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Abstract：
OBJECTIVE: Patients with intrahepatic cholangiocarcinoma (iCCA) respond poorly to immune checkpoint blockades (ICBs). In this study, we aimed to dissect the potential mechanisms underlying poor response to ICBs and explore a rational ICB-based combination therapy in iCCA.
METHODS: scRNA-seq dataset GSE151530 was analyzed to investigate the differentially expressed genes in malignant cells following ICBs therapy. RNA-seq analysis and western blot assays were performed to examine the upstream and downstream signaling pathways of CD73. Subcutaneous tumor xenograft models were utilized to investigate the impact of CD73 on iCCA growth. Plasmid AKT/NICD-induced spontaneous murine iCCAs were used to explore the therapeutic efficacy of CD73 enzymatic inhibitor AB680 combined with PD-1 blockade. Time-of-flight mass cytometry (CyTOF) was conducted to identify the tumor-infiltrating immune cell populations and their functional changes in murine iCCAs treated with AB680 in combination with PD-1 antibody.
RESULTS: scRNA-seq analysis identified elevated CD73 expression in malignant cells in response to ICBs therapy. Mechanistically, ICBs therapy upregulated CD73 expression in malignant cells via TNF-α/NF-κB signaling pathway. In vivo studies revealed that CD73 inhibition suppressed the growth of subcutaneous tumors, and achieved synergistic depression effects with gemcitabine and cisplatin (GC). Adenosine produced by CD73 activates AKT/GSK3β/β-catenin signaling axis in iCCA cells. CD73 inhibitor AB680 potentiates anti-tumor efficacy of PD-1 antibody in murine iCCAs. CyTOF analysis showed that AB680 combined with anti-PD-1 therapy promoted the infiltration of CD8+ T, CD4+ T cells, and NK cells in murine iCCAs, while simultaneously decreased the proportions of macrophages and neutrophils. Moreover, AB680 combined with anti-PD-1 significantly upregulated the expression of Granzyme B, Tbet and co-stimulatory molecule ICOS in infiltrating CD8+ T cells.
CONCLUSIONS: CD73 inhibitor AB680 limits tumor progression and potentiates therapeutic efficacy of GC chemotherapy or anti-PD-1 treatment in iCCA. AB680 combined with anti-PD-1 therapy effectively elicits anti-tumor immune response.
METHODS: scRNA-seq dataset GSE151530 was analyzed to investigate the differentially expressed genes in malignant cells following ICBs therapy. RNA-seq analysis and western blot assays were performed to examine the upstream and downstream signaling pathways of CD73. Subcutaneous tumor xenograft models were utilized to investigate the impact of CD73 on iCCA growth. Plasmid AKT/NICD-induced spontaneous murine iCCAs were used to explore the therapeutic efficacy of CD73 enzymatic inhibitor AB680 combined with PD-1 blockade. Time-of-flight mass cytometry (CyTOF) was conducted to identify the tumor-infiltrating immune cell populations and their functional changes in murine iCCAs treated with AB680 in combination with PD-1 antibody.
RESULTS: scRNA-seq analysis identified elevated CD73 expression in malignant cells in response to ICBs therapy. Mechanistically, ICBs therapy upregulated CD73 expression in malignant cells via TNF-α/NF-κB signaling pathway. In vivo studies revealed that CD73 inhibition suppressed the growth of subcutaneous tumors, and achieved synergistic depression effects with gemcitabine and cisplatin (GC). Adenosine produced by CD73 activates AKT/GSK3β/β-catenin signaling axis in iCCA cells. CD73 inhibitor AB680 potentiates anti-tumor efficacy of PD-1 antibody in murine iCCAs. CyTOF analysis showed that AB680 combined with anti-PD-1 therapy promoted the infiltration of CD8+ T, CD4+ T cells, and NK cells in murine iCCAs, while simultaneously decreased the proportions of macrophages and neutrophils. Moreover, AB680 combined with anti-PD-1 significantly upregulated the expression of Granzyme B, Tbet and co-stimulatory molecule ICOS in infiltrating CD8+ T cells.
