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Posts Tagged ‘Zebrafish’


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

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

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 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.

Highlights

  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

http://www.cell.com/cms/attachment/2053320047/2060147607/fx1.jpg

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

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. 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
http://dx.doi.org/10.1155/2016/6261721


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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Single Cell Shines Light on Cell Malignant Transformation

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Single Cell Lights Beacon to Cancer Origins

http://www.genengnews.com/gen-news-highlights/single-cell-lights-beacon-to-cancer-origins/81252303/

http://www.nytimes.com/2016/01/29/health/how-skin-cancer-develops-melanoma-zebra-fish.html?ref=todayspaper

For the first time scientists are able to visualize the origin of cancer cells and track their progression through tumorigenesis. [Dr. Leonard Zon, Boston Children’s Hospital].

Conundrums exist in every facet of our daily lives and are often dismissed as either general annoyances or fleeting curiosities. Yet for scientists, solving common scientific enigmas can often lead to a watershed moment within their respective discipline.  For instance, researchers have been perplexed for decades as to why the vast number of cells that have cancer genes never morph into cancerous tumors.

Researchers at Boston Children’s Hospital have published results from a new study, which visualized the origins of cancer from the first affected cell and watched its spread within a live animal. The research team is hopeful that their findings could change the way scientists understand melanoma and other cancers, as well as open doors to new therapeutic interventions.

“An important mystery has been why some cells in the body already have mutations seen in cancer, but do not yet fully behave like the cancer,” stated lead author Charles Kaufman, M.D., Ph.D., a postdoctoral fellow in the laboratory of Howard Hughes Medical Institute investigator Leonard Zon, M.D. at Boston Children’s Hospital. “We found that the beginning of cancer occurs after activation of an oncogene or loss of a tumor suppressor, and involves a change that takes a single cell back to a stem cell state.”

The findings from this study were published online today in Science through an article entitled “A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation.”

The Boston researchers tracked the development of melanoma in live zebrafish that had been genetically engineered to have the human cancer mutation BRAFV600E—found in most benign moles—and the p53 tumor suppressor gene removed. Moreover, the fish contained a GFP-crestin gene that would light up if it was turned on, a beacon indicating activation of a genetic program characteristic of stem cells. Typically this program is turned off after embryonic development. Occasionally, however, crestin and other genes in the program turn back on in individual cells.

“Every so often we would see a green spot on a fish,” noted Dr. Zon, who is the director of the Stem Cell Research Program at Boston Children’s, member of the Harvard Stem Cell Institute, and senior author on the current study. “When we followed them, they became tumors 100 percent of the time.”

When Dr. Zon and his colleagues looked to see what was different in these early cancer cells, they found that crestin and the other activated genes are the same ones turned on during zebrafish embryonic development, specifically in the stem cells that give rise to the pigment cells known as melanocytes within a structure called the neural crest.

“What’s cool about this group of genes is that they also get turned on in human melanoma,” Dr. Zon said. “It’s a change in cell fate, back to neural crest status.”

While trying to track these glowing cells in live fish was exceedingly difficult, Dr. Kaufman managed to isolate and characterize 30 fish with a small green glowing cluster of cells. Interestingly, in all 30 cases, the clusters grew into melanomas. In two fish, Dr. Kaufman was able to see a single green-glowing cell and watch it divide to ultimately become a tumor mass.

“It’s estimated that only one in tens or hundreds of millions of cells in a mole eventually become a melanoma,” explained Dr. Kaufman. “Because we can also efficiently breed many fish, we can look for these very rare events. The rarity is very similar in both humans and fish, which suggests that the underlying process of melanoma formation is probably much the same in humans.”

The investigators are optimistic that the results from their study could lead to new genetic tests for suspicious moles to see whether the cells are behaving like neural crest cells, indicating that the stem-cell program has been turned on. Additionally, the scientists are investigating the regulatory elements that turn on the genetic program, often called super-enhancers. These control elements have analogous epigenetic functions in zebrafish and human melanoma, potentially providing druggable targets to stop a mole from becoming cancerous.

https://vimeo.com/152633925

 

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation
Visualizing the beginnings of melanoma

In cancer biology, a tumor begins from a single cell within a group of precancerous cells that share genetic mutations. Kaufman et al. used a zebrafish melanoma model to visualize cancer initiation (see the Perspective by Boumahdi and Blanpain). They used a fluorescent reporter that specifically lit up neural crest progenitors that are only present during embryogenesis or during adult melanoma tumor formation. The appearance of this tumor correlated with a set of gene regulatory elements, called super-enhancers, whose identification and manipulation may prove beneficial in detecting and preventing melanoma initiation.

Science, this issue p. 10.1126/science.aad3867; see also p. 453

 

Structured Abstract

INTRODUCTION

The “cancerized field” concept posits that cells in a given tissue sharing an oncogenic mutation are cancer-prone, yet only discreet clones within the field initiate tumors. Studying the process of cancer initiation has remained challenging because of (i) the rarity of these events, (ii) the difficulty of visiualizing initiating clones in living organisms, and (iii) the transient nature of a newly transformed clone emerging before it expands to form an early tumor. A more complete understanding of the molecular processes that regulate cancer initiation could provide important prognostic information about which precancerous lesions are most prone to becoming cancer and also implicate druggable molecular pathways that, when inhibited, may prevent the cancer from ever starting.

RATIONALE

The majority of benign nevi carry oncogenic BRAFV600E mutations and can be considered a cancerized field of melanocytes, but they only rarely convert to melanoma. In an effort to define events that initiate cancer, we used a melanoma model in the zebrafish in which the humanBRAFV600E oncogene is driven by the melanocyte-specific mitfa promoter. When bred into a p53mutant background, these fish develop melanoma tumors over the course of many months. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward.

RESULTS

We developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors. We show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAFV600Emutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. Early crestin+ patches of cells expand and are transplantable in a manner consistent with their possessing tumorigenic activity, and they exhibit a gene expression pattern consistent with the NCP identity readout by the crestin reporter. Thecrestin element is regulated by NCP transcription factors, including sox10. Forced sox10overexpression in melanocytes accelerated melanoma formation, whereas CRISPR/Cas9 targeting of sox10 delayed melanoma onset. We show activation of super-enhancers at NCP genes in both zebrafish and human melanomas, identifying an epigenetic mechanism for control of this NCP signature leading to melanoma.

CONCLUSION

This work using our zebrafish melanoma model and in vivo reporter of NCP identity allows us to see cancer from its birth as a single cell and shows the importance of NCP-state reemergence as a key event in melanoma initiation from a field of cancer-prone melanocytes. Thus, in addition to the typical fixed genetic alterations in oncogenes and tumor supressors that are required for cancer development, the reemergence of progenitor identity may be an additional rate-limiting step in the formation of melanoma. Preventing NCP reemergence in a field of cancer-prone melanocytes may thus prove therapeutically useful, and the association of NCP genes with super-enhancer regulatory elements implicates the associated druggable epigenetic machinery in this process.

 

 

Neural crest reporter expression in melanoma.

The crestin:EGFP transgene is specifically expressed in melanoma inBRAFV600E/p53 mutant melanoma-prone zebrafish. (Top) A single cell expressingcrestin:EGFP expands into a small patch of cells over the course of 2 weeks, capturing the initiation of melanoma formation (bracket). (Bottom) A fully formed melanoma specifically expresses crestin:EGFP, whereas the rest of the fish remains EGFP-negative.

 

Neural crest reporter expression in melanoma.

The crestin:EGFP transgene is specifically expressed in melanoma inBRAFV600E/p53 mutant melanoma-prone zebrafish. (Top) A single cell expands into a small patch of cells over the course of 2 weeks, capturing the initiation of melanoma formation (bracket). (Bottom) A fully formed melanoma specifically expresses crestin:EGFP, whereas the rest of the fish remains EGFP-negative.

The “cancerized field” concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAFV600E mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAFV600Emutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.

 

A Cancer’s Surprise Origins, Caught in Action

Harvard University

Most of the time skin moles are harmless, but they occasionally turn into melanoma, a life-threatening skin cancer. Leonard Zon and colleagues found that this happens when a single cell regresses back to a stem cell state and starts to divide and invade the surrounding tissue. (Image: Courtesy of Boston Children's Hospital)

http://www.biosciencetechnology.com/sites/biosciencetechnology.com/files/bt1601_harvard_melenoma.jpg

Most of the time skin moles are harmless, but they occasionally turn into melanoma, a life-threatening skin cancer. Leonard Zon and colleagues found that this happens when a single cell regresses back to a stem cell state and starts to divide and invade the surrounding tissue. (Image: Courtesy of Boston Children’s Hospital)

 

Researchers at Harvard-affiliated Boston Children’s Hospital have, for the first time, visualized the origins of cancer from the first affected cell and watched its spread in a live animal. Their work, published in the Jan. 29 issue of Science, could change the way scientists understand melanoma and other cancers and lead to new, early treatments before the cancer has taken hold.

“An important mystery has been why some cells in the body already have mutations seen in cancer, but do not yet fully behave like the cancer,” said the paper’s first author, Charles Kaufman, a postdoctoral fellow in the Zon Laboratory at Boston Children’s Hospital. “We found that the beginning of cancer occurs after activation of an oncogene or loss of a tumor suppressor, and involves a change that takes a single cell back to a stem cell state.”

That change, Kaufman and colleagues found, involves a set of genes that could be targeted to stop cancer from ever starting.

The study imaged live zebrafish over time to track the development of melanoma. All the fish had the human cancer mutation BRAFV600E — found in most benign moles — and had also lost the tumor suppressor gene p53.

Kaufman and colleagues engineered the fish to light up in fluorescent green if a gene called crestin was turned on — a “beacon” indicating activation of a genetic program characteristic of stem cells. This program normally shuts off after embryonic development, but occasionally, in certain cells and for reasons not yet known, crestin and other genes in the program turn back on.

“Every so often we would see a green spot on a fish,” said Leonard Zon, director of the Stem Cell Research Program at Boston Children’s and senior investigator on the study. “When we followed them, they became tumors 100 percent of the time.”

