Background: Cancer cell-induced changes in tumor microenvironment (TME) include epigenetic changes of TME cell populations. Since tongue cancer has became a global epidemic with an annual increasing rate of 4%, we investigated epigenetic changes in the TME of tongue cancer and how these changes correlate with TME -cancer-associated fibroblasts (CAFs) and with clinical outcomes.
M&M: Tongue cancer samples (N=50) were immunohistochemically stained with anti-5-methylcytosine (5-MC) to assess global DNA methylation and with anti-α-smooth muscle actin (αSMA) to identify TME-CAFs. Sections were examined for expression of nuclear 5-MC immunostaining in tumor cells and in TME with scores from 0 to 3, 0: no staining, 3: > 25% stained cells. CAF density was scored from 0 to 4: 0 -no stained cells, 4: dense CAFs staining within TME. Cases were classified according to the density of CAFs (CAF-poor = scores 0 and 1, CAF-intermediate = score 2, CAF-rich = score 3) and correlated with 5-MC scores. Both parameters were correlated with clinical outcomes. A preliminary in-vitro study was performed using a human tongue cancer cell line (HSC-3) co-cultured with human gingival fibroblasts (GFs) on collagen gel and cells were assessed for 5-MC expression.
Results: In the TME, 5-MC expression was higher in the CAF-intermediate (p=0.021) and CAF-rich (p=0.0009) than in CAF-poor cases. In the tumor cells, 5-MC was higher in the CAF-rich than in the CAF-poor cases (p=0.0191). Expression of 5-MC in the TME was associated with recurrence (p=0.027) and that of CAFs with recurrence (p=0.004) and survival (p=0.011). In the in-vitro experiments, expression of 5-MC was observed in both HSC-3 cells and GFs.
Conclusion: Epigenetic changes in the TME of tongue cancer are related to CAF density and both parameters are associated with poor prognosis.
