial lipase (LIPG) fulfils this function. LIPG expression allows the import of lipid precursors, thereby contributing to BCa proliferation. LIPG stands out as an essential component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. LIPG is ubiquitously and highly expressed under the control of FoxA1 or FoxA2 in all BCa subtypes. The downregulation of either LIPG or FoxA in transformed cells results in decreased proliferation and impaired synthesis of intracellular lipids.

FoxA1 and FoxA2 in BCa growth

The importance of FoxA1 in BCa cells differentiation and its contribution to controlling the expression of metabolic genes in several other tissues makes this transcription factor a highly attractive target to explain the metabolic alterations reported in BCa. For these reason, we decided to ascertain the metabolic processes controlled by FoxA1 in BCa. We first confirmed the association between high FoxA1 expression (mRNA and protein) and luminal subtype (Fig. 1a). To this end, we used two cohorts of primary breast tumours with annotated clinical features and follow-up. The MSKCC/EMC BCa data set is based on gene expression profiles from an original series of 560 cases10, whereas the Spanish BCa data set (n=439) is a tissue microarray of formalin-fixed paraffin-embedded stage I–III breast tumour specimens11 (details provided in Methods Section). High FoxA1 gene expression significantly correlated with high expression of well-established luminal markers, such as GATA3 and ESR1, in primary tumours (Supplementary Fig. 1a). Next we explored FoxA1 expression beyond the luminal subtype. Lower FoxA1 expression was observed in non-luminal tumours (Fig. 1a,b); however, a subset also expressed higher FoxA1 levels (Supplementary Fig. 1b and Supplementary Table 1). Given that FoxA2, in conjunction with FoxA1, is also involved in the regulation of several metabolic pathways, we determined the expression of this factor in BCa samples. Unfortunately, no FoxA2 probes in the Affymetrix platform used in the MSKCC/EMC data set provided a reliable interpretation. To overcome this limitation, we used tissue arrays of early BCa samples (Spanish BCa set). Histological examination of FoxA2-stained tissue microarray slides from the Spanish BCa set revealed the expression of this factor in six non-luminal samples, which were scored as FoxA1 (examples in Fig. 1b and summarized inSupplementary Table 1). Collectively, the number of FoxA+ BCa samples detected by immunohistochemistry accounted for 81.3% of all samples in the Spanish BCa set (Supplementary Table 1), which represent a significant proportion of BCa and point to the participation of FoxA in this disease, beyond to its involvement in differentiation and control of hormonal responses.

Figure 1: FoxA1 and FoxA2 in BCa growth.


(a, top) FoxA1 mRNA expression in the MSKCC/EMC set. BCa samples were stratified in Luminal A, Luminal B, Her2, triple negative and unknown subgroups. The unknown group represents specimens that were not classified in any group. (bottom) FoxA1 protein levels by IHC staining in Luminal, Her2 and triple negative samples in the Spanish BCa set (cohort of 439 BCa patients). Data is average±s.d. (b) FoxA1 and FoxA2 IHC staining in FFPE human specimens representative of the different BCa subtypes. Six independent cases are depicted. FoxA1 and FoxA2 are expressed mainly in the nuclei of tumour cells. Scale bar, 50μm. (c) FoxA1 and FoxA2 mRNA expression analysis by qRT-PCR and protein expression by western blot in human BCa cell lines compared with HMECs. T-test was used. Data are average±s.e.m.; n= 3. Of note, MDA435 are of melanoma origin. (d) FoxA1 and FoxA2 expression in MCF7, MDA231 and their derivatives cells by qRT-PCR and western blot. FoxA1 and FoxA2 depletion was achieved with a doxycycline-inducible short hairpin vector. FoxA-depleted cells were rescued by expression of FoxA2 in MCF7 cells or FoxA1 in MDA231 cells. Cell populations were cultured in the presence or absence of doxycycline for 6 days. P value is the result of T-test. Data are average±s.e.m.;n=3. *P≤0.05, ***P≤0.001 (e, left) Schematic representation of MDA231 and MCF7 cells grown without doxycycline and inoculated in Balb/c nude mice treated with or without doxycycline to induce the expression of the indicated FoxA short hairpins. All tumour cell lines have GFP constitutive expression, and tRFP concomitantly with the short hairpin were expressed in doxycycline treated tumours. (right) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points. P value is the result of T-test. Data are average±s.e.m.; n= 5–8 tumours. *P≤0.05,**P≤0.01, ***P≤0.001. FFPE, formalin-fixed paraffin-embedded.

Next, we extended our analysis to BCa cell lines for further mechanistic studies. We compared FoxA1 and FoxA2 mRNA expression in four estrogen receptor positive (ER+) (MCF7, T47D, BT474 and ZR75) and four estrogen receptor negative (ER−) (SKBR3, MDA468, BT20 and MDA231) BCa cell lines, a cell line of melanoma origin (MDA435), and human mammary epithelial cells (HMECs). Of note, two of the BCa lines tested were HER2+ (BT474 and SKBR3) (Fig. 1c). All ER+ BCa cells (MCF7, T47D, BT474 and ZR75), the ER−/HER2+ SKBR3 and both triple negative-like MDA468 and BT20 cell lines expressed FoxA1. Interestingly, MDA231 triple negative-like cells expressed high levels of FoxA2 but not FoxA1, and the non-tumour HMECs did not express these factors (Fig. 1c). No BCa cells co-expressed these two proteins (Fig. 1c). Our results suggest that the expression of FoxA transcription factors is a common feature of breast tumours, as well as of BCa cell lines. This notion implies that FoxA factors play a major role in BCa growth, independently of luminal fate specification.

To examine the molecular basis of the contribution of FoxA1 and FoxA2 to BCa growth, we engineered constitutive GFP-luciferase-expressing MCF7 and MDA231 cells with a doxycycline-inducible short-hairpin RNA (sh-RNA) vector targeting either FoxA1 or FoxA2. Doxycycline addition to the cell culture media decreased FoxA expression in both cell lines compared with control cells (ShControl (Dox+) and Sh FoxA1 or Sh FoxA2 (Dox−))(Fig. 1d), with the concomitant expression of tRFP (Supplementary Fig. 1c). Of note, there was no gain of expression of FoxA2 in FoxA1-depleted cells or vice versa (Fig. 1d). Interestingly, cancer cell proliferation was impaired in vitroupon depletion of either FoxA1 or FoxA2 in MCF7 and MDA231 cells, respectively (Supplementary Fig. 1d,e). Similarly, when Balb/c nude mice implanted with xenograft tumours from the above described cellular populations were treated with doxycycline and the short hairpins were induced, striking differences in tumour growth were observed. FoxA1-depleted MCF7 and FoxA2-depleted MDA231 tumour growth was blunted (Fig. 1e and additional controls in Supplementary Fig. 1f. Experimental details in the Supplementary Methods Section). Collectively, these observations confirm that FoxA1 or FoxA2 expression is required for BCa growth.

Previous studies indicate that FoxA1 and FoxA2 transcriptionally regulate common genes in the liver and pancreas that are central to development and metabolism. We therefore hypothesized that crossed expression of FoxA factors could rescue tumour growth by restoring the expression of essential metabolic genes. To this end, we engineered doxycycline-driven shFoxA1 MCF7 cells to express exogenous FoxA2 and doxycycline-driven shFoxA2 MDA231 cells to express exogenous FoxA1 (Fig. 1d). Interestingly, when these BCa modified cells were implanted in Balb/c nude mice and FoxA depletion was induced with doxycycline, the sustained expression of another FoxA factor (FoxA2 in MCF7 and FoxA1 in MDA231 cells) was sufficient for tumours to continuously grow (Fig. 1e and additional controls in Supplementary Fig. 1f). Quantitative real-time PCR (qRT-PCR) analysis confirmed FoxA expression in the distinct tumour populations ex-vivo (Supplementary Fig. 1g). These results showed that retention of minimal levels of FoxA1 or FoxA2 expression is necessary for BCa cell growth.

FoxA1- and FoxA2-regulated transcripts for BCa growth

Figure 2: A genomic approach to identify FoxA1- and FoxA2-regulated transcripts in MCF7 and MDA231 cells.


(a) FACS profiling of MCF7 and MDA231 cells derived from tumours isolated from mice on the basis of the expression of GFP+ and RFP− (control group) or GFP+ and tRFP+ (knockdown and rescue groups). (b) Representation of the transcripts up- and downregulated by FoxA in MCF7 and MDA231 cells isolated from tumours. Up- and downregulated transcripts present a Bayesian false discovery rate below 5% and fold change >2.5. (c) LIPG, Bcl2 and Cdh11OB mRNA levels of the indicated genetically modified MCF7 and MDA231 tumour xenografts analysed by qRT-PCR. P value is the result of T-test. Data are average±s.e.m.; n= 5–8 tumours. *P≤0.05, ***P≤0.001. (d) LIPG protein expression in constitutive shFoxA1 MCF7 or shFoxA2 MDA231 cells. (e) Promoter reporter assay in HEK 293 cells. Cells were transfected with LIPG promoter reporter and FoxA1 or FoxA2 expressing vectors when indicated. P value is the result of T-test. Data are average±s.e.m.; n=3. ****P≤0.0001.

LIPG expression in BCa

Next, we showed that LIPG expression in primary tumours was specific to BCa tumour cells and not to other stroma cellular entities (Fig. 3a). Subsequently, we tested LIPG expression in normal breast epithelia and interrogated 20 samples from mammoplasty reductions. Normal breast epithelial cells showed a lower expression of LIPG than cells from tumour specimens (Fig. 3b). Similar results were obtained for LIPG protein levels in a panel from BCa lines compared with HMEC cells. Of the cellular populations tested, the eight BCa cell lines expressing FoxA1 or FoxA2 had very high levels of LIPG protein compared with the melanoma MDA435 cell line and the human epithelial cell (Fig. 3c). Consistent with this observation, 83.8% of BCa samples in the Spanish tumour cohort were LIPG+ (Fig. 3d and Supplementary Table 3), and LIPG expression correlated with FoxA expression (Spearman correlation; r=0.477, P=0.000001; Fig. 3e). Further analysis showed that LIPG expression levels in primary tumours do not have the capacity to stratify patients for differential risk of overall or disease-free survival (Supplementary Fig. 2a) and are not dependent on estrogen signalling (Supplementary Fig. 2b), thus reinforcing the notion that LIPG is essential for BCa growth.

Figure 3: LIPG contributes to BCa growth.


a) Representative LIPG IHC staining on primary BCa tissues (cohort of 439 BCa patients). LIPG is expressed in the cytoplasm of tumour cells. Faint staining is also detected in the extracellular area. Scale bar, 50μm. (b) Representative LIPG IHC staining in normal breast tissue from mammoplasty reductions. Weak LIPG expression occurs in epithelial cells from ducts and lobuli. Scale bar, 50μm. (c) LIPG protein expression in human cancer cell lines compared with HMECs. Actin was used as loading control.*Unspecific band. Of note, MDA435 are of melanoma origin. (d) LIPG protein levels by IHC staining in Luminal, Her2, and triple negative samples in the Spanish BCa set (cohort of 439 BCa patients). Data is average±s.d. (e) Spearman correlation (P=0.000001) between FoxA and LIPG IHC staining intensities in Spanish BCa set (cohort of 439 BCa patients). (f) Left panel, in vitro proliferation curves of MCF7 and MDA231 cells transduced with a control or a LIPG short hairpin. Data are average±s.e.m.; n=3. (right) LIPG protein expression in shLIPG MCF7 and shLIPG MDA231 cells. The blot shown is representative of three independent experiments. P value is the result of T-test.**P≤0.01, ***P≤0.001. (g) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points.P value is the result of T-test. Data are average±s.e.m.; n= 6–8 tumours. *P≤0.05.

LIPG is a phospholipase located in the cytosol and cellular membrane and has been shown to hydrolyse extracellular phospholipids from high-density lipoprotein that are afterwards incorporated into intracellular lipid species thus providing lipid precursors of cell metabolism17, 18. Thus we questioned whether LIPG regulates essential lipid intake in BCa and whether it is necessary for proliferation. To validate this hypothesis, we genetically downregulated the expression of this protein in MCF7 and MDA231 cells by means of sh-RNA (Fig. 3f and Supplementary Fig. 2c). LIPG depletion blunted BCa cell capacity to proliferate in vitro (Fig. 3f), as previously observed in FoxA-depleted cells (Supplementary Fig. 1d,e), and caused a reduction in invasion and self-renewal properties (Supplementary Fig. 3a–d). Similarly, LIPG-depleted cells were unable to grow tumours in vivo (Fig. 3g).