The cell that caused melanoma

When Kaufman, Zon, and colleagues looked to see what was different about these early cancer cells, they found that crestin and the other activated genes were the same ones turned on during zebrafish embryonic development — specifically, in the stem cells that give rise to the pigment cells known as melanocytes, within a structure called the neural crest.

“What’s cool about this group of genes is that they also get turned on in human melanoma,” said Zon, who is also a member of the Harvard Stem Cell Institute and a Howard Hughes Medical Institute investigator. “It’s a change in cell fate, back to neural crest status.”

Finding these cancer-originating cells was tedious. Wearing goggles and using a microscope with a fluorescent filter, Kaufman examined the fish as they swam around, shooting video with his iPhone. Scanning 50 fish could take two to three hours. In 30 fish, Kaufman spotted a small cluster of green-glowing cells about the size of the head of a Sharpie marker — and in all 30 cases, these spots grew into melanomas. In two cases, he was able to see on a single green-glowing cell and watch it divide and ultimately become a tumor mass.

“It’s estimated that only one in tens or hundreds of millions of cells in a mole eventually becomes a melanoma,” said Kaufman, who is also an instructor at the Harvard-affiliated Dana-Farber Cancer Institute. “Because we can also efficiently breed many fish, we can look for these very rare events. The rarity is very similar in both humans and fish, which suggests that the underlying process of melanoma formation is probably much the same in humans.”

Zon, the Grousbeck Professor of Pediatric Medicine at Harvard Medical School, and Kaufman believe that their findings could lead to a new genetic test for suspicious moles to see whether the cells are behaving like neural crest cells, indicating that the stem-cell program has been turned on. They are also investigating the regulatory elements that turn on the genetic program (known as super-enhancers). These DNA elements have epigenetic functions that are similar in zebrafish and human melanoma, and could potentially be targeted with drugs to stop a mole from becoming cancerous.

A paradigm shift for cancer?

Zon and Kaufman posit a new model for cancer formation, going back to a decades-old concept of “field cancerization.” They propose that normal tissue becomes primed for cancer when oncogenes are activated and tumor suppressor genes are silenced or lost, but that cancer develops only when a cell in the tissue reverts to a more primitive, embryonic state and starts dividing. They believe this model may apply to most if not all cancers, not just melanoma.

The study was supported by the National Institutes of Health, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, the Ellison Foundation, the Melanoma Research Alliance, the V Foundation, and the Howard Hughes Medical Institute. Zon is a founder and stockholder of Fate, Inc., and Scholar Rock.

Source: Harvard Gazette

 

 

The little fish that could

Zebrafish help HSCI researchers fight human disease

Leonard Zon, MD, could be called HSCI’s most prolific aquarist. The Executive Committee Chair estimates that he has over 300,000 fish spread throughout his laboratories at the Harvard Department of Stem Cell and Regenerative Biology and Boston Children’s Hospital

His collection isn’t very diverse, composed as it is entirely of zebrafish—minnow-like, freshwater, tropical fish that grow about an inch-and-a-half long. But Zon isn’t interested in winning best in show. Instead, he is leading a scientific movement to show that his striped fish could be humanity’s new animal of choice for drug discovery and studying disease.

Not only does the zebrafish require fewer facility resources than science’s go-to organism, the mouse, but fish are quicker to reproduce, easier to experimentally manipulate, and at least 70 percent of human protein-coding genes have analogs in the zebrafish, including those related to skin cancer, muscular dystrophy, and T cell leukemia.

“The zebrafish is now emerging as another powerful organism for the modeling and study of human diseases, and it is conceivable that zebrafish models will complement mouse models in the future,” Zon wrote in the December issue of Trends in Cell Biology, pointing out the accelerating increase in studies on zebrafish, from about 150 in 1995 to over 2,000 in 2013.

As a pediatric oncologist, Zon’s main purpose in using zebrafish is to help cancer patients. He is the first scientist to successfully apply basic research from the zebrafish to develop an FDA-approved treatment for human melanoma, and is now applying similar methods to find therapies for muscle and blood cancers.

One cancer Zon is pursuing is rhabdomyosarcoma, a rare muscle cancer diagnosed primarily in early childhood. Zebrafish, under certain conditions, develop tumors very similar to the human disease. He is currently using zebrafish embryos to identify which genes transform a normal muscle stem cell into a malignant tumor, as well as searching for factors that might suppress the cancer.

“One of the things that’s really interesting in zebrafish is that the embryos are completely transparent and you can watch the tumors invade the normal tissues,”
he said. “That’s a process that you can’t study in any other organism.”

Postdocs in Zon’s lab can expose the zebrafish embryos to multiple chemicals and literally watch changes in the fish’s development. In 2007, this technique led to the discovery of a type of prostaglandin that expands blood stem cells about 300 to 400 percent. Last fall, the prostaglandin found in Zon’s lab passed Phase Ib clinical trials as a therapy that increases the success of cord blood transplants.

A similar screen led to a major paper last November, in which Zon and fellow HSCI Executive Committee member Amy Wagers, PhD, showed that the same chemicals that stimulate muscle development in zebrafish can also be used to differentiate human stem cells into muscle cells in the laboratory, an historically challenging task that, now overcome, makes muscle cell therapy a more realistic possibility.

“This research demonstrates that over 300 million years of evolution, the pathways used in the fish are conserved through vertebrates all the way up to the human,” Zon said.

His passion for and success with zebrafish has helped make the animal a staple in HSCI faculty member laboratories across Boston, and an unexpected symbol for stem cell research.

Human muscle stem cell therapy gets help from zebrafish

November 7, 2013

Harvard Stem Cell Scientists have discovered that the same chemicals that stimulate muscle development in zebrafish can also be used to differentiate human stem cells into muscle cells in the laboratory, an historically challenging task that, now overcome, makes muscle cell therapy a more realistic clinical possibility.

The work, published this week in the journal Cell, began with a discovery by Boston Children’s Hospital researchers, led by Leonard Zon, MD, and graduate student Cong (Tony) Xu, who tested 2,400 different chemicals in cultures of zebrafish embryo cells to determine if any could increase the numbers of muscle cells formed. Using fluorescent reporter fish in which muscle cells were visible during their creation, the researchers found six chemicals that were very effective at promoting muscle formation.

Zon shared his results with Harvard Department of Stem Cell and Regenerative Biologyprofessor Amy Wagers, PhD, and Mohammadsharif Tabebordbar, a graduate student in her laboratory, who tested the six chemicals in mice. One of the six, called forskolin, was found to increase the numbers of muscle stem cells from mice that could be obtained when these cells were grown in laboratory dishes. Moreover, the cultured cells successfully integrated into muscle when transplanted back into mice.

Inspired by the successful application of these chemicals in mice, Salvatore Iovino, PhD, a joint postdoctoral fellow in the Wagers lab and the lab of C. Ronald Kahn, MD, at the Joslin Diabetes Center, investigated whether the chemicals would also affect human cells and found that a combination of three chemicals, including forskolin, could induce differentiation of human induced pluripotent stem (iPS) cells, made by reprogramming skin cells. Exposure of iPS cells to these chemicals converted them into skeletal muscle, an outcome the Wagers and Kahn labs had been striving to achieve for years using conventional methods. When transplanted into a mouse, the human iPS-derived muscle cells also contributed to muscle repair, offering early promise that this protocol could provide a route to muscle stem cell therapy in humans.

The interdisciplinary, cross-laboratory collaboration between Zon, Wagers, and Kahn highlights the advantage of open exchange between researchers. “If we had done this screen directly on human iPS cells, it would have taken at least 10 times as long and cost 100 times as much,” said Wagers. “The zebrafish gave us a big advantage here because it has a fast generation time, rapid development, and can be easily and relatively cheaply screened in a culture dish.”

“This research demonstrates that over 300 million years of evolution, the pathways used in the fish are conserved through vertebrates all the way up to the human,” said Wagers’ fellow HSCRB professor Leonard Zon, chair of the Harvard Stem Cell InstituteExecutive Committee and director of the stem cell program at Boston Children’s Hospital. “We can now make enough human muscle progenitors in a dish to allow us to model diseases of the muscle lineage, like Duchenne muscular dystrophy, conduct drug screens to find chemicals that correct those disease, and in the long term, efficiently transplant muscle stem cells into a patient.”

In a similar biomedical application, Kahn, who is chief academic officer at the Joslin, plans to apply the new ability to quickly produce muscle stem cells for diabetes research. His lab will generate iPS-derived muscle cells from people who are at risk for diabetes and people who have diabetes to identify alterations that lead to insulin resistance in the muscle.

Going forward, Zon plans to apply this platform of cross-species discovery to other stem cell lines, including those involved in blood and eye development. “We have a new system to use to study tissue development, and it’s not just muscle that can be studied, every single organ can be studied in the zebrafish system,” he said.

The research was funded by the Harvard Stem Cell Institute, the Howard Hughes Medical Institute, the National Institutes of Health, the Novo Nordisk Diabetes Center, and the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center.

Photo:Newly created muscle progenitor cells (green) and muscle fibers (red) in a zebrafish embryo. (credit: Zon Lab)

 

Dynamics and heterogeneity of a fate determinant during transition towards cell differentiation

Yan is an ETS-domain transcription factor responsible for maintaining Drosophila eye cells in a multipotent state. Yan is at the core of a regulatory network that determines the time and place in which cells transit from multipotency to one of several differentiated lineages. Using a fluorescent reporter for Yan expression, we observed a biphasic distribution of Yan in multipotent cells, with a rapid inductive phase and slow decay phase. Transitions to various differentiated states occurred over the course of this dynamic process, suggesting that Yan expression level does not strongly determine cell potential. Consistent with this conclusion, perturbing Yan expression by varying gene dosage had no effect on cell fate transitions. However, we observed that as cells transited to differentiation, Yan expression became highly heterogeneous and this heterogeneity was transient. Signals received via the EGF Receptor were necessary for the transience in Yan noise since genetic loss caused sustained noise. Since these signals are essential for eye cells to differentiate, we suggest that dynamic heterogeneity of Yan is a necessary element of the transition process, and cell states are stabilized through noise reduction.

As animal cells develop, they pass through different states to mature into specific cell types. For some cells, this development depends on the cell’s ability to switch between two stable states, a property called bistability.

