Table 1_IFNλ1 is a STING-dependent mediator of DNA damage and induces immune activation in lung cancer.docx

IntroductionThe importance of the cGAS-STING pathway and type I interferon (IFN) in anti-tumor immunity has been widely studied. However, there is limited knowledge about the role of type III IFNs in cancer settings. Type III IFNs, comprising IFNλ1-4, are opposite to type I IFN only expressed by a few cell types, including epithelial cells, and the receptor subunit IFNLR1, is equally only expressed on limited types of cells.
MethodsGene and protein expression of the cGAS-STING signaling pathway was characterized in a series of non-small cell lung cancer (NSCLC) cell lines. Herring-testis DNA stimulation and chemotherapy drugs (doxorubicin and cisplatin) were used to activate the cGAS-STING pathway, and the level of activation was determined by measuring changes in the transcriptomic profile as well as type I and III IFNs by ELISA. Re-expression of IFNLR1 on cancer cell lines was achieved using CRISPR activation (CRISPRa) followed by evaluating chemotherapy-induced apoptosis using flow cytometry assays.
ResultsSTING was not broadly expressed across the NSCLC cell lines. Those cancer cell lines expressing all relevant factors supporting the cGAS-STING pathway secreted IFNλ following STING activation whereas only few of them expressed IFNβ. Treatment with chemotherapy drugs likewise preferentially induced IFNλ, which was abrogated in CRISPR-Cas9 STING knock-out cells. Expression of IFNLR1 was found downregulated in the cancer cell lines compared to the benign epithelial cell line Nuli-1. Rescuing IFNLR1 expression by CRISPRa in multiple cancer cell lines sensitization them to IFNλ-stimulation and resulted in significant reduction in cell viability.
ConclusionDownregulation of IFNLR1 can be an immune evasion mechanism developed by cancer cells to avoid responding to endogenous type III IFNs. Thus, rescuing IFNLR1 expression in NSCLC in conjunction to chemotherapy may potentially be harnessed to elevate the anti-tumoral responses.

【引言】cGAS-STING通路与I型干扰素（type I interferon, IFN）在抗肿瘤免疫中的重要性已得到广泛研究，但目前学界对III型干扰素（type III IFNs）在肿瘤环境中的作用认知仍较为有限。III型干扰素包含IFNλ1-4亚型，与I型干扰素不同，其仅在少数细胞类型（包括上皮细胞）中表达；其受体亚基IFNLR1同样仅在有限的细胞类型中表达。

【方法】本研究在一系列非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞系中对cGAS-STING信号通路的基因与蛋白表达进行了表征。采用青鱼精巢DNA刺激与化疗药物（多柔比星、顺铂）激活cGAS-STING通路，并通过转录组谱变化以及酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）检测I型与III型干扰素的水平，以确定通路激活程度。通过CRISPR激活（CRISPR activation, CRISPRa）技术在癌细胞系中实现IFNLR1的重新表达，随后采用流式细胞术检测化疗诱导的细胞凋亡情况。

【结果】STING在NSCLC细胞系中并非广泛表达。在具备cGAS-STING通路所需全部相关因子的癌细胞系中，STING激活后会分泌IFNλ，但其中仅少数细胞系可表达IFNβ。化疗药物处理同样优先诱导IFNλ的产生，该效应在CRISPR-Cas9介导的STING敲除细胞中被完全阻断。与良性上皮细胞系Nuli-1相比，癌细胞系中IFNLR1的表达显著下调。通过CRISPRa在多种癌细胞系中恢复IFNLR1的表达，可使细胞对IFNλ刺激产生致敏性，并显著降低细胞活力。

【结论】IFNLR1的下调可能是癌细胞为逃避内源性III型干扰素应答而演化出的免疫逃逸机制。因此，在非小细胞肺癌中联合化疗恢复IFNLR1的表达，有望用于增强抗肿瘤免疫应答。