A2AR Pathway Mediates CXCL5 Secretion By Tumor Associated Macrophages In Promoting NSCLC Progression

Background and objectivesCancer immunotherapy has achieved great clinical advances in recent years,especially in the application of adoptive cell transfer therapy and immune checkpoint blockade(ICB).Among various of immunotherapies,although,ICB targeting programmed cell death 1/programmed cell death ligand 1(PD1/PDL1)axis is the most famous and performed well in clinical trials,most of the patients who received the therapy could not be benefit from the therapy because of the primary resistance.Previous researches have dedicated to exploring mechanisms of antitumor immunity and tried to improve the effect of the therapy.Researchers have found that the primary resistance is largely owing to the immunosuppressive atmosphere in the tumor microenvironment.The incidence of lung cancer ranks high in China.Among them,non-small cell lung cancer(NSCLC)is the most common type.Surgery and chemotherapy showed limited effects in the treatment of advance NSCLC.Recently,PD1 inhibitors have achieved certain effects in clinical applications and approved for the first-line treatment of NSCLC.However,primary resistance is still a difficult problem to solve.Due to primary resistance,the overall response rate of anti-PD1/PDL1 therapy is only 20%.A large number of patients did not respond to anti-PD1/PDL1 treatment,and some patients finally developed acquired resistance after initial response to the treatment.The underlying mechanism behind this phenomenon still needs further research.Studies have shown that in patients who were resistant to PD1/PDL1 therapy,the presence of tumor associated macrophages(TAMs)makes it difficult to improve the immunosuppressive microenvironment in the tumor,even when blocking PD1/PDL1 axis,the anti-tumor activity of T cells is still difficult to recover and may exhausted again.Macrophages can be divided into two types:anti-tumor M1 type and pro-tumor M2 type.TAMS are generally considered to be M2 type.TAMs are abundant in tumors which can interact with tumor cells to promote the formation of immunosuppressive microenvironment and tumor progression.Cytokines derived from TAMs,on the one hand,upregulate the expression of tumor epithelial mesenchymal transition(EMT)related genes and help maintain the sternness of tumor cells;on the other hand,they have influences on other immune cells to promote the formation of tumor immunosuppressive microenvironment(TIME).Therapies targeting TAMs are expected to reprogram TIME,enhance the efficacy of anti-PD1/PDL1 therapy,and abolish drug resistance.Many studies have been devoted to exploring targets on TAMs,but the effects are still limited.The interaction between TAMs and tumor cells,as well as its correlation with anti-PD1/PDL1 therapies need to be further explored.The potential critical targets may be discovered on TAMs,which will help to develop more effective treatment approaches and strengthen the power of existing immunotherapies.This research explored the interaction between tumor cells and TAMs through the co-incubation model of NSCLC cells and monocyte-derived macrophages.In our previous study,we found that the CXCL5 in the supernatant that generated from coculturing system of NSCLC cells and macrophages was significantly increased.Subsequently,we verified this phenomenon in clinical specimens,and studied the mechanisms and effects of CXCL5 upregulation.Adenosine can stimulate the activation of A2AR on macrophages to promote the expression of immunosuppressive cytokines,which consequently promote the formation of TIME.We found that A2AR was activated on macrophages and regulated the exppression of CXCL5 through NFκB mediated pathway.Finally,we initially discussed whether CXCL5 had an effect on PD1/PDL1 axis.In summary,we used the co-incubation model to simulate the interaction between tumor cells and TAMs,explore the mechanisms of CXCL5 upregulation,and the effect of CXCL5 in NSCLC.We aimed to find potential therapeutic targets which could reprogram the immunosuppressive microenvironment,and provide a theoretical basis for improving the effect of existing immunotherapies.Materials and methods1.Transwell chambers was used to establish an in vitro co-incubation model to simulate the cross-talk of tumor cells and macrophages in tumor microenvironment.2.ELISA was used to detect the concentration of CXCL5 in the supernatant of coincubation system,tumor cells and macrophages.3.RT-PCR and immunofluorescence were used to detect the expression of CXCL5 in both mRNA and protein levels.4.Expression of CD 163,CXCL5 and CD39 on macrophages,CD73 on tumor cells,and functions of T cells was analyzed by flow cytometry.5.Transcriptome sequencing was conducted to screen the changes in signaling pathways on tumor cells and macrophages after co-incubation in vitro.6.Western blot was conducted to detect changes of signaling pathway.7.Dual luciferase reporter gene assay was performed to confirm the transcription factor of CXCL5.8.Chemotaxis experiment was conducted to verify the chemotaxis effect of CXCL5 on neutrophil.9.Neutrophil extracellular traps(NETs)was observed by fluorescence microscope after fluorescence staining.Results1.CXCL5 in the culture supernatant increased when NSCLC were incubated with M2 macrophages for 24h.2.TAMs expressed high level of CXCL5 in both the co-incubation system and the TME of NSCLC patients.Macrophage abundance was positively associated with CXCL5 expression.3.The adenosine receptor A2AR in macrophages,which was upregulated after coculture,regulated the expression of CXCL5 in macrophages.The transcription factor NF-κB mediated the regulation of CXCL5.4.CXCL5 upregulated the expression of N-cadherin and PDL1 in tumor cells.5.The chemotaxis of neutrophil driven by CXCL5 was inhibited by CXCL5 antibody.6.CXCL5 induced the formation of NETs to suppress T cell function.7.The level of CXCL5 expression was correlated with poor prognosis.Immune subtypes with poor prognosis expressed high level of CXCL5.Conclusion1.TAMs express high level of CXCL5 in NSCLC,tumor cells promote the secretion of CXCL5 on TAMs through activating the adenosine receptor A2AR pathway in NF-κB dependent manner.2.CXCL5 upregulates the expression of N-cadherin and PDL1 in NSCLC cells,and induce neutrophils to produce NETs which can inhibit the function of CD8+T cells and promote their exhaustion.3.Advanced NSCLC express high level of CXCL5 which is related to the poor prognosis and immune infiltration classification.