Influences Of Autophagic Regulations Of Tomor Associated Macrophages On The Radiosensitivity Of Gastric Cancer And Their Potential Mechanisms

Purposes: We aimed to investigate the possibility and the potential mechanisms of influencing radiosensitivity of gastric cancer by regulating the autophagic status of the cocultured Tumor associated Macrophages(TAMs), and to investigate the possibility of improving the therapeutic effects of radiotherapy on gastric cancer through regulation of autophagy in TAMs by observing the influences of the autophagically regulated TAMs on the radiosensitivity of transplanted tumor.Methods: 1)Via alternatively active pathway, Human mononuclear cells THP-1 were sequential treated with PMA、recombinant Human IL-4, after the M2 type tumor associated macrophage were obtained, both autophagic inducer(Rapamycin and Bafiloycin A1) acting on M2 type TAMs were employed. The flow cytometry were used to identify the biomarker(CD68、CD204、CD206)on the surface of M2. The expression of autophagy was monitored through monodansylcadaverin(MDC)staining and immunefluorescence about MAP1 LC3. Lyso-Tracker Red and the Mito-Traker Green staining were used to detect the expression of mitochondria and lysosome, transmission electron microscope were used to observe cell morphology of tumor associated macrophages in each group with the regulation of autophagy. 2)Using non-contact cocultivation, autophagy upreglation group M2 tumor associated macrophages, the blank group, autophagy downregulation group M 2 tumor associated macrophages and human gastric cancer MGC803 cells were co-cultured 48 h. We detected the the radiation reaction expression of gastric cancer MGC803 cells after irradiation with the treatment of autophagy regulator, MDC staining, Lyso-Tracker Red and Mito-Traker Green staining were used to detect the expression of mitochondria and lysosome, immunofluorescence of autophagic marker MAP1 LC3 and transmission electron microscope were used to observe cell morphology of MGC803 cells in each group with the regulation of autophagy. Western Blot were used to detect cathepasin B、D、E、L and caspase 3、7 protein expression in gastric cancer cells of each group respectively.3)Rapamycin pretreated TAMs, Bafilomycin A1 pretreated TAMs and untreated TAMs were coinjected with gastric cancer cells MGC803 with a ratio of 3:1 into the right flank of nude mice. Cancer cells alone were injected without TAMs as control. The formation of tumor mass, weight and sizes of tumor, pathological findings and protein expressions such as COX-2, PDL1, PTEN and TNF-αwere compared after exposure to 8Gy X-ray irradiation.Results: The human mononuclear cells THP-1 were treated with PMA(200nM)/ PMA(200nM)+IL-4(10ng/ml)for 72 h, after the treatment, the expression of CD68/ CD 204/CD206 on surface of the two kind of adherent cells were detected by flow cytometry, which were higher than the THP-1 and have significant difference between themselves(P<0.05). The immune fluorescence expressions of MAP1 LC3 and the detection of autophagic vacuoles in M2-TAMs showed that the expression of MAP1 LC3 and autophagic vacuoles were remarkable. Combining with the results of MDC experiments, Lyso-Tracker Red and Mito-Traker Green double Staining, after the pretreatment of autophagy regulator in M2-TAMs, and the autophagy state would maintain sustainable for a period of time, so it can be used as a trained research reagents. 2)After a certain dose(4gy)irradiation, MGC803 cells with the treatment of different autophagy regulator in M2-TAMs.It was shown that autophagy upregulation group, the number of clone forming increased, and autophagy downregulation group decreased in clone forming experiments.In radiation sensitivity, the autophagy of M2-TAMs promoted MGC803 cell clone formation rate, inhibit the apoptosis of MGC803 cells when autophagy of M2-TAMs upregulated. By contrast, the influence of the inhibition of the M2-TAMs co-culture system in autophagy is expected to improve the radiation sensitivity of gastric cancer cells. Induction of autophagy in TAMs upregulated the expression of cathepsin B、D、E and downregulated the expression caspase-3、7 and cathepsin L in irradiated MGC803. Inhibition of autophagy in TAMs showed the opposite results in MGC803. 3)Mice in the upregulation of autophagy in TAMs(NC)group had the largest volume of tumor, whereas autophagically up-regulated group had the strongest expression of COX-2、PD-L1 and TNF-α. The autophagic down-regulattion group had the lowest expression of COX-2, PD-L1 and TNF-αand yielded the smallest tumor.Conclusions:Autophagic down-regulation in TAMs can increase the radiosensitivity of cocultured gastric cancer cells and up-regulation led to radioresistance. TAMs are potently tumorigenic when coinoculated with cancer cells regardless of their autophagic status. The autophagy of TAMs may be a potential target for radiosensitization in cancer therapy and autophagic inhibition of TAMs may improve the radiosensitivity of cancer, which merits further study.