Research Center

Modeling the Role of Lymph Node Metastases in Tumor-Mediated Immunosuppression

Overview

Center Title

Stanford University Center for Cancer Systems Biology

Overall Project Title

Modeling the role of lymph node metastases in tumor-mediated immune suppression

Center Website

http://ccsb.stanford.edu

Center Summary

Our Research Center aims to identify the mechanisms in which tumor cells instruct the immune system to tolerant them by focusing on the understudied role of lymph node invasion in tumor-mediated immunosuppression.

Overall Distant metastasis is the primary cause of cancer-related death. To colonize distant tissues, cancer cells must migrate while evading elimination by the immune system. Evidence suggests that key steps in the induction process of immune tolerance occur early in the metastatic cascade, located at regional lymph nodes. However, the nature of the interactions between tumors and immune cells remains poorly understood, particularly for those occurring within the lymph nodes. Even though lymph nodes are in fact commonly assessed in cancer patients to determine disease stage and treatment plan, they are understudied in the context of metastatic progression.

We hypothesize that lymph node metastasis constitutes an essential, first step in the metastatic cascade of cancer progression. Based on our preliminary findings, we speculate that such metastases act locally upon the adaptive immune system within the nodes to begin to induce systemic tolerance of the tumor. We will explore, compare and test this hypothesis in two malignancies: (i) melanoma and (ii) head and neck squamous cell carcinoma. We have assembled a multidisciplinary team whose coordinated efforts will involve the application of genomic and single-cell in-situ imaging technologies on preclinical and human samples to explore the evidence and mechanisms of the induction of immunosuppression in the lymph nodes. We propose three inter-connected Research Projects that focus our scientific theme on different platforms: murine models (Project 1), high-dimensional in-situ imaging (Project 2), and integrative computational analysis (Project 3). All three projects will utilize a shared resource core dedicated to the acquisition of patient samples and associated clinical annotation and data management (Biospecimen and Data Management Core. These efforts will yield highly multiplexed, multi-scale datasets which will be analyzed by novel bio-computational methods to reconstruct intracellular and intercellular molecular interaction networks in order to identify, then functionally validate, critical mediators of tumor immunosuppression.

Our ultimate objective is to advance our understanding of the systemic consequences of lymph node metastases and identify new therapeutic approaches to cancer immunotherapy. Our findings promise to provide critical insights into blocking metastatic progression and thereby preventing cancer-related deaths.

Investigators

Principal Investigators

Sylvia Plevritis, Ph.D.

Sylvia Plevritis, Ph.D. is a Professor in the Department of Radiology in the Stanford School of Medicine. Dr. Plevritis holds a PhD in Electrical Engineering (Stanford, 1992) with concentration on MRI spectroscopic imaging of tumors. She also holds an MS in Health Services Research (Stanford, 1996), with concentration on the evaluation of cancer screening programs on reducing cancer mortality. Dr. Plevritis is the Director of the Stanford Center for Cancer Systems Biology (CCSB), Director of the Cancer Systems Biology Scholars (CSBS) Program, and the co-Section Chief of the Integrative Biomedical Imaging Informatics at Stanford (IBIIS). She has developed integrative cancer research programs that bridge genomics, imaging and population sciences to understand cancer progression and treatment response. She served as PI in the NCI Integrative Cancer Biology Program for the past 10 years, currently as the Director of the Stanford Center in Cancer Systems Biology (CCSB).

Dr. Plevritis is also Director of the Stanford Postdoctoral Scholars Program in Cancer Systems Biology which aims to train the next generation of scholars at the interface of molecular cancer biology and biocomputation. She serves as the coDirector of Integrative Biomedical Imaging Informatics at Stanford (IBIIS, ibiis.stanford.edu), a section in the Department of Radiology that promotes the development and application of novel biocomputational tools to integrate imaging, molecular and clinical data. She has been a Principal Investigator with the NCI Cancer Intervention Surveillance Network (CISNET, cisnet.cancer.gov) for over fifteen years and currently serves on the Executive Committee of Stanford Biomedical Data Sciences Initiative which promotes interdisciplinary research opportunities for “big data” sciences in precision medicine at Stanford University. Having pursued a career path that spans multiple disciplines, she was drawn to cancer systems biology research as it propels the intersection of diverse scientific concepts with the goal of unraveling the complexity of cancer and ultimately defeating this disease through improvements in early detection and treatment strategies.

