Eric Lin

Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers

Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the neoplastic transformation process. Here we engineered lymphoma-prone mice with chromosomal instability to assess the usefulness of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Along with targeted re-sequencing, our comparative oncogenomic studies identified FBXW7 and PTEN to be commonly deleted both in murine lymphomas and in human T-cell acute lymphoblastic leukaemia/lymphoma (T-ALL). The murine cancers acquire widespread recurrent amplifications and deletions targeting loci syntenic to those not only in human T-ALL but also in diverse human haematopoietic, mesenchymal and epithelial tumours. These results indicate that murine and human tumours experience common biological processes driven by orthologous genetic events in their malignant evolution. The highly concordant nature of genomic events encourages the use of genomically unstable murine cancer models in the discovery of biological driver events in the human oncogenome.

Enrichment of sequencing targets from the human genome by solution hybridization

To exploit fully the potential of current sequencing technologies for population-based studies, one must enrich for loci from the human genome. Here we evaluate the hybridization-based approach by using oligonucleotide capture probes in solution to enrich for approximately 3.9 Mb of sequence target. We demonstrate that the tiling probe frequency is important for generating sequence data with high uniform coverage of targets. We obtained 93% sensitivity to detect SNPs, with a calling accuracy greater than 99%.

The tumor microenvironment in esophageal cancer

Esophageal cancer is a deadly disease, ranking sixth among all cancers in mortality. Despite incremental advances in diagnostics and therapeutics, esophageal cancer still carries a poor prognosis, and thus, there remains a need to elucidate the molecular mechanisms underlying this disease. There is accumulating evidence that a comprehensive understanding of the molecular composition of esophageal cancer requires attention to not only tumor cells but also the tumor microenvironment (TME), which contains diverse cell populations, signaling factors and structural molecules that interact with tumor cells and support all stages of tumorigenesis. In esophageal cancer, environmental exposures can trigger chronic inflammation, which leads to constitutive activation of pro-inflammatory signaling pathways that promote survival and proliferation. Antitumor immunity is attenuated by cell populations such as myeloid-derived suppressor cells and regulatory T cells, as well as immune checkpoints like programmed death-1. Other immune cells such as tumor-associated macrophages can have other pro-tumorigenic functions, including the induction of angiogenesis and tumor cell invasion. Cancer-associated fibroblasts secrete growth factors and alter the extracellular matrix to create a tumor niche and enhance tumor cell migration and metastasis. Further study of how these TME components relate to the different stages of tumor progression in each esophageal cancer subtype will lead to development of novel and specific TME-targeting therapeutic strategies, which offer considerable potential especially in the setting of combination therapy.

Comparative transcriptomes of adenocarcinomas and squamous cell carcinomas reveal molecular similarities that span classical anatomic boundaries

Advances in genomics in recent years have provided key insights into defining cancer subtypes “within-a-tissue”—that is, respecting traditional anatomically driven divisions of medicine. However, there remains a dearth of data regarding molecular profiles that are shared across tissues, an understanding of which could lead to the development of highly versatile, broadly applicable therapies. Using data acquired from The Cancer Genome Atlas (TCGA), we performed a transcriptomics-centered analysis on 1494 patient samples, comparing the two major histological subtypes of solid tumors (adenocarcinomas and squamous cell carcinomas) across organs, with a focus on tissues in which both subtypes arise: esophagus, lung, and uterine cervix. Via principal component and hierarchical clustering analysis, we discovered that histology-driven differences accounted for a greater degree of inherent molecular variation in the tumors than did tissue of origin. We then analyzed differential gene expression, DNA methylation, and non-coding RNA expression between adenocarcinomas and squamous cell carcinomas and found 1733 genes, 346 CpG sites, and 42 microRNAs in common between organ sites, indicating specific adenocarcinoma-associated and squamous cell carcinoma-associated molecular patterns that were conserved across tissues. We then identified specific pathways that may be critical to the development of adenocarcinomas and squamous cell carcinomas, including Liver X receptor activation, which was upregulated in adenocarcinomas but downregulated in squamous cell carcinomas, possibly indicating important differences in cancer cell metabolism between these two histological subtypes of cancer. In addition, we highlighted genes that may be common drivers of adenocarcinomas specifically, such as IGF2BP1, which suggests a possible link between embryonic development and tumor subtype. Altogether, we demonstrate the need to consider biological similarities that transcend anatomical boundaries to inform the development of novel therapeutic strategies. All data sets from our analysis are available as a resource for further investigation.

