Autophagy Inhibition Induces The Repolarisation Of Tumour-associated Macrophages And Enhances Chemosensitivity Of Laryngeal Cancer Cells To Cisplatin

Research background:Tumor-associated macrophages(TAMs)are one of the major cellular components and key regulators in the tumor microenvironment.They are involved in tumor growth,angiogenesis and lymphangiogenesis,tumor invasion,metastasis and immunosuppression of many cancers,including laryngeal cancer.In the last decade,TAMs were found to contribute to the chemotherapy resistance.Chemotherapeutic agents such as paclitaxel and platinum disrupt the mitotic process and induce apoptosis of the cancerous cells.The death of the tumor cells would stimulate TAM expansion and recruit their circulating precursors,and the proliferated TAMs would secrete cytokines to accelerate cancer stem cell replication and supress apoptosis.It has been shown that in colon cancer,pancreatic cancer and breast cancer,targeting TAMs could suppress tumor progression and improve chemotherapy efficacy.Macrophages are immune cells that have a high degree of phenotypical and functional heterogeneity.They are often simplified into two subtypes,M1 and M2.Ml macrophages are 'classically' activated,pro-inflammatory and tumor-inhibitory,while M2 macrophages are ' alternatively' activated,anti-inflammatory and tumor-promotive.The composition of macrophage subtypes in a tumor tissue determines the inhibitory or promotive role of TAMs.In many cancers,TAMs are dominated by M2 macrophages and thus play a tumor-promotive role.Hence,re-educating TAMs from M2 to M1 phenotype is a promising anticancer strategy.Autophagy is a process of phagocytic cytoplasmic proteins or organelles that are engulfed into vesicles and fused with lysosomes to form autophagic lysosomes that degrade the contents of their enclosures,thereby achieving the metabolic needs of the cell itself and the renewal of certain organelles.This process is highly conserved.Autophagy is an essential cellular function for macrophages.This process is not only involved in intracellular quality control and metabolic adaptation,but also crucial for cell differentiation and remodelling.Accumulating evidence suggests that macrophage is one of the bridges connecting autophagy and immunity.Autophagy regulates cellular development of monocytes,resulting in the disturbance of macrophage differentiation.The activation of autophagy leads to the recycling ofcellular components and ATP,which are exactly what macrophages requires in their energy architecture,especially during activation.It has been revealed that the down-regulation of autophagy level limits the M2 macrophage differentiation.Based on this knowledge,herein,we hypothesized that chloroquine(CQ),an autophagy inhibitor,can repolarise TAMs from M2 macrophages to M1 phenotype.First,we proved that CQ could repolarise M2 macrophages to M1 phenotype in vitro.Then,the effects of repolarised macrophages on Hep-2 laryngeal tumor cells and cisplatin(CDDP)resistance were examined.Finally,we assessed the effects of CQ treatment in vivo.It was demonstrated that CQ could reduce laryngeal tumor growth and effectively enhance CDDP sensitivity,and TAMs were successfully repolarised during this process.Research purposes:We found that chloroquine(CQ),an autophagy inhibitor,was able to repolarise M2 macrophages to the anti-tumor M1 phenotype.Therefore,we investigated the mechanism of CQ-induced macrophage repolarization inhibiting tumor growth in vitro and in vivo.Research methods:1.Inducing macrophage repolarization in vitroMurine BMDMs at MO stage were used to generate M1 and M2 macrophages.The isolated BMDMs from C57BL/6 were induced with IFN-y and LPS for the M1 polarisation,and IL-4 for the M2 polarisation.qRT-PCR was performed to detect transcription levels of arginase 1(Argl),mannose receptor 1(MRC1),interleukin-12(il-12)and nitric oxide synthase(NOS2)in polarized macrophages to assess the polarisation state.2.To prove that CQ could repolarise M2 macrophages to Ml phenotype in vitroBased on LC3-? level is positively correlated with the autophagic activity in a cell,the LC3-? level in different macrophage states was examined using Western blot to assess the autophagic activity.Then,in order to test whether autophagy inhibition could repolarise M2 macrophages to M1 phenotype,the polarised M2 macrophages were treated with 20 ?M CQ,an autophagy inhibitor,for 24 hrs.PBS was used to treat the control group for 24 hrs.The RNA and protein expression levels of Argl,Mrc1,IL-12 and NOS2 in CQ-treated group and control group were detected by qRT-PCR and WB,respectively.To furtherly confirm the repolarisation of the macrophages,fluorescence-activated cell sorting(FACS)was used to analyse the resultant cells populations for CD206(M2 marker)and MHCII(M1 marker),respectively.3.To evaluate the effect of M2 macrophage repolarization on macrophage phagocytosis and whether it affects the drug sensitivity of human laryngeal cancer cells to CDDPHuman laryngeal cancer cell Hep-2 cell line was used to test the anti-tumor ability of the repolarised macrophages.It was first assessed whether M2-to-Ml repolarisation enhances direct tumor cell killing by phagocytosis.M2 macrophages were pre-treated with CQ for M2-to-M1 repolarisation,and the repolarised macrophages were co-cultured with GFP-labelled Hep-2 cells for 24 hrs(fig.3A).Flow cytometry was used to detect the GFP signal concurrent with F4/80 pan-macrophage selection marker.The concurrence of the signals will indicate the phagocytosis of Hep-2-GFP cell by macrophages.In addition,Hep-2 