Targeting DNA-PKcs promotes anti-tumoral immunity via triggering cytosolic DNA sensing and inducing an inflamed tumor immuno-microenvironment in metastatic triple negative breast cancer

Based on clinical samples from the TCGA database, we analyzed the expression of PRKDC in various types of cancers using Timer online analysis software and compared them with the normal group. In addition we also used Timer to analyze the immune cell infiltration in breast invasive carcinomas with different amplified copy numbers of PRKDC, which provided clinical data to support the results of our subsequent experiments.In cellular experiments, we treated the human-derived triple-negative breast cancer cell line BT549 with the DNA-PKcs inhibitor NU7441 and performed transcriptome sequencing analysis of the drug-treated BT549 cells compared to the untreated group.In addition, we also examined the changes of cytoplasmic nucleic acid sensing pathways in three cell lines, BT549, MDA-MB-231, and CH12F3, after drug addition using MTT, ELISA, Western blot, RNA Extraction and Quantitative PCR and other experimental methods.In animal experiments, we treated normal BALB/c female mice with NU7441 and examined the effect of the drug on immunity by ELISA, flow cytometry and other experimental methods.In addition, we also used mouse breast cancer cell line 4T1 to construct Xenograft mouse model in Female immune competent BALB/c mice and athymic nude BALB/c mice.NU7441 treatment was followed by ELISA, Immunohistochemistry, flow cytometry and other assays to detect the effects of drug treatment on tumor immune cell infiltration and anti-tumor immunity.本研究基于癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库的临床样本，借助Timer在线分析软件（Timer online analysis software），对多种癌症中PRKDC基因的表达水平进行了分析，并与正常对照组进行对比。此外，本研究还利用Timer软件分析了不同PRKDC基因拷贝数扩增型浸润性乳腺癌的免疫细胞浸润情况，为后续实验结果提供了临床数据支撑。在细胞实验部分，我们使用DNA依赖蛋白激酶催化亚基（DNA-PKcs）抑制剂NU7441处理人源三阴性乳腺癌细胞系BT549，并对给药组与未给药组的BT549细胞开展转录组测序分析。此外，我们还通过MTT比色法、酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）、蛋白质免疫印迹（Western blot）、RNA提取以及定量PCR等实验手段，检测了BT549、MDA-MB-231与CH12F3三种细胞系在给药后细胞质核酸感知通路的变化情况。在动物实验部分，我们对正常雌性BALB/c小鼠施以NU7441处理，并通过ELISA、流式细胞术（flow cytometry）等实验手段检测该药物对小鼠免疫功能的影响。此外，我们利用小鼠乳腺癌细胞系4T1，分别在免疫健全雌性BALB/c小鼠与无胸腺裸BALB/c小鼠体内构建异种移植瘤模型。随后通过ELISA、免疫组织化学（Immunohistochemistry）、流式细胞术等实验手段，检测NU7441给药处理对肿瘤免疫细胞浸润及抗肿瘤免疫的影响。