Characterizing Immune Signaling in the Melanoma Microenvironment

Abstract

Melanoma is the deadliest form of skin cancer, arising from malignant transformation and proliferation of melanocytes. Dysregulation of immune signaling pathways, including IFN, Wnt, and TGFβ, is common in melanoma and contributes to immune suppression within the tumor microenvironment (TME). To define the temporal and spatial dynamics of pathway activation during melanomagenesis, I leveraged high-resolution confocal microscopy in a zebrafish melanoma model optimized for live imaging, integrating signaling pathway reporters and cell-type-specific reporters to resolve pathway activity at single-cell resolution. Using a Wnt-inducible reporter (WIE:EGFP), I identified a Wnt-signaled tumoral macrophage (TAM) population that localizes to crevices adjacent to scale edges within tumors. Transcriptomic and proteomic analyses revealed that Wnt-signaled TAMs upregulate lipid metabolism, ferroptosis resistance, and anti-inflammatory signatures. Genetic activation of Wnt signaling in TAMs promotes melanoma onset, likely through the maintenance of localized Wnt signaling hubs that suppress infiltrating T cells. Additional imaging with an in vivo TGFβ-inducible reporter (TIE:EGFP) revealed the formation of TGFβ signaling hubs in which anti-inflammatory TAMs are either endogenously TGFβ- signaled or phagocytose TGFβ-signaled tumor cells via glycan-mediated clearance. In parallel with these macrophage-centric studies, a systematic analysis of global immune signaling patterns across melanoma genotypes uncovered distinct pathway activation states associated with common driver mutations. In vivo imaging of an IFN-inducible reporter (isg15:EGFP) demonstrated that PTEN-mutant tumors exhibit reduced IFN signaling compared to p53-mutant melanomas, YAP1 overexpression induces regionalized IFN activation, and NRAS-mutant tumors display elevated TGFβ and Wnt signaling. Together, this work reveals spatially organized immunosuppressive macrophage populations that contribute to localized immune regulation within the melanoma TME and identifies genotype-specific patterns of immune signaling dysregulation. These findings provide insight into how IFN, Wnt, and TGFβ pathways may be strategically targeted in combination with immune checkpoint blockade therapies in the future.