LIPG induces BCa cells lipid metabolic reprograming

Figure 4: LIPG regulates the uptake of lipids in BCa cells inducing a lipid metabolic reprograming.

LIPG regulates the uptake of lipids in BCa cells inducing a lipid metabolic reprograming.


(a) Schematic representation of LIPG action. (b) Heat map representation of the downregulated (blue) lipids identified by MS/MS in the cell homogenates of MCF7 or MDA231 LIPG-depleted cells compared with shControl cells. Depicted lipids have a fold change >1.5 and P value<0.05 using the Welch’s t-testn=5. (c) Downregulated lipid species (previously identified in b) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG (blue box). P values are <0.05 and calculated using Welch’s t-test, n=5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. (d) Heat map representation of the upregulated (red) lipids identified by MS/MS in the media of MCF7 or MDA231 LIPG-depleted cells compared with the corresponding shControl cells. Characterized lipids have a fold change >1.5 and P value<0.05 using the Welch’s t-test n=5. (e) Upregulated lipid species in the media (previously identified in d) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG cells (blue box). P values are <0.05 and calculated using Welch’s t-test, n=5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. (f) Heat map representation of the MS/MS downregulated (blue) lipids in the cell media of MCF7/MDA231 LIPG-depleted or shControl cells (as described in d) compared with fresh medium (without cell incubation). Depicted lipid species have a log2 fold change>1.5 and P value<0.05 using the Welch’s t-test n=5. (g) MDA231 and MCF7 cell growth for 48h in complete medium: medium containing 10% FBS 10%); lipoprotein-free medium: medium containing 10% free lipoprotein FBS; and LPC (18:0): medium containing 10% free lipoprotein FBS and 20μM of LPC (18:0). P value is the result of T-test. Data are average±s.e.m.; n=3. **P≤0.01, ***P≤0.001, ****P≤0.0001. (h) Above, schematic representation of the experimental protocol used. (bottom) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice treated with high-fat diet (HFD) are determined at the indicated time points. P value is the result of T-test. Data are average±s.e.m.; n= 6–8 tumours. *P≤0.05, **P≤0.01. Inside graph, plasma cholesterol levels of animals treated with standard diet (SD) or HFD. P value is the result of T-test. Data are average±s.e.m.; n= 4 animals per group. **P≤0.01, ***P≤0.001.

LIPG location has been shown to be functional on the outer face of the cellular membrane (Fig. 4a)18, thus we postulated the possibility that BCa cells are dependent on LIPG function to access extracellular lipids to support their growth needs. To test this notion, we profiled the media of control and LIPG-depleted MCF7 and MDA231 cells following the same liquid chromatography-mass spectrometry-based untargeted lipidomic approach as for cell homogenates. LIPG depletion prevented the absorption of particular lipids from the media (Supplementary Fig. 4a). The structural identification of the lipids by MS/MS confirms the absence of degradation of glycerophospholipids belonging to the LPC class in both MCF7 and MDA231 cells, which is depicted by higher levels in the media of these species in LIPG-depleted when compared with control cells (Fig. 4d,e). Interestingly when we analysed the LPCs species in the media of control and LIPG-depleted cells and compared with fresh media (without cells), all LPC species from control cell media were decreased. This reduction was weaker in the media of Sh LIPG cells, indicating that LIPG-depleted cells have a defect in processing and importing of pre-existing lipid species from the medium (Fig. 4f).

Finally, we evaluated which of the commonly identified potential substrates of LIPG sustains BCa cell proliferation. Initially, we confirmed that the growth of MCF7 and MDA231 cells is impaired when grown in vitro in lipoprotein-depleted media (Fig. 4g). Next we tested the capacity of LPC (18:0) to rescue BCa cell growth in the absence of lipoproteins and confirmed that this lysophosphatidylcholine was able to restore the cells’ capacity to proliferate (Fig. 4g). In accordance, this process was dependent on LIPG expression (Fig. 4g). Similarly, LIPG-depleted cells were not able to grow in vivo in animals fed with high-fat diet (Fig. 4h) indicating that LIPG is indispensable to process the extracellular lipids and mediate their uptake by the cells, irrespectively of the concentration of lipid substrates in circulation, a phenotype also observed in FoxA-depleted cells (Fig. 4h).

LIPG activity supports BCa growth

Figure 5: LIPG activity is essential for BCa growth.

LIPG activity is essential for BCa growth.


(a, top) Homology 3D structural model of LIPG (backbone coloured according to the QMEANlocal parameter values; red residues with low error). The heavy atoms of the three catalytic residues are shown explicitly and the residue mutated in this study is shown in green (Asp 193). (b) FoxA1, FoxA2 and LIPG protein expression in MCF7, MDA231 and their derivative cells determine by western blot. FoxA1 and FoxA2 depletion was achieved with a doxycycline-inducible short hairpin vector. FoxA-depleted cells were rescued by expression of a WT or Inactive LIPG. Cell populations were cultured in the presence or absence of doxycycline for 6 days. *blots represent different exposition times. (c) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points. Pvalue is the result of T-test. Data are average±s.e.m.; n=5–8 tumours. *P≤0.05, **P≤0.01. (d) MDA231 and MCF7 cell growth for 48h treated with DMSO (control), FAS inhibitor (C75) and/or lipase inhibitor (Orlistat). For MDA231 cells C75 was used at a final concentration of 10μgml−1 and for MCF7 cells 8μgml−1. Orlistat was used at a final concentration of 30 or 10μgml−1 in MCF7 or MD231 respectively. Pvalue is the result of T-test. Data are average±s.e.m.; n=3.*P≤0.05, **P≤0.01, ***P≤0.001. (e) Forty-eight hours cell growth of MDA231 or MCF7 cells overexpressing exogenous WT or Inactive LIPG. Cells were treated with DMSO (control) and FAS inhibitor (C75) at a final concentration of 20μgml−1. P value is the result of T-test. Data are average±s.e.m.; n=3.***P≤0.001, ****P≤0.0001 (f) Schematic representation showing how FoxA controls LIPG and lipid metabolism to support tumour growth.

As previous reports showed that de novo lipid metabolism is necessary for BCa growth3, 22, we next questioned whether this lipid synthesis was sufficient or, instead, whether exogenous sources are also required to support BCa cell growth and proliferation, as suggested by our experimental data. To this end, we inhibited the activity of fatty acid synthase (FAS) in BCa cells by means of the chemical inhibitor C75 (ref. 23). FAS activity is crucial for de novo lipid synthesis in cancer cells3,22. To test the complementarity of both de novo and/or exogenous lipid supplies, we used a C75 concentration causing a 50% reduction in BCa cell growth in vitro 48h post incubation (Fig. 5d andSupplementary Fig. 5d). Similarly, we tested the contribution of LIPG inhibition by means of treatment with a lipase inhibitor, Orlistat21. A specific dose causing a 50% reduction in the growth of each BCa cell line was further used (Fig. 5d and Supplementary Fig. 5d). Interestingly, concomitant treatment with FAS and LIPG inhibitors caused an additive effect, blunting BCa cell growth (Fig. 5d). Next, we evaluated whether LIPG activity was sufficient to rescue the chemical inhibition of FAS. To this end, we overexpressed WT and inactive LIPG and grew MCF7 and MDA231 cells in the presence or absence of a high dose of C75 (20mgml−1), which blocks cell growth (Supplementary Fig. 5d). Complete blockade of FAS was not rescued by LIPG (Fig. 5e). Collectively, our results suggest that both exogenous lipid precursors provided by means of LIPG activity and de novo lipid synthesis mediated by FAS are necessary for BCa cell growth.


Here we reveal that FoxA factors provide a central metabolic growth function by specifically regulating LIPG expression, thereby allowing the acquisition of indispensable extracellular lipids for BCa tumour proliferation. FoxA family of transcription factors are expressed in the vast majority of BCa and FoxA1 is expressed across various BCa subtypes. Moreover we show that, in some cases, its absence is associated with the expression of FoxA2. Interestingly, in addition of FoxA1 contribution to luminal commitment24, 25, 26, 27 the factor may drive BCa growth by specifically regulating LIPG levels.

The catalytic activity of LIPG generates extracellular lipid precursors that are imported to fulfill the intracellular production of lipid species (Fig. 5f). LIPG downregulation blocks BCa cell growth, thereby indicating that the import of extracellular lipid precursors is important for the proliferation of these cells. This is a striking observation given that it is generally believed that de novo fatty acid synthesis is the main driver of tumour growth22. Indeed, our experimental data with LIPG-depleted BCa cells revealed a massive decrease of most intracellular glycerolipid intermediates in the synthesis of TG (PC, PE, PG and DG) and their derivatives (LPC and LPE). Accordingly, certain lipid species (LPC) in the media were not decreased in LIPG-depleted cells as much as in control cells, thus indicating that extracellular lipids are the substrates for intracellular lipid production. In particular, we demonstrate the relevance of extracellular LPC (18:0) for BCa cell proliferation in a lipoprotein-depleted medium, a process dependent on LIPG. In this context, a high-fat diet was shown to rescue the absence of a critical intracellular lipase, Monoacylglycerol lipase, for cancer pathogenesis given cancer cells ability to uptake lipids from the extracellular compartment was functional19. Herein, we showed that this rescue mechanism is not functional in BCa cells in the absence of FoxA2 or LIPG. In support of this notion, it is worth noting that extracellular LIPG activity releases fatty acids from high-density lipoprotein phospholipids and these acids are further employed for intracellular lipid production in the human hepatic cell line HepG2 (refs 28, 29).

In conclusion, BCa cells are dependent on a mechanism to supply precursors derived from extracellular sources for intracellular lipid production, and LIPG fulfills this function. Therefore, LIPG stands out as an important component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. Our results also suggest thatde novo lipid synthesis is necessary but not sufficient to support lipid production for BCa tumour growth. Accordingly, recent clinical studies demonstrate the association between lipids and lipoproteins in circulation and risk of BCa in women with extensive mammographic density. This observation implies that interventions aimed to reduce them may have effect on BCa risk30. All together, these observations make LIPG activity an Achilles heel of luminal and, more importantly, of triple negative/basal-like breast tumours, for which limited therapeutic options are currently available.

In normal cells, the glucose carbon flow is directed into a de novo lipogenic pathway that is regulated, in part, via phosphoinositide-3 kinase (PI-3K)-dependent activation of ATP citrate lyase (ACL), a key rate-limiting, enzyme in de novo lipogenesis. ACL is a cytosolic enzyme that catalyzes the generation of acetyl CoA from citrate. Inhibition of ACL results in a loss of B-cell growth and cell viability [10] .
The plasma membrane and its constituent phosphoinositides form the basis of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway, which is crucial for cell proliferation and survival. Phosphatase and tensin-homolog deleted on chromosome 10 (PTEN) is a tumor-suppressor protein that regulates phosphatidylinositol 3-kinase (PI3-K) signaling by binding to the plasma membrane and hydrolyzing the 3′ phosphate from phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) to form phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Several loss-of-function mutations in PTEN that impair lipid phosphatase activity and membrane binding are oncogenic, leading to the development of a variety of cancers. Of these three residues, R335 was observed to interact with the membrane to the greatest extent across all of the simulations. R335L, in common with several other germline mutations, has been associated with the inherited cancer [11] .
ACLY is up-regulated or activated in several types of cancers, and its inhibition is known to induce proliferation arrest in cancer cells both in vitro and in vivo. The last studies were showed that BCR-mediated signaling is regulated in part by the amount of membrane cholesterol. It was observed that statins (Lovostatin), the pharmacological inhibitors of cholesterol synthesis, induce apoptosis of CLL cells in vitro and in vivo. Also the ectopic expression of CD5 in a B-cell line stimulates the transcription of genes involved in the synthesis of cholesterol [12] .

[10] Zaidi N, Swinnen JV, Smans K. ATP-citrate lyase: a key player in cancer metabolism Cancer Res; 2012 (11): 3709-14.

[11] Craig N, Mark S.P. Sansom. Defining the Membrane-Associated State of the PTEN Tumor Suppressor Protein. Biophys J 2013; 5; 104(3: 613–21.

[12] Tomowiak C, Kennel A, Gary-Gouy, Hadife N. High Membrane Cholesterol in CLL B-Cells and Differential Expression of Cholesterol Synthesis Genes in IG GENE Unmutated vs Mutated Cells. British Journal of Medicine & Medical Research 2012; 2(3): 313-26.


