Title: Investigating Spontaneous Regression in Large Congenital Melanocytic Nevus: A Single-Cell Transcriptomic Study

Abstract Background Large congenital melanocytic nevus (LCMN) is a lifelong and potentially malignant skin melanocytic tumor. Case reports have documented spontaneous hypopigmentation of congenital melanocytic nevi, with histological evidence of immune cell infiltration in the lesions. Objective To explore the mechanism behind the spontaneous regression of LCMN. Methods Using Sanger sequencing and 10x Genomics single-cell RNA sequencing technology, we analyzed the genotype and cell population transformations during the spontaneous regression of LCMN. Results Our findings highlight the pivotal role of IFN-II in regressing LCMN by reversing immune evasion. IFN-II stimulates melanocytes, enhancing their antigen presentation function to recruit cytotoxic immune cells. Endothelial cells and fibroblasts further amplify the inflammatory response and help reconstruct the tumor microenvironment. The regressed LCMN displayed a marked reduction in melanocytes, increased immune cell infiltration, and remodeling of the tumor microenvironment, characterized by angiogenesis and fibroblast activity. Conclusion This study is the first to apply single-cell RNA sequencing technology to LCMN. Our unique sample provides novel insights into the tumor microenvironment of LCMN and its immune mechanisms, offering potential avenues for immunotherapy. Our findings suggest that IFN-II therapy, TIL therapy, and DC immunotherapy could be promising treatment options for LCMN, pending further validation in larger patient cohorts and transgenic animal models.