Tumor derived exosomal ENTPD2 impair CD8 T cell function in colon cancer through ATP-adenosine metabolism reprogramming.

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.

Conclusion: 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.