Dihydroartemisinin Inhibits Lewis Lung Carcinoma Progression By Inducing Macrophages M1 Polarization Via AKT/mTOR Pathway

Objective: Preclinical and clinical data show a close relationship between high infiltration of tumor-associated macrophages(TAMs)and a poor prognosis in most types of tumors,thus targeting TAMs stands out as promising anticancer immunotherapies.Recent studies have demonstrated the anti-tumor effects of artemisinin via enhancing anti-tumor immunity within tumor microenvironment,but the underlying mechanism is still not clear.This study mainly explores the role and mechanism of dihydro artemisinin(DHA)in anti-tumor immune response and regulation of TAM polarization in Lewis lung carcinoma model mice.Methods: We established LLC tumor models in C57BL/6.During the experiment,body weights of Control group and DHA group mice were monitored;then tumor tissues were disassembled into single cells to evaluate the infiltration and polarization of macrophages by flow cytometry(FCM)analysis.In vitro studies,RAW264.7 and PMs was stimulated under M1 stimulation condition(LPS + IFN-γ).Then,M1 phenotype-related molecules CD86、i NOS and COX-2,production of M1-related inflammatory cytokines IL-6,TNF-α,MCP-1 and IL-1β were detected by flow cytometry,WB,ELISA,RT-PCR assasy.In addition,we utilized flow cytometry and fluorescence microscope imaging to count the fluorescence-labelled latex beads internalized by RAW264.7.Peritoneal macrophages(PMs)from C57BL/6mice,was induced by IL-4 + IL-13.Then,M2 phenotype-related molecules CD206,CD86,Arg-1 and M2-related inflammatory cytokines IL-10 were detected by flow cytometry,WB,RT-PCR.For mechanism research,RAW264.7 and PMs were induced with DHA under M1 and M2 stimulation conditions respectively and phosphorylation level of AKT and mTOR were detected by western blot assay.Then,Rapamycin was used in RAW264.7 cells which have been induced under M1 stimuli plus DHA,and the phosphorylation of mTOR and the effector molecules expression were assessed.Results: We found that DHA inhibited Lewis Lung carcinoma progress,moderately decreased the frequencies of TAMs within tumor stroma,and significantly increased CD86 expression while decreased CD206 expression on TAMs which indicates the role of DHA in polarizing TAMs into a M1-like phenotype.Then,our in vitro data confirmed that DHA dose-dependently promoted macrophage M1 phenotype transition by increasing M1 phenotype-related molecules,meanwhile decreasing the expression of M2 phenotype-related molecules and reprogram M2 macrophages into M1 macrophages.In addition,DHA increased proinflammatory cytokine production,enhanced the phagocytic capacity while decreased antiinflammatory cytokine production.Finally,in order to prove that AKT/mTOR signaling potentially mediated DHA-induced macrophage differentiation,we used rapamycin to specifically block the activity of mTOR and stimulated macrophages under M1 stimuli.Our data clearly showed that rapamycin significantly decreased DHA-induced M1-related phenotypes and proinflammatory cytokine expression.Conclusions: We uncovered an important role of dihydroartemisinin(DHA)in regulating intratumoral TAM polarization and anti-tumor immune responses in mouse Lewis Lung carcinoma model.In summary,our study highlighted DHA as one of future potential therapeutic options for the development of novel anticancer immunotherapies in lung cancer.