Study On The Mechanism Of AuNPs In Anti-tumor Growth Based On Autophagy Regulating Phenotypic Polarization Of Tumor-associated Macrophages

【Background and Objective】Macrophages(MΦ)can be polarized to two distinct phenotypes under different conditions,classically activated M1 phenotype and alternatively activated M2phenotype.M1 macrophages are considered as killing phenotype which can secrete inflammatory factor,such as IL-12,iNOS and TNFα,to inhibit tumor growth.While M2 macrophages can secrete anti-inflammatory factors and immunosuppressive factor,such as IL-10,VEGF and TGF-β,to promote tumor angiogenesis and tumor growth,and are considered as healing phenotype.Tumor-associated macrophages(TAMs)originates from bone marrow-derived blood monocytes,which are drived by CCL2,CCL4 and CSF-1.TAMs may represent up 50%-80%of the tumor mass and be a vital part of tumor microenvironment(TEM).Studys found that TAMs showed the M2phenotype in many malignant tumors and promoted the tumor growth,invasion and metastasis.The formation of TAMs is closely related to the TEM,which can drive macrophages polarizing to M2 phenotype by oxygen-deficient environment and expressing of cytokines,immunologic factors.Therefore,studys on the relationship between TAMs and TEM and reprogrammeing the phenotype of TAMs are significant for cancer therapy.With the further understanding of tumor composition and tumor matrix,more and more nanocarriers have been applied in the treatment of targeting TEM.Immune response induced by delivery nanocarriers into tumor matrix may be an effective way to eliminate tumor.Nanocarriers are naturally targeted to macrophages which can easily phagocytize them.Studies have found that certain nanomaterials have regulatory functions on macrophages by themselves.For example,iron oxide nanoparticles inhibit tumor growrh by inducing pro-inflammatory macrophage polarization in tumor tissues;carboxyl-and amino-functionalized polystyrene nanoparticles both can inhibit the expression of CD163 and release of IL-10,which are M2 markers;Glycocalyx-Mimicking nanoparticles improve cancer immunotherapy through reversion of tumor-associated macrophages from M2 to M1.In our previous work,we studied on size dependent localization and penetration of gold nanoparticles in macrophages,and found that gold nanoparticles could affect the secretion of inflammatory cytokines of macrophages.In this study,we systematically studied the regulation of gold nanoparticles on the expression of macrophage phenotype cytokines under TEM condition,for the purpose of investigating the effect of gold nanoparticles on phenotypic polarization of tumor-associated macrophages.Autophagy is a conservative intracellular catabolic process that relies on lysosomes to degrade excess or damaged organelles and proteins to maintain cell homeostasis.Autophagy is an important intracellular metabolic mechanism.Studies have shown that autophagy has a broad regulatory effect on macrophages and may affect the function of TAMs in tumors.Autophagy can regulate the self-renewal and maintenance of HSCs,the differentiation of monocytes into macrophages,and the recruitment of monocytes/macrophages at tumor sites.Therefore,autophagy plays a key role in the production and differentiation of macrophages.Stimulated by different drugs or under various pathological and physiological environment,autophagy may play opposite role on macrophage polarization.Since the phenotype of macrophages is a key event that promotes tumor growth and development,the relationship between autophagy and macrophage phenotype polarization in tumor microenvironment needs to be clarified.At the same time,many studies have confirmed that in a variety of biological models,nanocarriers can interfere with autophagy pathways and induce or block autophagy in cells.Based on the research background above,this study aims to explore the effect of gold nanoparticles on macrophage phenotype polarization and autophagy in tumor microenvironment,further as well as the relationship between autophagy and macrophage phenotype regulation,to clarifiy the anti-tumor mechanism of gold nanoparticles targeting tumor microenvironment.【Methods】1.Preparation,characterization and cytotoxicity of PEG-coated AuNPsPEG-modified gold nanoparticles with size of 20nm were prepared by sodium borohydride reduction and S-Au coordination.The AuNPs were characterized by Malvern laser particle size analyzer,transmission electron microscope and uv-vis spectrophotometer.The cytotoxic effects of AuNPs on RAW264.7 macrophages and HEPA 1-6 mouse hepatoma cells were investigated by cck-8 assay.2.Investigate the effect of AuNPs on the phenotype of tumor-associated macrophages in vitro.(1)Estabilishment of TAMs in vitro by tumor culture supernatant(TSN)The supernatant of HEPA 1-6 cultured for 3 days was collected and centrifuged.TSN medium was prepared by mixing the supernatant with ordinary DMEM medium at a volume ratio of 1:1.5.Macrophages were cultured with TSN medium for 48 h.The number of CD206 positive macrophages was determined by flow cytometry.The secretion of IL-10 by macrophages was detected by ELISA kit.The expression of M2-related factors Arg1 and Mgl1 were detected by Real-time PCR.