Garry Nolan, Ph.D.

Garry Nolan, Ph.D. is the Rachford and Carlota A. Harris Professor in the Department of Microbiology and Immunology at Stanford University School of Medicine. He trained with Leonard Herzenberg (for his Ph.D.) and Nobelist Dr. David Baltimore (for postdoctoral work for the first cloning/characterization of NF-kB p65/ RelA and the development of rapid retroviral production systems). He has published over 180 research articles and is the holder of 20 US patents, and has been honored as one of the top 25 inventors at Stanford University. Dr. Nolan is the first recipient of the Teal Innovator Award (2012) from the Department of Defense (a $3.3 million grant for advanced studies in ovarian cancer), the first recipient of an FDA BAAA, for “Bio-agent protection” grant, $3million, from the FDA for a “Cross-Species Immune System Reference”, and received the award for “Outstanding Research Achievement in 2011” from the Nature Publishing Group for his development of CyTOF applications in the immune system. Dr. Nolan has new efforts in the study of Ebola, having developed instrument platforms to deploy in the field in Africa to study Ebola samples safely with the need to transport them to overseas labs (funded by a new $3.5 million grant from the FDA).

Dr. Nolan is an outspoken proponent of translating public investment in basic research to serve public welfare. Dr. Nolan was the founder of Rigel Inc. (NASDAQ: RIGL), and Nodality, Inc. (a diagnostics development company), BINA (a genomics computational infrastructure company sold to Roche Diagnostics), and serves on the Boards of Directors of several companies as well as consults for other biotechnology companies. DVS Sciences, on which he was Chair of the Scientific Advisory Board, recently sold to Fluidigm for $207 million dollars (2014) on an investment of $14 million.
His areas of research include hematopoiesis, cancer and leukemia, autoimmunity and inflammation, and computational approaches for network and systems immunology. Dr. Nolan’s recent efforts are focused on a single cell analysis advance using a mass spectrometry-flow cytometry hybrid device, the socall “CyTOF” and the “Multiparameter Ion Beam Imager” (MIBI) developed by Dr. Mike Angelo in his lab (Dr. Angelo is now an Assistant Professor in the Dept of Pathology at Stanford). The approaches uses an advanced ion plasma source to determine the levels of tagged reagents bound to cells enabling a vast increase in the number of parameters that can be measured per cell either as flow cytometry devices (CyTOF) or imaging platforms for cancer (MIBI).Further efforts are being develop with another imaging platform termed CODEX that inexpensively converts fluorescence scopes to high dimensional imaging platforms.Dr. Nolan’s efforts are to enable a deeper understanding not only of normal immune function, trauma, pathogen infection, and other inflammatory events but also detailed substructures of leukemias and solid cancers which will enable wholly new understandings that will enable better management of disease and clinical outcomes.

Ed Engleman, M.D., Ph.D.

Ed Engleman, M.D., Ph.D. is Professor of Pathology and of Medicine Immunology and Rheumatology). His laboratory studies the biology of immune cells and their roles in the pathogenesis of cancers and other life-threatening diseases. By applying new and more precise analytical tools for assessing this system in mice and humans, they have been successful at identifying disease-promoting immune abnormalities. By targeting the cells responsible for or affected by these abnormalities, they have succeeded in reversing the abnormalities and ameliorating the diseases they cause.
Dr Engleman’s lab has been particularly interested in the biology and functions of dendritic cells (DC), which are potent antigen presenting cells that can either induce or suppress immunity. Their first generation methods for isolating and arming human DC with tumor antigens provided the basis for the Sipuleucel-T vaccine that was approved by the FDA in 2010 for the treatment of metastatic prostate cancer. More recently, the lab developed a novel immunotherapeutic strategy that targets tumoral DC in vivo. In addition, they have been using newer technologies, including high dimensional single cell proteomic technology (CyTOF) and deep gene sequencing to investigate the immune system in cancer. A key goal is to identify and understand the key cellular and molecular mechanisms required for tumor elimination. The lab makes extensive use of mouse models for in depth mechanistic studies in addition to studying human samples.