IL-6 Mediates Cross-Talk between Tumor Cells and Activated Fibroblasts in the Tumor Microenvironment

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR.

CD38+ M-MDSC expansion characterizes a subset of advanced colorectal cancer patients

BACKGROUNDnMyeloid-derived suppressor cells (MDSCs) are a population of immature immune cells with several protumorigenic functions. CD38 is a transmembrane receptor-ectoenzyme expressed by MDSCs in murine models of esophageal cancer. We hypothesized that CD38 could be expressed on MDSCs in human colorectal cancer (CRC), which might allow for a new perspective on therapeutic targeting of human MDSCs with anti-CD38 monoclonal antibodies in this cancer.nnnMETHODSnBlood samples were collected from 41 CRC patients and 8 healthy donors, followed by peripheral blood mononuclear cell (PBMC) separation. Polymorphonuclear (PMN-) and monocytic (M-) MDSCs and CD38 expression levels were quantified by flow cytometry. The immunosuppressive capacity of M-MDSCs from 10 CRC patients was validated in a mixed lymphocyte reaction (MLR) assay.nnnRESULTSnA significant expansion of CD38+ M-MDSCs and a trend of expansion of CD38+ PMN-MDSCs (accompanied by a trend of increased CD38 expression on both M- and PMN-MDSCs) were observed in PBMCs of CRC patients when compared with healthy donors. The CD38+ M-MDSCs from CRC patients were found to be immunosuppressive when compared with mature monocytes. CD38+ M- and PMN-MDSC frequencies were significantly higher in CRC patients who previously received treatment when compared with treatment-naive patients.nnnCONCLUSIONSnThis study provides a rationale for an attempt to target M-MDSCs with an anti-CD38 monoclonal antibody in metastatic CRC patients.nnnFUNDINGnNCI P01-CA14305603, the American Cancer Society, Scott and Suzi Lustgarten Family Colon Cancer Research Fund, Hansen Foundation, and Janssen Research and Development.

Abstract A37: The role of IL-6 in the interaction between fibroblasts and tumor cells in esophageal adenocarcinoma

Often described as wounds that do not heal, cancer cells depend on interactions with the surrounding stroma to develop and progress. Among the various stromal components, cancer-associated fibroblasts (CAFs) play a critical role in promoting tumor growth and invasion, leading to treatment resistance and poor survival in a number of cancers, including esophageal adenocarcinoma. Mechanistically, CAFs communicate with tumor cells in large part via secreted signaling factors. One such factor, interleukin-6 (IL-6), has been shown to be secreted by CAFs to promote angiogenesis, epithelial-to-mesenchymal transition (EMT), tumor cell stemness, and treatment resistance in adenocarcinomas of the colon, pancreas, stomach, and breast. In EAC, the upregulation of IL-6 signaling among tumor cells, particularly as a result of exposure to reflux, is well characterized. However, the role of IL-6 as a potential mediator of the CAF-tumor cell interaction in EAC remains poorly understood. To confirm that IL-6 is indeed involved in this interaction, we used ELISA to study the dynamics of IL-6 secretion by activated fibroblasts (FEF3303, FEF3 and PDF.G.P) and EAC cells (OE-19 and OE-33) in the setting of mono- and co-culture. We found that the interaction of CAFs and EAC cells in co-culture dramatically increased IL-6 levels compared to either EAC cells or fibroblasts alone. Next, we considered the relevance of these findings to human disease by examining human EAC biopsy specimens for patterns of IL-6 expression via immunohistochemical staining. IL-6 was strongly expressed in the tumor-associated stroma of 10/10 EAC biopsy specimens, with 5/10 showing tumor expression as well, which in several cases was localized to the invasive edge near the tumor-stromal interface. In addition, to assess for IL-6 signaling, we stained the sections for downstream mediators of IL-6 signaling, namely STAT3 and ERK1/2. We found nuclear localization of STAT3 and ERK in 10/10 and 7/10 samples, respectively, indicating activation of these pathways. In summary, our findings indicate that IL-6 is involved in the communication between CAFs and tumor cells in EAC. Furthermore, we have reason to believe, especially in cases where both tumor cells and stroma have strong IL-6 expression, that IL-6 may be a mediator of a bidirectional “crosstalk” between these two cell types. Additional investigation, for instance with the use of a 3-D organotypic model of EAC, will help to further characterize the functional role of IL-6 in this context. This is the first report describing a potential role for IL-6 in mediating crosstalk between CAFs and tumor cells in EAC. Citation Format: Eric W. Lin, Tatiana A. Karakasheva, Sarah Derks, Kwok K. Wong, Adam J. Bass, Anil K. Rustgi. The role of IL-6 in the interaction between fibroblasts and tumor cells in esophageal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A37.

