WGBS of control and DAC-treated tumor tissue from PDX model

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: Fresh tumor samples from patients identified as MSS were selected for PDX model establishment. At 28 days post-inoculation, we administered DAC 20 µg to each mouse for 5 consecutive days by intraperitoneal injection. The control groups were given the same volumes of saline in the same manner. On the seventh day after the end of the drug treatment, the tumors were collected and conducted extraction of DNA and RNA.

PD-1阻断疗法（PD-1 blockade）在高微卫星不稳定性（microsatellite instability, MSI）结直肠癌中已展现出卓越的临床疗效。然而，针对低微卫星不稳定性或微卫星稳定型肿瘤患者的治疗效果仍有待进一步提升。
本研究证实，低剂量地西他滨（decitabine，DAC）可上调主要组织相容性复合体基因（major histocompatibility complex genes）、细胞因子相关基因（cytokine-related genes）等免疫相关基因的表达，并增加CT26结直肠癌荷瘤小鼠肿瘤部位的淋巴细胞浸润数量。相较于单独使用地西他滨或PD-1阻断疗法的小鼠，联合应用PD-1阻断疗法与地西他滨的CT26小鼠模型中，肿瘤生长抑制效果更为显著，生存期也得以延长。低剂量地西他滨可增强PD-1阻断疗法的抗肿瘤作用。
对经地西他滨处理的CT26细胞以及患者来源的微卫星稳定型肿瘤异种移植（patient tumor-derived xenograft，PDX）模型中的肿瘤样本开展RNA测序（RNA sequencing）与全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing）后发现，地西他滨处理后，包括抗原加工基因与抗原呈递基因在内的众多免疫相关基因表达上调，而启动子去甲基化（promoter demethylation）水平则出现下调。
因此，基于地西他滨的肿瘤微环境重塑策略可改善PD-1阻断疗法的疗效。将地西他滨应用于基于PD-1阻断疗法的免疫治疗，可诱发更强效的免疫应答，从而为低微卫星不稳定性或微卫星稳定型结直肠癌患者带来临床获益。
实验设计：本研究选取经鉴定为微卫星稳定（microsatellite stability, MSS）的患者新鲜肿瘤样本构建PDX模型。接种后第28天，通过腹腔注射向每只小鼠给予20 μg地西他滨，连续给药5天。对照组以相同方式给予等体积生理盐水。给药结束后第7天，收集肿瘤样本并提取DNA与RNA。