Single-cell landscape of melanoma reveals ETV5-driven C3 ID4+ tumor subpopulation with extracellular vesicle-associated immunosuppressive and pro-metastatic potential

Background: Malignant melanoma is characterized by marked intratumoral heterogeneity and an immunosuppressive tumor microenvironment (TME), and resistance to immunotherapy remains common. We hypothesized that melanoma contains a poorly differentiated tumor subpopulation characterized by an ETV5-centered transcriptional program and TGF-β-associated intercellular crosstalk, which may contribute to malignant progression and immune evasion. EV-related signaling was explored as a potential, but unvalidated, component of this phenotype. Methods: We performed integrative single-cell transcriptomic analyses of single-cell RNA sequencing (scRNA-seq) data from 10 Stage I/III melanoma specimens to deconvolute the tumor microenvironment and tumor heterogeneity. Analyses included clustering, differential expression, stemness prediction (CytoTRACE), pseudotime trajectory reconstruction, cell-cell communication (CellChat), and transcriptional regulatory network inference (SCENIC). Functional validation was performed using CRISPR/Cas9-mediated ETV5 knockdown, followed by migration, proliferation, apoptosis, and RT-qPCR assays. Results: TCs through the TGFB2-(TGFBR1+TGFBR2) signaling axis. ETV5 was further identified as a key transcriptional regulator in this subpopulation. Functional validation showed that ETV5 knockdown reduced melanoma-cell migration and proliferation and promoted apoptosis. Conclusion: TCs as a critical driver of melanoma progression, associated with metabolic reprogramming, immune evasion, and TGF-β-related intercellular crosstalk. We further demonstrate that the transcriptional regulator ETV5 is functionally required to maintain this aggressive phenotype. These findings nominate ETV5 and TGF-β-associated signaling as candidate therapeutic vulnerabilities in melanoma enriched for this high-risk subpopulation.