Research About Antitumor Efficacy And Mechanism Of Combining Irradiation And A2AR Antagonism In Murine Models

Background Immune system is close required to ensure the efficacy of anti-tumor therapy.However,tumor cells can avoid recognition and destruction of the immune system in a variety of ways.Anti-PD-1/PD-L1 antibody is a representative agent of immunotherapy and considerable achievements have been achieved.However,the response rate of immunotherapy is less than 30%.A large number of patients still cannot benefit from immune treatment.On the other hand,immune system is complicated.Relative co-stimulating or co-inhibiting regulation immuno-target are continually understood in recent years.Adenosine is a hypoxia-driven cell metabolite which has an immunosuppressive effect in tumor environment.Adenosine has been found to be able to bind to a variety of receptors,including A1,A2A,A2B and A3.A2A receptor is major receptor associated with immunosuppression.A2A receptor can be expressed on tumor cells,endothelial cells,macrophages,NK cells and T lymphocytes.After binding to A2A receptor,adenosine can inhibit immune function,promote the proliferation as well as differentiation of immunosuppressive cells(such as Treg,MDSCs,M2,etc.),thus affect the prognosis of patients.Adenosine is accumulated in tumor environments.Accumulated adenosine significantly inhibits immune function.A2A receptor antagonism is a new anti-tumor therapy strategy.So far,related studies are few and all of them are in early stages.Radiotherapy has attracted much attention in the field of immunotherapy due to its unique immune effect.Therefore,this study was designed to explore the antitumor effect and its mechanism of combination treatment with A2A receptor antagonism and radiotherapy.Methods:For in vitro assays,Intracellular phosphorylation of CREB levels was used to analyze the activation and blocking effect of adenosine A2A receptor.HPLC method was used to determine the concentration of adenosine in the supernatant of cell culture medium or dialysate of tumor tissue before and after radiotherapy.For in vivo assays,mouse colon cancer model MC38 and mouse triple-negative breast cancer model 4T1 were used in this study.A2A receptor antagonism or radiotherapy monotherapy as well as combination therapy were used and tumor volumes were measured every 2-3 days.CD4+,CD8+,Foxp3+T lymphocytes in collected tumor samples was measured by immunohistochem ical and the proportion of selected subgroup was analyzed.Expression of overall IFN-? in tumor samples was determined by in situ hybridization assay.Results:Brand A2AR antagonism DZD2269 can inhibit CREB phosphorylation induced by the adenosine analogue NEC A(1 ?m)and relieve adenosine-mediated immunosuppression effect in a dose-dependent way.Radiotherapy can cause a rapid increase in adenosine concentration in the tumor environment,and the peak value of adenosine concentration measured in this study was up to 600nM.Comparing to monotherapy,in murine models,combination treatment with radiotherapy can significantly inhibit tumor growth and improve anti-tumor efficacy.A-single-5Gy radiotherapy can cause increased infiltration of various lymphocyte subtypes in the tumor environment,including CD4+,CD8+and Foxp3+subsets.Combining therapy can improve the tumor immune microenvironment after radiotherapy,including reduction of infiltration of Tregs and enhance the expression of IFN-y in the tumor environment.Conclusion:This study is the first preclinical study to investigate the synergistic anticancer effects of radiotherapy combined with adenosine A2A receptor antagonism.The results of this study indicated that adenosine A2A receptor antagonism DZD2269 monotherapy could inhibit the growth rate of tumor in mouse model and further improve the anti-tumor efficacy after radiotherapy.Compared with radiotherapy alone,adenosine A2A receptor blocker can improve the tumor microenvironment after radiotherapy,reduce Treg infiltration,and increase the overall interferon expression.Moreover,the experimental mice showed good tolerance to the treatment without obvious adverse reactions.The results showed that radiotherapy combined with adenosine A2A receptor antagonism was safe and effective in mouse tumor-bearing model and could be used as a new anti-tumor treatment strategy in the future.