Abstract A10: Biomarker independant microfluidic Labyrinth for the high throughput enrichment and characterization of circulating tumor cells in pancreatic cancer

Introduction and Objective: Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 6%. By the time of diagnosis, less than 15% of patients have surgically resectable tumors, which is one reason why it has the highest mortality rate among all cancer types. Emerging evidence has pointed the importance of circulating tumor cells (CTCs) in the spread of cancers and metastasis. CTCs are believed to be the most promising cancer biomarkers available in blood. Recent reports also show that these cells could be a good surrogate biomarker for not only prognosis, but also for cancer detection and development of personalized treatment. We have developed an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs. Methods: To investigate the isolation efficiency of our technology before running cancer patients’ samples, PANC-1 cells labeled with cell tracker dye were spiked into 1 mL of whole blood. Using this optimized platform we isolate CTCs from pancreatic cancer patients, followed by downstream analysis . CTCs are characterized using immunocytochemistry for heterogeneity studies among CTC population. Results: Our microfluidic device consists of a size based separation platform in which CTCs are separated from other blood components. This high throughput label free separation device was designed and developed by our lab. After more than 20 generations of device revision, an optimized flow rate of 2.5 ml/min for PANC-1 pancreatic cancer cell line has yielded a 95% recovery of these cells from whole blood, with cancer cells traveling to the second outlet and 80% of the White Blood Cells (WBC) to the first outlet. We have successfully isolated CTCs from over 100 pancreatic cancer patients. Among several patients we were able to characterize different subpopulations using the epithelial marker CK-19 and EpCAM, and the EMT-like marker ZEB1. Conclusion: The implementation of our engineering technology to the study of pancreatic cancer can present novel ways to confront major hurdles in cancer research, such as early detection and the lack of effective therapeutics. The isolation and expansion of CTCs, the heterogeneous population of cells that promote metastasis, can provide meaningful information to elucidate the process of pancreatic tumorigenesis to preempt its fatal result. Citation Format: Lianette Rivera-Baez, Eric Lin, Meghna Waghray, Shamileh Fouladdel, Ebrahim Azizi, Max Wicha, Vaibhav Sahai, Diane Simeone, Sunitha Nagrath.{Authors}. Biomarker independant microfluidic Labyrinth for the high throughput enrichment and characterization of circulating tumor cells in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A10.

Abstract IA13: The interplay between tumor cells, cancer associated fibroblasts, and immature myeloid cells in the esophageal tumor microenvironment

Esophageal cancer has two major subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC is more common worldwide and illustrative of other SCCs (e.g. head/neck, lung) from a molecular pathogenesis viewpoint. EAC is rising in incidence in the United States and Western Europe. Unfortunately, 5-year survival remains dismal for both types, at about 15%. Interrogation of the esophageal tumor microenvironment, replete with desmoplasia, CAFs and diverse immune cells, may afford opportunities to reveal new therapeutic targets, especially in a combinatorial fashion. We have generated previously a conditional knockout model of p120-catenin whereby mice develop preneoplastic and neoplastic lesions in the esophagus. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells (myeloid deriver suppressor cells or MDSCs) comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts. We have identified CD38 as playing a vital role in MDSC biology. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor–derived factors, specifically IL-6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38+ MDSCs in peripheral blood of advanced-stage cancer patients. Additional work reveals cross-talk between CAFs and tumor cells through the induction of IL-6 and RANTES, suggesting there is interaction between tumor cells, CAFs and MDSCs. In aggregate, we find that CD38 (on MDSCs), IL-6 and RANTES (secreted by tumor cells and CAFs) serve to provide novel approaches in therapy. Citation Format: Tatiana Karakasheva, Eric Lin, Phil Hicks, Todd Waldron, Anil K. Rustgi. The interplay between tumor cells, cancer associated fibroblasts, and immature myeloid cells in the esophageal tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA13.

