Modulation of CCL۵ and CXCR۴ as EMT signaling biomarkers by cold atmospheric plasma and Anti-PD-۱ combination therapy in melanoma: Insights from integrated bioinformatics and in vitro and in vivo validation

Objective(s): 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-۱ 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-۱, and their combination on cell viability and gene expression were evaluated in B۱۶F۱۰ melanoma cells, L۹۲۹ fibroblasts, and a syngeneic mouse melanoma model.Results: WGCNA highlighted CCL۵ and CXCR۴ as hub genes enriched in EMT-related pathways. MTT assays showed that CAP reduced B۱۶F۱۰ cell viability, an effect further enhanced by Anti-PD-۱, while sparing L۹۲۹ fibroblasts. In tumor-bearing mice, combination therapy produced the most pronounced tumor regression and down-regulation of CCL۵ and CXCR۴ compared with single treatments. Minimal viability or expression changes were observed in normal fibroblasts or untreated controls.Conclusion: CAP and Anti-PD-۱ 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 CCL۵ and CXCR۴ 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.

کلیدواژه ها:

Anti-PD-۱ ، C-X-C chemokine receptor - type ۴ (CXCR۴) ، C-C motif chemokine ligand - ۵ (CCL۵) ، Cold atmospheric plasma - (CAP) ، Epithelial-mesenchymal - transition (EMT) ، Melanoma