Targeting Melanoma-Associated Fibroblasts to Overcome Cancer Stem Cell-Driven Drug Resistance.

Acquired resistance to BRAF V600E inhibitors remains a major obstacle in melanoma treatment. We hypothesized that melanoma-initiating cells (MICs), regulated by cancer-associated fibroblasts in the tumor microenvironment, mediate drug resistance through stromal Notch1 signaling. Patient-derived melanoma-associated fibroblasts (MAFs) were engineered to activate Notch1 signaling (MAF N1IC-GFP ) via lentiviral transduction. Drug resistance was assessed using BRAF inhibitor-resistant melanoma cells (451LuBR) in 3-dimensional spheroid co-culture assays with PLX4720 treatment. In vivo efficacy was evaluated in NOD scid gamma mice cografted with 451LuBR cells and MAF N1IC-GFP or control MAF GFP (n = 8/group). Primary endpoints included tumor growth, angiogenesis, and CD271⁺ MIC populations assessed by immunofluorescence and quantitative imaging. In vitro, MAF N1IC-GFP suppressed drug-resistant melanoma spheroid formation across PLX4720 concentrations (1 to 100 μM, p < 0.01). In vivo, MAF N1IC-GFP cografting reduced tumor growth by 50% (mean tumor weight, p < 0.01) and bioluminescence signal (p < 0.05). Mechanistically, MAF N1IC-GFP selectively depleted CD271⁺ MICs (~50% reduction in fluorescence intensity, p < 0.01), decreased their proliferation (p < 0.01), and induced apoptosis (p < 0.01). Activating Notch1 signaling in MAFs overcomes BRAF inhibitor resistance by disrupting cancer stem cell niches. This stromal-targeted approach represents a novel therapeutic strategy to complement surgical and systemic therapies in melanoma patients with drug-resistant disease.