FoxP3 demethylation is increased in human colorectal cancer and rat cholangiocarcinoma tissue

• Our novel assay for rat FoxP3 demethylation can be used for detection of Tregs.
• The demethylation assays described could be used with fresh frozen material.
• We found a 3.5-fold increase of FoxP3 promoter demethylation in CRC and ICC.
• The CD3/FoxP3 ratio was significantly lower in CRC than in normal tissue.

ObjectivesFoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC).Design and methodsBisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n = 6 ICC induced by thioacetamide and n = 4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay.ResultsA significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P < 0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n = 11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n = 6) and in tumor tissue (4.14%, n = 6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P < 0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n = 6) in comparison to controls (2.14%, n = 4).ConclusionFoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.