(2)Effect of AuNPs on macrophages phenotype in TSN cultureMacrophages cultured in TSN were set as control group.Macrophages cultured in TSN medium with high and low concentrations(10 M and 50 M)of AuNPs were set as the experimental group.All of the macrophages were cultured for 48h.The M1 related markers of CD80,IL-12,iNOS and TNFαwere detected,as well as the M2 related markers of CD206,IL-10,Arg1 and Mgl1.3.Investigate the effect of AuNPs on the phenotype of tumor-associated macrophages and tumor growth in vivoA mouse subcutaneous tumor model was established by co-injection of gold nanoparticles and tumor cells.The volume of tumor growth for 20 days was observed and graphed.Mice were sacrificed on day 7 and day 14,and tumors were isolated and identified by immunofluorescence.The macrophages in tumor tissues were isolated and purified by constant density gradient centrifugation and magnetic bead separation.The expression of M2 phenotypic related factors in tumor-associated macrophages was detected by flow cytometry and real-time PCR.4.Effects of gold nanoparticles on autophagy in tumor-associated macrophagesMacrophages cultured in TSN were set as control group.Macrophages cultured in TSN medium with high and low concentrations(10 M and 50 M)of AuNPs were set as the experimental group.Western blot was used to detect the expressions of autophagy related proteins LC3-I/II,p62 and Beclin 1 in macrophages.Macrophages were transfected with autophagy LC3 double-labeled adenovirus(mRFP-GFP-LC3)and were observed under Laser Scanning Confocal Microscope for the changes of autophagy flow flux.Besides,lysosomal function in macrophages were detected by LysoSensor Green DND-189 probe and acridine orange dye(AO).5.The role of autophagy in regulating the phenotype of tumor-associated macrophages(1)Autophagy level changes of macrophages in TSN cultureMacrophages were cultured in normal medium and TSN medium for 48h.Western blot was used to detect the expressions of autophagy related proteins LC3-I/II,p62 and Beclin 1 in macrophages.Macrophages were transfected with autophagy LC3 double-labeled adenovirus(mRFP-GFP-LC3)and were observed under Laser Scanning Confocal Microscope for the changes of autophagy flow flux.(2)The effects of autophagy inhibitors and autophagy inducers on TSN-induced macrophage phenotype polarizationMacrophages were cultured in normal medium and TSN medium respectively and autophagy of macrophages were inhibited by Atg5 siRNA and CQ.The expression of M2-related factors of macrophages was used to detect by Real-time PCR before and after autophagy inhibited.The autophagy activator Rapamycin(Rapa)were added in normal medium-and TSN medium-cultured macrophages.The expression of M2-related factors of macrophages was used to detect by Real-time PCR before and after autophagy activated.【Results】1.The prepared AuNPs had uniform particle size and satisfying dispersion.AuNPs of 50μM or less had no adverse effects on RAW264.7 macrophages and HEPA 1-6 tumor cells,which are meet the requirements for safty.2.The number of CD206 positive macrophages was more in TSN-cultured than normal cultured.The secretion of IL-10 and the expression of Arg1 and Mgl mRNA were both significantly increased in TSN-cultured macrophages.3.The number of CD206 positive macrophages decreased and the expression of IL-10,Arg1 and Mgl1 were inhibited in TSN-cultured macrophages incubated with AuNPs,with concentration-dependent.While the number of CD80 positive macrophages and the expression of IL-12、iNOS and TNFαhad no difference.4.The tumor growth curve of mice injected with gold nanoparticles in vivo slowed down significantly after 20 days.The immunofluorescence results of tumor tissues on day 7 and day 14 showed that the infiltration of CD206 positive macrophages in the tumors of the gold nanoparticle injection group was significantly less than that of the control group.However,with the growth of the tumor,the infiltration of CD206 positive macrophages increased gradually.Tumor-associated macrophages were isolated and purified for flow cytometry detection,and the result showed that CD206~+/F4/80~+macrophages were significantly reduced in the AuNPs injection groups compared with the control group.Moreover,the inhibition effect of high dose AuNPs was more obvious.Real-time PCR results showed that,compared with the control group,the expression of Arg1,Mgl1 and IL-10 in tumor-associated macrophages in the AuNPs injection groups was significantly decreased,especially in the high-dose group.5.Western blot results shown that p62 protein and total LC3-I/II protein of macrophages in the AuNPs groups showed a cumulative increase compared with the control group,while the Beclin 1 protein decreased.Autophagy flux detected by mRFP-GFP-LC3 indicated that autophagy was blocked in AuNPs groups.The result of lysosomal function indicated the pH of macrophage lysosomes in the AuNPs groups was increased and the AO fluorescence intensity was decreased.6.Western blot results shown that total LC3-I/II protein and Beclin 1 protein were more in TSN-cultured macrophages than normal-cultured macrophages,while p62protein was less.Autophagy flux detected by mRFP-GFP-LC3 indicated that autophagy was activated in TSN-cultured macrophages.7.Real-time PCR results showed that,after inhibition of autophagy,the expression of M2-related factors decreased in the TSN-cultured macrophages.And activation of autophagy by Rapamycin made the expression of M2-related factors increasing in the TSN-cultured macrophages.【Conclusions】1.With TSN-cultured medium,macrophages were polarized toward M2 TAMs in vitro.2.AuNPs inhibited the M2 polarization of macrophages in TSN-cultured medium in vitro.3.AuNPs inhibited the M2 phenotype polarization of tumor-associated macrophages in vivo and play an anti-tumor role.4.AuNPs blocked the autophagy flux of macrophages in TSN-cultured medium and then inhibit its autophagy.5.Macrophage autophagy was activated under TSN-cultured condition.6.The autophagy inhibitor antagonized the M2 polarization of TSN-cutured macrophages.While the autophagy inducer promoted the M2 polarization of TSN-cutured macrophages.