Cellular and Genetic Dynamics of the Melanoma Microenvironment

Abstract

The tumor microenvironment is comprised of a diverse assemblage of cells. These populations dynamically interact with one another in varied ways that modify the individual cells and shape the overall trajectory of the tumor. Here, we examine the ways CD8+ T cells, cancer-associated fibroblasts, and endothelial cells engage with tumors and are reshaped using novel imaging and genetic techniques in an autochthonous, zebrafish model of melanoma. Using in vivo live imaging, we visualized the native T cell response throughout tumor development. We identified crater-like structures on the surface of melanoma tumors wherein CD8+ T cells preferentially accumulate and engage with dendritic cells and melanoma cells. Upon immune stimulation with CpG ODN or TGF-inhibitor, crater-resident CD8+ T cells were found engaging with apoptotic melanoma cells along the crater edge and expressing ifng in tight clusters, respectively. These craters were conserved in human melanoma tumors in perivascular spaces and at the stromal border and similarly harbor accumulations of CD8+ T cells. We then used sequencing-based approaches to assess the transformation of stromal populations in the context of zebrafish melanoma. We identified a cancer-activated fibroblast population that exhibited a distinct transcriptional signature and a unique chromatin landscape. Zebrafish melanoma cancer-activated fibroblasts specifically upregulated a variety of secreted factors, including cxcl8b.1 (IL8). We found that overexpression of cxcl8b.1 in zebrafish melanoma induces a robust change in tumor vascularization indicative of increased angiogenesis and a trend toward CD8- T cell infiltration. Finally, we used genetic lineage tracing to assess clonal dynamics of tumor, endothelial, and stromal cells in zebrafish melanoma. We identified clonal expansion of tumor cells in BRAF- and NRAS-mutant melanoma. While endothelial cells did not exhibit significant changes in clonal dominance or diversity, stromal cells in aged NRAS tumor-bearing fish displayed a trend toward decreasing clone number and dominance of 1-2 clones, suggesting that tumor stroma is selectively shaped by the tumor or surrounding tissue. Together this work identifies novel cellular dynamics between CD8+ T cells, stromal cells, endothelial cells, and tumor cells and highlights ways in which cellular interactions shape the activity of the tumor niche.