Exploring the adenosine system by cancer-associated fibroblasts in the context of radiotherapy

Abstract

Background of the study: Cancer-associated fibroblasts (CAFs) are a heterogeneous population of cells that are mainly found in solid malignancies and play vital roles in tumor progression and metastasis, including important immunoregulatory functions. In this study, our objective was to explore the immunosuppressive adenosine system within the CAFs isolated from non-small cell lung carcinomas (NSCLC) and to examine the impact of radiation on the CD73/adenosine system in CAFs. Methods: The study was conducted using primary cultures of CAFs isolated from freshly resected NSCLC samples (n=5), as well as Peripheral Blood Mononuclear Cells (PBMCs) isolated from randomly selected healthy volunteers. The effect of different radiation doses (1x6 Gy and 1x18 Gy) on the expression of ectonucleotidases CD73, CD39, and CD38 by CAFs was assessed through various techniques, including flow cytometry, western blotting, and ELISA using both irradiated and non-irradiated CAFs and CAF-conditioned medium (CAF-CM). An adenosine conversion assay was performed to examine cell-associated and extracellular CD73 activity in CAFs lysates and supernatants. Additionally, the immunosuppressive effect of CAF-CM on activated T-cells was investigated by blockade of CAF-derived soluble CD73 with AB630 inhibitor. Results: Our findings indicate that CAFs may play a more important role in adenosine (ADO)-mediated immunosuppression compared to tumor cells. Following exposure to ionizing radiation (HD-IR), CAFs displayed enhanced CD73 expression, although the changes measured on the surface and in whole-cell expression were not statistically significant. After exposure to ionizing radiation, the surface expression of CD39 and CD38 was enhanced in CAFs, while the total cellular expression of CD39 did not show significant changes and CD38 expression remained undetected. On the other hand, HD-IR was found to have a significant effect on the upregulation of sCD73. Furthermore, the contribution of CAF to ADO generation through sCD73 was increased in cultured supernatants exposed to HD-IR. Soluble factors derived from CAFs exerted strong immune inhibitory effects on Phytohaemagglutinin-L (PHA-L) stimulated T-cells, leading to significant inhibition of their proliferation function (P<0.001). Notably, this inhibition was partially restored by targeting CD73 in CAF-CM. Conclusion: This study demonstrated that CAFs are major contributors to adenosine generation by the CD39/CD73 system in vitro and that the expression of ectoenzymes is enhanced upon exposure to radiation, especially after high radiation doses. Importantly, HD-IR triggered the release of sCD73 and the concomitant elevation of adenosine production in the extracellular space. This study also demonstrates that CAF-mediated immunosuppressive effects over activated T-cells can be partially counteracted by blocking ADO-conversion.

Keywords: Cancer-associated fibroblasts, CAFs, Ionizing radiation, Radiotherapy, Tumor microenvironment, TME, CD73, Adenosine