The Role and Mechanism of Upregulation of HO-1 Expression By Activating of Adenosine-2a Receptor in the Tumor Immune Microenvironment with Diffuse Large B-Cell Lymphoma

Objective: In the previous study, we found a high proportion expression of HO-1 and Foxp3 in tumor tissues of patients with DLBCL result in a poor prognosis, and there is a positive correlation between HO-1 and Foxp3. Activation of A2aR not only increases the number of Treg cells but also directly enhances their immunosuppression to tumor cells. on the other hand, A2aR on the surface of tumor cells was also induced, considering a positive correlation between HO-1 and Foxp3, it is hypothesized that activation of A2aR in tumor cells results in upregulated HO-1 expression. And HO-1 play a crucial role in the maintenance and survival of tumor cells in the tumor immune microenvironment, so this study aimed to prove that adenosine activates the A2aR receptor of tumor cells to upregulate the expression of HO-1. Further investigating the regulation and mechanism of the interaction between A2aR and HO-1, elucidating the role of A2aR receptor activation in the immune microenvironment of patients with DLBCL. Methods: Multi-color immunohistochemical staining (mIHC) was performed on paraffin sections of clinical cases with DLBCL to make a quantitative analysis the expression of biomarkers of the immune microenvironment, K-M survival curve and Log-Rank test were used for statistical analysis. Oci-ly10 and Oci-ly19 cell lines were used as the DLBCL cell model, were treated with A2aR receptor agonist, adenosine and NECA. NECA-pretreated cells were treated by anticancer drugs Cisplatin and Vincristine (VCR). Western blot, CCK8 test and FACS were performed on measure the expression of HO-1, cell viability and apoptosis, respectively. NOD-SCID mouse bearing Oci-ly10 cells were established in to investigate the effect of induced HO-1 by A2aR activation on tumor growth inhibition and drug-resistance in vivo. Results: The results of multicolor immunohistochemistry showed that lower expression of HO-1, Foxp3 and PD-L1 and higher expression of CD8 obtained a higher overall survival(p<0.05). There was no significant difference in the OS with CD4 and CD68 (p>0.05). There was a positive correlation between the HO-1 and Foxp3 cells among the tumor microenvironment markers (p<0.05). Western blotting results showed that both NECA and adenosine induced the upregulation expression of HO-1 through the activation of A2aR receptors in Oci-ly10 and Oci-ly19 cell lines in a concentration- and time-dependent manner (p<0.05). 0.5μmol of the inhibitor preladenant inhibited the activation of HO-1 expression mediated by A2aR receptor activation (p<0.05). The knockdown A2aR expression by si-A2aR resulted in the above induction. siRNA rescue experiments showed that the cells regained the ability of NECA to induce HO-1 upregulation by reset the A2aR expression. Pretreatment with NECA reduced the sensitivity of cells to cisplatin or VCR, and the concentration of IC50 increased significantly (p<0.05). However, the above changes were not significant after preladenant-pretreatment. FACS results showed that NECA significantly increased anti-apoptotic ability of cisplatin in Oci-ly10 cells, but preladenant-pretreatment blocked the enhancement of anti-apoptotic effect (p<0.05). When HO-1 was knockdown, NECA activation of A2aR receptor could not induce HO-1 up-regulation, and the apoptosis rate was not significantly different from that of cisplatin-treated group (p>0.05) NECA further phosphorylated p38 MAPK through activation of A2aR. Downstream transcription factors regulate the induction of HO-1 expression. The results showed that the tumor growth of cisplatin and preladenant combination group was the most significantly inhibited (p<0.05), while cisplatin had limited effects in inhibiting tumor growth. In addition, tumor growth inhibition was significantly reduced (p<0.05) by NECA-pretreatment. The positive rates of HO-1 and Foxp3 cells in the cisplatin plus NECA group were significantly higher than those in the cisplatin group after NECA stimulation of the A2aR receptor (p<0.05). In contrast, after the inhibition of A2aR receptor activation, the positive rate of HO-1 cells in the cisplatin plus preladenant group was lower than that in the cisplatin group (p<0.05). Conclusion: It is essential for the maintenance and survival of tumor cells in the tumor immune microenvironment. Upregulation of HO-1 by A2aR receptor activation plays an important role in the immune microenvironment of DLBCL tumors.