ABSTRACT The melanoma niche facilitates growth, differentiation, and drug resistance of cancer cells. Although many of the cell types, including immune, endothelial, and stromal cells, have been characterized, the interactions of the many signals that directly regulates these niche cells have not been evaluated in vivo. The niche cells also could expand or alter their differentiation in response to the tumor cells or extracellular signals, and such changes could trigger clonal expansion. We have utilized the zebrafish to study melanoma. Recently we developed zebrafish reporters for the signaling pathways TGF β , Wnt, and Type I Interferons (IFN). Surprisingly, we see certain regions of tumors that express the reporters and the Wnt and IFN reporters co-localize. Using a new system to image tumors continuously for up to 24 hours, we noted small indentations of the surface of melanomas that we termed “craters”. These regions contain CD8+ T cells that are both activated and exhausted with dendritic cells. These immune hubs function as a conduit for T cells to enter tumors. We have found craters also in human melanoma samples. In zebrafish melanomas, we specifically found Wnt positive macrophages localized to craters. We plan to probe the function of the Wnt positive macrophages and to examine the role of Wnt in the craters. Macrophages also phagocytose TGFβ-signaled and some Wnt-signaling melanoma cells. We plan to probe the “eat-me” signals that are involved. We recently have examined clonality using a variety of cellular barcoding approaches in zebrafish and human tumors. In the zebrafish, we have a Brainbow approach that uses colors to examine clonality and a CRISPR based DNA barcode system called GESTALT. Using single cell analysis, we found that the cancer associated fibroblasts secrete high IL-8 levels. Clonal analyses of tumors overexpressing IL-8 revealed that IL8 mediates endothelial cell clonal expansion which is associated with a decrease of CD8+T cell infiltration. We have used mitochondrial barcoding to examine single cell clonality, gene expression and chromatin accessibility in human melanomas. We plan to compare zebrafish and human tumors, examining spatial transcriptomics and multiplex imaging, coupled with clonality. The clonality of the blood and immune cells in melanoma will also be assessed, independent of the TCR sequencing. Our studies should shed light on how multiple signals in cancer niche intersect and function and will relate these effects to clonal growth. This work with have impact on the choice of primary therapies or when drug resistance occurs.