Cancer’s Vanguard

Exosomes are emerging as key players in metastasis.

By Catherine Offord | April 1, 2016   http://www.the-scientist.com/?articles.view/articleNo/45577/title/Cancer-s-Vanguard/


PREPARING THE TURF: Before tumor cells arrive at their metastatic destination, part of the site is readied for them. One recent study of liver metastasis in mice found that resident macrophages called Kupffer cells take up exosomes from the original tumor (1). Additionally, macrophages from the bone marrow show up upon the release of fibronectin by other liver cells called stellate cells (2). A current proposal for additional steps in metastatic niche development includes the recruitment of epithelial cells and fibroblasts, which contribute to angiogenesis, and, finally, the arrival of tumor cells themselves (3).© IKUMI KAYAMA/STUDIO KAYAMA

In 2005, David Lyden noticed something unexpected. He and his colleagues at Weill Cornell Medical College had been researching metastasis—the spread of cancer from one part of the body to another. The team had shown that bone marrow–derived cells (BMDCs) were recruited to future metastatic sites before the arrival of tumor cells, confirming that metastasis occurred after a habitable microenvironment, or “premetastatic niche,” had been prepared.1

But carefully studying images of this microenvironment in the lung tissue of mice, Lyden saw something else. Amongst the BMDCs, the micrographs showed tiny specks, far too small to be cells, gathering at the future site of metastasis. “I said, ‘What are these viruses doing here?’” recalls Lyden. “I had no idea about exosomes, microvesicles, and microparticles.”

Those specks, Lyden would come to realize, were in fact primary tumor–derived exosomes. These membrane-enclosed vesicles packed full of molecules are now attracting growing attention as important mediators of intercellular communication, particularly when it comes to cancer’s insidious capacity to spread from one organ to another.

Preparing the ground

Tumors require a community of support cells, including fibroblasts, BMDCs, and endothelial cells, to provide functional and structural assistance and to modulate immune system behavior. Bringing together the first members of this community before the arrival of tumor cells is all part of cancer’s survival strategy, says Joshua Hood, a cancer researcher at the University of Louisville.

“It wouldn’t be efficient for tumor cells to strike out on their own, and just say, ‘Oh, here we are!’” he says. “They would run the risk of being destroyed.” Preparing a “nest” in advance makes the process much safer. “Then the tumor can just efficiently come along and set up shop without ever having to fight much of a battle with the immune system.”

But although Lyden’s group had shown that this preparation was taking place, it remained unclear how such a process might be regulated. For the next few years, many cancer researchers believed that tumor cells must communicate with the premetastatic niche primarily through tumor-secreted signaling molecules such as cytokines.

Meanwhile, research into extracellular vesicles, previously considered biological garbage bags, was revealing new modes of intercellular communication. In 2007, a group of scientists in Sweden discovered that exosomes, tiny vesicles measuring just 30 nanometers to 100 nanometers across, transport mRNA and microRNAs intercellularly, with the potential to effect changes in protein synthesis in recipient cells.2 A new means for tumors to regulate distant cellular environments came into focus, and research on exosomes exploded. In 2011, Hood and his colleagues showed that exosomes facilitate melanoma metastasis through the lymphatic system.3 The following year, Lyden’s group demonstrated that tumor-derived exosomes can direct BMDCs to one of melanoma’s most common sites of metastasis, the lung.4 Exosomes, it seemed, had been underestimated.

Tiny terraformers

Armed with the knowledge that exosomes are involved in multiple stages of melanoma metastasis, Lyden’s lab went searching for the vesicles’ potential role in the metastasis of other cancers. Turning to pancreatic ductal adenocarcinoma (PDAC)—one of the most lethal cancers in humans—postdoctoral researcher Bruno Costa-Silva led a series of exhaustive in vitro and in vivo experiments in mouse models to detail the process of premetastatic niche formation in the liver, PDAC’s most common destination. The team’s results, published last May, reveal an intricate series of sequential steps—mediated by PDAC-derived exosomes (Nature Cell Biol, 17:816-26, 2015).

Using fluorescence labeling, Lyden’s group observed that PDAC-derived exosomes are taken up by Kupffer cells, specialized macrophages lining the outer walls of blood vessels in the liver. There, the exosomes trigger the cells’ secretion of transforming growth factor β (a type of cytokine involved in cell proliferation), plus the production of fibronectin by neighboring hepatic stellate cells, and the recruitment of BMDCs.

The researchers also showed that this cascade of events could be inhibited by depleting exosomal macrophage migratory inhibitory factor (MIF), an abundant protein in PDAC exosomes. “If you target the specific proteins of exosomes, you can reduce metastasis,” explains coauthor Héctor Peinado, leader of the microenvironment and metastasis group at the Spanish National Cancer Research Center.

For Hood, the findings add to a developing picture of exosomes’ vital role as “vanguard” in the progression of cancer. “It’s like the colonization of a new planet,” he says. “They’re terraforming the environment to make it hospitable.”



  • B. Costa-Silva et al., “Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver,”Nature Cell Biol, 17:816-26, 2015.
  • A. Hoshino et al., “Tumour exosome integrins determine organotropic metastasis,” Nature, 527:329-35, 2015.
  • L. Zhang et al., “Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth,”Nature, 527:100-04, 2015.

Internal mail

Although research was revealing the steps involved in forming premetastatic sites, it was less clear how these sites were being selected. “This has always been a great mystery in cancer,” says Ayuko Hoshino, a research associate in Lyden’s lab. “Why do certain cancers metastasize to certain organs?”

One theory, proposed in 1928 by pathologist James Ewing, suggested that anatomical and mechanical factors explained organ specificity in metastasis. The premetastatic niche, then, might form wherever exosomes are likely to land. But this couldn’t be the whole story, says Hoshino. “For instance, there’s eye melanoma. Thinking about that site, you could imagine it metastasizing to the brain. But actually, it almost only metastasizes to the liver.”

Because exosomes arrive at metastatic sites before tumor cells, the team reasoned, perhaps the exosomes themselves were organotropic (i.e., attracted to particular organs or tissues). Sure enough, Lyden says, when Hoshino and Costa-Silva began injecting tumor-derived exosomes into mice, “their preliminary findings were that wherever they injected the exosomes, the pancreatic cancer ones were ending up in the liver and the breast metastasis exosomes would end up in the lung.”

Using mass spectrometry, the researchers analyzed the protein content of exosomes from lung-tropic, liver-tropic, and brain-tropic tumors. They found that the composition of exosomes’ integrins—membrane proteins involved in cell adhesion—was destination-specific (Nature, 527:329-35, 2015). Exosomes bearing integrin α6β4, for example, were directed to the lung, where they could prepare a premetastatic niche potent enough even for normally bone-tropic tumor cells to colonize. Integrin αvβ5, meanwhile, directed metastasis to the liver.

The researchers also showed that exosomal integrins didn’t necessarily correspond to the parent-cell proteins, making exosomes potentially better indicators of where a cancer will spread than the tumor cells themselves. “We can show that an integrin that’s high in the tumor cell might be completely absent in the tumor exosome or vice versa,” says Lyden, adding that, taken together, the results point to a role for exosomes in “dictating the future sites of metastasis.”

“It’s a beautiful story,” says Dihua Yu, a molecular and cellular oncologist at the University of Texas MD Anderson Cancer Center. “This is a very novel finding that gives really good indicators for potential strategies to intervene in metastasis.”

Metastatic crosstalk

In the same month that Lyden’s group published its work on organotropism, Yu’s own lab published a different exosome study—one that told another side of the story.

Yu and her colleagues had found that when tumor cells in mice metastasized to the brain, they downregulated expression of a tumor suppressor gene called PTEN, and became primed for growth at the metastatic site. When the tumor cells were taken out of the microenvironment and put in culture, however, they restored normal PTEN expression.

The researchers demonstrated that a microRNA from astrocytes—star-shape glial cells in the brain—reversibly downregulated the levels of PTEN transcripts in the tumor cells, but they couldn’t figure out how the microRNA was getting into the tumor. Blocking “obvious signaling pathways,” such as gap junctions, failed to have an effect, Yu says.

Scrutinizing astrocyte-conditioned media using electron microscopy, the researchers identified spherical vesicles between 30 nanometers and 100 nanometers in diameter—the defining size of exosomes. Exposing mouse tumor cells to these vesicles increased cell microRNA content and reduced PTENexpression (Nature, 527:100-04, 2015). The study revealed yet another role for exosomes in the communication between tumors and their microenvironment.

The findings were a surprise, says Yu, not least because they showed a different perspective from the bulk of recent research. “We’re talking about astrocytes in the brain secreting exosomes to give welcome help to the cancer cells,” she says.

“I find it an extremely interesting paper because it shows that the astrocytes can change the whole phenotype of the tumor in the brain,” says Lyden. He adds that the results underline the importance of studying the mutational status of tumors at various sites. “All this work in exosomes, it adds to the complexity,” he says. “We can’t just target tumor cells at the primary site. We’ll have to understand all the details of metastasis if we’re really going to tackle it.”

What’s next?

The discovery of multiple roles for exosomes in metastasis has generated excitement about the potential for their use in diagnostics and treatment. As protective containers of tumor-derived genetic material, exosomes could provide information about the status of cancer progression. And as mediators of premetastatic niche formation, they make obvious targets for inhibition. (See “Banking on Blood Tests,”here.)

Exosomes might even be useful as vehicles to deliver drugs because they’re patient-matched and “naturally designed to function in a biocompatible way with living systems,” says Hood. “You could take them out of people, and at some point down the road try to have patients be their own nanofactory, using their own particles for treatment purposes.”

Pancreatic cancer exosomes initiate pre-metastatic nihe formation in the liver

Bruno Costa-SilvaNicole M. AielloAllyson J. Ocean, et al.   Nature Cell Biology 2015; 17,816–826   http://dx.doi.org:/10.1038/ncb3169

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Ayuko HoshinoBruno Costa-SilvaTang-Long ShenGoncalo RodriguesAyako HashimotoMilica Tesic Mark, et al. Nature Nov 2015; 527,329–335  http://dx.doi.org:/10.1038/nature15756

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

  1. Paget, S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 8, 98101 (1989)
  2. Hart, I. R. & Fidler, I. J. Role of organ selectivity in the determination of metastatic patterns of B16 melanoma. Cancer Res. 40, 22812287 (1980)
  3. Müller, A. et al. Involvement of chemokine receptors in breast cancer metastasis. Nature410, 5056 (2001)
  4. Weilbaecher, K. N., Guise, T. A. & McCauley, L. K. Cancer to bone: a fatal attraction. Nature Rev. Cancer 11, 411425 (2011)
  5. Zhou, W. et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell 25, 501515 (2014)
  6. Chang, Q. et al. The IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis.Neoplasia 15, 848862 (2013)
  7. Lu, X. & Kang, Y. Organotropism of breast cancer metastasis. J. Mammary Gland Biol. Neoplasia 12, 153162 (2007)


Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth

Lin ZhangSiyuan ZhangJun YaoFrank J. LoweryQingling ZhangWen-Chien Huang, et al.  Nature  Nov 2015; 527,100–104   http://dx.doi.org:/10.1038/nature15376

The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells’ adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites1. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs2. Clearly, the dynamic interaction between metastatic tumour cells and extrinsic signals at individual metastatic organ sites critically effects the subsequent metastatic outgrowth3, 4. Yet, it is unclear when and how disseminated tumour cells acquire the essential traits from the microenvironment of metastatic organs that prime their subsequent outgrowth. Here we show that both human and mouse tumour cells with normal expression of PTEN, an important tumour suppressor, lose PTEN expression after dissemination to the brain, but not to other organs. The PTEN level in PTEN-loss brain metastatic tumour cells is restored after leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN messenger RNA and protein downregulation is epigenetically regulated by microRNAs from brain astrocytes. Mechanistically, astrocyte-derived exosomes mediate an intercellular transfer of PTEN-targeting microRNAs to metastatic tumour cells, while astrocyte-specific depletion of PTEN-targeting microRNAs or blockade of astrocyte exosome secretion rescues the PTEN loss and suppresses brain metastasis in vivo. Furthermore, this adaptive PTEN loss in brain metastatic tumour cells leads to an increased secretion of the chemokine CCL2, which recruits IBA1-expressing myeloid cells that reciprocally enhance the outgrowth of brain metastatic tumour cells via enhanced proliferation and reduced apoptosis. Our findings demonstrate a remarkable plasticity of PTEN expression in metastatic tumour cells in response to different organ microenvironments, underpinning an essential role of co-evolution between the metastatic cells and their microenvironment during the adaptive metastatic outgrowth. Our findings signify the dynamic and reciprocal cross-talk between tumour cells and the metastatic niche; importantly, they provide new opportunities for effective anti-metastasis therapies, especially of consequence for brain metastasis patients.