cells were treated with CQ for 48 h,and the cell viability was detedcted by MTT assay.We also assessed whether M2-to-M1 repolarisation could increase chemotherapeutic efficacy.The CQ-treated M2 macrophages and the vehicle-treated M2 macrophages were pre-cultured for 24 hrs,and the conditioned media were collected to grow the Hep-2 tumor cells.CDDP was added to the medium,and the apoptosis of Hep-2 cells was measured using Annexin V/Propidium Iodide apoptosis assay after 48 hrs incubation.4.To investigate the anti-tumor activity of CDDP,CQ and CQ/CCDP combined treatment in vivoHep-2 laryngeal tumor models were established on 6-8 weeks old male nu/nu mice.The Hep-2 cells were subcutaneously injected under the skin of the right back of the nu/nu mice.After the sizes of the tumors reached 50 mm3,CDDP,CQ,CDDP+CQ or the control treatment was given every three days for five times.The mean volume and mean weight of the tumors of each group was used to plot the tumor growth curves.PCNA analysis and TUNEL analysis were conducted to dissect the mechanism of the anti-tumor effects.5.To investigate whether CQ affects tumor growth in vivo by inhibiting autophagy and inducing TAMs repolarizationWe determined the expression level of LC3-II by WB to characterize the autophagy activity in tumor tissues.Flow cytometry was used to detect the CDllb+f4/80+CD206+M2 macrophages and CD11b+f4/80+MHCII+M1 macrophages in tumor tissues treated with PBS,CQ,CDDP and CQ+CDDP.Research results:1.Ml and M2 macrophages polarisation in vitroARG1 and MRC1 are characteristic to M2 macrophages,while IL-12 and NOS2 are characteristic to M1 macrophages.It was shown that the IL-4 induced macrophages expressed Argl and Mrcl significantly higher than the LPS/IFN-y induced macrophages(P<0.0001****).In contrast,the former expressed 1112 and Nos2 significantly lower than the later(P<0.0001****).This confirmed that LPS/IFN-y and IL-4 successfully induced Ml and M2 macrophage polarisation.2.Autophagy inhibitor chloroquine(CQ)repolarises M2 macrophages to Ml phenotype in vitroThe WB densitometric analysis demonstrated that M2 macrophages contained significantly higher level of LC3-II than M1 macrophages(P<0.001***),indicating that M2 macrophages do have higher level of autophagic activity.The qRT-PCR and WB results analysis both revealed that after CQ treatment,the transcription of M2 characteristic genes Argl and Mrcl was down-regulated(P<0.005**),while the transcription of Ml characteristic genes IL-12 and NOS2 was up-regulated(P<0.001***).The FACS demonstrated that compared to the control group,the CQ-treated M2 macrophages expressed less CD206 but more MHCII,indicating a skew to Ml phenotype.These results suggested that CQ could be an M2-to-Ml repolarising agent.3.Repolarization of M2 macrophage enhances the phagocytosis of macrophages and increase sensitivity of human laryngeal cancer cells to CDDPThe FACS analysis demonstrated that the occurrence of GFP was significantly more frequent for the CQ-treated M2 macrophages than the vehicle-treated M2 macrophages suggesting that the M2-to-M1 repolarisation led to more active phagocytosis toward the tumor cells.Annexin V/Propidium Iodide apoptosis assay demonstrated that M2 macrophages reduces CDDP sensitivity of Hep-2 cells,significantly reducing the apoptosis of Hep-2 cells.However,M2-to-M1 repolarisation could reverse this effect,significantly increasing the apoptosis of Hep-2 cells again.And the apoptosis of Hep-2 cells was unlikely attributed to the tumoricidal effect of CQ in the media.4.Anti-tumor activity of CDDP,CQ and CQ/CDDP combined therapy in vivoIt was observed that CQ alone significantly supressed the tumor volume and the tumor weight growth compared to the control group.The CQ/CDDP combined treatment had a significantly better tumor suppression effect compared to the CDDP group as well.PCNA quantitative analysis showed that there were less PCNA-positive cells in the CQ/CDDP combined treatment group,indicating a more effective cell proliferation inhibition.Meanwhile,there were significantly more TUNEL-positive cells in the CQ/CDDP treatment group,indicating an enhanced cell apoptosis.These data suggested the therapeutic potential of CQ,as it inhibited tumor growth and improved CDDP efficacy.5.CQ treatment inhibits autophagy and repolarises TAMs to M1 phenotype in Hep-2 tumorsThe WB results illustrated that the level of LC3-II significantly increased in the CQ treated group(P<0.001***)and the CQ/CDDP combined therapy treated group(P<0.001***).The elevated level of LC3-? indicated a generally impeded autophagy process in the tumor tissue.The FACS revealed that CQ treatment significantly reduced the percentage of M2 macrophages in the cell population by more than 50%(CQ:P<0.005**;CQ+CDDP:P<0.001***).Meanwhile,it increased the percentage of M1 macrophages by more than 200%(CQ:P<0.005**;CQ+CDDP:P<0.005**).Taking together,these data indicated that CQ reduced the autophagy in the tumour tissue and repolarised M2 macrophages to create an M1-dominated TAM population.Conclusions:We demonstrated that CQ,an autophagy inhibitor,could repolarise TAMs from M2 to the anti-tumor M1 phenotype.CQ treatment reduced Hep-2 laryngeal tumor growth and greatly improved the CDDP treatment outcome in the mouse model.This result might be attributed to the CQ-induced TAM repolarisation and direct autophagy inhibition in the tumor cells.Further tests are required before translating these findings to clinical practice.It needs to be investigated whether this strategy is applicable to other cancer types and different cancer stages.