Leaders in Pharmaceutical Business Intelligence (LPBI) Group

New insights in cancer, cancer immunogenesis and circulating cancer cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

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

http://www.the-scientist.com/?articles.view/articleNo/45883/title/Circulating-Tumor-Cells-Traverse-Tiny-Vasculature

http://www.the-scientist.com/images/News/April2016/tumorclusters1.gif

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.

http://www.the-scientist.com/images/News/April2016/tumorclusters2.gif

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

http://www.the-scientist.com/images/News/April2016/tumorclusters3.gif

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

http://www.genengnews.com/gen-news-highlights/signal-loop-pulls-healthy-cells-into-cancer-s-echo-chamber/81252618/

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 KRAS^G12D signaling in pancreatic ductal adenocarcinoma (PDA) cells,” wrote the authors of the Cell article. “Tumor cell KRAS^G12D 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 KRAS^G12D.”

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 KRAS^G12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS^G12D 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

New Genomic Analysis of Immune Cell Infiltration in Colorectal Cancer

http://www.genengnews.com/gen-news-highlights/new-genomic-analysis-of-immune-cell-infiltration-in-colorectal-cancer/81252616/

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. Tumor-infiltrating immune cells and prognosis: the potential link …

   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. Immune Cells in Colorectal Cancer: Prognostic Relevance and Role …

   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 immune–cell 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
http://dx.doi.org/10.1155/2016/6261721

Not Your Average Circulating Tumor Cells   

Translational Scientists Profile Cancer Cells That Have Gone on the Lam

• Richard A. Stein, M.D., Ph.D

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.”