Many bistable systems operate during development and often feature proteins called transcription factors that regulate a few cell states in specific tissues. These proteins activate or repress a range of target genes, and cells can adjust the levels of the transcription factors to bring about transitions between states. In fruit flies, two transcription factors, called Yan and Pnt, regulate numerous processes throughout development.

In the developing embryo of a fruit fly, Yan and Pnt are regulated by signals induced by a receptor called EGFR. When EGFR is activated, Pnt is produced and Yan is degraded. When this receptor is not activated, Yan is produced and represses Pnt. Mathematical modelling of these interactions has concluded that this is a bistable system: that is, cells should either have high levels of Yan and low levels Pnt, or low Yan levels and high Pnt levels. However, larval eye cells first activate, and then turn off, both proteins together. This argues against bistability and raises questions about how these proteins regulate cell fate transitions in the eye, and perhaps in other organs.

To investigate this question, Peláez et al. tagged Yan with a fluorescent marker to track its activity in the eyes of fruit fly larvae as they develop. A combination of fluorescence-based microscopy and an automated imaging analysis system were then used to score fluorescence in thousands of individual eye cells and assess the changes in the levels of Yan over time. This approach revealed some unexpected results.

Yan levels were seen to vary in both immature and maturing cells. Thus eye cells transition between states as Yan levels increase rapidly, suggesting the need for a mechanism that is distinct from bistability. Peláez et al. suggest that larger changes in the seemingly random fluctuations in the levels of the transcription factor (also known as “expression noise”) might play a role in this mechanism. In particular, Yan expression noise briefly peaks as cells transition to a more mature state, and the transient nature of this ‘noisy’ response requires the activation of EGFR.

One possible explanation for these observations is that Yan’s effect on cell states depends on this variability in its levels, which might prime cells to change states when they receive another signal. These findings also raise many questions for future studies to explore, including how this increase in the noise level of Yan is triggered as cells begin their transitions towards specific cell types.

Introduction

Cells within complex organisms exist in different states that confer specific functionalities to each cell. Cellular states can be organized into cyclic cascades such as the G1-S-G2-M cell cycle, or into linear cascades as observed in cell differentiation. A common feature of cells that undergo state transitions is the apparent irreversibility of the transitions even when such transitions are triggered by transient stimuli. Modeling of these transitions often assumes system bistability, in which cells can be resting in one of two stable states.

Animal cells frequently utilize transcription factors to enforce a given state, and transitions to another state are driven by increasing or decreasing the levels of these transcription factors (Yao et al., 2008;Kueh et al., 2013; Laslo et al., 2006; Park et al., 2012). The Rb-E2F pathway generates bistability in E2F expression, which dictates the transition from G1 to S phase (Yao et al., 2008). Expression of the transcription factor PU.1 determines lymphoid versus myeloid hematopoietic cell lineages (Kueh et al., 2013; Laslo et al., 2006). Adipocyte differentiation is controlled by differential expression of C/EBP and PPARγ proteins (Park et al., 2012). Regulation by positive feedback is a hallmark of bistable systems, and in all of the above cases, the transcription factors act in one or more positive feedback circuits. In some systems, positive feedback is generated by two transcription factors that mutually repress each other’s expression. In these scenarios, cells in one state continually express a transcription factor that represses a second transcription factor, and when these cells transit to another state, they continually express the second transcription factor, which represses its antagonist. Fate restriction in hematopoietic, neural, pancreatic, and muscle cell lineages is regulated by such double-negative feedback circuits (Briscoe et al., 2000; Laslo et al., 2006; Olguin et al., 2007; Schaffer et al., 2010).

Most examples of this type of bistable mechanism have transcription factors that act specifically within a handful of cell states limited to a single tissue or organ system. One remarkable exception to this rule is found in Drosophila. There, two ETS-domain transcription factors act in a wide assortment of cell types across the body and across the life cycle. Yan and Pointed (Pnt) act downstream of signals mediated by receptor tyrosine kinases (RTKs) that regulate cell differentiation, migration, and division in tissues ranging from ovarian follicular cells, dorsal and ventral neuroectoderm, embryonic mesoderm, the embryonic trachea, and the post-embryonic compound eye (Dumstrei et al., 1998;Gabay et al., 1996; Halfon et al., 2000; Jurgens et al., 1984; Morimoto et al., 1996; O’Neill et al., 1994; Ohshiro et al., 2002; Yao et al., 2008) It is thought that Yan and Pnt control such diverse cell states by acting in concert with tissue-specific transcription factors to regulate transcription of appropriate target genes. For instance, transcription of even-skipped occurs in mesoderm only if Yan/Pnt act in combination with Tinman and Twist proteins (Halfon et al., 2000), whereas transcription of prospero (pros) in the eye only occurs if Yan/Pnt act in combination with Lozenge, Sine Oculis, and Glass proteins (Hayashi et al., 2008; Xu et al., 2000).

Several tissues show mutually exclusive expression of Yan and Pnt, suggestive of cross-repression (Boisclair Lachance et al., 2014). The best characterized is the embryonic ventral ectoderm, where ventral-most cells express Pnt and more lateral cells express Yan (Gabay et al., 1996). This pattern is established by secretion of a ligand for the EGF Receptor (EGFR) from the ventral midline (Golembo et al., 1996). EGFR activation in nearby ventral cells induces the Ras-MAPK pathway to express Pnt and degrade Yan (Gabay et al., 1996; Melen et al., 2005). Cells with insufficient EGFR activation express Yan, which represses Pnt. Mathematical modeling has described this as a bistable system in which cells are either in a High Yan/Low Pnt state or a Low Yan/High Pnt state (Graham et al., 2010; Melen et al., 2005). Transition from one state to the other is ultrasensitive to the strength of EGFR activation (Melen et al., 2005).

Paradoxically, other Drosophila tissues show co-expression of Yan and Pnt (Boisclair Lachance et al., 2014). The larval eye is one such tissue. Retinal progenitor cells initiate expression of both proteins, and when they transit to differentiated photoreceptor fates, these cells reduce expression of both proteins. In contrast, when retinal progenitor cells transit to differentiated cone cell fates, they maintain their expression of both proteins. These observations are at odds with long-standing genetic studies that support a standard bistable mechanism acting in the eye (Lai and Rubin, 1992;O’Neill et al., 1994; Rebay and Rubin, 1995). Thus, new approaches to studying these transitions in the eye are needed.

Here, we have adopted a systems-level approach to study Yan dynamics in the larval eye. A yellow fluorescent protein (YFP) — based isoform of Yan was developed as a reporter for Yan protein levels. Fluorescence-based microscopic imaging of cells was coupled with automated high-throughput image analysis to score fluorescence in each cell and annotate the data in a quantitative and unbiased fashion. Yan exhibits monostability, both in progenitor and differentiating cells, with Yan levels varying in cells in either state. Cell state transitions occur independent of absolute Yan concentrations, suggesting that some other mechanism allows Yan to regulate transitions. One such mechanism might be the noise in Yan levels, which undergoes a transient spike as cells begin to transition to differentiated states. Loss of EGFR signaling, which prevents cells from differentiating, causes these cells to have prolonged noisy Yan expression, and suggests that Yan noise is key for cell state transitions in the eye.

Results

The compound eye epithelium is established during embryogenesis as an internal disc of cells called the eye imaginal disc (Wolff and Ready, 1993). During the larval phase of the life cycle, the disc grows in size by asynchronous cell division. During the final 50 hr of the larval phase, a morphogenetic furrow (MF) moves across the eye disc from posterior to anterior (Figure 1A,B). All cells arrest in G1 phase within five cell diameters anterior to the furrow, and then as the furrow passes through them, periodic clusters of cells express the proneural gene atonal (Jarman et al., 1994). Atonal expression is subsequently restricted to one cell per cluster, which becomes the R8 photoreceptor. Each R8 cell then secretes an EGFR ligand that activates the receptor in neighboring cells and causes them to transit from multipotent progenitor to differentiated states (Figure 1C) (Freeman, 1996). Transitions occur in a sequence of symmetric pairs of multipotent progenitor cells that differentiate into R2/R5, R3/R4, and R1/R6 photoreceptors (Figure 1C) (Wolff and Ready, 1993). Thereafter, a single progenitor transits to a R7 photoreceptor fate followed by two pairs of cells, C1/C2 and C3/C4, that differentiate into cone cells. These cone cells are non-neuronal and form the simple lens that overlies each cluster of eight photoreceptors. The furrow induces the nearly simultaneous differentiation of a column of R8 cells, with repeated column inductions producing approximately 800 units or ommatidia as the furrow moves across the eye.

Figure 1.Development and patterning of the compound eye.

(A) Differentiation is initiated in the developing eye by the MF, which moves across the eye epithelium. On the furrow’s posterior side, G1-arrested progenitor cells undergo differentiation (light blue). On the anterior side, progenitor cells are still proliferating (dark blue). The large grey rectangle outlines the region that was analyzed for Yan levels; the small rectangle corresponds to the developmental sequence outlined in panel C (B) A maximal projection of Yan-YFP fluorescence in an eye. Bar = 100 μm. (C) Top, an apical view of the sequential differentiation of eight photoreceptor (R1-R8) and four cone cell types (C1-C4) from multipotent progenitor cells (grey) in an ommatidium. Arrows denote inductive signal transmitted from the R8 to activate EGFR on nearby cells. Bottom, a cross-section view through an eye disc adapted after Wolff and Ready (1993). Note the progenitor cell nuclei are basally positioned, and as they transition into a differentiated state, their nuclei migrate apically. C1/C2 cells are positioned anterior and posterior in the ommatidium while C3/C4 cells are positioned equatorial and polar in the ommatidium. (D) Top, an optical slice of H2Av-mRFP fluorescence in an eye disc at a plane that bisects progenitor cell nuclei. Bottom, the same optical slice imaged for Yan-YFP fluorescence. Bars = 8 μm

A central tenet of the bistable model of cell differentiation in the eye posits that differentiation is marked by a transition from high Yan protein levels in multipotent progenitor cells to low Yan levels in differentiating cells (Graham et al., 2010). Formulation of this model stemmed from studies of R7 cell differentiation, the final photoreceptor recruited to each ommatidium. Reduced Yan causes inappropriate expression of the R7 determinant pros and ectopic R7 cells in yan hypomorphic mutants (Kauffmann et al., 1996; Lai and Rubin, 1992). Conversely, a Yan isoform that is resistant to MAPK-dependent degradation, blocks R7 differentiation and pros expression (Kauffmann et al., 1996; Rebay and Rubin, 1995).

