Spondin 1 Enriched in p16^Ink4a Reporter-Labeled Fibroblasts Promotes Melanoma Cell Proliferation in Vitro and Is Associated with Poor Prognosis

Abstract

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 p16^Ink4a in melanoma remain poorly defined. We combined p16-Cre^ERT2-tdTomato lineage tracing with single-cell RNA sequencing in a syngeneic YUMMER1.7-H2B-GFP5 melanoma model to characterize p16^Ink4a-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 p16^INK4A. 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.