Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP–ADO pathway amplified by HCC–macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP–adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP–adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC.

中文翻译：

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通过肿瘤 - 巨噬细胞相互作用放大空间分离的腺苷途径诱导肝细胞癌中的抗 PD1 抗性

免疫检查点阻断抵抗缩小了癌症免疫疗法的疗效，但潜在的机制仍然难以捉摸。描述抗 PD1 耐药的内在机制对于改善晚期 HCC 患者的预后非常重要。通过 RNA 测序在接受抗 PD1 治疗的 HCC 患者中测量 cricTMEM181 的水平，然后通过 qPCR 和 Sanger 测序确认。通过流式细胞术和 IHC 评估 HCC 患者或小鼠模型的肿瘤微环境中的免疫状态。通过超速离心分离来自 HCC 细胞系的外泌体，并通过免疫荧光证实它们被巨噬细胞内化。SILAC、RNA FISH 和 RNA 免疫沉淀证实了 HCC 衍生的外泌体 circTMEM181 作用于巨噬细胞的潜在机制。通过 ATP、AMP 和 ADO 测量以及巨噬细胞特异性 CD39 敲除小鼠评估了由 HCC-巨噬细胞相互作用放大的 ATP-ADO 途径。在我们的回顾性 HCC 队列中，还确定了 circTMEM181 在抗 PD1 治疗中的作用及其临床意义。在这里，我们发现 circTMEM181 在对抗 PD1 治疗反应不佳的肝细胞癌 (HCC) 患者和术后预后不良的 HCC 患者中升高。此外，我们还发现高外泌体 circTMEM181 有利于免疫抑制微环境，并赋予 HCC 抗 PD1 抗性。机制上，外泌体 circTMEM181 吸收 miR-488-3p 并上调巨噬细胞中 CD39 的表达。使用巨噬细胞特异性 CD39 敲除小鼠和药理学方法，我们揭示了一种新的 HCC 抗 PD1 耐药模式。我们发现巨噬细胞中的细胞特异性 CD39 表达和 HCC 细胞中的 CD73 表达协同激活 eATP-腺苷途径并产生更多的腺苷，从而损害 CD8+ T 细胞功能并驱动抗 PD1 抗性。总之，HCC 衍生的外泌体 circTMEM181 通过提高 CD39 表达来促进免疫抑制和抗 PD1 抗性，通过靶向巨噬细胞上的 CD39 抑制 ATP-腺苷途径可以挽救 HCC 中的抗 PD1 治疗抗性。