Department of Molecular Oncology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo, 113-8519, Japan.
Department of Gastrointestinal Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
J Gastroenterol. 2023 Jun;58(6):540-553. doi: 10.1007/s00535-023-01969-w. Epub 2023 Mar 2.
Recent advances in immune checkpoint blockade (ICB) have improved patient prognosis in mismatch repair-deficient and microsatellite instability-high colorectal cancer (dMMR/MSI-H CRC); however, PD-1 blockade has faced a challenge in early progressive disease. We aimed to understand the early event in ICB resistance using an in vivo model.
We subcutaneously transplanted the MC38 colon cancer cells into C57BL/6 mice, intraperitoneally injected anti-PD-1 antibody and then isolated ICB-resistant subclones from the recurrent tumors.
Comparative gene expression analysis discovered seven genes significantly downregulated in the ICB-resistant cells. Tumorigenicity assay of the MC38 cells knocked out each of the seven candidate genes into C57BL/6 mice treated with anti-PD-1 antibody and bioinformatics analysis of the relationship between the expression of the seven candidate genes and the outcome of cancer patients receiving immunotherapy identified Rtp4, an interferon-stimulated gene and a chaperon protein of G protein-coupled receptors, as a gene involved in ICB resistance. Immunohistochemical analysis of transplanted tumor tissues demonstrated that anti-PD-1 antibody failed to recruit T lymphocytes in the Rtp4-KO MC38 cells. Mouse and human RTP4 expression could be silenced via histone H3 lysine 9 (H3K9) trimethylation, and public transcriptome data indicated the high expression level of RTP4 in most but not all of dMMR/MSI-H CRC.
We clarified that RTP4 could be silenced by histone H3K9 methylation as the early event of ICB resistance. RTP4 expression could be a promising biomarker for predicting ICB response, and the combination of epigenetic drugs and immune checkpoint inhibitors might exhibit synergistic effects on dMMR/MSI-H CRC.
免疫检查点阻断（ICB）的最新进展改善了错配修复缺陷和微卫星不稳定高的结直肠癌（dMMR/MSI-H CRC）患者的预后；然而，PD-1 阻断在早期进行性疾病中面临挑战。我们旨在使用体内模型了解 ICB 耐药的早期事件。
我们将 MC38 结肠癌细胞皮下移植到 C57BL/6 小鼠中，腹腔内注射抗 PD-1 抗体，然后从复发性肿瘤中分离 ICB 耐药亚克隆。
比较基因表达分析发现 ICB 耐药细胞中七个基因显著下调。将这七个候选基因中的每一个敲除的 MC38 细胞的致瘤性测定移植到接受抗 PD-1 抗体治疗的 C57BL/6 小鼠中，并对七个候选基因的表达与接受免疫治疗的癌症患者结局之间的关系进行生物信息学分析，确定干扰素刺激基因和 G 蛋白偶联受体伴侣蛋白 Rtp4 是参与 ICB 耐药的基因。移植肿瘤组织的免疫组织化学分析表明，抗 PD-1 抗体未能在 Rtp4-KO MC38 细胞中招募 T 淋巴细胞。可以通过组蛋白 H3 赖氨酸 9（H3K9）三甲基化沉默小鼠和人 RTP4 的表达，并且公共转录组数据表明大多数但不是所有 dMMR/MSI-H CRC 中 RTP4 的高表达水平。
我们阐明了 RTP4 可以通过组蛋白 H3K9 甲基化沉默作为 ICB 耐药的早期事件。RTP4 表达可能是预测 ICB 反应的有前途的生物标志物，并且表观遗传药物和免疫检查点抑制剂的联合可能对 dMMR/MSI-H CRC 表现出协同作用。
