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Abstract：
BACKGROUND: Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell-derived ATPases in the development and progression of colon cancer.
METHODS: Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues.
RESULTS: We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells.
CONCLUSIONS: Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.
METHODS: Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues.
RESULTS: We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells.
CONCLUSIONS: Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.
摘要：
背景：细胞外ATP-AMP-腺苷代谢在调节肿瘤免疫应答中起关键作用。以前的研究表明，ATP向AMP的转化主要由三磷酸三磷酸二磷酸水解酶1（ENTPD1/CD39）催化，一种被广泛研究的ATP酶,在肿瘤相关免疫细胞中表达。然而,来自肿瘤细胞本身的ATP酶的功能仍然知之甚少。目的探讨结肠癌细胞源性ATPases在结肠癌发生发展中的作用。
方法：进行生物信息学和组织微阵列分析以研究ATPase家族成员在结肠癌中的表达。ATP水解试验,高效液相色谱(HPLC),CCK8和集落形成试验用于确定ENTPD2对结肠癌细胞生物学功能的影响。流式细胞术和RNA-seq分析用于探索CD8+T细胞的功能。采用免疫电镜和免疫印迹法评价ENTPD2在外泌体中的表达。采用双标记免疫荧光法和免疫印迹法检测血清外泌体和结肠癌组织中ENTPD2的表达。
结果：我们发现ENTPD2，而不是众所周知的ATPaseCD39，在癌细胞中高表达，并且与结肠癌患者的不良预后显著正相关。ENTPD2在癌细胞中的过表达通过抑制CD8+T细胞的功能增强免疫活性小鼠的肿瘤进展。此外，ENTPD2主要位于外泌体内。一方面,外泌体ENTPD2降低细胞外ATP水平，从而抑制P2X7R介导的NFATc1核转录;另一方面,它促进ATP转化为腺苷的增加，因此促进腺苷-A2AR途径活性。在结肠癌患者中，外泌体ENTPD2的血清水平与晚期TNM分期和高肿瘤浸润深度呈正相关。此外，结肠癌患者血清外泌体中ENTPD2水平与配对结肠癌组织中ENTPD2表达水平呈正相关,血清外泌体和组织中的ENTPD2水平与肿瘤浸润性CD8+T细胞中的ENTPD2表达水平显著负相关。
结论：我们的研究表明，外泌体ENTPD2起源于结肠癌细胞，通过促进ATP-腺苷代谢有助于免疫抑制微环境。这些发现强调了外泌体衍生的水解酶作为塑造肿瘤免疫微环境的独立实体的重要性。
方法：进行生物信息学和组织微阵列分析以研究ATPase家族成员在结肠癌中的表达。ATP水解试验,高效液相色谱(HPLC),CCK8和集落形成试验用于确定ENTPD2对结肠癌细胞生物学功能的影响。流式细胞术和RNA-seq分析用于探索CD8+T细胞的功能。采用免疫电镜和免疫印迹法评价ENTPD2在外泌体中的表达。采用双标记免疫荧光法和免疫印迹法检测血清外泌体和结肠癌组织中ENTPD2的表达。
结果：我们发现ENTPD2，而不是众所周知的ATPaseCD39，在癌细胞中高表达，并且与结肠癌患者的不良预后显著正相关。ENTPD2在癌细胞中的过表达通过抑制CD8+T细胞的功能增强免疫活性小鼠的肿瘤进展。此外，ENTPD2主要位于外泌体内。一方面,外泌体ENTPD2降低细胞外ATP水平，从而抑制P2X7R介导的NFATc1核转录;另一方面,它促进ATP转化为腺苷的增加，因此促进腺苷-A2AR途径活性。在结肠癌患者中，外泌体ENTPD2的血清水平与晚期TNM分期和高肿瘤浸润深度呈正相关。此外，结肠癌患者血清外泌体中ENTPD2水平与配对结肠癌组织中ENTPD2表达水平呈正相关,血清外泌体和组织中的ENTPD2水平与肿瘤浸润性CD8+T细胞中的ENTPD2表达水平显著负相关。
结论：我们的研究表明，外泌体ENTPD2起源于结肠癌细胞，通过促进ATP-腺苷代谢有助于免疫抑制微环境。这些发现强调了外泌体衍生的水解酶作为塑造肿瘤免疫微环境的独立实体的重要性。
