Modulation of CCL5 and CXCR4 as EMT signaling biomarkers by cold atmospheric plasma and Anti-PD-1 combination therapy in melanoma: Insights from integrated bioinformatics and in vitro and in vivo validation.

OBJECTIVES: Cold atmospheric plasma (CAP) has emerged as a promising non-thermal modality with anticancer effects. Combining CAP with immune checkpoint blockade (ICB) may enhance therapeutic efficacy, yet the molecular targets underlying this synergy remain incompletely understood. MATERIALS AND METHODS: Epithelial--mesenchymal transition (EMT)--associated genes responsive to CAP and Anti-PD-1 therapy were identified by integrating bioinformatics analyses of melanoma transcriptomic data with in vitro and in vivo experiments. Weighted gene co-expression network analysis (WGCNA), GO, and KEGG enrichment identified key modules and candidate genes. The effects of CAP, Anti-PD-1, and their combination on cell viability and gene expression were evaluated in B16F10 melanoma cells, L929 fibroblasts, and a syngeneic mouse melanoma model. RESULTS: WGCNA highlighted CCL5 and CXCR4 as hub genes enriched in EMT-related pathways. MTT assays showed that CAP reduced B16F10 cell viability, an effect further enhanced by Anti-PD-1, while sparing L929 fibroblasts. In tumor-bearing mice, combination therapy produced the most pronounced tumor regression and down-regulation of CCL5 and CXCR4 compared with single treatments. Minimal viability or expression changes were observed in normal fibroblasts or untreated controls. CONCLUSION: CAP and Anti-PD-1 combination therapy effectively suppressed melanoma cell viability and modulated EMT-associated gene expression both in vitro and in vivo. We further explored a potential molecular mechanism underlying this therapeutic effect, revealing that the EMT-related genes CCL5 and CXCR4 play a vital role in this response. These findings highlight the relevance of these pathways and support the potential of combining CAP with ICB as a promising approach for melanoma treatment.