Effect of Indirect Co-Culture With Gingival Mesenchymal Stem Cells on Cytokine Secretion in Primary Oral Squamous Cell Carcinoma Cells

Background:This study evaluated whether indirect paracrine exposure to gingival mesenchymal stem cells (GMSCs) alters inflammatory cytokine signaling in low-passage primary epithelial-enriched cells derived from histopathologically confirmed oral squamous cell carcinoma (OSCC) tissue. Material/Methods:Two experimental models were used: (1) treatment with 50% and 100% GMSC-conditioned medium (GMSC-CM), and (2) indirect Transwell co-culture with live GMSCs (0.4-μm pore size).Primary OSCC cultures were established from 3 independent histopathologically confirmed OSCC donor specimens and used at passage 1. GMSCs were isolated from healthy gingival tissue from 3 donors, expanded in culture, immunophenotypically verified, and used at passage 4. Each biological experiment was performed in triplicate.Outcomes assessed were cytokine secretion determined by cytokine bead array (CBA), and inflammatory and gene expression assessed by RT-qPCR, and we performed phase-contrast morphology computational/statistical analysis. Results:GMSCs demonstrated a fibroblast-like spindle morphology with positive CD73/CD90 and negative CD34/CD45 expression, consistent with MSC identity.OSCC Control released high levels of pro-inflammatory cytokines, including IL-6, IL-8, MCP-1/CCL2, and TNF-a.RT-qPCR showed increased IL10 and CXCL10 and decreased TNF, CCL2, and IL6 following indirect GMSC exposure, consistent with altered inflammatory cytokine signaling in vitro.Spearman analyses showed strong associations between transcript levels and corresponding secreted cytokines. Conclusions:Indirect GMSC signaling (Transwell co-culture or conditioned medium) attenuated pro-inflammatory cytokine secretion (IL-6, TNF-a, MCP-1/CCL2) and increased IL-10 and CXCL10/IP-10 expression in primary OSCC cells, indicating reshaping of inflammatory cytokine signaling in vitro.Functional tumor assays and immune-cell-inclusive models are needed to determine downstream effects on OSCC behavior and immune interactions.