RNA sequencing of control and DAC-treated CT26 cells.

PD-1 blockade has demonstrated impressive clinical outcomes in colorectal cancers that have high microsatellite instability. However, the therapeutic efficacy for patients with tumors with low microsatellite instability or stable microsatellites needs further improvement. Here, we have demonstrated that low-dose decitabine could increase the expression of immune-related genes such as major histocompatibility complex genes and cytokine-related genes as well as the number of lymphocytes at the tumor site in CT26 colorectal cancer-bearing mice. A more significant inhibition of tumor growth and a prolongation of survival were observed in the CT26 mouse model after treatment with a combination of PD-1 blockade and decitabine than in mice treated with decitabine or PD-1 blockade alone. The anti-tumor effect of the PD-1 blockade was enhanced by low-dose decitabine. The results of RNA sequencing and whole-genome bisulfite sequencing of decitabine-treated CT26 cells and tumor samples with microsatellite stability from the patient tumor-derived xenograft model have shown that many immune-related genes, including antigen processing and antigen-presenting genes, were upregulated, whereas the promoter demethylation was downregulated after decitabine exposure. Therefore, decitabine-based tumor microenvironment re-modulation could improve the effect of the PD-1 blockade. The application of decitabine in PD-1 blockade-based immunotherapy may elicit more potent immune responses, which can provide clinical benefits to the colorectal cancer patients with low microsatellite instability or stable microsatellites. Overall design: CT26 cells were seeded at a density of 1 × 105 cells/ml in a six_x0002_well culture plate. After 24 h, the medium was replaced with complete medium containing 1 µM DAC every 24 h for 3 days.After the third DAC treatment, the medium was replaced with fresh complete medium without DAC, and the cells were cultured for an additional 24 h and then used for subsequent experiments.Control groups were treated synchronously under the same conditions except for the absence of DAC.