Quantifying Yan dynamics

To quantitatively test the bistable model, we used BAC recombineering to insert fast-fold yellow fluorescent protein (YFP) in-frame at the carboxy-terminus of the yan coding sequence (Webber et al., 2013). The Yan-YFP transgene fully complemented null mutations in the endogenous yan gene and completely restored normal eye development (Figure 1—figure supplement 1), demonstrating that the YFP tag does not compromise Yan function and that all essential genomic regulatory sequences are included. The pattern of Yan-YFP protein expression was qualitatively similar to that of endogenous Yan (Figure 1—figure supplement 1).

We used histone His2Av-mRFP fluorescence in fixed specimens to mark all eye cell nuclei for automated segmentation (Figure 1D—figure supplement 2). Nuclei were manually classified into the different cell types of the eye, which is possible because every cell undergoing differentiation can be unambiguously identified by its position and nuclear morphology without the need of cell-specific markers (Ready et al., 1976; Tomlinson, 1985; Tomlinson and Ready, 1987; Wolff and Ready, 1993). To validate our identification of all cell types using this method, we cross-checked with specific cell-specific markers, and found that our classification was accurate over 98% of the time (Figure 1—figure supplement 3). Cells were scored for nuclear Yan-YFP concentration and their exact position within 3D coordinate space of each fixed eye sample (Figure 1—figure supplement 2). Yan-YFP protein levels were normalized to His2Av-mRFP, which provided some control over measurement variation (Figure 1—figure supplement 4). We then mapped each cell’s spatial position in the x-y plane of the eye disc onto time. Two reasons made this possible. First, the furrow moves at an approximately constant velocity forming one column of R8 cells every two hours (Basler and Hafen, 1989; Campos-Ortega and Hofbauer, 1977). Second, each column of R8 cells induces the other cell fates at a constant rate (Lebovitz and Ready, 1986). Thus even in a fixed specimen, the temporal dynamics of cell state transitions are visible in the repetitive physical organization of ommatidia relative to the furrow (Figure 1C). Together these features allow the estimation of time based on a cell’s position relative to the furrow (Figure 2—figure supplement 1). This can be applied repeatedly to hundreds of cells in a single sample, creating a composite picture of the dynamics (Figure 2B–F). Although the developmental progression of an individual cell cannot be measured by this approach, it nevertheless provides a dynamic view of hundreds of cells across a developing epithelium as they respond to signaling processes. From this information, individual cell behaviors can be reconstructed and modeled.

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Figure 2.Dynamics of Yan-YFP in eye cells.

(A) Average time at which initiation of differentiation is first detected by apical migration of committing cell nuclei. Time zero is set to when R8 differentiation initiates. Differentiation proceeds over a time course after commitment is initiated (horizontal arrows) (B) Yan-YFP fluorescence in multipotent progenitor cells. We fit a Hill function (blue curve) to the inductive phase and an exponential decay (black curve) to the decay phase. (C-F) Scatter plots of Yan-YFP levels in individual cells for R2/R5 (C), R3/R4 (D), R7 (E), and C3/C4 (F) cells. These are overlaid with scatter plots of Yan-YFP in multipotent cells at times preceding and coincident with the appearance of the relevant differentiated cells. Note the similar Yan-YFP levels between multipotent cells and differentiating cells at their first appearance. (G) Moving averages of Yan-YFP levels for multipotent and photoreceptor cells. Gaps between the multipotent and photoreceptor curves are due to the window size for line averaging. (H) Moving averages of Yan-YFP in multipotent and cone cells.

Yan-YFP expression in multipotent progentior cells was biphasic (Figure 2B). Cells anterior to the furrow expressed a basal level of Yan-YFP, but this level dramatically increased in cells immediately anterior to the furrow, starting eight hours before the first R8 cells were identifiable. Approximately eight hours after R8 definition, Yan-YFP levels peaked, and thereafter gradually decayed until Yan-YFP approached its basal level again. The results are surprising in two ways. First, Yan-YFP is not maintained at a stable steady state within progenitor cells, which would have been predicted by the bistable model. Rather, its dynamics are reminiscent of monostable responses to transient stimuli, with a single basal steady state. Second, at the peak of Yan-YFP expression, there is remarkably large heterogeneity in Yan-YFP levels across cells.

We also followed Yan-YFP dynamics in cells as they transited into a differentiated state and thereafter. Again, the results did not follow the expectations predicted by the bistable model. First, progenitors transited to a differentiated state at levels of Yan-YFP that varied, depending upon the type of differentiated state being adopted (Figure 2C–H). Cells entering the R3/R4 and R1/R6 states began with Yan-YFP levels that were approximately two-fold greater than cells entering the R2/R5 states. Cells entering the R7 state were intermediate between these two extremes. Despite these differences at the transition point, Yan-YFP levels decayed to a similar basal steady state irrespective of the photoreceptor type, and this basal level was at least three-fold lower than that which the progenitor cells achieved (Figure 2G). Thus, rather than the single high Yan progenitor state previously modeled, our results suggest a dynamic range of high Yan states from which different cell fates are specified according to the spatio-temporal sequence of differentiation.

We noted that for most photoreceptors, it took approximately 20 hr for Yan-YFP to decay to the basal steady state (Figure 2G), significantly longer than had been previously modeled (Graham et al., 2010). Since expression of several neural-specific genes is detected 2–8 hr after the transition (Tomlinson and Ready, 1987; Van Vactor et al., 1988), the slower than anticipated Yan decay indicates that early differentiation does not require cells to have assumed a basal steady-state level of Yan-YFP.

The last group of progenitors to differentiate form the non-neuronal cone cells. We also measured Yan-YFP in those cells. Yan-YFP dynamics in cone cells were more similar to progenitor cells over the same time period (Figure 2F,H). This behavior was in contrast to photoreceptors, which exhibited different decay dynamics from progenitor cells. Thus, accelerated degradation of Yan-YFP is not essential for cells to transition to all retinal cell states.

EGFR-ras signaling regulates Yan-YFP dynamics

The bistable model posits that the switch from one state to another is triggered by a signal that progenitor cells receive though the EGFR protein. Given the unanticipated Yan-YFP dynamics, we wanted to ask whether and how they were influenced by EGFR signaling. EGFR null mutants are inviable; however, a temperature sensitive (ts) allele of EGFR enables controlled inactivation of the RTK’s activity (Kumar et al., 1998). We grew EGFRts mutant larvae at a restrictive temperature for 18 hr before analyzing effects on Yan-YFP. Surprisingly, progenitor cells exhibited biphasic expression of Yan-YFP over time, but the amplitude of the pulse in expression was significantly reduced (Figure 3A). This suggests that EGFR signaling contributes to the stimuli that induce the Yan-YFP peak. To further test this hypothesis, we misexpressed a constitutively active form of Ras protein in eye cells. The peak of Yan-YFP in progenitors was now prolonged (Figure 3B). Together, these results suggest that EGFR-Ras signaling stimulates the transient appearance of Yan-YFP in progenitor cells, and that the decline in Yan-YFP within older progenitor cells is linked to a loss of signal reception by these cells over time.

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Figure 3.EGFR/Ras and Pnt regulate Yan-YFP levels.

(A–D) Moving averages of Yan-YFP in different cell types. Shown with shading is the standard error of the mean for each moving average. (A,C) Wildtype and EGFRts mutants incubated at the non-permissive temperature and analyzed for progenitors (A) and R2/R5 cells (C). (B,D) Wildtype and sev>Rasv12 mutants were analyzed for progenitors (B) and R2/R5 cells (D). (E) Optical slice through progenitor cell nuclei in a disc that contains clones ofpnt2 mutant cells. Left, fluorescence of RFP, which positively marks wildtype cells and not pnt2 mutant cells. Middle, Yan-YFP fluorescence, showing reduced levels in pnt2mutant clones. Arrows highlight apoptotic nuclei. Right, merged image with Yan-YFP in green and RFP in purple. Clone borders are outlined. (F,G) Moving averages of Yan-YFP in R3/R4 cells that ectopically express PntP1 (F) or PntP2-VP16 (G) due to LongGMR-Gal4 driving the UAS transgenes. Since PntP2 requires MAPK phosphorylation to become transcriptionally active, we misexpressed a VP16 fusion of PntP2. PntP1 is constitutively active (Brunner et al., 1994Gabay et al., 1996).

We next examined the effects of EGFR and Ras on Yan-YFP dynamics in cells as they differentiate. The bistable model predicts that EGFR is required for the loss of Yan-YFP in photoreceptors. Indeed,EGFRts mutant R2/R5 cells delayed their initial decline in Yan-YFP levels (Figure 3C). Conversely, misexpression of constitutively active Ras caused a premature decline in Yan-YFP (Figure 3D). These results are consistent with EGFR-Ras stimulating the degradation of Yan-YFP as cells switch their states. However, Yan-YFP dropped to below-normal levels in EGFRts mutant R2/R5 cells (Figure 3C). These complex effects suggest a dual role for EGFR signaling in photoreceptors. In the first few hours as cells transit to a photoreceptor state, EGFR stimulates the accelerated decay of Yan-YFP. Thereafter, EGFR inhibits the decay of Yan-YFP in a manner that might be related to that role that EGFR plays in boosting Yan-YFP levels in progenitor cells.

The source of EGFR ligand originates from the R8 cell (Freeman, 1996). If this diffusive ligand is responsible for controlling Yan-YFP levels in other photoreceptors as they are recruited to an ommatidium, we would predict a correlation between Yan-YFP levels in differentiating cells and their distances from adjacent R8 cells. Indeed, at the times when R2/R5 cells differentiate (~0–15 hr), their Yan-YFP levels are positively correlated (p<0.01) with their physical distance to the nearest R8 cells (Figure 3—figure supplement 1). These correlations are absent in EGFRts mutants, providing evidence that R8 cells act through EGFR to control Yan-YFP dynamics in differentiating cells.

