微卫星稳定（MSS）结直肠癌对免疫检查点抑制剂缺乏敏感性，增敏剂的探索将使更多结直肠癌患者获益于PD-1抗体治疗。我们前期研究发现：IFNγ对MSS结直肠癌PD-1抗体治疗具有增敏作用，其机制与STAT3介导下肿瘤细胞PD-L1表达、趋化因子CXCL9相关CD8+T淋巴细胞募集有关。本项目在前期研究的基础上拟开展：利用CRISPR/Cas9技术构建STAT3敲除MSS细胞（MSS-KO），了解IFNγ作用下MSS-KO细胞PD-L1、CXCL9及JAK-STAT分子的表达。建立MSS-KO皮下成瘤动物模型，观察外源性IFNγ对PD-1抗体治疗效果的影响；检测瘤体组织E-cadherin+/CD8+细胞数量、凋亡及PD-L1、CXCL9/CXCR3的表达，并评价瘤体组织免疫微环境。本研究将有助于明确IFNγ通过STAT3-PD-L1/CXCL9对微卫星稳定结直肠癌PD-1抗体治疗的增敏作用。

Patients with microsatellite stable (MSS) colorectal cancer lack sensitivity to immune checkpoint inhibitors. Exploration of sensitizers will enable more colorectal cancer patients to benefit from PD-1 antibody therapy. Our early experiments found that IFNγ as a sensitizer promoted the effect of PD-1 antibody treatment in MSS colorectal cancer, and the mechanism was related to the expression of PD-L1 on tumor cells mediated by STAT3 and CD8+ T Lymphocyte recruitment involved with chemokine CXCL9. This project is planned to construct STAT3 knockout MSS cells (MSS-KO) using CRISPR/Cas9 technology. The expression of PD-L1, CXCL9 and JAK-STAT signals in MSS-KO cells with treatment of IFNγ are further analyzed. We establish animal models challenged with MSS-KO subcutaneously injection and observe the effect of STAT3 on the responsiveness of anti-PD-1 immunotherapy in MSS colorectal cancer. We further detect the number of E-cadherin+/CD8+ cells as well as PD-L1 and CXCL9/CXCR3 in tumor tissues. The immune microenvironment of tumor tissue is also evaluated. The purpose of this study is to clarify the involved STAT3-PD-L1/CXCL9 mechanism of that IFNγ promotes the responsiveness of anti-PD-1 immunotherapy in MSS colorectal cancer.