Abstract A32: Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment

Desmoplasia, or deposition of connective tissue proper in the stroma, is a key feature of esophageal squamous cell carcinoma (ESCC), a common malignancy worldwide. The cellular component of connective tissue proper is comprised of fibroblasts, and cancer-associated fibroblasts (CAFs) are widely recognized as a major constituent of the tumor microenvironment (TME). The purpose of this study was to investigate the cross-talk between CAFs and tumor cells in ESCC, as well as identify factors that mediate this cross-talk and evaluate these factors as therapeutic targets in ESCC and potentially other squamous cell carcinomas. We have observed that co-culture with CAFs prompts ESCC cells to acquire a more mesenchymal phenotype and express potent mediators of cell migration and invasion, such as matricellular proteins (periostin, fibrillin, osteonectin) and growth factors (EGF, HGF, VEGF). Furthermore, co-culture of ESCC cells with CAFs promotes tumor cell migration in our 3D organotypic culture, while co-transplantation of ESCC cells with fibroblasts leads to enhanced tumor growth in vivo , compared to tumors transplanted without fibroblasts. In order to identify potential mediators of ESCC-CAF cross-talk, we have performed a comprehensive cytokine array and found that interleukin 6 (IL-6) is significantly overexpressed in conditioned media from co-culture of esophageal CAFs and ESCC cells, compared to mono-culture. IL-6 is known to play important roles in the development of multiple types of cancer, mostly via activation of the STAT3 signaling pathway. To confirm relevance of our in vitro findings, we have performed immunohistochemical staining of tissue samples from ESCC patients and found that, compared to normal esophagus, expression of IL-6 is enhanced in ESCC in both epithelial cells and fibroblasts. Interestingly, knockdown of IL-6 resulted in altered morphology of ESCC cells in 3D organotypic culture. This was accompanied by restored membrane localization of E-cadherin and reduced activation of STAT3 signaling. In order to investigate the importance of STAT3 and MEK/ERK signaling (the latter being another pathway activated by IL-6 in cancer) on ESCC cell biology, we have treated 3D organotypic cultures with stattic and trametinib (inhibitors of STAT3 and MEK, respectively). This treatment resulted in impaired invasion, reduced proliferation and enhanced apoptosis of ESCC cells, accompanied by decreased expression of the basal cell marker p63. Furthermore, treatment with stattic and trametinib leads to reduced secretion of IL-6 by these cultures. These effects were more pronounced upon treatment with combination of stattic and trametinib. Since we found IL-6 to be important for ESCC progression in vitro , we have conducted a therapeutic study utilizing tocilizumab (an FDA-approved anti-human IL-6R neutralizing antibody). Subcutaneous xenograft tumors comprised of human ESCC and CAF cell lines, were treated with tocilizumab, which resulted in suppressed tumor growth, accompanied by decreased activation of STAT3 and ERK within the tumor, compared to control IgG-treated tumors. Furthermore, tocilizumab-treated animals had reduced numbers of splenic monocytic immature myeloid cells (CD11b + Ly-6C + ), which is in agreement with previously published findings regarding MDSCs in murine models of ESCC. In summary, our findings indicate that IL-6, at least in part, is responsible for migration, proliferation, survival and poor differentiation of ESCC cells, promoted by CAFs in the TME. We also report that these effects of IL-6 are mediated by STAT3 and ERK signaling. Finally, we demonstrate that inhibition of IL-6 signaling suppresses ESCC tumor growth in vivo . This is the first report of anti-tumorigenic activity of tocilizumab in ESCC, which makes it a promising candidate for ESCC therapy. Supported by NCI P01 CA098101. Citation Format: Tatiana A. Karakasheva, Monica Soni, Todd J. Waldron, Eric W. Lin, Philip D. Hicks, Andres Klein-Szanto, Kwok K. Wong, Adam J. Bass, Anil K. Rustgi. Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A32.