  1. Quail, D. F. & Joyce, J.A. Microenvironmental regulation of tumor progression and metastasis. Nature Med. 19, 14231437 (2013)
  2. Park, E. S. et al. Cross-species hybridization of microarrays for studying tumor transcriptome of brain metastasis. Proc. Natl Acad. Sci. USA 108, 1745617461 (2011)
  3. Joyce, J. A. & Pollard, J. W. Microenvironmental regulation of metastasis. Nature Rev. Cancer 9, 239252 (2009)
  4. Vanharanta, S. & Massagué, J. Origins of metastatic traits. Cancer Cell 24, 410421 (2013)
  5. Gray, J. Cancer: genomics of metastasis. Nature 464, 989990 (2010)
  6. Friedl, P. & Alexander, S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147, 9921009 (2011)

Banking on Blood Tests

How close are liquid biopsies to replacing current diagnostics?

By Jyoti Madhusoodanan | April 1, 2016  http://www.the-scientist.com/?articles.view/articleNo/45584/title/Banking-on-Blood-Tests/

No matter where a tumor lurks in the body, its secrets circulate in the blood. Stray tumor cells begin metastatic migrations by slipping into the vasculature. Vesicles secreted by cancer cells and free-floating DNA are also released into the bloodstream. Because these bits of cellular debris are a grab-bag of biomarkers that could both signal a cancer’s presence and predict its progression and response to treatment, the use of blood-based tests, or liquid biopsies, to detect and evaluate them is now drawing significant commercial interest.

Last year, San Diego–based Pathway Genomics began advertising a screen “for the early detection of up to 10 different cancer types in high-risk populations.” But the screen had only been tested in already-diagnosed patients, not in at-risk individuals, and within weeks of making it commercially available, the company received an FDA notice to provide more information about their promotional claims before further marketing. “We . . . have not found any published evidence that this test or any similar test has been clinically validated as a screening tool for early detection of cancer in high risk individuals,” the agency wrote.

The Forces of Cancer

A tumor’s physical environment fuels its growth and causes treatment resistance.

By Lance L. Munn and Rakesh K. Jain | April 1, 2016   http://www.the-scientist.com/?articles.view/articleNo/45603/title/The-Forces-of-Cancer/

Ahelium balloon tugs gently at the end of its string. The tension in the string resists the buoyant force of the helium, and the elastic nature of the balloon’s rubber contains the helium gas as it tries   to expand. Cutting the string or poking the rubber with a pin reveals the precarious balance between the forces, upsets the equilibrium, and sets the system into motion.

Some biological tissues also exist in such a state of offsetting forces. The most familiar example is the balance between blood pressure and the elastic tension in the cardiovascular system that contains and conveys blood without bursting or collapsing. And in tumors, both solid and fluid forces are generated that make the cancerous tissue a lot like that helium balloon: cut a tumor with a scalpel and it rapidly swells and deforms as pent-up forces break free from structural elements that are severed.1

One force that is notably higher in tumors than in healthy tissues is fluid pressure, resulting from hyperpermeable, leaky blood vessels and a dearth of draining lymphatic vessels. Researchers have known since the 1950s that tumors exhibit elevated fluid pressure, but the implications for tumor progression and drug delivery were not realized until the late 1980s. That was when we (R.K.J. and colleagues) used a mathematical model to predict—and subsequently validate in animal and human tumors—that a precipitous drop in fluid pressure at the tumor–normal tissue interface causes interstitial fluid to ooze out of the tumor.2 This seeping fluid pushes drugs, growth factors, and cancer cells into the surrounding tissue and lymphatics, reducing drug delivery and facilitating local tumor invasion and distant metastasis.

Based on this insight, we suggested in 2001 that anti-angiogenic drugs could be used to lower a tumor’s fluid pressure and improve treatment outcome.3 This hypothesis changed the thinking about how existing anti-angiogenesis therapies actually work and spurred research into other physical forces acting in cancer.4 In the last 15 years, researchers have identified diverse sources of increased pressure in tumors, which may serve as possible targets for cancer therapy.5 For example, solid forces exerted by the extracellular matrix can be reduced by treatment with drugs approved by the US Food and Drug Administration (FDA) for controlling hypertension (angiotensin blockers) or diabetes (metformin). Retrospective clinical studies have found improved survival in cancer patients who were treated with these agents, which are now being tested in prospective trials for a variety of solid tumors.6,7

Tumors under pressure

In vitro experiments showing that cancer cells actively migrate in response to fluid flow have supported the hypothesis that fluid escaping from the boundary of a tumor may guide the invasive migration of cancer cells toward lymphatic or blood vessels, potentially encouraging metastasis. There remains controversy over how the fluid forces induce the migration; the cells may respond to chemical gradients created by the cells and distorted by the flowing fluid,8 or the fluid may activate cell mechanosensors.9 Because of the potential for new therapeutic interventions, the transduction of mechanical fluid forces into biochemical signals by cell mechanosensors is an active area of investigation. In a more direct manner, the fluid flow can physically carry cancer cells to lymph nodes.

Fluid forces may also promote tumor progression by recruiting blood vessels into the cancerous mass.10 Because tumor blood vessels are leaky, plasma can pass freely between vessels that have different pressures. When this happens at the periphery of a tumor, where angiogenic growth factors are prevalent, there can be synergistic induction of new vessel sprouts.


And fluid pressure is just one of the many forces in a tumor that can influence its development and progression. Tumors also develop increased solid pressure, as compared with normal tissue, stemming from the uncontrolled division of cancer cells and from the infiltration and proliferation of stromal and immune cells from the surrounding tissue and circulation. High-molecular-weight polysaccharides known as hydrogels found in the extracellular matrix (ECM) also add pressure on a tumor. The most well-studied of these hydrogels is hyaluronan; when the polysaccharide absorbs water, it swells, pressing on surrounding cells and structural elements of the tissue.

The ECM contains a highly interconnected network of collagen and other fibers and is normally very good at resisting and containing such tension. It also has support from infiltrating myofibroblasts, which detect areas where the ECM density or tension is not normal and initiate actomyosin-based contraction of collagen and elastin matrix structures to restore tensional homeostasis. But while this repair effort is typically effective in healthy tissues, uncooperative tumor cells interfere with these efforts, both by themselves generating pressure and by hyperactivating cancer-associated fibroblasts to produce more ECM and thus produce even more force.11

Because cell growth and ECM composition are not spatially uniform in cancer, tumors are subjected to multiple, dispersed sources of pressure associated with matrix “containers” of various sizes. This solid pressure from within the tumor deforms the surrounding normal tissue, potentially facilitating the metastatic escape of cancer cells. The physical forces also compress blood vessels and lymphatic vessels in the tumor and adjacent normal tissue,12 increasing the fluid pressure in the tumor13  and interrupting the delivery of nutrients, removal of waste, and entry of tumor-targeted drugs via the blood.4 Insufficient blood flow also results in poor oxygenation, which has been linked to immunosuppression, inflammation, invasion, and metastasis, as well as lowered efficacy of chemo-, radio-, and immunotherapies.4 These are all indirect consequences of solid stresses in and on tumors.

Such forces can also have direct effects on cancer cells, and may serve as independent triggers for tumor invasion. Mechanical forces are central to many of our sense systems, such as hearing, touch, and pain, and to tissue maintenance programs, such as bone regeneration and blood vessel remodeling. In these systems, mechanical forces are transduced by mechanosensors to activate downstream biochemical and genetic pathways. (See “Full Speed Ahead,” The Scientist, December 2009.) Cancer cells may similarly be able to sense and respond to dynamic forces in tumors. We have shown, for example, that metastatic cancer cells exposed to compressive stresses in a culture dish undergo a phenotypic transformation to become more invasive,14 and others have shown that compressive forces applied in vivo can also induce oncogenes in normal epithelium of the mouse colon.15

It is thus becoming quite clear that the physical environment can influence a tumor’s development and spread, and it may even be possible for physical forces to kick-start cancerous growth.


Full Speed Ahead

Physical forces acting in and around cells are fast—and making waves in the world of molecular biology.

By Jef Akst | December 1, 2009    http://www.the-scientist.com/?articles.view/articleNo/27816/title/Full-Speed-Ahead/

When it comes to survival, few things are more important than being able to respond quickly to a change of circumstances. And when it comes to fast-acting indicators, it turns out that signals induced by physical forces acting in and around cells, appropriately dubbed biomechanical signals, are the champions of the cellular world.

“If you look at this mechanical signaling, it’s about 30 meters per second—that’s very fast,” says bioengineer Ning Wang of the University of Illinois at Urbana-Champaign. That’s faster than most family-owned speedboats, and second only to electrical (e.g., nerve) impulses in biological signaling. By comparison, small chemicals moving by diffusion average a mere 2 micrometers per second—a speed even the slowest row boater could easily top.

Indeed, when the two signal types were pitted against each other in a cellular race last year, the mechanical signals left chemical signals in their wake, activating proteins at distant sites in the cytoplasm in just a fraction of a second, at least 40 times faster than their growth factor opponent.1 Mechanical signals are so fast, Wang adds, they are “beyond our resolution,” meaning that current imaging techniques cannot capture the very first cellular changes that result from mechanical stress, which occur within nanoseconds.

For centuries, scientists have scrutinized the molecular inner workings of the body, with little or no regard to the physical environment in which these biological reactions take place. But the growing realization that physical forces have a pervasive presence in physiology (operating in a variety of bodily systems in thebone, blood, kidney, and ear, for instance), and act with astonishing speed, has caused many to consider the possibility that mechanical signaling may be just as important as chemical communication in the life of a cell.

“Biologists have traditionally ignored the role of mechanics in biology,” says biomechanical engineer Mohammad Mofrad of the University of California, Berkley, “[but] biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”

The wave within: Mechanical forces acting inside the cell

Once believed to be little more than sacks of chemically active goop, cells didn’t seem capable of transmitting physical forces into their depths, and researchers largely limited their search for molecules or structures that respond to physical forces, or mechanosensors, to the plasma membrane.

Mechanical signaling may be just as important as chemical communication in the life of a cell.

In the late 1990s, however, closer examination revealed that the cell’s interior is in fact a highly structured environment, composed of a network of filaments.2 Pull on one side of the cell, and these filaments will transmit the force all the way to other side, tugging on and bumping into a variety of cellular structures along the way—similar to how a boat’s wake sends a series of small waves lapping up on a distant and otherwise peaceful shoreline. Scientists are now realizing the potential of such intracellular jostling to induce molecular changes throughout the cell, and the search for mechanosensing molecules has escalated dramatically in scope, including, for example, several proteins of the nucleus.

It’s a search that will likely last a while, predicts cell biologist Donald Ingber, director of the Wyss Institute for Biologically Inspired Engineering at Harvard University. “To try to find out what’s the mechanosensor is kind of crazy at this point,” he says. As scientists are now learning, “the whole cell is the mechanosensor.”

A key player, most agree, is the cytoskeleton, which is comprised of a variety of microfilaments, including rigid actin filaments and active myosin motors—the two principle components of muscle. Activation of the so-called nonmuscle myosins causes the cytoskeleton to contract, much like an arm muscle does when it lifts a heavy object.

The first intimation that the cytoskeleton could go beyond its established inner-cell duties (molecule transport and cell movement and division) came in 1997, when Ingber did the logical (in hindsight, at least) experiment of pulling on the cells to see what happened inside.2 Using a tiny glass micropipette coated in ligands, Ingber and his team gently probed the surface proteins known as integrins, which secure the cell to the extracellular matrix. When they quickly pulled the micropipette away, they saw an immediate cellular makeover: cytoskeletal elements turned 90 degrees, the nucleus distorted, and the nucleolus—a small, dense structure within the nucleus that functions primarily in ribosome assembly—aligned itself with the direction of the applied force.

“That kind of blew people away,” Ingber recalls. “It revealed that cells have incredible levels of structure not only in the cytoplasm but in the nucleus as well.”