Pnt regulates Yan dynamics

Pnt proteins have been hypothesized to cross-repress Yan expression, and this double negative feedback loop is thought to be necessary for bistability (Graham et al., 2010; Shilo, 2014). At odds with this view, Pnt and Yan proteins are co-expressed in progenitor and differentiating cells of the eye (Boisclair Lachance et al., 2014). Since Pnt proteins act downstream of many RTKs including EGFR, we wondered if Pnt mediated the positive effects of EGFR-Ras signaling on Yan-YFP in progenitor cells. Null mutants of the pnt gene are embryonic inviable; therefore we generated clones of eye cells that were null mutant for pnt in an otherwise wildtype animal. Mutant progenitor cells showed a reduction in Yan-YFP levels as they progressed through their biphasic expression pattern (Figure 3E). Thus, Pnt possibly mediates the positive effect of EGFR signaling on Yan-YFP expression in progenitors. We also wished to know if Pnt mediates the complex effects of EGFR in differentiating photoreceptors. Pnt proteins are rapidly cleared in photoreceptors (Boisclair Lachance et al., 2014) and so loss-of-function mutant analysis would be uninformative. Therefore, we overexpressed PntP1 or constitutively-active PntP2 in cells as they transited into a photoreceptor state and beyond. The early phase of Yan-YFP decay was accelerated while the later phase of Yan-YFP decay was inhibited (Figure 3F,G). These complex effects are precisely the opposite to those caused by loss of EGFR signaling, as would be expected if Pnt mediated EGFR’s complex effects on Yan-YFP dynamics in photoreceptor cells.

Cells are indifferent to absolute levels of Yan

The bistable model predicts that different cell states depend upon discrete absolute concentration of Yan present inside cells. To test this idea, we varied the number of Yan-YFP gene copies. In general, protein output is proportional to gene copy number in Drosophila (Lucchesi and Rawls, 1973). We increased Yan-YFP copy number from its normal diploid number to tetraploid, and monitored Yan-YFP in progenitors and differentiating cells. As expected, four copies caused a higher steady-state level of Yan-YFP in progenitor cells, though this increase was less than two-fold (Figure 4A). The amplitude of the Yan-YFP pulse was also increased as progenitor cells aged. Strikingly, as four-copy progenitor cells transited to a differentiated state, the onset of Yan-YFP decay occurred at the same time as it occurred for two-copy cells (Figure 4A–C). Yan-YFP levels were much greater in four-copy cells compared to two-copy cells making their transit into the identical cell states. To confirm that absolute Yan-YFP concentration had little effect on cell state transitions, we examined expression of a direct target of Yan in R7 cells: the pros gene (Kauffmann et al., 1996; Xu et al., 2000). Expression was monitored with an antibody specific for Pros protein. We observed at most a one hour delay in the onset of Pros expression in R7 cells containing four copies of Yan-YFP (Figure 4D), far less than the ten-hour delay predicted if absolute concentration of Yan-YFP dictated when Pros expression begins (Figure 4D).

Figure 4.Cell state transitions are unaffected by Yan-YFP gene copy number.

(A–C) Moving averages of Yan-YFP in eye discs containing two versus four copies of the Yan-YFP transgene. (A) R2/R5 and progenitor cells. (B) R3/R4 and progenitor cells. (C) R7 and progenitor cells. (D) Moving averages of Yan-YFP and Pros proteins in R7 cells containing either two vs. four copies of the Yan-YFP transgene.

Possibly, absolute concentration of Yan is unimportant when a cell transits to a different state, but the switch to a constant basal Yan level is robustly maintained regardless of starting concentration. An examination of Yan-YFP decay in photoreceptor cells makes that possibility less likely; Yan-YFP decays to different basal levels in two- versus four-copy differentiated cells (Figure 4A–C). To further test this notion, we fit the data to several plausible functional forms. We found that exponentially decaying functions systematically best-fit to the data (Figure 4—figure supplement 1). Thus, for each cell state we fit an exponential decay function to its Yan-YFP temporal profile (Figure 4—figure supplement 2). From these fits, we derived the average decay rate constants and half-lives of Yan-YFP for cells carrying two, four, or six copies of yan. As expected, we found that Yan-YFP half-life was different between progenitors and differentiating photoreceptors (Figure 5—figure supplement 1). The half-life in photoreceptors was two-fold lower than in progenitors, accounting for the more rapid loss of Yan-YFP in the former cells. Strikingly, Yan-YFP half-life was not significantly affected byyan copy number within either progenitor or photoreceptor cells (Figure 5). Thus, Yan-YFP concentration only affected its decay rate as a first-order function, implying that there is no higher order mechanism to accelerate Yan-YFP decay when cells contain greater concentrations of Yan-YFP.

http://cdn-site.elifesciences.org/content/elife/4/e08924/F13.medium.gif

Figure 5.Yan protein half-life is largely unaffected by yan gene copy number.

Exponential decay of Yan-YFP levels. Note the robustness of Yan-YFP half-lives across replicates and yan gene copy number. Note also how half-lives are nearly twice as long for progenitor cells versus photoreceptor neurons.

As a final test of the effects of Yan-YFP levels on cell states, we allowed 4X and 6X yan animals to complete eye development and then examined the external patterning of the fully differentiated compound eye. The compound eyes were completely normal in appearance (Figure 5—figure supplement 2), implying that differentiation was unaffected by the absolute concentrations of Yan inside eye cells.

Yan expression noise spikes during cell state transitions

Our results indicate that Yan’s effects on retinal cell states are not dictated by uniform and stable concentrations of Yan protein. One potential explanation is that Yan’s effect on cell states actually depends on variability in Yan protein levels. A growing body of evidence is pointing to the importance of transient fluctuations in expression of factors to control cell states (Cahan and Daley, 2013;Torres-Padilla and Chambers, 2014). Key regulators of stem cells fluctuate in abundance over time, and this is correlated with stem cells existing in multiple connected microstates, with the overall structure of the population remaining in a stable pluripotent macrostate (MacArthur and Lemischka, 2013). Heterogeneity among cells is not simply due to independent noise in expression of individual genes but is due to fluctuating networks of regulatory genes (Chang et al., 2008; Kumar et al., 2014). Such fluctuations appear to be stochastic in nature, priming cells to transit into differentiated states with a certain probability that is guided by extrinsic signals.

Our data revealed considerable heterogeneity in the level of Yan-YFP among cells at similar developmental times (Figure 2B). To quantify the noise, we used developmental time to bin cells of similar age, and measured detrended fluctuations of Yan-YFP by calculating residuals to the fitted function and normalizing binned residuals to the function (Goldberger et al., 2002). Progenitor cells showed a spike in Yan-YFP noise as they began to induce Yan-YFP expression (Figure 6A). The noise spike was short-lived (approximately 17 hr), and noise thereafter returned to a basal level with secondary minor spikes. The major peak in noise magnitude coincided with the time at which R8 cells are formed.

Figure 6. Noise in Yan-YFP expression is highly dynamic.

Moving averages of Yan-YFP levels and noise (detrended fluctuations) for (A) progenitors, (B) R2/R5, (C) R3/R4, (D) R1/R6, and (E) R7 cells. (F) Comparative noise dynamics for all cells analyzed in (AE). (G) Moving averages of Yan-YFP noise (coefficient of variation) in R2/R5 cells sampled from wildtype and EGFRts mutant eyes at the non-permissive temperature. Shown with shading is the standard error of the mean for each moving average. (H) Moving averages of Yan-YFP noise (coefficient of variation) in R2/R5 cells sampled from wildtype and sev>Rasv12 mutant eyes. Shown with shading is the standard error of the mean for each moving average.

Photoreceptor cells showed a large spike in Yan-YFP noise as they began to differentiate (Figure 6B–E). The magnitude of each noise peak varied with the photoreceptor cell state; R3 and R4 cells exhibited the greatest amplitude in noise (Figure 6F). These noise spikes showed a distinct temporal relationship, with spikes coinciding with the times at which individual cell states were switched (Figure 6F). Thus, the noise spikes are not a simple consequence of a global stimulus synchronously affecting noise in all cells. Thereafter, all cells reduced Yan-YFP noise to a basal level that was comparable to basal noise in the progenitor cells. However, each cell type exhibited a secondary minor spike at 30–35 hr, which might reflect a synchronous stimulus.

Detrended fluctuation is one method to measure expression variability, but it can suffer from distortion if the model fitting is not adequately weighted. Therefore, we also calculated the coefficient of variation, that is, the standard deviation of Yan-YFP intensity within a sliding window divided by its mean. This method yielded noise profiles with transient spikes for each cell type that was consistent with calculations using detrended fluctuation (Figure 6—figure supplements 1 and 2). However, while the coefficient of variation yielded results that varied strongly with bin width, the detrended fluctuations yielded profiles that were generally robust against changes in bin width (Figure 6—figure supplement 1).

To rule out the possibility that these unexpectedly dynamic features of Yan-YFP were caused by its transgenic origins or fusion with YFP, we compared Yan-YFP dynamics to those of endogenous Yan protein that was bound with an anti-Yan antibody. The profiles of Yan-YFP protein levels and noise were highly similar to endogenous Yan protein levels and noise, in both multipotent and differentiating cells (Figure 6—figure supplement 3). Thus, transient spikes of expression heterogeneity are a fundamental feature of Yan protein.

Since Yan regulates Pros expression in R7 cells, it was possible that Pros showed a transient noise spike as a consequence. Therefore, we measured Pros protein heterogeneity and found that its dynamics did not resemble that of Yan (Figure 6—figure supplement 4). Instead, Pros noise was high starting at the onset of expression, and thereafter gradually declined as Pros protein levels increased in R7 cells. We conclude that noise spikes are not a general feature of gene expression in the developing eye but might reflect unique roles of Yan in mediating cell state transitions.

We wondered what might cause these spikes in Yan-YFP noise during cell state transitions. Because EGFR signaling is important for regulating Yan-YFP concentration during these transitions (Figure 3), we analyzed Yan-YFP noise when EGFR signaling was inhibited in EGFRts mutant animals raised at a non-permissive temperature. The noise spike in progenitor cells was not significantly affected by loss of EGFR signaling (data not shown). We also examined the effects of EGFRts on noise in differentiating photoreceptor cells. Interestingly, noise increased at the normal time of transition but the elevated noise did not quickly drop to basal levels (Figure 6G). Rather, high noise was extended for an additional 10 to 15 hr. Conversely, misexpressing constitutively active Ras within differentiating cells caused a premature dropdown in Yan-YFP noise (Figure 6H). These results indicate that EGFR/Ras signaling is required for the rapid drop in Yan-YFP noise after it has peaked, creating a transient spike.