In addition to cancer, the Engleman lab have been studying the role of immune cells in autoimmune diseases, metabolic diseases, graft versus host disease and transplantation tolerance. Recently, the group has developed novel tools for studying microglia in the brain and has begun to use these tools to analyze the role of these rare immune cells in chronic neurodegenerative disorders such as Alzheimer’s Disease, Parkinson’s Disease and amyotrophic lateral sclerosis (ALS). Preliminary findings in mouse models suggest that abnormal metabolism affecting these cells may contribute to the development of these disorders.

John Sunwoo M.D., Ph.D.

John Sunwoo M.D., Ph.D. is Professor of Otolaryngology (Head and Neck Surgery). Dr Sunwoo received his undergraduate degree from Brown University in Providence, Rhode Island and his medical degree from Washington University in St. Louis, Missouri. He completed his training in Otolaryngology – Head and Neck Surgery at Washington University. Dr. Sunwoo has been at Stanford University since 2008, and his clinical focus is on the surgical management of head and neck cancer, specifically focusing on melanoma and neoplasms of the thyroid and parathyroid glands. He is a member of the Pigmented Lesions and Melanoma Clinic and the Melanoma Working Group at Stanford. He is also the co-founder of the Stanford Thyroid and Parathyroid Tumor Board.
In addition to his clinical work, Dr. Sunwoo is the Director of Head and Neck Cancer Research at Stanford University and the principal investigator of an NIH-funded laboratory in the Stanford Cancer Institute. His research is focused on three primary areas: (1) the immune response to cancer, particularly a tumorigenic population of cells within malignancies called cancer stem cells; (2) the biology and developmental programs of a special lymphocyte population involved in innate immunity called natural killer (NK) cells; and (3) intra-tumor and inter-tumor heterogeneity in head and neck cancer. A major focus of the Sunwoo lab is the study of natural killer (NK) cells, a special lymphocyte population critically involved in the innate immune response to viral infections and malignancy. They are interested in understanding the regulation of NK cell development, homeostasis, and effector functions. One such regulator of these processes is the aryl hydrocarbon receptor (AhR), a cytoplasmic receptor that binds numerous endogenous and exogenous ligands. They recently showed that these small molecule ligands can modulate NK cell homeostasis and anti-tumor functions.

An overarching goal of the lab is to understand how the immune system interfaces with and protects against developing and metastasizing tumor cells, especially a rare population of tumor-initiating cells called cancer stem cells. In these studies, they utilize human and mouse models of head and neck squamous cell carcinoma (HNSCC) and melanoma. The lab is investigating novel mechanisms by which malignant cells can alert the immune system at the earliest stages of transformation. The lab also studies how regulators of genetic networks control the behavioral features of a subset of cells with stem cell–like properties. These cells are more resilient and have the ability to metastasize and are more resistant to chemotherapy and radiation therapy. A particularl interest is identifying mechanisms by which these tumor-initiating cells can selectively suppress the host immune response and understanding how to overcome these immunosuppressive properties.

Participating Investigators

Christina Kong, M.D.

Christina Kong, M.D. is Professor of Pathology. Her group is interested in improving the accuracy of cytologic diagnosis through refining diagnostic criteria and the use of ancillary techniques (e.g. immunoperoxidase stains, flow cytometry, in situ hybridization, PCR) on specimens obtained by the minimally invasive technique of fine needle aspiration biopsy. They are also working on identifying potential indicators of prognosis in head and neck squamous cell carcinomas. Additional interests include evaluating the utility of immunohistochemical stains in refining the diagnosis of squamous dysplasia of the cervix, vulva, and head and neck.

Robert Tibshirani, Ph.D.

Robert Tibshirani, Ph.D. is Professor of Statistics and of Biomedical Data Sciences. His main interests are in applied statistics, biostatistics, and data mining. He is co-author of the books Generalized Additive Models (with T. Hastie), An Introduction to the Bootstrap (with B. Efron), and Elements of Statistical Learning (with T. Hastie and J. Friedman). His current research focuses on problems in biology and genomics, medicine, and industry. With collaborator Balasubramanian Narasimhan, he also develops software packages for genomics and proteomics.

