DataSheet_1_Integrin-Linked Kinase Expression Characterizes the Immunosuppressive Tumor Microenvironment in Colorectal Cancer and Regulates PD-L1 Expression and Immune Cell Cytotoxicity.docx

Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon. However, a role for ILK in the tumor microenvironment (TME) and immune evasion has not been investigated. Here, we show a correlation of ILK expression with the immunosuppressive TME and cancer prognosis. We also uncover a role for ILK in the regulation of programmed death-ligand 1 (PD-L1) expression and immune cell cytotoxicity. Interrogation of web-based data-mining platforms, showed upregulation of ILK expression in tumors and adjacent-non tumor tissue of colorectal cancer (CRC) associated with poor survival and advanced stages. ILK expression was correlated with cancer-associated fibroblast (CAFs) and immunosuppressive cell infiltration including regulatory T cells (Treg) and M2 macrophages (M2) in addition to their gene markers. ILK expression was also significantly correlated with the expression of different cytokines and chemokines. ILK expression showed pronounced association with different important immune checkpoints including PD-L1. Deletion of the ILK gene in PD-L1 positive CRC cell lines using a doxycycline inducible-CRISPR/Cas9, resulted in suppression of both the basal and IFNγ-induced PD-L1 expression via downregulating NF-κB p65. This subsequently sensitized the CRC cells to NK92 immune cell cytotoxicity. These findings suggest that ILK can be used as a biomarker for prognosis and immune cell infiltration in colon cancer. Moreover, ILK could provide a therapeutic target to prevent immune evasion mediated by the expression of PD-L1.

整合素连接激酶（Integrin-linked kinase, ILK）已被证实为结肠炎症与肿瘤发生的分子驱动因子及介导因子。然而，ILK在肿瘤微环境（Tumor microenvironment, TME）及免疫逃逸中的作用尚未得到研究。本研究揭示了ILK表达与免疫抑制性肿瘤微环境及癌症预后的相关性，同时阐明了ILK在调控程序性死亡受体配体1（PD-L1）表达及免疫细胞细胞毒性中的作用。通过对网络数据挖掘平台的分析，本研究发现结直肠癌（Colorectal cancer, CRC）患者的肿瘤组织及癌旁非肿瘤组织中ILK表达上调，且该现象与不良生存结局及疾病晚期分期相关；ILK表达与癌相关成纤维细胞（Cancer-associated fibroblast, CAFs）、包括调节性T细胞（Regulatory T cells, Treg）和M2型巨噬细胞在内的免疫抑制细胞浸润及其基因标志物表达均存在相关性，同时还与多种细胞因子及趋化因子的表达显著相关，且与包括PD-L1在内的多种关键免疫检查点均存在显著关联。采用强力霉素诱导型CRISPR/Cas9系统，在PD-L1阳性的结直肠癌细胞系中敲除ILK基因，可通过下调核因子κB p65（NF-κB p65）的表达，抑制基础状态及干扰素γ（IFN-γ）诱导的PD-L1表达，进而使结直肠癌细胞对NK92免疫细胞的细胞毒性敏感性升高。上述研究结果表明，ILK可作为结肠癌预后评估及免疫细胞浸润情况的生物标志物；此外，ILK还有望成为靶向阻断PD-L1表达介导的免疫逃逸的治疗靶点。