| Objective:As a cancer with extremely high mortality,the effective treatment of lung adenocarcinoma(LUAD)has always been a challenge.Although PD-1/PD-L1 inhibitors are effective drugs for the treatment of solid tumors,they are also helpless for some refractory tumors.A2 a R antagonists,an adenosine inhibitory tumor immune microenvironment regulating drug,are gradually entered people’s field of vision because of their great potential to treat solid tumors.This article attempts to explore the potential function and therapeutic potential of A2 a R in the immune microenvironment of LUAD.Methods:Analyzing the survival significance of A2 a R expression by using network database.Following this,the cellular localization of A2 a R was observed by tissue immunofluorescence staining.The migration level of macrophages in the presence/absence A2 a R antagonist(ZM241385)was detected by Transwell method.Then,the expression levels of A2 a R and the expression of chemokines(CCL2,CCL5)and inflammatory factors(IL6,TGF-β)in five different LUAD cells were detected by RT-q PCR.The expression levels of macrophage surface-associated receptors(CCR2,CCR5,IL-6R and TGF-βR)in different culture conditions were detected by RT-q PCR.Co-transcription factors were screened by TRRUST database analysis,and further verified by cellular immunofluorescence and RT-q PCR.The signaling pathways affected by A2 a R were detected by Western Blotting.The animal experiment was designed to detect the effect of A2 a R antagonist on ectopic implanted lung cancer cell in mice,and it was further verified by immunofluorescence staining on human tissue specimens and RT-q PCR at the cellular level.Results:Bioinformatics analysis suggested that A2 a R was closely associated with the prognosis of LUAD patients.Tissue immunofluorescence staining showed that A2 a R was mainly localized in tumor cells of LUAD,and was positively correlated with the distribution of macrophages(P=0.0067).The results of Transwell experiment showed that the migration of macrophages depended on the high expression of A2 a R,and this chemotaxis was inhibited by A2 a R antagonist(ZM241385).In co-culture/conditioned culture,RT-q PCR showed that LUAD with high expression of A2 a R expressed higher levels of chemokines(CCL2,CCL5)and inflammatory factors(IL6,TGF-β),and macrophages expressed higher levels of CCR2,CCR5,IL-6R and TGF-βR.Through TRRUST database analysis,cellular immunofluorescence and RT-q PCR further screening,NF-κB was confirmed to be the co-transcription factor of the above chemokines and inflammatory factors.Western Blotting results showed that antagonizing A2 a R could down-regulate the protein expression of PI3 K,AKT and NF-κB in LUAD cells.In vivo experiments demonstrated that A2 a R antagonist can effectively reduce the tumor growth of mice and inhibit the transformation of macrophages around the tumor into M2 type.Human tissue immunofluorescence staining also proved that A2 a R was positively correlated with M2 type macrophages.In vitro experiments showed that supernatant of LUAD tumor cells antagonizing A2 a R could reduce the expression of M2 type markers in macrophages and increase the expression of M1 type markers in macrophages.Conclusion:In this study,we found that A2 a R is closely and positively correlated with macrophage in LUAD,and the tumor cells with high A2 a R expression can promote macrophages migration.A2 a R regulates the expression of CCL2,CCL5 and IL6,TGF-β in LUAD tumor cells mainly through PI3K/AKT/NF-κB pathway,and further promotes the migration of macrophage by increasing the expression of CCR2,CCR5,IL-6R and TGF-βR in macrophage.In addition,antagonizing A2 a R can effectively inhibit tumor growth and conversion of macrophages to M2 type in mice.Therefore,the development of immune checkpoints targeting A2 a R is expected to be a new strategy for LUAD treatment.Figure [22] Table [8] Reference [38]... |
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