Wang (once a postdoc in Ingber’s lab at the Harvard School of Public Health) and other collaborators combined a similar technique with fluorescent imaging technology to visualize how these forces were channeled within the cell’s interior. Upping the resolution and further refining these techniques, Wang began mapping these intracellular forces as they made their way through the cell. In 2005, the maps confirmed the physical connection between the cell-surface integrins and the nucleus, and showed that these external forces follow a nonrandom path dictated by the tension of the cytoskeletal elements.3

“Biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”
–Mohammad Mofrad

The end point of these mechanical pathways is likely a mechanosensitive protein, which changes shape in response to the force, thereby exposing new binding areas or otherwise changing the protein’s function. In mitochondria, for example, mechanical forces may trigger the release of reactive oxygen species and activation of signaling molecules that contribute to inflammation and atherosclerosis.

Similarly, proteins on the nuclear membrane may pass mechanical signals into the nucleus by way of a specialized structure known as LINC (linker of nucleoskeleton and cytoskeleton), which physically links the actin cytoskeleton to proteins important in nuclear organization and gene function. To determine if mechanical forces directly affect gene expression, last year scientists began exploiting the increasingly popular fluorescence resonance energy transfer (FRET) technology,1 in which energy emitted by one fluorescent molecule can stimulate another, resulting in a visible energy transfer that can track enzymatic activities in live cells. By combining FRET technology with the techniques that apply physical forces to specific cell membrane proteins, scientists can visualize entire mechanochemical transduction pathways, Wang says.

“The big issue right now in the field of mechanotransduction is whether the genes in the nucleus can be directly activated by forces applied to the cell surface,” Wang explains. While the physical maps of the cytoskeleton tentatively sketch out a path that supports this possibility, confirmatory data is lacking. This combination of new technologies will be “tremendously” helpful in answering that question, he says, and “push the field” towards a more complete understanding of how mechanical forces can influence cellular life.

An early start: Mechanical forces in development

In the world of developmental biology, the cytoskeleton’s role in biomechanics really comes into its own. As the embryo develops, the cells themselves are the force generators, and by contracting at critical times, the cytoskeleton can initiate many key developmental steps, from invagination and gastrulation to proliferation and differentiation, and overall cellular organization.

The idea that physical forces play a role in development is not a new one. In the early 20th century, back when Albert Einstein was first developing the molecular basis of viscosity and scientists were realizing molecules are distinct particles, biologist and mathematician D’Arcy Thompson of the University of Dundee in Scotland suggested that mechanical strain is a key player in morphogenesis. Now, nearly a century later, biologists are finally beginning to agree.

Because Thompson “couldn’t measure [the forces] at that time, that kind of thinking got pushed to the wayside as genetic thinking took over biology,” says bioengineer Christopher Chen of the University of Pennsylvania. That is, until 2003, when Emmanuel Farge of the Curie Institute in France squeezedDrosophila embryos to mimic the compression experienced during early development and activated twist—a critical gene in the formation of the digestive tract.4 These results gave weight to Thompson’s idea that stress in the embryo stimulates development and growth, and inspired developmental scientists to begin considering mechanical effects, Chen says. “Now we’re at the stage where there’s a lot of interest and willingness to consider the fact that mechanical forces are not only shaping the embryo, but are linked to the differentiation programs that are going on.”

Again, the cytoskeleton is a key player in this process. In fruit flies and frogs, for example, nonmuscle myosins contract the actin filaments to generate the compressive forces necessary for successful gastrulation—the first major shape-changing event of development. Myosins similarly influence proliferation in the development of the Drosophila egg chamber, with increased myosin activity resulting in increased cell division.

Cytoskeleton contractility also appears to direct stem cell differentiation. In 2006, Dennis Discher of the University of Pennsylvania demonstrated that the tension of the substrate on which cells are grown in culture is important for determining what type of tissue the cells will form.5 Cells grown on soft matrices that mimic brain tissue tended to grow into neural cells, while cells grown on stiffer matrices grew into muscle cell precursors, and hard matrices yielded bone. In this case, it seems that stiffer substrates increased the expression of nonmuscle myosin, generating greater tension in the actin cytoskeleton and affecting differentiation. (Altering or inhibiting myosin contraction can also affect differentiation.)

“To try to find out what’s the mechanosensor is kind of crazy at this point. As scientists are now learning, the whole cell is the mechanosensor.”
–Donald Ingber
Shaping a tumor

In addition to the influence of physical forces on cancer growth and invasion, forces can alter a tumor’s mechanical properties, and vice versa. Tumors are more rigid, or stiffer, than surrounding tissues, usually because they contain excess collagen in the ECM,5 and this can contain and amplify local forces produced by proliferating cancer cells. On the other hand, tumor rigidity can be further enhanced if the cells exert tension on ECM collagen fibers by pulling on them, or by stretching them, as occurs when tumors grow uncontrollably. Fluid forces can also influence the assembly of collagen fibers within and around tumors,8potentially increasing stiffness.

Importantly, tumor stiffness tends to be associated with poor prognoses, though the reasons for this are not fully understood. Cells are known to differentiate into different lineages depending on the local rigidity;16 for example, stem cells differentiate into bone on stiff substrates, but make adipose (fat) cells on softer substrates. Similar mechanisms are thought to affect tumor progression when the ECM changes rigidity, inducing cancer cells to become more invasive as well as more likely to metastasize. Indeed, longer collagen fibers in the matrix are associated with increased invasion and metastasis, as well as reduced survival, in mice.17

In addition, the abnormal ECM in tumors can affect cancer progression by activating normal stromal cells, such as macrophages and fibroblasts, that accelerate tumor growth and treatment resistance. These activated stromal cells further strengthen and stretch the ECM, causing a snowball effect.

The biochemical composition and organization of the ECM also influences tumor biology. Dysregulation of normal matrix signals can lead to tumor progression, characterized by excessive cell proliferation, immortality, enhanced migration, changes in metabolism, and evasion of the immune response. More research is needed to dissect the relationships between the ECM’s mechanical properties, forces, and cell signaling pathways.

Targeting the ECM

Because unchecked proliferation of cancer cells increases solid stress in the tumor, anticancer therapies should decrease the compressive forces in tumors and reopen collapsed blood and lymphatic vessels.11 This is exactly what happens when tumors are treated with certain doses of paclitaxel or docetaxel, two widely used cancer drugs. Shrinking tumors increases blood flow and allows more efficient fluid movement through the extravascular space, lowering the tumor interstitial fluid pressure in mouse models and in patients with breast cancer.5 However, cancer cells invariably develop resistance to treatment and begin to regrow, increasing solid stress again. As a result, other targets for reducing solid stresses are needed.

Because of its role in containing and concentrating the forces in a tumor, the collagen matrix within and around the tumor is another potential target for relieving tumor-related stresses. Indeed, solid stress in tumors can be reduced by drugs that selectively reprogram activated fibroblasts or modify the assembly of matrix components such as collagen and hyaluronan. In rodent studies, targeting these force-altering components in the tumor microenvironment has been shown to decrease solid stress, improve blood perfusion and drug delivery, and improve tumor response to chemotherapy and animal survival.6 We have found, for example, that injecting tumors with a collagen-digesting enzyme increases the diffusion of antibodies and viral particles and improves drug penetration in the tumor. Similarly, treatments that target transforming growth factor–beta (TGF-β), which controls the production of collagen by myofibroblasts, increase perfusion, improve the delivery of drugs of all sizes in mammary tumors, and improve treatment outcomes in mice.5

A class of drugs that is widely used to control blood pressure in hypertensive patients also blocks the TGF-β pathway. These drugs, known as angiotensin receptor 1 blockers, can reduce collagen production in and around the tumor by reducing the activity of TGF-β, as well as by blocking the function of connective tissue growth factor (CTGF), which is involved in stabilizing collagen and inducing resistance to chemotherapy.6Losartan and other angiotensin inhibitors reduce levels of collagen in various experimental models of fibrosis, and decrease renal and cardiac fibrosis in hypertensive patients. When given to mice with one of four different types of tumors characterized by high levels of cancer-associated fibroblasts (CAFs) and excess extracellular matrix—pancreatic ductal adenocarcinoma, breast cancer, sarcoma, and melanoma—losartan treatment caused a decrease in collagen content in a dose-dependent manner, enhanced penetration of nanoparticles into the tumor, and improved efficacy of diverse anticancer drugs. This is supported by a number of retrospective studies in patients with pancreatic, lung, and kidney cancers.6Researchers at Massachusetts General Hospital are now running a Phase 1/2 clinical trial to test losartan in pancreatic cancer patients.


THE TUMOR ENVIRONMENT: The extracellular matrix and stromal cells within a tumor’s microenvironment influence the physical forces a tumor experiences. Left: The immunofluorescent image shows stromal cells (red and green) surrounding tumor cells (red cluster with blue nuclei); the cells were isolated from a mouse model of lung adenocarcinoma. Right: In this immunofluorescent image of triple-negative breast cancer, tumor cells (blue) are in close contact with matrix collagen (purple). Immune cells are labeled in red and green.VASILENA GOCHEVA, JACKS LAB, KOCH INSTITUTE AT MIT; DONGMEI ZUO, LABORATORY DR. MORAG PARK

Another potential cancer treatment target is hyaluronan, which is abundant in 20 percent to 30 percent of human tumors, most notably breast, colon, and prostate cancers. In addition to its role as a pressure-creating gel, hyaluronan can sequester growth factors and inhibit interstitial fluid movement within the tumor. Hyaluronidase, an enzyme that digests hyaluronan, reduces mechanical stress in tumors grown in mice.1 And San Diego–based Halozyme Therapeutics’s PEGPH20, a formulation of hyaluronidase coated with polyethylene glycol to enhance bioavailability, can decompress blood vessels and improve treatment outcome in genetically engineered mouse models of pancreatic ductal adenocarcinoma. Based on these studies, Halozyme researchers are now testing PEGPH20 in a randomized clinical trial of pancreatic cancer patients. Another matrix-altering drug is the widely-prescribed antidiabetic drug metformin, which has been shown to decrease collagen and hyaluronan levels in pancreatic tumors in obese mice and patients.7 Metformin is currently being tested in more than 200 clinical trials worldwide as a treatment for different types of cancer.

Clearly, tumors should be studied not only in light of their biochemical processes and genetic underpinnings, but also for the specific physical forces and mechanical properties that may influence progression. Understanding the physical microenvironment of tumors, as well as its interplay with the biochemical environment, is necessary to improve cancer detection, prevention, and treatment.

  1. T. Stylianopoulos et al., “Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors,” PNAS, 109:15101-08, 2012.
  2. R.K. Jain, L.T. Baxter, “Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: Significance of elevated interstitial pressure,” Cancer Res, 48:7022-32, 1988.
  3. R.K. Jain, “Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy,”Science, 307:58-62, 2005.
  4. R.K. Jain, “Antiangiogenesis strategies revisited: From starving tumors to alleviating hypoxia,”Cancer Cell, 26:605-22, 2014.
  5. R.K. Jain et al., “The role of mechanical forces in tumor growth and therapy,” Annu Rev Biomed Eng, 16:321-46, 2014.
  6. V.P. Chauhan et al., “Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels,” Nat Commun, 4:2516, 2013.
  7. J. Incio et al., “Metformin reduces desmoplasia in pancreatic cancer by reprogramming stellate cells and tumor-associated macrophages,” PLOS ONE, 10:e0141392, 2015.
  8. M.A. Swartz, A.W. Lund, “Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with immunity,” Nat Rev Cancer, 12:210-19, 2012.
  9. H. Qazi et al., “Cancer cell glycocalyx mediates mechanotransduction and flow-regulated invasion,”Integr Biol, 5:1334-43, 2013.
  10. J.W. Song, L.L. Munn, “Fluid forces control endothelial sprouting,” PNAS, 108:15342-47, 2011.

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Curbing Cancer Cell Growth & Metastasis-on-a-Chip’ Models Cancer’s Spread

Curator: Larry H. Bernstein, MD, FCAP


New Approach to Curbing Cancer Cell Growth


Using a new approach, scientists at The Scripps Research Institute (TSRI) and collaborating institutions have discovered a novel drug candidate that could be used to treat certain types of breast cancer, lung cancer and melanoma.

The new study focused on serine, one of the 20 amino acids (protein building blocks) found in nature. Many types of cancer require synthesis of serine to sustain rapid, constant and unregulated growth.

To find a drug candidate that interfered with this pathway, the team screened a large library of compounds from a variety of sources, searching for molecules that inhibited a specific enzyme known as 3-phosphoglycerate dehydrogenase (PHGDH), which is responsible for the first committed step in serine biosynthesis.

