Spondin 1 Enriched in p16Ink4a Reporter-Labeled Fibroblasts Promotes Melanoma Cell Proliferation in Vitro and Is Associated with Poor Prognosis

The tumor microenvironment contains phenotypically diverse stromal cells that can support tumor progression; however, the identity and function of stromal populations expressing the senescence marker p16Ink4a in melanoma remain poorly defined. We combined p16-CreERT2-tdTomato lineage tracing with single-cell RNA sequencing in a syngeneic YUMMER1.7-H2B-GFP5 melanoma model to characterize p16Ink4a-lineage-positive [p16-Lin(+)] stromal cells and identify candidate mediators of tumor-stroma interactions. Immunofluorescence detected p16-Lin(+) cells across multiple stromal populations, while transcriptomic analysis identified fibroblasts as a major p16-Lin(+) stromal population. The integration of differentially expressed genes in p16-Lin(+) fibroblasts with a secretome database and survival data from The Cancer Genome Atlas Skin Cutaneous Melanoma cohort identified spondin 1 (SPON1) as a candidate extracellular mediator. Spon1 was preferentially expressed in fibroblasts, upregulated in the p16-Lin(+) fraction, and nearly undetectable in YUMMER1.7-H2B-GFP5 cells. Immobilized recombinant SPON1 promoted YUMM1.7 cell proliferation in a dose-dependent manner in vitro. In an independent human skin tumor single-cell dataset, SPON1 expression was enriched in fibroblasts, with higher enrichment observed in those expressing CDKN2A, which encodes p16INK4A. Moreover, high SPON1 expression was independently associated with shorter overall survival after adjusting for tumor type, age, and sex. Together, these findings identify SPON1 as a candidate factor enriched in p16-Lin(+) fibroblasts, with potential functional and prognostic relevance in melanoma.