目的:抗程序性细胞死亡蛋白1(PD-1)治疗在结直肠癌(CRC)中显示出临床益处。然而,CRC对免疫治疗的应答率低是亟待解决的问题.
方法:在7周龄雄性C57BL/6小鼠的侧腹皮下攻击MC-38肿瘤细胞。将小鼠随机分为3组,每2天腹膜内注射200μg/小鼠抗PD-1抗体和100mg/kgRAS-嗜睡致死3(RSL)或磷酸盐缓冲盐水(PBS)。通过蛋白质印迹法检测氧化应激和铁凋亡相关基因的表达,实时逆转录聚合酶链反应,普鲁士蓝染色,和酶联免疫吸附测定。
结果:抗PD-1治疗无效的肿瘤显示出比反应性肿瘤更强的免疫抑制。值得注意的是,反应性肿瘤显示出更高水平的H2O2和活性氧,两者都可能损害细胞毒性CD8+T细胞的抗肿瘤作用。抗PD-1治疗反应性肿瘤比抗PD-1无反应性肿瘤显示更高的促铁凋亡基因表达和Fe2积累,表明铁凋亡在抗PD-1治疗疗效中的潜在作用。在MC-38同源肿瘤模型中,(1S,3R)-RSL3(RSL),谷胱甘肽过氧化物酶4抑制剂,有效促进抗PD-1治疗的体内抗肿瘤作用。然而,抗PD-1治疗不影响肿瘤模型中铁凋亡相关基因的水平.机械上,RSL处理显著上调增殖(ki67+)和细胞毒性(GZMB+)CD8+T细胞的频率。此外,在RSL+抗PD-1治疗后,肿瘤新抗原特异性干扰素(IFN)-γCD8+T细胞的频率显着增加.
结论:RSL可能是增强抗PD-1治疗CRC的抗肿瘤功效的有希望的药物。
OBJECTIVE: Anti-programmed cell death protein 1 (PD-1) treatment has exhibited clinical benefits in colorectal cancer (CRC). However, the low response rate of CRC to immunotherapy is an urgent problem that needs to be solved.
METHODS: MC-38 tumor cells was challenged subcutaneously in the flank of 7-week-old male C57BL/6 mice. The mice were randomly divided into 3 groups, and 200µg/mouse anti-PD-1 antibody and 100 mg/kg RAS-Seletive Lethal 3 (RSL) or phosphate buffer saline (PBS) were intraperitoneally injected every 2 days. The expression of oxidative stress and ferroptosis-related genes was measured by Western blotting, real-time reverse transcription-polymerase chain reaction, Prussian blue staining, and enzyme-linked immunosorbent assay.
RESULTS: Anti-PD-1 treatment-unresponsive tumors showed stronger immunosuppression than responsive tumors. Notably, the responsive tumors showed higher levels of H2O2 and reactive oxygen species, both of which could impair the antitumor effect of cytotoxic CD8+ T cells. The anti-PD-1 treatment-responsive tumors showed a higher expression of pro-ferroptosis genes and Fe2+ accumulation than those of anti-PD-1 nonresponsive tumors, indicating the potential role of ferroptosis in the efficacy of anti-PD-1 treatment. In MC-38 syngeneic tumor model, (1S, 3R)-RSL3 (RSL), a glutathione peroxidase 4 inhibitor, effectively promoted the antitumor effect of anti-PD-1 treatment in vivo. However, anti-PD-1 treatment did not affect the levels of ferroptosis-related genes in tumor model. Mechanistically, RSL treatment significantly upregulated the frequency of proliferating (ki67+) and cytotoxic (GZMB+) CD8+ T cells. Furthermore, the frequency of tumor neoantigen-specific interferon (IFN)-γ CD8+ T cells showed a significant increase after RSL plus anti-PD-1 treatment.
CONCLUSIONS: RSL may be a promising drug for potentiating the antitumor efficiency of anti-PD-1 treatment in CRC.