“In addition to discovering an inhibitor that targets cancer metabolism, we also now have a tool to help answer interesting questions about serine metabolism,” said Luke L. Lairson, assistant professor of chemistry at TSRI and principal investigator of cell biology at the California Institute for Biomedical Research (CALIBR).

Lairson was senior author of the study, published recently in the Proceedings of the National Academy of Sciences (PNAS), with Lewis Cantley of Weill Cornell Medical College and Costas Lyssiotis of the University of Michigan.

Addicted to Serine

Serine is necessary for nucleotide, protein and lipid biosynthesis in all cells. Cells use two main routes for acquiring serine: through import from the extracellular environment or through conversion of 3-phosphoglycerate (a glycolytic intermediate) by PHGDH.

“Since the late 1950s, it has been known that cancer cells use the process of aerobic glycolysis to generate metabolites needed for proliferative growth,” said Lairson.

This process can lead to an overproduction of serine. The genetic basis for this abundance had remained mysterious until recently, when it was demonstrated that some cancers acquire mutations that increased the expression of PHGDH; reducing PHGDH in these “serine-addicted” cancer cells also inhibited their growth.

The labs of Lewis C. Cantley at Weill Cornell Medical College (in work published in Nature Genetics) and David Sabatini at the Whitehead Institute (in work published in Nature) suggested PHGDH as a potential drug target for cancer types that overexpress the enzyme.

Lairson and colleagues hypothesized that a small molecule drug candidate that inhibited PHGDH could interfere with cancer metabolism and point the way to the development of an effective cancer therapeutic. Importantly, this drug candidate would be inactive against normal cells because they would be able to import enough serine to support ordinary growth.

As Easy as 1-2-800,000

Lairson, in collaboration with colleagues including Cantley, Lyssiotis, Edouard Mullarky of Weill Cornell and Harvard Medical School and Natasha Lucki of CALIBR, screened through a library of 800,000 small molecules using a high-throughput in vitro enzyme assay to detect inhibition of PHGDH. The group identified 408 candidates and further narrowed this list down based on cell-type specific anti-proliferative activity and by eliminating those inhibitors that broadly targeted other dehydrogenases.

With the successful identification of seven candidate inhibitors, the team sought to determine if these molecules could inhibit PHGDH in the complex cellular environment. To do so, the team used a mass spectrometry-based assay (test) to measure newly synthesized serine in a cell in the presence of the drug candidates.

One of the seven small molecules tested, named CBR-5884, was able to specifically inhibit serine synthesis by 30 percent, suggesting that the molecule specifically targeted PHGDH. The group went on to show that CBR-5884 was able to inhibit cell proliferation of breast cancer and melanoma cells lines that overexpress PHGDH.

As expected, CBR-5884 did not inhibit cancer cells that did not overexpress PHGDH, as they can import serine; however, when incubated in media lacking serine, the presence of CBR-5884 decreased growth in these cells.

The group anticipates much optimization work before this drug candidate can become an effective therapeutic. In pursuit of this goal, the researchers plan to take a medicinal chemistry approach to improve potency and metabolic stability.


How Cancer Stem Cells Thrive When Oxygen Is Scarce

(Image: Shutterstock)
image: Shutterstock

Working with human breast cancer cells and mice, scientists at The Johns Hopkins University say new experiments explain how certain cancer stem cells thrive in low oxygen conditions. Proliferation of such cells, which tend to resist chemotherapy and help tumors spread, are considered a major roadblock to successful cancer treatment.

The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say.

“There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells,” said Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center. “That gives us a few more possible targets for drugs that diminish their threat in human cancer.”

A summary of the findings was published online March 21 in the Proceedings of the National Academy of Sciences.

“Aggressive cancers contain regions where the cancer cells are starved for oxygen and die off, yet patients with these tumors generally have the worst outcome. Our new findings tell us that low oxygen conditions actually encourage certain cancer stem cells to multiply through the same mechanism used by embryonic stem cells.”

All stem cells are immature cells known for their ability to multiply indefinitely and give rise to progenitor cells that mature into specific cell types that populate the body’s tissues during embryonic development. They also replenish tissues throughout the life of an organism. But stem cells found in tumors use those same attributes and twist them to maintain and enhance the survival of cancers.

Recent studies showed that low oxygen conditions increase levels of a family of proteins known as HIFs, or hypoxia-inducible factors, that turn on hundreds of genes, including one called NANOG that instructs cells to become stem cells.

Studies of embryonic stem cells revealed that NANOG protein levels can be lowered by a chemical process known as methylation, which involves putting a methyl group chemical tag on a protein’s messenger RNA (mRNA) precursor. Semenza said methylation leads to the destruction of NANOG’s mRNA so that no protein is made, which in turn causes the embryonic stem cells to abandon their stem cell state and mature into different cell types.

Zeroing in on NANOG, the scientists found that low oxygen conditions increased NANOG’s mRNA levels through the action of HIF proteins, which turned on the gene for ALKBH5, which decreased the methylation and subsequent destruction of NANOG’s mRNA. When they prevented the cells from making ALKBH5, NANOG levels and the number of cancer stem cells decreased. When the researchers manipulated the cell’s genetics to increase levels of ALKBH5 without exposing them to low oxygen, they found this also decreased methylation of NANOG mRNA and increased the numbers of breast cancer stem cells.

Finally, using live mice, the scientists injected 1,000 triple-negative breast cancer cells into their mammary fat pads, where the mouse version of breast cancer forms. Unaltered cells created tumors in all seven mice injected with such cells, but when cells missing ALKBH5 were used, they caused tumors in only 43 percent (six out of 14) of mice. “That confirmed for us that ALKBH5 helps preserve cancer stem cells and their tumor-forming abilities,” Semenza said.

How cancer stem cells thrive when oxygen is scarce    https://www.sciencedaily.com/releases/2016/03/160328100159.htm

The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say.

“There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells,” says Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center.

Chuanzhao Zhang, Debangshu Samanta, Haiquan Lu, John W. Bullen, Huimin Zhang, Ivan Chen, Xiaoshun He, Gregg L. Semenza.
Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA.
Proceedings of the National Academy of Sciences, 2016; 201602883     DOI: 10.1073/pnas.1602883113


Pluripotency factors, such as NANOG, play a critical role in the maintenance and specification of cancer stem cells, which are required for primary tumor formation and metastasis. In this study, we report that exposure of breast cancer cells to hypoxia (i.e., reduced O2 availability), which is a critical feature of the tumor microenvironment, induces N6-methyladenosine (m6A) demethylation and stabilization of NANOG mRNA, thereby promoting the breast cancer stem cell (BCSC) phenotype. We show that inhibiting the expression of AlkB homolog 5 (ALKBH5), which demethylates m6A, or the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α, which activate ALKBH5 gene transcription in hypoxic breast cancer cells, is an effective strategy to decrease NANOG expression and target BCSCs in vivo.

N6-methyladenosine (m6A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m6A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α–dependent expression of AlkB homolog 5 (ALKBH5), an m6A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m6A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.

Specific Proteins Found to Jump Start Spread of Cancer Cells


Metastatic breast cancer cells. [National Cancer Institute]

Scientists at the University of California, San Diego School of Medicine and Moores Cancer Center, with colleagues in Spain and Germany, have discovered how elevated levels of particular proteins in cancer cells trigger hyperactivity in other proteins, fueling the growth and spread of a variety of cancers. Their study (“Prognostic Impact of Modulators of G Proteins in Circulating Tumor Cells from Patients with Metastatic Colorectal Cancer”) is published in Scientific Reports.

Specifically, the international team, led by senior author Pradipta Ghosh, M.D., associate professor at the University of California San Diego School of Medicine, found that increased levels of expression of some members of a protein family called guanine nucleotide exchange factors (GEFs) triggered unsuspected hyperactivation of G proteins and subsequent progression or metastasis of cancer.

The discovery suggests GEFs offer a new and more precise indicator of disease state and prognosis. “We found that elevated expression of each GEF is associated with a shorter, progression-free survival in patients with metastatic colorectal cancer,” said Dr. Ghosh. “The GEFs fared better as prognostic markers than two well-known markers of cancer progression, and the clustering of all GEFs together improved the predictive accuracy of each individual family member.”

In recent years, circulating tumor cells (CTCs), which are shed from primary tumors into the bloodstream and act as seeds for new tumors taking root in other parts of the body, have become a prognostic and predictive biomarker. The presence of CTCs is used to monitor the efficacy of therapies and detect early signs of metastasis.

But counting CTCs in the bloodstream has limited utility, said Dr. Ghosh. “Enumeration alone does not capture the particular characteristics of CTCs that are actually tumorigenic and most likely to cause additional malignancies.”

Numerous efforts are underway to improve the value and precision of CTC analysis. According to Dr. Ghosh the new findings are a step in that direction. First, GEFs activate trimeric G proteins, and second, G protein signaling is involved in CTCs. G proteins are ubiquitous and essential molecular switches involved in transmitting external signals from stimuli into cells’ interiors. They have been a subject of heightened scientific interest for many years.

Dr. Ghosh and colleagues found that elevated expression of nonreceptor GEFs activates Gαi proteins, fueling CTCs and ultimately impacting the disease course and survival of cancer patients.

“Our work shows the prognostic impact of elevated expression of individual and clustered GEFs on survival and the benefit of transcriptome analysis of G protein regulatory proteins in cancer biology,” said Dr. Ghosh. “The next step will be to carry this technology into the clinic where it can be applied directly to deciphering a patient’s state of cancer and how best to treat.”

Metastasis-on-a-Chip’ Models Cancer’s Spread


In the journal Biotechnology Bioengineering, the team reports on its “metastasis-on-a-chip” system believed to be one of the first laboratory models of cancer spreading from one 3D tissue to another.

The current version of the system models a colorectal tumor spreading from the colon to the liver, the most common site of metastasis. Skardal said future versions could include additional organs, such as the lung and bone marrow, which are also potential sites of metastasis. The team also plans to model other types of cancer, such as the deadly brain tumor glioblastoma

To create the system, researchers encapsulated human intestine and colorectal cancer cells inside a biocompatible gel-like material to make a mini-organ. A mini-liver composed of human liver cells was made in the same way. These organoids were placed in a “chip” system made up of a set of micro-channels and chambers etched into the chip’s surface to mimic a simplified version of the body’s circulatory system. The tumor cells were tagged with fluorescent molecules so their activity could be viewed under a microscope.

To test whether the system could model metastasis, the researchers first used highly aggressive cancer cells in the colon organoid. Under the microscope, they saw the tumor grow in the colon organoid until the cells broke free, entered the circulatory system and then invaded the liver tissue, where another tumor formed and grew. When a less aggressive form of colon cancer was used in the system, the tumor did not metastasize, but continued to grow in the colon.

To test the system’s potential for screening drugs, the team introduced Marimastat, a drug used to inhibit metastasis in human patients, into the system and found that it significantly prevented the migration of metastatic cells over a 10-day period. Likewise, the team also tested 5-fluorouracil, a common colorectal cancer drug, which reduced the metabolic activity of the tumor cells.

“We are currently exploring whether other established anti-cancer drugs have the same effects in the system as they do in patients,” said Skardal. “If this link can be validated and expanded, we believe the system can be used to screen drug candidates for patients as a tool in personalized medicine. If we can create the same model systems, only with tumor cells from an actual patient, then we believe we can use this platform to determine the best therapy for any individual patient.”

The scientists are currently working to refine their system. They plan to use 3D printing to create organoids more similar in function to natural organs. And they aim to make the process of metastasis more realistic. When cancer spreads in the human body, the tumor cells must break through blood vessels to enter the blood steam and reach other organs. The scientists plan to add a barrier of endothelial cells, the cells that line blood vessels, to the model.

This concept of modeling the body’s processes on a miniature level is made possible because of advances in micro-tissue engineering and micro-fluidics technologies. It is similar to advances in the electronics industry made possible by miniaturizing electronics on a chip.

Scientists Synthesize Anti-Cancer Agent

A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University
A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University  http://www.dddmag.com/sites/dddmag.com/files/ddd1603_rice-anticancer.jpg

A team led by Rice University synthetic organic chemist K.C. Nicolaou has developed a new process for the synthesis of a series of potent anti-cancer agents originally found in bacteria.

The Nicolaou lab finds ways to replicate rare, naturally occurring compounds in larger amounts so they can be studied by biologists and clinicians as potential new medications. It also seeks to fine-tune the molecular structures of these compounds through analog design and synthesis to improve their disease-fighting properties and lessen their side effects.