Jinah Kim, M.D., Ph.D.

Jinah Kim, M.D., Ph.D. is Assistant Professor, Pathology, and Dermatology, Stanford University Medical Center; Medical Director, Dermatopathology Service, Stanford Hospital and Clinics. Dr. Kim is a clinical pathologist in melanoma. She brings to the CCSB her considerable expertise to provide a pathological interpretation of the ABSeq (CODEX) imaging analysis of the melanoma samples. Research: Melanocytic lesions, fibrohistiocytic neoplasms, and genodermatoses.

Andrew Gentles, Ph.D.

Andrew Gentles, Ph.D. is Assistant Professor of Medicine (Biomedical Informatics Research) Dr. Gentles has been part of the Stanford ICBP for the past 8 years serving as Scientific Program Manager for the past 5 years. He lead the CCSB Data Integration and Analytics Core, creating a focal point for sharing data and facilitating collaborations between the different groups in the previous CCSB center and he co-leads the Data Core for the new center. Dr. Gentles developed prognostic signatures based on genomic data for AML, specifically the influence of leukemic stem cells, lung cancer, and large cell lymphoma. His research interests are in systems biology of cancer, integration/analysis of proteomic and genomic data to computationally infer biological insights about molecular pathways, analysis of next-generation sequencing data.

Projects

Project 1: Murine modeling of tumor-mediated immunosuppression

We hypothesize that lymph node (LN) metastasis constitutes an essential step in the metastatic cascade of melanomas and head and neck tumors in that such metastases act locally upon the adaptive immune system within the nodes to induce tolerance to the tumor and that leukocytes recirculating from these nodes carry the tolerance to distant sites. Our objectives are to establish whether LN metastases induce perturbations in anti-tumor immunity and to identify the mechanisms of these perturbations. We will a) characterize differences in local and systemic immune responses to metastatic tumors; b) identify differential regulators of tolerance induction by metastatic cells through the use of genomic profiling; and c) identify the molecular mediators of metastatic tolerance induction in mice and humans. Through the use of serial in vivo passaging, we have developed a panel of syngeneic melanoma cell lines that exhibit enhanced LN metastatic potential. We will compare the activation states of these immune cells, cytokine profiles, T cell polarization, and cytolytic activity toward tumor cells using single cell proteomic methods (Project 2). Using cytokine profiling and RNA sequencing on the lines, we will apply computational systems biology approaches (Project 3) to identify the molecules relevant for induction of tolerance. If our hypothesis is proven correct that LN metastasis is an obligate step in the generation of systemic disease due to tolerance induction, targeting the molecules responsible for LN metastasis induced tolerance could prevent and treat metastatic disease.

Project 2: Spatial architecture of tumor-mediated immunosuppression

Beyond the internal genomic and epigenetic events that occur to drive a cell towards outright carcinogenesis and then metastasis, there co-exist the ordered events a cancer imposes on immune cells it encounters on its progression towards advanced disease. Induction of tolerance, avoidance of apoptosis, and even recruitment of the immune system to aid a tumor’s growth are all poorly understood processes. We propose to undertake deep phenotyping of the 2D and 3D architecture of tumour-lymph node micro-environment—wherein it is expected some of the initial phases of the tumor’s avoidance and recruitment mechanisms are implemented. How is the architecture of the immune environment disrupted in the face of tumor metastasis? Are their micro-communities (as defined by particular cell-cell interactions) whose presence or absence defines an outcome in progression of the tumor? To this end we have developed a technology (ABSeq) that enables us to sensitively and quantitatively image tumors with 60 markers per 3 hours (scalable to 480 in a time-dependent manner) with markers selected from a range of intracellular or surface epitopes (recognized by antibodies) or RNAs. The hypothesis is that an orchestrated corruption of immune surveillance is initiated by cancers as they progress, and that the micro-scale architecture of the lymph node (by way of which cells are talking to whom and what broader effects occur across the lymph node and beyond) is disrupted in a defined manner. A major aim of the research is to, with ABSeq, define the 2D and 3D architecture and communities of immune and cancer cells in draining lymph nodes from 2 cancers (melanoma and head and neck cancer) in murine models and with human samples. Databases of 2D and 3D microenvironments will be publicly created and mined for associations that define the architectural changes that occur as tumors progress and initiate tolerance.

