Project 2: Contribution of the Stromal Microenvironment to Early Dissemination

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PROJECT 2 – SUMMARY Metastasis initiates early in melanoma, and coincides with phenotypic and functional changes in tumor cells, surrounding stromal cells, and extracellular matrix components. Amongst the stromal cell types, cancer associated fibroblasts (CAFs) are emerging as a particularly relevant cell population. Although they are genetically stable, CAFs are phenotypically and functionally diverse and distinct subsets of CAFs were found to support or inhibit cancerous growth in pancreatic cancer models. Although these findings suggest functionally diverse sets of CAFs could also exist in other cancers, they have yet to be defined in most other cancer types. Furthermore, because specific CAFs might be valuable prognostic markers or therapeutic targets, it is becoming increasingly important to better understand their development and regulation. Here, we propose to investigate how signals between melanoma cells and fibroblasts create heterogeneity within tumors and how this heterogeneity unleashes metastatic behaviors in a subset of melanoma cells. To test this hypothesis, we developed genetically engineered congenic mouse models that allow us to genetically manipulate melanocyte stem cells to initiate tumors that faithfully recapitulate the pathology of human melanoma and to independently inhibit specific genes in melanoma cells or CAFs. These models will uniquely enable us to investigate how specific signaling mechanisms influence CAF functions in melanoma development and metastasis. We propose to couple our mouse models with single-cell RNA sequencing, spatial transcriptomics, ATAC-seq, and ChIP-seq approaches to transcriptionally define distinctive CAF states. We will also use them along with melanoma specimens from patients with and without metastatic progression to map specific CAFs and their proximity to other cell types with highly multiplexed immuno-histochemistry (Aim 1). Next, we will use these tools and data to discover cell state specific receptor-ligand interactions and study how IGF1-IGF1R signaling between melanoma cells and CAFs –one model pathway our preliminary studies identified – influences intratumor heterogeneity and metastatic progression in vivo (Aim 2). Project 2 will leverage the pathological, technological, and analytical resources of Cores B and C. It will synergize with Project 1 to determine whether and how melanoma cells interact with distinctive CAFs and how these interactions allow for metastatic dissemination. Project 2 will also enhance Project 3 by shedding new light onto CAF-secreted cytokines that modulate tumor draining lymph nodes and make immune cells tolerant to metastasizing melanoma cells. Once we understand these processes on a molecular level and link them to clinical characteristics, we can begin to inform patient selection for adjuvant therapy in early-stage melanoma and catalyze a rational development of targeted therapies that prevent or treat metastatic dissemination in early- stage melanoma and other cancer types.