Such is the case with their synthesis of trioxacarcins, reported this month in the Journal of the American Chemical Society.

“Not only does this synthesis render these valuable molecules readily available for biological investigation, but it also allows the previously unknown full structural elucidation of one of them,” Nicolaou said. “The newly developed synthetic technologies will allow us to construct variations for biological evaluation as part of a program to optimize their pharmacological profiles.”

At present, there are no drugs based on trioxacarcins, which damage DNA through a novel mechanism, Nicolaou said.

Trioxacarcins were discovered in the fermentation broth of the bacterial strain Streptomyces bottropensis. They disrupt the replication of cancer cells by binding and chemically modifying their genetic material.

“These molecules are endowed with powerful anti-tumor properties,” Nicolaou said. “They are not as potent as shishijimicin, which we also synthesized recently, but they are more powerful than taxol, the widely used anti-cancer drug. Our objective is to make it more powerful through fine-tuning its structure.”

He said his lab is working with a biotechnology partner to pair these cytotoxic compounds (called payloads) to cancer cell-targeting antibodies through chemical linkers. The process produces so-called antibody-drug conjugates as drugs to treat cancer patients. “It’s one of the latest frontiers in personalized targeting chemotherapies,” said Nicolaou, who earlier this year won the prestigious Wolf Prize in Chemistry.

Fluorescent Nanoparticle Tracks Cancer Treatment’s Effectiveness in Hours

Bevin Fletcher, Associate Editor    http://www.biosciencetechnology.com/news/2016/03/fluorescent-nanoparticle-tracks-cancer-treatments-effectiveness-hours

Using reporter nanoparticles loaded with either a chemotherapy or immunotherapy, researchers could distinguish between drug-sensitive and drug-resistant tumors in a pre-clinical model of prostate cancer. (Source: Brigham and Women's Hospital)

Using reporter nanoparticles loaded with either a chemotherapy or immunotherapy, researchers could distinguish between drug-sensitive and drug-resistant tumors in a pre-clinical model of prostate cancer. (Source: Brigham and Women’s Hospital)

Bioengineers at Brigham and Women’s Hospital have developed a new technique to help determine if chemotherapy is working in as few as eight hours after treatment. The new approach, which can also be used for monitoring the effectiveness of immunotherapy, has shown success in pre-clinical models.

The technology utilizes a nanoparticle, carrying anti-cancer drugs, that glows green when cancer cells begin dying. Researchers, using  the “reporter nanoparticles” that responds to a particular enzyme known as caspase, which is activated when cells die, were able to distinguish between a tumor that is drug-sensitive or drug-resistant much faster than conventional detection methods such as PET scans, CT and MRI.  The findings were published online March 28 in the Proceedings of the National Academy of Sciences.

“Using this approach, the cells light up the moment a cancer drug starts working,” co-corresponding author Shiladitya Sengupta, Ph.D., principal investigator in BWH’s Division of Bioengineering, said in a prepared statement.  “We can determine if a cancer therapy is effective within hours of treatment.  Our long-term goal is to find a way to monitor outcomes very early so that we don’t give a chemotherapy drug to patients who are not responding to it.”

Cancer killers send signal of success

Nanoparticles deliver drug, then give real-time feedback when tumor cells die   BY   SARAH SCHWARTZ

New lab-made nanoparticles deliver cancer drugs into tumors, then report their effects in real time by lighting up in response to proteins produced by dying cells. More light (right, green) indicates a tumor is responding to chemotherapy.

Tiny biochemical bundles carry chemotherapy drugs into tumors and light up when surrounding cancer cells start dying. Future iterations of these lab-made particles could allow doctors to monitor the effects of cancer treatment in real time, researchers report the week of March 28 in theProceedings of the National Academy of Sciences.

“This is the first system that allows you to read out whether your drug is working or not,” says study coauthor Shiladitya Sengupta, a bioengineer at Brigham and Women’s Hospital in Boston.

Each roughly 100-nanometer-wide particle consists of a drug and a fluorescent dye linked to a coiled molecular chain. Before the particles enter cells, the dye is tethered to a “quencher” molecule that prevents it from lighting up. When injected into the bloodstream of a mouse with cancer, the nanoparticles accumulate in tumor cells and release the drug, which activates a protein that tears a cancer cell apart. This cell-splitting protein not only kills the tumor cell, but also severs the link between the dye and the quencher, allowing the nanoparticles to glow under infrared light.

Reporter nanoparticle that monitors its anticancer efficacy in real time

Ashish Kulkarnia,b,1,Poornima Raoa,b,Siva Natarajana,b,Aaron Goldman, et al.

The ability to identify responders and nonresponders very early during chemotherapy by direct visualization of the activity of the anticancer treatment and to switch, if necessary, to a regimen that is effective can have a significant effect on the outcome as well as quality of life. Current approaches to quantify response rely on imaging techniques that fail to detect very early responses. In the case of immunotherapy, the early anatomical readout is often discordant with the biological response. This study describes a self-reporting nanomedicine that not only delivers chemotherapy or immunotherapy to the tumor but also reports back on its efficacy in real time, thereby identifying responders and nonresponders early on

The ability to monitor the efficacy of an anticancer treatment in real time can have a critical effect on the outcome. Currently, clinical readouts of efficacy rely on indirect or anatomic measurements, which occur over prolonged time scales postchemotherapy or postimmunotherapy and may not be concordant with the actual effect. Here we describe the biology-inspired engineering of a simple 2-in-1 reporter nanoparticle that not only delivers a cytotoxic or an immunotherapy payload to the tumor but also reports back on the efficacy in real time. The reporter nanoparticles are engineered from a novel two-staged stimuli-responsive polymeric material with an optimal ratio of an enzyme-cleavable drug or immunotherapy (effector elements) and a drug function-activatable reporter element. The spatiotemporally constrained delivery of the effector and the reporter elements in a single nanoparticle produces maximum signal enhancement due to the availability of the reporter element in the same cell as the drug, thereby effectively capturing the temporal apoptosis process. Using chemotherapy-sensitive and chemotherapy-resistant tumors in vivo, we show that the reporter nanoparticles can provide a real-time noninvasive readout of tumor response to chemotherapy. The reporter nanoparticle can also monitor the efficacy of immune checkpoint inhibition in melanoma. The self-reporting capability, for the first time to our knowledge, captures an anticancer nanoparticle in action in vivo.


Cancer Treatment’s New Direction  
Genetic testing helps oncologists target tumors and tailor treatments

Evan Johnson had battled a cold for weeks, endured occasional nosebleeds and felt so fatigued he struggled to finish his workouts at the gym. But it was the unexplained bruises and chest pain that ultimately sent the then 23-year-old senior at the University of North Dakota to the Mayo Clinic. There a genetic test revealed a particularly aggressive form of acute myeloid leukemia. That was two years ago.

The harrowing roller-coaster that followed for Mr. Johnson and his family highlights new directions oncologists are taking with genetic testing to find and attack cancer. Tumors can evolve to resist treatments, and doctors are beginning to turn such setbacks into possible advantages by identifying new targets to attack as the tumors change.

His course involved a failed stem cell transplant, a half-dozen different drug regimens, four relapses and life-threatening side effects related to his treatment.

Nine months in, his leukemia had evolved to develop a surprising new mutation. The change meant the cancer escaped one treatment, but the new anomaly provided doctors with a fresh target, one susceptible to drugs approved for other cancers. Doctors adjusted Mr. Johnson’s treatment accordingly, knocked out the disease and paved the way for a second, more successful stem cell transplant. He has now been free of leukemia for a year.

Now patients with advanced cancer who are treated at major centers can expect to have their tumors sequenced, in hopes of finding a match in a growing medicine chest of drugs that precisely target mutations that drive cancer’s growth. When they work, such matches can have a dramatic effect on tumors. But these “precision medicines” aren’t cures. They are often foiled when tumors evolve, pushing doctors to take the next step to identify new mutations in hopes of attacking them with an effective treatment.

Dr. Kasi and his Mayo colleagues—Naseema Gangat, a hematologist, and Shahrukh Hashmi, a transplant specialist—are among the authors of an account of Mr. Johnson’s case published in January in the journal Leukemia Research Reports.

Before qualifying for a transplant, a patient’s blasts need to be under 5%.

To get under 5%, he started on a standard chemotherapy regimen and almost immediately, things went south. His blast cells plummeted, but “the chemo just wiped out my immune system,”

Then as mysteriously as it began, a serious mycotic throat infection stopped. But Mr. Johnson couldn’t tolerate the chemo, and his blast cells were on the rise. A two-drug combination that included the liver cancer drug Nexavar, which targets the FLT3 mutation, knocked back the blast cells. But the stem cell transplant in May, which came from one of his brothers, failed to take, and he relapsed after 67 days, around late July.

He was put into a clinical trial of an experimental AML drug being developed by Astellas Pharma of Japan. He started to regain weight. In November 2014, doctors spotted the initial signs in blood tests that Mr. Johnson’s cancer was evolving to acquire a new mutation. By late January, he relapsed again , but there was a Philadelphia chromosome mutation,  a well-known genetic alteration associated with chronic myeloid leukemia. It also is a target of the blockbuster cancer drug Gleevec and several other medicines.

Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3) inhibitor therapy with evolving actionable targets

Naseema GangatMark R. LitzowMrinal M. PatnaikShahrukh K. HashmiNaseema Gangat

•   The article reports on a case of AML that underwent clonal evolution.
•   We report on novel acquisition of the Philadelphia t(9;22) translocation in AML.
•   Next generation sequencing maybe helpful in these refractory/relapse cases.
•   Novel FLT3-inhibitor targeted therapies are another option in patients with AML.
•   Personalizing cancer treatment based on evolving targets is a viable option.

For acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215) to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈https://clinicaltrials.gov/ct2/show/NCT02014558〉)

The development of kinase inhibitors for the treatment of leukemia has revolutionized the care of these patients. Since the introduction of imatinib for the treatment of chronic myeloid leukemia, multiple other tyrosine kinase inhibitors (TKIs) have become available[1]. Additionally, for acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors [2], [3], [4] and [5]. The article herein reports a unique case of AML that underwent clonal evolution while on a novel FLT3-inhibitor clinical trial.

Our work herein presents clinical and next generation sequencing data at the time of progression to illustrate these important concepts stemming from Darwinian evolution [6]. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process.

Our work focuses on a 23-year-old male who presented with 3 months history of fatigue and easy bruising, a white blood count of 22.0×109/L with 51% circulating blasts, hemoglobin 7.6 g/dL, and a platelet count of 43×109/L. A bone marrow biopsy confirmed a diagnosis of AML. Initial cytogenetic studies identified trisomy 8 in all the twenty metaphases examined. Mutational analysis revealed an internal tandem duplication of the FLT3 gene (FLT3-ITD).

He received standard induction chemotherapy (7+3) with cytarabine (ARA-C; 100 mg/m2for 7 days) and daunorubicin (DNM; 60 mg/m2 for 3 days). His induction chemotherapy was complicated by severe palatine and uvular necrosis of indeterminate etiology (possible mucormycosis).

Bone marrow biopsy at day 28 demonstrated persistent disease with 10% bone marrow blasts (Fig. 1). Due to his complicated clinical course and the presence of a FLT3-ITD, salvage therapy with 5-azacitidine (5-AZA) and sorafenib (SFN) was instituted. Table 1.
The highlighted therapies were employed in this particular case at various time points as shown in Fig. 1.



    • [1]
    • J.E. Cortes, D.W. Kim, J. Pinilla-Ibarz, et al.
    • A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias
    • New Engl. J. Med., 369 (19) (2013), pp. 1783–1796
    • [2]
    • F. Ravandi, M.L. Alattar, M.R. Grunwald, et al.
    • Phase 2 study of azacytidine plus sorafenib in patients with acute myeloid leukemia and FLT-3 internal tandem duplication mutation
    • Blood, 121 (23) (2013), pp. 4655–4662
    • [3]
    • N.P. Shah, M. Talpaz, M.W. Deininger, et al.
    • Ponatinib in patients with refractory acute myeloid leukaemia: findings from a phase 1 study
    • Br. J. Haematol., 162 (4) (2013), pp. 548–552
    • [4]
    • Y. Alvarado, H.M. Kantarjian, R. Luthra, et al.
    • Treatment with FLT3 inhibitor in patients with FLT3-mutated acute myeloid leukemia is associated with development of secondary FLT3-tyrosine kinase domain mutations
    • Cancer, 120 (14) (2014), pp. 2142–2149
    • [5]
    • C.C. Smith, C. Zhang, K.C. Lin, et al.
    • Characterizing and overriding the structural mechanism of the Quizartinib-Resistant FLT3 “Gatekeeper” F691L mutation with PLX3397
    • Cancer Discov. (2015)
    • [6]
    • M. Greaves, C.C. Maley
    • Clonal evolution in cancer
    • Nature, 481 (7381) (2012), pp. 306–313




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Lymphatic Cancer Metastasis Model

Larry H. Bernstein, MD, FCAP, Curator



How Stress Affects Cancer’s Spread

A mouse study reveals how chronic stress remodels lymphatic vasculature to facilitate the spread of tumor cells.

