Targeting Neuropilin‐1 to Enhance Immunotherapy in Melanoma: Reducing Peripheral Treg‐Mediated Immunosuppression and Tumour Progression

Melanoma, a highly aggressive skin cancer, ranks fifth among Caucasians and has a high mutation rate. Despite advances in immunotherapy, over 40% of patients face immune-related side effects. Neuropilin-1 (NRP1), which is overexpressed in various cancers, enhances the function of Treg cells and promotes tumour growth, thereby contributing to poor patient outcomes. This study investigated the effects of NRP1 inhibition in B16-F10 melanoma, with a focus on its impact on immune responses regulated by peripheral T regulatory cells (Tregs). NRP1 was found to be overexpressed in the majority of cancer patient samples and cancer cell lines, including melanoma, as indicated by cBioPortal and the human protein atlas database analysis, establishing a positive correlation between NRP1 and various cancers. mRNA studies revealed elevated NRP1 expression in tumorigenic B16-F10 cells compared with non-tumorigenic NIH-3T3 cells. Inhibition of NRP1 induced apoptosis in B16-F10 cells without affecting NIH-3T3 cells. It also reversed the immunosuppression caused by B16-F10-cell culture supernatant (B16-F10-CS), reducing the Treg population (NRP1, NKG2A, and FOXP3), Treg suppressive activity and the secretion of immunomodulatory cytokines (IL-10 and IL-17A) while increasing T cell proliferation and the secretion of effector cytokines (TNF, IFN-γ, IL-6, and IL-2). Additionally, NRP1 inhibition suppressed the STAT, ERK MAPK, and Smad2/3 pathways and activated the PI3K/AKT pathway in Tregs cultured in B16-F10-CS in vitro. In tumour-bearing mice, NRP1 inhibition decreased Treg and Th2 population marker expression and enhanced T cell proliferation and Th1 population marker expression in peripheral Tregs. It also reduced lung metastasis, decreased tumour size, and improved survival. These findings suggest that targeting NRP1 could slow melanoma progression and reduce peripheral Treg-mediated immunosuppression, making it a promising approach for future cancer immunotherapies, especially in combination with other treatments.