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Background  The influence of natural regulatory T cells (nTregs) on the patients with colon cancer is unclear. Demethylated status of the Treg-specific demethylated region (TSDR) of the  FOXP3  gene was reported to be a potential biomarker for the identification of nTregs.    Methods  The demethylation rate of the TSDR (TSDR-DMR) was calculated by using methylation-specific quantitative polymerase chain reaction (MS-qPCR) assay. The expression of TSDR-DMR and  FOXP3  mRNA was investigated in various colorectal cancer cell lines. A total of 130 colon carcinoma samples were utilized to study the DMR at tumor sites (DMR T ) and adjacent normal tissue (DMR N ). The correlations between DMRs and clinicopathological variables of patients with colon cancer were studied.    Results  The TSDR-DMRs varied dramatically among nTregs (97.920 ± 0.466%) and iTregs (3.917 ± 0.750%). Significantly, DMR T  (3.296 ± 0.213%) was higher than DMR N  (1.605 ± 0.146%) (n = 130,  p  = 0.000). Higher DMR N  levels were found in female patients ( p  = 0.001) and those with distant metastases ( p  = 0.017), and were also associated with worse recurrence-free survival in non-stage IV patients (low vs. high,  p  = 0.022). However, further Cox multivariate analysis revealed that the  FOXP3 -TSDR status does not have prognostic value.    Conclusion  MS-qPCR assays of  FOXP3 -TSDR can efficiently distinguish nTregs from non-nTregs. Abnormal recruitment of nTregs occurs in the local tumor microenvironment. Infiltration of tissue-resident nTregs may have a negative role in anti-tumor effects in patients with colon cancer; however, this role is limited and complicated.

Higher FOXP3-TSDR demethylation rates in adjacent normal tissues in patients with colon cancer were associated with worse survival