CONCLUSIONS: CD73 inhibitor AB680 limits tumor progression and potentiates therapeutic efficacy of GC chemotherapy or anti-PD-1 treatment in iCCA. AB680 combined with anti-PD-1 therapy effectively elicits anti-tumor immune response.
摘要：
目的：肝内胆管癌（iCCA）患者对免疫检查点阻断（ICBs）反应较差。在这项研究中,我们旨在剖析iCCA对ICBs反应不佳的潜在机制,并探索合理的基于ICB的联合治疗.
方法：分析了scRNA-seq数据集GSE151530，以研究ICBs治疗后恶性细胞中差异表达的基因。进行RNA-seq分析和蛋白质印迹测定以检查CD73的上游和下游信号传导途径。使用皮下肿瘤异种移植模型来研究CD73对iCCA生长的影响。使用质粒AKT/NICD诱导的自发性鼠iCCAs来探索CD73酶抑制剂AB680与PD-1阻断组合的治疗功效。进行飞行时间质量细胞计数(CyTOF)以鉴定用AB680与PD-1抗体组合处理的鼠iCCAs中的肿瘤浸润性免疫细胞群及其功能变化。
结果：scRNA-seq分析确定了对ICBs治疗反应的恶性细胞中CD73表达升高。机械上,ICBs治疗通过TNF-α/NF-κB信号通路上调恶性细胞CD73表达。体内研究表明，CD73抑制抑制皮下肿瘤的生长，与吉西他滨和顺铂（GC）的协同抑制作用。CD73产生的腺苷激活iCCA细胞中的AKT/GSK3β/β-连环蛋白信号轴。CD73抑制剂AB680增强PD-1抗体在鼠iCCA中的抗肿瘤功效。CyTOF分析显示AB680联合抗PD-1治疗促进CD8+T细胞浸润,CD4+T细胞,和NK细胞在小鼠iCCAs中，同时降低巨噬细胞和中性粒细胞的比例。此外，AB680联合抗PD-1显著上调颗粒酶B的表达,浸润CD8+T细胞中的Tbet和共刺激分子ICOS。
结论：CD73抑制剂AB680限制了肿瘤进展，并增强了GC化疗或抗PD-1治疗在iCCA中的疗效。AB680联合抗PD-1治疗可有效引发抗肿瘤免疫反应。
方法：分析了scRNA-seq数据集GSE151530，以研究ICBs治疗后恶性细胞中差异表达的基因。进行RNA-seq分析和蛋白质印迹测定以检查CD73的上游和下游信号传导途径。使用皮下肿瘤异种移植模型来研究CD73对iCCA生长的影响。使用质粒AKT/NICD诱导的自发性鼠iCCAs来探索CD73酶抑制剂AB680与PD-1阻断组合的治疗功效。进行飞行时间质量细胞计数(CyTOF)以鉴定用AB680与PD-1抗体组合处理的鼠iCCAs中的肿瘤浸润性免疫细胞群及其功能变化。
结果：scRNA-seq分析确定了对ICBs治疗反应的恶性细胞中CD73表达升高。机械上,ICBs治疗通过TNF-α/NF-κB信号通路上调恶性细胞CD73表达。体内研究表明，CD73抑制抑制皮下肿瘤的生长，与吉西他滨和顺铂（GC）的协同抑制作用。CD73产生的腺苷激活iCCA细胞中的AKT/GSK3β/β-连环蛋白信号轴。CD73抑制剂AB680增强PD-1抗体在鼠iCCA中的抗肿瘤功效。CyTOF分析显示AB680联合抗PD-1治疗促进CD8+T细胞浸润,CD4+T细胞,和NK细胞在小鼠iCCAs中，同时降低巨噬细胞和中性粒细胞的比例。此外，AB680联合抗PD-1显著上调颗粒酶B的表达,浸润CD8+T细胞中的Tbet和共刺激分子ICOS。
结论：CD73抑制剂AB680限制了肿瘤进展，并增强了GC化疗或抗PD-1治疗在iCCA中的疗效。AB680联合抗PD-1治疗可有效引发抗肿瘤免疫反应。