Discussion

This study relied upon a set of methods that enable systems-level analysis of Yan expression dynamics in a developing animal tissue. Transgene recombineering was used to insert YFP into a genomic rescue fragment of the yan gene, which fully replaced endogenous yan. Yan-YFP protein was quantified in thousands of individual cells by fluorescence confocal microscopy and automated cell segmentation analysis. Based on the unique features of Drosophila eye development, every analyzed cell was assigned an age, and composites of cells across a time-spectrum of ages were derived. This allowed us to reconstruct the temporal dynamics of Yan protein expression in cells as they transited from one state to another or were maintained in a given state. The fact that both Yan concentration and noise were easily measured using our approach indicates that it provides a powerful method for studying how other molecular determinants regulate cell states.

Contrary to what is currently believed, the expression of Yan in progenitor cells has many hallmarks of monostability. A stable basal state exists in cells anterior to the furrow, and when the furrow passes, Yan rises and falls to form a biphasic profile (Figure 7A). If cells transit towards differentiation, then the fall in Yan is accelerated but the fundamental biphasic profile is preserved. This monostable-like behavior is not like a behavior where progenitor cells exist in a high Yan stable-state and switch to a low Yan stable-state when they transit towards differentiation (Figure 7A). Other lines of evidence also point away from a bistable switch mechanism. Yan reaches its basal steady state many hours after cells have adopted their differentiated photoreceptor state and are executing specialized gene expression programs. Thus, Yan levels are variable at the time when cells actually become differentiated.

 Figure 7.

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Figure 7.Summary of analysis.

(A) Top: a hypothetical bistable behavior would be where Yan is in a stable high state within progenitor cells and in a stable low state within differentiated cells. Bottom: the observed behavior of Yan appears monostable, with both progenitors and differentiated cells in unstable Yan states. (B) Heterogeneity in Yan expression is maximal when progenitor cells enter a transition state that resolves to a more homogeneous differentiated state, dependent upon EGFR signaling. This heterogeneous transition state may be a primary mechanism for Yan’s effect on cells, independent of the absolute Yan concentration within progenitors.

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Illuminating a Cancer’s Origins

Researchers have developed a technique to visualize the origin of melanoma in zebrafish, throwing light on a genetic switch for cancer.

By Catherine Offord | February 1, 2016

Many cells containing cancer-associated genes never become tumors. Using a fluorescent reporter to highlight cells activating stem cell-like patterns of gene expression, researchers at Boston Children’s Hospital have now developed a technique to visualize the origins of tumors in zebrafish. The team’s findings were published last week (January 29) in Science.

“An important mystery has been why some cells in the body already have mutations seen in cancer, but do not yet fully behave like the cancer,” Charles Kaufman of Boston Children’s Hospital said in a statement. “We found that the beginning of cancer occurs after activation of an oncogene or loss of a tumor suppressor, and involves a change that takes a single cell back to a stem cell state.”

The researchers focused on a gene present in vertebrates called crestin. This gene is expressed normally only in neural crest progenitor cells, which give rise to a number of other cell types during embryogenesis. However the gene is also expressed in melanoma tumor cells, making it a clear marker of the onset of cancer.

Injecting a fluorescent crestin reporter into transparent zebrafish carrying mutations in melanoma genes, the researchers found they were able to visualize the moment that precancerous cells activated stem cell-like patterns of gene expression.

“Every so often we would see a green spot on a fish,” said study coauthor Leonard Zon of Boston Children’s Hospital in the statement. “When we followed them, they became tumors 100 percent of the time.”

“It’s a significant advance in the field,” Ze’ev Ronai, the scientific director of the Sanford Burnham Prebys Medical Discovery Institute in La Jolla, California, told The New York Times. “They provide a pretty strong demonstration that that pathway is correct.”

The team will now focus on the pathways regulating the switch in gene expression, according to the statement—research that Kornelia Polyak of the Dana-Farber Cancer Institute told the Times would be essential to develop drugs targeting melanoma.

“What is the stimulus that allows a single cell out of millions to become cancerous?” she asked. “What is the trigger and why does it happen so rarely? Why, why is this happening?”

Fluorescence, nucleic acid strands help show computing operations inside a living cell

Using strands of nucleic acid and a fluorescence reporter, scientists at the Georgia Institute of Technology (Georgia Tech; Atlanta, GA) and colleagues have demonstrated basic computing operations inside a living mammalian cell. The research could lead to an artificial sensing system that could control a cell’s behavior in response to such stimuli as the presence of toxins or the development of cancer.

Related: Non-damaging, light-emitting nanoprobes enable long-term study of living cells

The research uses DNA strand displacement, a technology that has been widely used outside of cells for the design of molecular circuits, motors, and sensors. Researchers modified the process to provide both “AND” and “OR” logic gates able to operate inside the living cells and interact with native messenger RNA (mRNA).

The tools they developed could provide a foundation for biocomputers able to sense, analyze, and modulate molecular information at the cellular level. Philip Santangelo, an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, says the devices could sense an aberrant RNA, for instance, and then shut down cellular translation or induce cell death.

Strand displacement reactions are the biological equivalent of the switches or gates that form the foundation for silicon-based computing. They can be programmed to turn on or off in response to an external stimuli such as a molecule. An “AND” gate, for example, would switch when both conditions were met, while an “OR” gate would switch when either condition was met.

In the switches the researchers used, a fluorescence reporter molecule and its complementary quenching molecule were placed side-by-side to create an “off” mode. Binding of RNA in one of the strands then displaced a portion of nucleic acid, separating the molecules and allowing generation of a signal that created an “on” mode. Two “on” modes on adjacent nucleic acid strands created an “AND” gate.

http://www.bioopticsworld.com/content/dam/bow/online-articles/2016/01/nucleicacid_web.jpg

Image shows activation of “AND” gates in cells as observed by fluorescence microscopy. (Credit: Chiara Zurla, Georgia Tech)

Georg Seelig, assistant professor of computer science and engineering and electrical engineering at the University of Washington, says that in the longer term, the research team wants to expand this technology to create circuits with many inputs, such as those they have constructed in cell-free settings.

The researchers used ligands designed to bind to specific portions of the nucleic acid strands, which can be created as desired and produced by commercial suppliers.

Challenges in the research team’s work were getting the devices into the cells without triggering the switches, providing operation rapid enough to be useful and not killing the human cell lines that the researchers used in the lab.

The nucleic acid computers ultimately operated as desired, and the next step is to use their switching to trigger the production of signaling chemicals that would prompt the desired reaction from the cells. Cellular activity is normally controlled by the production of proteins, so the nucleic acid switches will have to be given the ability to produce enough signaling molecules to induce a change.

Cells, of course, already know how to sense toxic molecules and the development malignant tendencies, and to then take action. But those safeguards can be turned off by viruses or cancer cells that know how to circumvent natural cellular processes.

Full details of the work appear in the journal Nature Nanotechnology; for more information, please visit http://dx.doi.org/10.1038/nnano.2015.278.

 

Computing in mammalian cells with nucleic acid strand exchange

Benjamin GrovesYuan-Jyue ChenChiara Zurla, Sergii PochekailovJonathan L. KirschmanPhilip J. Santangelo & Georg Seelig

Nature Nanotechnology(2015)    http://dx.doi.org:/10.1038/nnano.2015.278

DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution.

Subject terms:  DNA computingRNA nanotechnology

Figure 1: Empirical design parameters determine in-cell performance.close

Empirical design parameters determine in-cell performance.

Decisions made at the design level, such as the choice of gate architecture, nucleic acid modifications and delivery method have a strong impact on reaction kinetics, stability against nuclease degradation and subcellular localization..

 

aurelianu2007 commented on Single Cell Shines Light on Cell Malignant Transformation

Single Cell Shines Light on Cell Malignant Transformation Larry H. Bernstein, MD, FCAP, Curator LPBI Single Cell …

The lack of oxygen inhibits the transport of protons and thereby causes a decrease in membrane potential. Cell survival under conditions of mild hypoxia is mediated by phosphoinositide-3 kinase (PIK3) using severe hypoxia or anoxia, and then cells initiate a
cascade of events that lead to apoptosis. After DNA damage, a very important regulator of apoptosis is the p53 protein. This tumor suppressor gene has mutations in over 60% of human tumors and acts as a suppressor of cell division. The growth-suppressive effects of p53 are considered to be mediated through the transcriptional trans-activation activity of the protein. In addition to the maturational state of the clonal tumor, the prognosis of patients with CLL is dependent of genetic changes within the neoplastic cell population.
The genetic changes can be identified by fluorescent probes to chromosomal using a technique referred to as fluorescent in situ hybridization (FISH). Chromosomal evaluation using FISH can identify certain chromosomal abnormalities of CLL that have prognostic significance. Deletion of part of the short arm of chromosome 17 (del 17 q), with target the cell cycle regulating protein p53, is particularly deleterious. This abnormality is found in 10% of patients with CCL and has a pour
prognosis. Deletion of long arm of chromosome 13(del 13q) is the most common geneticabnormality in CLL with roughly 50% of patients exhibiting this effect. These patients have the best prognosis and most will live many years without the need for therapy. Agents damaging DNA may increase the expression of p53 and its trans-activation activity, suggesting that p53 acts to protect cells against the accumulation of mutants and their subsequent conversion to a malignant
status.
Protein p53, in its normal form, acts in stopping the cell division whenever damage to a cell’s DNA is detected, thus giving the cells the possibility of repairing DNA before the errors would duplicate and be passed on to the daughter cells. Antibodies to human p53 have been detected in patients with cancer. These antibodies are highly specific for malignant diseases and are rarely detected in healthy donors or patients having benign diseases. This immune response is correlated with the presence of a p53 gene mutation, leading to the accumulation of an ineffectivep53 protein in tumor cells[ 8] with either tridimensional structure;Over-expression of normal p53 protein can result either in G1 arrest, mediated by p21 protein [ or in the induction of apoptosis [ 9 ]. Also hypoxia itself ca also prevent apoptosis by inducing the expression of the anti apoptotic protein IAP-2. A typical response to the hypoxic environment, by hypoxia inducible factor 1, [ 6] for example, is expression of insulin-independent GLUT [5] triggered by HIF 1α [6] insuring maximum glucose uptake for glycolytic ATP generation.