By Catherine Offord | March 1, 2016



Green fluorescently-tagged nanospheres flow through a lymph vessel from an unstressed mouse (top) and a mouse that has been administered the stress hormone norepinephrine (bottom). Scale bar: 20 μmNATURE COMMUNICATIONS, LE ET AL.

Stress is implicated in increased tumor progression risk and poor survival in cancer patients. A number of recent studies have linked these effects to the promotion of tumor cell dissemination through the bloodstream via stress-induced pathways. Now, a mouse study led by researchers in Australia has revealed the mechanisms by which stress modulates cancer’s spread through another transport network open to tumor cells—the lymphatic system. The findings were published today (March 1) in Nature Communications.

Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination

Caroline P. LeCameron J. NowellCorina Kim-FuchsEdoardo Botteri, …., Andreas MöllerSteven A. Stacker Erica K. Sloan
Nature Communications  7,  Article number:10634    doi:10.1038/ncomms10634

Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes.

In everyday life, we encounter stressful experiences that pose a threat to physiological homeostasis. These threats trigger stress responses, including activation of the sympathetic nervous system (SNS), which leads to elevated local and systemic levels of catecholaminergic neurotransmitters that signal to cells1. Stress-induced SNS signalling is important to enhance alertness and physiological functions for rapid reaction to threat2. However, chronic periods of stress can be detrimental to health by increasing inflammation and promoting the progression of diseases including cancer3, 4, 5, 6. Clinical studies have linked experience of stressful events to poor cancer survival7, 8. This is supported by preclinical studies that show chronic stress promotes cancer progression3, 4, 6. These studies found that stress recruits inflammatory cells to tumours and increases the formation of blood vessels3, 6, which may provide routes for tumour cell dissemination. In addition to dissemination through blood vessels, cancer cells also escape from tumours through lymphatic vasculature9, 10, 11.

The lymphatic system plays an important role in immune function and therefore can influence the trajectory of disease progression. Under normal physiological conditions, the lymphatic system maintains homeostasis by directing cells and solutes from the interstitial fluid of peripheral tissues through lymphatic vessels and into lymph nodes, where they undergo immune examination12, 13. In addition, the lymphatic system aids in the resolution of inflammation by transporting immune cells away from sites of infection14. In cancer, the lymphatic system contributes to disease progression by providing a pathway for tumour cell escape while also being a rich source of chemokines that can promote the invasive properties of tumour cells15. Furthermore, tumour-draining lymph nodes and associated lymphatic endothelium have been shown to develop an immunosuppressive environment, which promotes immune tolerance to the cancer and facilitates tumour growth and spread16, 17, 18. The importance of the lymphatic system in cancer progression is supported by vast clinical data that show tumour-associated lymphatic vessel density (LVD), tumour cell invasion into lymphatic vasculature and the presence of tumour cells in lymph nodes are each associated with increased clinical tumour stage and reduced disease-free survival19, 20,21.

The lymphatic system is innervated by fibres of the SNS22, and acute SNS activity has been shown to increase lymphatic vessel contraction23, 24 and lymphocyte output into lymphatic circulation25. However, little is known about whether stress-induced SNS signalling affects tumour lymphatic vasculature and the consequences this may have on cancer progression.

In this study, we show that chronic stress increases intratumoural LVD while also inducing dilation and increasing flow in lymphatic vessels that drain metastatic tumour cells into lymphatic circulation. Inhibition of COX2 activity blocked the effect of stress on lymphatic vascular remodelling, and showed a key role for macrophage-mediated inflammation in the effects of stress. In addition, we show a critical role for tumour cell-derived VEGFC in the effects of stress on lymphatic vasculature. In both clinical and preclinical studies we demonstrate that disrupting SNS regulation of lymphatics, by blocking β-adrenoceptor signalling, protects against lymphatic dissemination and cancer progression. These findings identify stress signalling as a regulator of lymphatic remodelling and provide evidence for the feasibility of clinically targeting SNS regulation of lymphatics to prevent tumour cell dissemination through lymphatic routes.

Figure 1: Chronic stress remodels tumour-associated lymphatic architecture to promote lymph node metastasis.

Chronic stress remodels tumour-associated lymphatic architecture to promote lymph node metastasis.


(a) Schematic representation of the chronic stress paradigm. (b) Quantification and representative images of tumour LVD (LYVE-1+, green; nuclear, blue) immunostaining of MDA-MB-231 orthotopic tumours. Scale bar, 200μm (n=5). (c) Quantification of MDA-MB-231 primary tumour size in control or stressed BALB/c nu/nu mice over time (n=5 at each time point). (d) Quantification and representative images of tumour-draining lymphatic vessel diameter (LV, blue) in mice with MDA-MB-231 tumours. Scale bar, 1mm (ngreater than or equal to7). (e) Left: skin flap preparation after injection of Patent Blue V dye into the primary tumour (PT) showing the dye taken up into the tumour-draining LV and into the tumour-draining axillary lymph node (AxLN). The LV is adjacent to a blood vessel (BV). Right top panel: epifluorescence image of mCherry-tagged MDA-MB-231 tumour cells (TCs, red) that had spontaneously disseminated from orthotopic PT and were present in the tumour-draining LV that contained microspheres (green) and was adjacent to an autofluorescent BV. Right lower panel: corresponding maximum projection of multiphoton image. Scale bar, 100μm (Supplementary Movie 1). (f) Representative in vivo bioluminescence image of orthotopic MDA-MB-231 breast cancer model showing PT, and spontaneous metastasis to draining lymph node (LN) and lung 21 days after tumour cell injection. (g) Representative images of LN and lung metastasis and quantification of metastasis by ex vivo bioluminescence (BLI) imaging in control versus stressed mice with MDA-MB-231 tumours (n=5). (h) Metastasis in vivo over time (n=5 at each time point). (i) LN metastasis in mice that were negative or positive for tumour cells in collecting lymphatic vessels (ngreater than or equal to13). (j) Ex vivo quantification of bioluminescence from LN at day 28 of 66cl4 tumour progression from control or stressed mice (n=5). (k) Area of lymph node metastasis when primary tumour diameter reached 12mm in control or stressed MMTV-PyMT mice (ngreater than or equal to8). Experiments were completed 2–4 times. All data represent mean±s.e. **P<0.01 and ***P<0.001 by Student’s t-test or Mann–Whitney U-test (post hocBonferroni correction).


Figure 7: Stress-induced lymphatic remodelling

Stress-induced lymphatic remodelling.


Stress remodels lymphatic vasculature through a tumour neural-inflammatory axis to promote lymphogenous tumour cell dissemination and metastasis. Tumour cell-derived VEGFC is necessary for stress-enhanced lymphatic remodelling but is not directly activated by β-adrenoceptor signalling. Tumour-associated macrophages respond to β-adrenoceptor signalling to produce inflammatory molecules such as PGE2, which may then signal to tumour cells to produce VEGFC required for lymphatic remodelling. These effects may be clinically blocked using BBs, anti-VEGFC therapeutics (αVEGFC) or COX2 inhibitors (COX2i). E, epinephrine; NE, norepinephrine; β-AR, β-adrenoceptor.


These findings suggest that it may be important to identify stressed individuals who may be particularly susceptible to lymphogenous dissemination. One approach may be through transcriptional profiling using a stress signature55. Alternatively, as cancer is often a highly stressful experience, it is plausible that SNS intervention may be generally useful to improve cancer outcome. In support of that contention, we found here that clinical BB use was linked to a significant reduction in lymph node metastasis (and reduced distant metastasis) in a cancer cohort without prior evaluation of stress levels.

Stress regulation of lymphatic vasculature may have evolved to promote survival during times of threat. Co-ordinated regulation of the fight-or-flight stress response with increased lymphatic function may have provided an evolutionary advantage by enhancing immune surveillance and activating a rapid immune response to physical threat. However, the findings presented here demonstrate that SNS-regulated lymphatic function can have adverse effects in the context of chronic diseases such as cancer. Importantly, these findings identify multiple points of clinical intervention to limit these adverse effects of stress.


“Stress not only affects your well-being, but it also affects your biology,” said study coauthor Erica Sloan, a cancer researcher at Monash University in Melbourne. “Our study particularly highlights the early steps of tumor cell dissemination into the lymphatic system.”

“This is an excellent contribution,” said Kari Alitalo, a professor of translational cancer biology at the University of Helsinki, Finland, who was not involved in the study. “It’s certainly a very refreshing, novel aspect of biology that they explore in this paper.”

Chronic stress, mediated partly through the sympathetic nervous system, has been associated in cancer patients with a number of physiological changes that promote metastasis (the spread of cancer), including the promotion of blood vessel formation and the recruitment of inflammatory cells like macrophages.

To investigate whether stress could also induce changes in lymph vasculature, the researchers subjected various types of mammary tumor–bearing mice—including strains genetically engineered to develop tumors spontaneously, as well as animals given tumor transplants—to a paradigm designed to induce chronic stress: confinement in a tight space. Comparing stressed mice to controls that bore the same cancerous tumors but had been kept in normal cage conditions, the researchers found no difference in primary tumor growth, but significant differences in lymph vasculature architecture and the frequency of metastases.

“We found that stress helps to build new lymphatic freeways out of the tumor [and] modulates how quickly lymph flows through lymph vessels,” said Sloan, adding that “stress increases the speed limit on these little lymphatic highways and helps cells transit more quickly out of the tumor.”

Since tumor cell dissemination is a key step in cancer metastasis, the team wanted to test whether dissemination through the lymphatic system could be reduced by blocking stress signaling pathways. The researchers turned to beta-blockers—cheap, widely available drugs commonly used to treat hypertension—which inhibit signaling of norepinephrine (or noadrenaline), a stress hormone already implicated in cancer progression risk.

Administering beta-blockers to tumor-bearing mice, the researchers were able to minimize changes in the density of lymph vessels at the primary tumor site, and subsequently reduce metastasis to the lymph nodes. By contrast, artificially stimulating norepinephrine receptors increased both lymph vessel density and metastasis. Through a series of further experiments, the team demonstrated important roles for macrophages involved in inflammatory signaling and a set of tumor-secreted vascular endothelial growth factors (VEGFs) in the regulation of lymph vasculature remodeling and tumor cell dissemination.

“It’s an important step in understanding how stress pathways can influence metastasis,” said Anil Sood, a professor of translational research at MD Anderson Cancer Center in Houston, Texas, who was not involved in the research. “It really helps us to understand the possible mechanisms by which sympathetic nervous system pathways can affect how lymphatics may be remodeled.”

The study also included an analysis of observational data from a cohort of nearly 1,000 breast cancer patients in Milan, which corroborate the team’s findings in mice: patients taking beta-blockers showed a significantly lower incidence of lymph node and distant metastases, even once potentially confounding factors such as age and treatment type had been taken into account.

But Alitalo cautioned against drawing strong conclusions from these data. “Stress biology is complex,” he said. “In laboratory conditions with mice, it’s easier to define and measure stress. In real life, these things fluctuate a lot, especially in cancer patients.” He added that beta-blockers show “no specificity to the lymphatic system, so [their] effects as such could be transduced via a variety of pathways.”

Sloan and colleagues are now working to further resolve the molecular mechanisms involved in stress-induced remodeling of the tumor microenvironment in mice, and are investigating potential interactions between beta-blockers and standard cancer treatments, with a view to using the drugs to tackle stress-related metastasis risk in the clinic.

“This is something that, when we treat cancer, we should be considering,” Sloan said. “By actually addressing stress in the patient, we’re giving our cancer therapies a better chance to work.”

C.P. Le et al., “Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination,” Nature Communications, http://dx.doi.org:/10.1038/ncomms10634,2016.

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