Project 3: Integrative computational modeling of tumor-mediated immunosuppression

We will develop and apply computational tools to integrate the complex datasets generated by our Center in order to identify candidate mediators of tumor-immune interactions that induce immunosuppression for functional validation. To enrich our ability for interpretation, we will explore signatures of the immune system in a pan-cancer analysis using the TCGA datasets annotated with time to distant metastasis, in the context of node-negative and nodepositive patients. We hypothesize that pan-cancer genes whose expression is strongly associated with time to distant metastasis are more likely to be associated with tumor-intrinsic or microenvironmental processes driving metastasis progression, thus we will prioritize these genes in our integrative computational analysis of our melanoma and head and neck squamous cell carcinoma datasets. Using the RNAseq data generated by our study, we will develop and apply novel network-based computational methods for reconstructing the interactions between malignant and immune subpopulations. Moreover, we will develop and apply new approaches to integrate the spatial information from high dimensional single cell in situ images from Project 2 with the gene expression datasets to further refine our inferences of candidate mediators of immunosuppression. The datasets and computational resources developed by our Project, and Center at large, will not only enable use to deeply explore the role of lymph nodes in tumor-mediated immunosuppression, but will also provide the community with powerful resources for understanding systemic influences on the forces governing metastatic dissemination.

Cores

Core 1: Biospecimen and Data Management Core

Our Biospecimen and Data Management Core will be utilized by all the Research Projects in our Center. All three Research Projects aim to identify mechanisms by which tumor cells, metastatic to regional lymph nodes, interface with the host immune system to perturb systemic immunity to induce global immune tolerance. While we will methodically study this process in murine models (Project 1), analysis of human tumor and lymph node specimens is paramount for two primary reasons. The first reason is to ensure the translation of the murine discovery approach to human disease. The second reason is to facilitate the discovery of critical mediators of tumor-induced immunosuppression directly from the human samples, then follow with functional validation in our murine models. Hence it is critical that we establish the necessary infrastructure to procure viable human tumor specimens and autologous immune tumorinfiltrating immune cells. Importantly, matched sets of fresh primary tumor cells, metastatic tumor cells, tumor-infiltrating immune cells (from the primary tumor and the lymph nodes) and circulating immune cells will be collected to address the hypotheses and goals of the Research Projects. Furthermore, in this shared core, a database of clinical annotation (aka clinical metadata) for these matched samples will be created and maintained. This clinical metadata is critical for interpretation of our molecular and imaging data given tumor and patient heterogeneity and will likely provide important insight into how the data generated by the proposed projects may be used to guide treatment and predict outcome. Our proposed Biospecimen and Data Management Core that will focus on collecting tissue and collating all the molecular data on our two index cancer models: melanoma and head and neck squamous cell carcinoma (HNSCC).

Core 2: Outreach Core

Stanford University has a diverse range of faculty in biostatistics, computer science, applied mathematics, cancer biology, immunology, biomedical engineering and many other disciplines. A number of transformative technologies such as DNA microarrays, flow cytometry, single cell transcriptomics, and applications of next generation sequencing had their genesis in labs here. We are therefore in a unique environment for promoting cancer systems biology to a wide audience of researchers, the relevance of whose expertise to the field may not be obvious a priori. We envisage that our efforts to promote our research and those of others to the Stanford community will seed new collaborations across disciplines. We will conduct outreach through regular seminar series and meetings, an annual symposium, funding for pilot projects, as well as participation in the CSBC Postdoctoral Exchange Program that will place researchers at reciprocal institutions to have short-term dense exposure to relevant research objectives and expertise at other institutions. And finally, important collaborative possibilities with the other CSBC Research Centers will be facilitated by their updates provided at annual meetings, as well as in our newsletter. The great value of this new NCI Consortium will be harnessed by ongoing building of an expansive investigator network across universities and research centers.