 

Mechanism of Tumor Suppressing Gene Uncovered

Published: Tuesday, January 26, 2016
Last Updated: Tuesday, January 26, 2016
The most commonly mutated gene in cancer,p53, works to prevent tumor formation by keeping mobile elements in check that otherwise lead to genomic instability, UT Southwestern Medical Center researchers have found.

The p53 gene long has been known to suppress tumor formation, but the mechanisms behind this function – and why disabling the gene allows tumors to form – were not fully understood.

Findings from the study answer some of these questions and could one day lead to new ways of diagnosing and treating cancer, said the study’s senior author, Dr. John Abrams, Professor of Cell Biology at UT Southwestern.

The investigators found that normal p53 gene action restrains transposons, mobile genetic elements called retroelements that can make copies of themselves and move to different positions on chromosomes. But, they discovered, when p53 is disabled by mutation, dramatic eruptions of these mobile elements occur. The study revealed that in mice with cancer and in human samples of two types of cancer (Wilms’ tumors and colon tumors) disabled for p53, transposons became very active.

In a healthy state, certain mechanisms work to keep these retroelements quiet and inactive, explained Dr. Abrams. One of those mechanisms is p53 action. Conversely, when p53 is mutated, retroelements can erupt.

“If you take the gene away, transposons can wreak havoc throughout the genome by causing it to become highly dysregulated, which can lead to disease,” Dr. Abrams said. “Our findings help explain why cancer genomes are so much more fluid and destabilized than normal genomes. They also provide a novel framework for understanding how normal cells become tumors.”

Although much more research is needed, Dr. Abrams said, the potential clinical implications of the team’s findings are significant.

“Understanding how p53 prevents tumors raises the prospect of therapeutic interventions to correct cases in which p53 is disabled,” Dr. Abrams said. “If retroelements are at the heart of certain p53-driven cancers, finding ways to suppress them could potentially allow us to prevent those cancers or intervene to keep them from progressing.”

This understanding also could lead to advances in diagnosing some cancers through biomarkers related to p53 and transposon activity.

“One possibility is that perhaps blood or urine tests could detect dysregulated retroelements that could be indicative of certain types of cancer,” Dr. Abrams said.

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Zebrafish—Susceptible to Cancer

Reporter: Larry H Bernstein, MD, FCAP

 

 

Models of Transparency

Researchers are taking advantage of small, transparent zebrafish embryos and larvae—and a special strain of see-through adults—to understand the development and spread of cancer.
By Joan K. Heath, David Langenau, Kirsten C. Sadler, and Richard White | April 1, 2013
LOOKING INSIDE DISEASE: The wild-type zebrafish larva on the left is stained for the two neuronal proteins (green) and membrane-trafficking proteins expressed near synapses (blue). On the right, the neurons of a transgenic zebrafish larva produce the dementia-associated Tau protein (red), a disease-specific form of which is stained in blue. Tubulin is stained in green.
COURTESY OF DOMINIK PAQUET, THE ROCKEFELLER UNIVERSITY, NEW YORK, USA
From frogs to dogs and people, cancer wreaks havoc across the animal kingdom—and fish are no exception. Coral trout, for example, develop melanoma from overexposure to sun, just as humans do. Rainbow trout develop liver cancer in response to environmental toxins. And zebrafish—small, striped fish indigenous to the rivers of India and a widely used model organism—are susceptible to both malignant and benign tumors of the brain, nervous system, blood, liver, pancreas, skin, muscle, and intestine.
Importantly, tumors that arise in the same organs in humans and fish look and behave alike, and the cancers often share common genetic underpinnings. As a result, most researchers believe that the basic mechanisms underlying tumor formation are conserved across species, allowing them to study the formation, expansion, and spread of tumors in animal models with the hope of eventually finding new insights into cancer in people.
Zebrafish are an increasingly popular choice among cancer biologists. Between 1995 and 2012, there was a 10-fold increase in the number of yearly PubMed citations of cancer studies in the species, with more than 200 research papers published last year.  Although dwarfed by cancer studies using human tissue and mouse models, the optical transparency of zebrafish embryos and larvae—and now, adult fish of a recently created strain—allows researchers to track tumors in a way that is not possible in other vertebrate models. Furthermore, their small size—embryos are small enough to be reared in 96-well plates—make them a more practical laboratory system than other cancer models. Indeed, researchers are now using these fish to identify druggable oncogenic drivers of specific tumor types, to tease apart the complex network of cancer genes that cooperate in tumor formation and progression, to probe the interplay between the genes that govern embryonic development and those that cause cancer, and to uncover how tumors metastasize and kill their host. The zebrafish model offers a major opportunity to discover important pathways underlying cancer and to identify novel therapies in high-throughput drug screens in a way that mice never could.

The zebrafish toolbox

Zebrafish (Danio rerio) have fast made their way from pet stores and home aquaria into research laboratories worldwide. Their weekly matings produce 100 to 200 embryos that rapidly and synchronously march through embryonic development, so that within 5 days of fertilization, they are mature, feeding larvae. Zebrafish are small and inexpensive to maintain in high numbers, facilitating large-scale experimentation and cheap in vivo drug screens. Famously, the fish are transparent during early larval stages, allowing investigators to directly observe internal development and making the fish a favorite of developmental biologists since the 1960s. But in recent years, the utility of zebrafish has been proven beyond developmental fields, and they are now being found in more and more laboratories studying behavior, diabetes, heart disease, regeneration, stem cell biology—and cancer.
Critically, zebrafish can be used to identify the important pathways and processes that cause cancer in people. Common organ systems and cell types are shared between human and zebrafish, and whether induced by transgenesis or carcinogens, cancers arising from the blood (leukemia and lymphoma), pigmented cells of the skin (melanoma), and the cells that line the bile ducts (cholangiocarcinoma) have microscopic features that are essentially indistinguishable between humans and zebrafish.
The zebrafish model offers a major opportunity to discover important pathways underlying cancer and to identify novel therapies in high-throughput drug screens, in a way that mice never could.
Comparing gene-expression profiles of tumors across various species provides a powerful mechanism for identifying genes that likely represent core functions of cancer. For example, microarray gene-expression analyses have compared the gene signatures of fish hepatocellular carcinoma to that of human liver, gastric, prostate, and lung tumors. Remarkably, this analysis revealed that fish and human liver tumors are more similar to each other than either tumor type is to human tumors derived from different tissues. Moreover, comparative studies can often be used to pinpoint pathways that are active in human disease. This is illustrated by work on a zebrafish model of rhabdomyosarcoma (RMS), a cancer of skeletal muscle, which revealed a gene signature that is also commonly found in human RMS, highlighting the importance of the RAS signaling pathway in the genesis of human RMS.1

A window into cancer

In 2003, the laboratory of A. Thomas Look at Children’s Hospital Boston was the first to realize the long-held dream of following the behavior of cancer cells as they initiate tumor growth and invade structures within live animals. Specifically, the researchers engineered leukemia-afflicted T cells to express green fluorescent protein (GFP) and visualized cancer onset within the zebrafish thymus.2 Moreover, these GFP-positive tumor cells were transplantable into recipient fish, a hallmark of the malignant cell type. Following up on this work, several researchers have now begun transplanting fluorescently labeled human cancer cells into zebrafish larvae to visualize tumor growth and spread in a manner not achievable in more common mouse xenograft models.
Capitalizing on the lack of an acquired immune system during larval stages and the ability to rear zebrafish at temperatures that mimic the human core temperature, Stefania Nicoli of the University of Brescia in Italy and colleagues implanted human cancer cells expressing high levels of vascular endothelial growth factor (VEGF) into zebrafish larvae with GFP-labeled blood vessels.3 VEGF is a factor commonly produced by growing cancers and is responsible for coaxing blood vessels to invade the developing tumor. Nicoli’s work allowed the direct visualization of vasculature remodeling and new vessel formation—and showed that it could be blocked by the addition of VEGF-inhibitory drugs to the water in which the larvae lived. Using similar approaches, many laboratories have successfully engrafted human cancer cells from a range of tumor types into zebrafish embryos.
Researchers have also used zebrafish to visualize the role of tumor heterogeneity within cancer over time. Work from one of our labs (David Langenau’s) has utilized a model of RAS-induced RMS to fluorescently label tumor cells based on differentiation status. This allowed the team to watch the never-before-visualized birth of cancer—the acquisition of invasive properties by normal muscle stem cells and the breakdown of normal muscle architecture, clearing the way for continued tumor expansion. The researchers also characterized two molecularly distinct cell populations, one that is responsible for tumor growth and another that drives cancer spread or metastasis. (See photos below—Imaging Blood Cancers; Solid Tumor Development.)
SEE-THROUGH SUBJECTS: Zebrafish embryos (bottom right), shown here at 28 hours, are naturally transparent, as are zebrafish larvae (bottom left) until around 3 weeks of age. A new strain of zebrafish, called casper, maintains its transparency into adulthood (top right), allowing researchers to observe cancer formation in adult fish. A wall of tanks (top left) at the Zebrafish Resource Center, Karlsruhe Institute of Technology.
CLOCKWISE FROM TOP: © MARTIN LOBER; COURTESY OF RICHARD WHITE; COURTESY OF GRAHAM SCOTT; COURTESY OF KIRSTEN EDEPLI
T-cell leukemia and RMS are pediatric diseases, favoring cancer development in early larval stages of these zebrafish models. However, cancer is predominantly a disease that affects adults, and zebrafish lose their transparency at around 3 weeks of age. To visualize tumor formation in older zebrafish, one of us (Richard White) and Len Zon of Children’s Hospital Boston have developed a strain of zebrafish called casper, which lacks pigment and is optically clear into adulthood.4 (See photo here.) Using these animals, investigators have implanted pigmented melanomas and witnessed local spread and metastasis over time. First described in 2008, casper is now the zebrafish strain of choice for imaging studies in the field.
Zebrafish have truly proven to be ideal organisms for visualizing cancer; there is no other animal system that allows researchers to literally watch tumors grow and spread. Because we can follow cells as they escape from the primary tumor, migrate, and form metastases in a variety of organs, zebrafish provide an unsurpassed model to describe the distinct steps of cancer progression, and researchers using this model are already contributing much to our understanding of cancer.

