Wnt3a-expressing cancer stem cells impair CD8 ⁺  TRM function, contributing to immunotherapy resistance in Melanoma

Immune checkpoint blockade (ICB) has revolutionized the treatment of advanced malignancies such as melanoma. However, a significant proportion of patients fail to respond, owing to mechanisms that remain poorly understood. Here, we developed murine melanoma cell lines (R1 and R2) derived from MO4 tumors and that are resistant to anti-PD-1 therapy. These resistant cells exhibited increased tumor initiation capacity <i>in vivo</i> correlating with enhanced spheroid formation <i>in vitro</i>, and greater invasiveness and metastatic potential in a lung macrometastasis model. Mechanistically, both R1- and R2- derived tumors showed enrichment of a cancer stem cell (CSC)-like population overexpressing Wnt3a, which correlated with reduced infiltration and impaired function of CD8⁺ tissue-resident memory (TRMs) T cells. This suggested a functional link between Wnt-driven cancer stemness and TRMs dysfunction. Pharmacological inhibition of Wnt/β-catenin signaling <i>in vitro</i> enhanced CD8⁺ T cell proliferation and expression of functional markers. Notably, Wnt/β-catenin inhibition <i>in vivo</i> reversed anti-PD-1 resistance in resistant tumors, coinciding with reduced OCT4⁺Wnt3a⁺ CSC-like cells and restoration of TNFα⁺CD8⁺ TRMs. Finally, transcriptomic analysis of publicly available melanoma cohorts showed that enrichment of the Wnt pathway correlates with poor prognosis and ICB resistance, highlighting this pathway as a potential druggable negative regulator of ICB efficacy in melanoma.