Next Generation Sequencing of tumor tissue (4T1 cell) of BALB/c

Immune checkpoint blockade (ICB), particularly programmed death 1 (PD-1) and its ligand (PD-L1), has shown considerable clinical benefits in patients with various cancers. Many studies show that PD-L1 expression may be biomarkers to help select responders for anti-PD-1 treatment. Therefore, it is necessary to elucidate the molecular mechanisms that control PD-L1 expression. As a potential chemosensitizer and anticancer drug, copper combined with disulfiram (DSF) kills tumor cells by regulating multiple signaling pathways and transcription factors in vitro and in vivo. Here, we showed that DSF increased PD-L1 expression in triple negative breast cancer (TNBC) cells. Through TCGA data analysis, we found that DNMT1 and IRF7 were highly expressed and the hypermethylation states in the IRF7 gene body promoter region in TNBC vs normal breast tissue (NBT). Then, we demonstrated that DSF affected PD-L1 expression via DNMT1-mediated IRF7 hypomethylation in two TNBC cell lines. In vivo experiments, DSF significantly enhanced the response to PD-1 antibody (Ab) in the triple-negative 4T1 BC mouse model. By analyzing the results of the tumor tissue sequencing, the DSF joint anti-PD-1 Ab could be a significant activation of anti-tumor immunity. And the inactive state of immune associated pathways was found to be reversed, such as Th1 and Th2 cell differentiation, antigen processing and presentation, natural killer cell-mediated cytotoxicity and T cell receptor signal transduction. In conclusion, we found that DSF could up-regulate PD-L1 in TNBC cells and elucidated its mechanism. Our findings revealed that the combination of DSF and anti-PD-1 Ab could activate the tumor immune microenvironment (TIME) to show much better antitumor efficacy than monotherapy. Overall design: mRNA profiles of 12 samples (4T1 tumor tissue, control/DSF treatment/anti-PD-1 treatment/combine, 3 replicates per group)

免疫检查点阻断（Immune checkpoint blockade, ICB），尤其是程序性死亡蛋白1（programmed death 1, PD-1）及其配体（PD-L1，programmed death-ligand 1），在多种癌症患者中已展现出可观的临床获益。诸多研究表明，PD-L1表达可作为筛选抗PD-1治疗响应者的生物标志物。因此，阐明调控PD-L1表达的分子机制具有重要意义。
作为潜在的化疗增敏剂与抗癌药物，铜联合双硫仑（disulfiram, DSF）可在体内外通过调控多条信号通路与转录因子杀伤肿瘤细胞。本研究发现，双硫仑可上调三阴性乳腺癌（triple negative breast cancer, TNBC）细胞中的PD-L1表达。通过癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据分析，我们发现相较于正常乳腺组织（normal breast tissue, NBT），三阴性乳腺癌组织中DNA甲基转移酶1（DNA methyltransferase 1, DNMT1）与干扰素调节因子7（interferon regulatory factor 7, IRF7）呈高表达，且IRF7基因体启动子区域存在高甲基化状态。
随后，我们在两株三阴性乳腺癌细胞系中证实，双硫仑通过DNMT1介导的IRF7低甲基化调控PD-L1的表达。体内实验结果显示，在三阴性4T1乳腺癌小鼠模型中，双硫仑可显著增强抗PD-1抗体（anti-PD-1 antibody, Ab）的治疗响应。通过对肿瘤组织测序结果的分析，双硫仑联合抗PD-1抗体可显著激活抗肿瘤免疫；原本处于失活状态的免疫相关通路得以逆转，包括Th1与Th2细胞分化、抗原加工呈递、自然杀伤细胞介导的细胞毒性以及T细胞受体信号转导。
综上，本研究证实双硫仑可在三阴性乳腺癌细胞中上调PD-L1的表达，并阐明了其背后的分子机制。研究结果揭示，双硫仑与抗PD-1抗体联合使用可激活肿瘤免疫微环境（tumor immune microenvironment, TIME），相较于单一疗法展现出更优异的抗肿瘤效果。
整体实验设计：本研究包含12份样本的mRNA表达谱，均取自4T1肿瘤组织，分为对照组、双硫仑处理组、抗PD-1处理组与联合处理组，每组设置3次生物学重复。