 Screening for drugs

In terms of drug discovery, zebrafish have emerged as the only vertebrate organism amenable to high-throughput and high-content chemical screening in vivo. The small size of freshly hatched zebrafish embryos means that up to 20 embryos can be dispensed into the individual wells of a 96-well plate, thereby providing a platform for the robotic delivery and testing of hundreds or thousands of compounds in living animals. Because of the parallels between embryonic development and cancer, compounds producing changes in the growth or proliferation of developing organs may also be relevant to cancer. However, to more directly search for small molecules capable of putting the brakes on cancerous growth, researchers are turning to several established tumor-prone zebrafish lines.
One prominent success from such endeavors is the identification of a drug to treat melanoma. Like human melanoma skin lesions, zebrafish melanomas exhibit a gene-expression signature characteristic of the embryonic neural crest, a multipotent group of stem cells that give rise to dozens of cell types, including pigmented skin cells called melanocytes. The genes in this signature, including sox10 and mitf, were hypothesized to be important for melanoma growth, so in 2011 White and Zon performed an in vivo screen to identify small molecules that suppressed the expression of these neural crest genes in developing embryos.5 After screening 2,000 molecules, they identified leflunomide, an approved treatment for rheumatoid arthritis. Importantly, leflunomide was then found to inhibit the growth of both zebrafish and human melanoma xenografts in vivo, and the drug moved from discovery to Phase 1/2 trials in only 4 years, demonstrating just how quickly discoveries in zebrafish can have clinical impact. Similar efforts to discover novel modulators of leukemia growth have recently been reported to work in both fish and humans, suggesting that this approach will be broadly applicable to a wide range of solid and liquid tumors.
Probing the cancer genome
The genesis of cancer generally depends on the inactivation of one or more tumor suppressor genes in conjunction with signaling from oncogenes. Indeed, rapid advances in sequencing technologies and efforts such as The Cancer Genome Atlas (TCGA) have revealed surprisingly few “driver” mutations capable of causing cancer alone. Instead, TCGA and other sequencing studies have identified vast genetic heterogeneity both across and within tumor types, with mutations extending well beyond genes likely to represent classical oncogenes or tumor suppressor genes. How these mutations influence tumor growth remains a major unanswered question in cancer biology.
IMAGING BLOOD CANCERS: Developing T lymphocytes in the thymus of a transgenic zebrafish (top right) express green fluorescent protein (GFP). A transgenic zebrafish (bottom right) that coexpresses the Myc oncogene with GFP shows signs of prominent leukemia, which has spread well beyond the boundaries of the thymus (T).
COURTESY OF DAVID LANGENAU
Enter zebrafish, and a range of high-throughput reverse-genetic techniques for cancer gene discovery. By transiently overexpressing each of 30 candidate genes in zebrafish larvae, for example, Craig Ceol and Yariv Houvras in Zon’s group identified a single cooperating oncogene, SETDB1, as a new player in melanoma.6 In this study, the researchers created and analyzed more than 3,000 transgenic animals. Because they used a transposon-based transgenic approach that leads to high-level, uniform expression, they could directly assess how the injected genes affected tumor onset without going through the lengthy process of germline transgenics, a major bottleneck in mouse genetics. This rapid screening approach is a prime example of how the mountains of data generated by TCGA can be quickly assessed for biological function, and zebrafish are the only in vivo whole-animal vertebrate system that enables researchers to rapidly sift through these data to understand which mutations drive cancer.
Other new approaches are advancing loss-of-function analyses. Until recently, such studies relied on TiLLING (targeting induced local lesions in genomes), in which a chemical carcinogen, ethylnitrosourea (ENU), introduces point mutations throughout the genome and high-throughput methods then look for mutations in genes of interest. This method yielded several valuable strains of tumor-prone zebrafish harboring clinically relevant mutations in the well-known tumor-suppressor genes p53, apc, and pten, and these have been pivotal to the development of multiple zebrafish cancer models. However, the unbiased nature of ENU mutagenesis makes TiLLING a labor-intensive and impractical business in most laboratory settings. Instead, precision editing of the genome has emerged as the method of choice for the systematic creation of knockout and mutant animals. Specifically, homology-based editing, using TALENs (transcription activator–like effector nucleases) and, more recently, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems, has revolutionized the field.7,8 Using relatively simple procedures, virtually any gene can now be mutated in zebrafish, allowing for very large-scale, in vivo assessments of novel cancer genes and the analysis of interacting mutations—one of the greatest challenges facing the cancer field in the coming decade.
SOLID TUMOR DEVELOPMENT: A transgenic zebrafish (bottom left) with fluorescent-labeled RAS-induced rhabdomyosacoma. Green fluorescent protein is expressed in the tumor-propagating cells, which drive continued tumor growth. Red fluorescent protein is localized to the nucleus and expressed in myoblast-like cells, while blue fluorescent protein is confined to terminally differentiated cancer cells that express myosin (magnified image, bottom right). Live-cell imaging permits dynamic visualization of the birth of cancer and the functional consequences of tumor cell heterogeneity within established tumors.
COURTESY OF DAVID LANGENAU
To complement approaches that directly inactivate genes within the genome, strategies to achieve interference RNA-mediated gene silencing in zebrafish have come of age as well. Expression of short hairpin RNAs, for example, have produced stable and tissue-specific knockdown in cancer-related genes such as chordin and wnt5b.9,10 Because of the ease of manipulating the genome as well as the large number of well-characterized zebrafish gene promoters, such strategies immediately afford the opportunity to knockdown known gene functions in a tissue-specific fashion, and it is likely that temporal control will be readily achievable as well.
In all, combining the descriptive data from TCGA with zebrafish transgenesis, high-throughput overexpression and knockout techniques, and unbiased genetic screens offers an unprecedented opportunity to functionally probe the cancer genome.

The future of the field

Given its power for imaging, transplantation, small-molecule screens, and high-throughput transgenesis, the zebrafish model should become a major platform for deeply interrogating cancer biology in vivo over the next decade. One major area where zebrafish are particularly valuable is in teasing apart the extreme complexity of cancer. Because combinations of genetic pathways can be assessed simultaneously, potentially dozens of genomic alterations found in human cancer could be tested for their effects in the fish, allowing us to sort biologically meaningful alterations from neutral ones. These techniques will also allow us to understand how numerous small changes, which on their own have little phenotypic effect, can combine to cause cancer.
The success of the field will depend upon improved funding for zebrafish cancer research, however. Currently, only a small fraction of National Institutes of Health RO1 grants for cancer research are awarded for zebrafish studies, with the vast majority going to work in mice, humans, and human cells. Consortium efforts analogous to the Mouse Model Consortium will be necessary to develop more faithful zebrafish models of human cancer, which can then be used as the basis for further screens. Whereas mouse models of cancer have delivered great insights into the biological mechanisms underlying human malignancy, we view zebrafish models as a springboard for the rapid launch of unbiased genetic and chemical screens.
With any cancer model, bridging the gap between the animals and human patients is the ultimate proof of its utility. For the zebrafish, this can occur not only through bringing drugs to the clinic, but also in the development of novel biomarkers and early detection methods. The next 10 years will be an exciting time, and we have great confidence that the zebrafish will contribute major discoveries to the treatment of human cancers.
Joan K. Heath is an associate professor in the ACRF Chemical Biology Division at the Walter and Eliza Hall Institute of Medical Research and the Department of Medical Biology at the University of  Melbourne, Australia, where her laboratory is studying the genetic regulation of  intestinal organogenesis and colorectal cancer.
David Langenau, an assistant professor of pathology at Harvard Medical School, studies the mechanisms that drive pediatric cancer relapse within the Molecular Pathology Unit and the Cancer Center at Massachusetts General Hospital.
Kirsten C. Sadler is an assistant professor in the Division of Liver Diseases/Department of Medicine and in Developmental and Regenerative Biology at the Icahn School of Medicine at Mount Sinai in New York City, where she studies the mechanisms of liver development, regeneration, and cancer.
Richard White is an assistant professor at the Memorial Sloan-Kettering Cancer Center and Weill Cornell Medical College in New York City. His laboratory studies the evolutionary mechanisms by which tumors develop the capacity for metastasis.

References

D.M. Langenau et al., “Effects of RAS on the genesis of embryonal rhabdomyosarcoma,” Genes Dev, 21:1382-95, 2007.
D.M. Langenau et al., “Myc-induced T cell leukemia in transgenic zebrafish,” Science, 299:887-90, 2003.
S. Nicoli et al., “Mammalian tumor xenografts induce neovascularization in zebrafish embryos,” Cancer Res, 67:2927-31, 2007.
R.M. White et al., “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell, 2:183-89, 2008.
R.M. White et al., “DHODH modulates transcriptional elongation in the neural crest and melanoma,” Nature, 471:518-22, 2011.
V.M. Bedell et al., “In vivo genome editing using a high-efficiency TALEN system,” Nature, 491:114-18, 2012.
W.Y. Hwang et al., “Efficient genome editing in zebrafish using a CRISPR-Cas system,” Nat Biotechnol, doi: 10.1038/nbt.2501, 2013.
M. Dong et al., “Heritable and lineage-specific gene knockdown in zebrafish embryo,” PLOS ONE, 4:e6125, 2009.
G. De Rienzo et al., “Efficient shRNA-mediated inhibition of gene expression in zebrafish,” Zebrafish, 9:97-107, 2012.
Tags
zebrafish, model organisms, cancer therapetics, cancer research, cancer genomics, cancer gene expression, cancer and animal models
Danio rerio, better known as the zebrafish

Danio rerio, better known as the zebrafish (Photo credit: Wikipedia)

Zebrafish embryo development

Zebrafish embryo development (Photo credit: Carl Zeiss Microscopy)

English: zebrafish (Danio rerio) ovary oocyte ...

English: zebrafish (Danio rerio) ovary oocyte maturation (Photo credit: Wikipedia)

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