Transcutaneous carbon dioxide application suppresses the expression of cancer-associated fibroblasts markers in oral squamous cell carcinoma xenograft mouse model

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Cancer-associated fibroblasts (CAFs) are the main stromal cells in the tumor microenvironment (TME). As CAFs promote tumor progression and hypoxia in the TME, regulating the conversion of normal fibroblasts (NFs) into CAFs is essential for improving the prognosis of patients with OSCC. We have previously reported the antitumor effects of transcutaneous carbon dioxide (CO(2)) application in OSCC. However, the effects of reducing hypoxia in the TME remain unclear. In this study, we investigated whether CO(2) administration improves the TME by evaluating CAFs marker expression. Human OSCC cells (HSC-3) and normal human dermal fibroblasts (NHDF) were coinjected subcutaneously into the dorsal region of mice. CO(2) gas was applied twice a week for 3 weeks. The tumors were harvested six times after transcutaneous CO(2) application. The expression of CAFs markers (α-SMA, FAP, PDPN, and TGF-β) were evaluated by using real-time polymerase chain reaction and immunohistochemical staining. The expression of α-SMA, FAP, PDPN, and TGF-β was significantly increased over time after co-injection. In the CO(2)-treated group, tumor growth was significantly suppressed after treatment initiation. In addition, the mRNA expression of these markers was significantly inhibited. Furthermore, immunohistochemical staining revealed a significant decrease in the protein expression of all CAFs markers in the CO(2)-treated group. We confirmed that transcutaneous CO(2) application suppressed CAFs marker expression and tumor growth in OSCC xenograft mouse model.