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 We identified 10 major cell types and 6 distinct tumor subpopulations. The C3 ID4 + tumor cell (TC) subpopulation was markedly enriched in Stage III tumors and showed a high-risk phenotype. It was characterized by enhanced proliferation, oxidative phosphorylation, stemness, and impaired immune activation. This subpopulation also expressed high levels of ID4, SPP1, and POSTN. Pseudotime analysis placed C3 ID4 + TCs at a poorly differentiated state along the developmental trajectory. Cell-cell communication analysis revealed extensive crosstalk between C3 ID4 + TCs and C0 GMPR + 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 Our integrative single-cell analysis identifies C3 ID4 + 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.