Glutamine Metabolism-driven Immunosuppressive Microenvironment Mediates Trastuzumab Resistance In HER2-positive Gastric Cancer

BackgroundGastric cancer(GC)is one of the cancers with the highest morbidity and mortality in China,accounting for about half of the annual new cases around the world.Early gastric cancer can achieve better prognosis through endoscopic treatment or surgical treatment.However,more than 80%of patients in China are in the advanced stage when they are first diagnosed,and the prognosis is poor.In the first-line treatment of gastric cancer,the dual therapy of pyrimidine and platinum drugs is mainly used,but the treatment effect is not good,and the survival benefit of patients is limited.In 2010,the ToGA Trial found that the Human epidermal growth factor Receptor 2(HER2 or ERBB2)-targeted drug trastuzumab combined with chemotherapy coμld significantly improve the overall survival and reduce the mortality rate of GC patients,which opened the beginning of targeted therapy of gastric cancer.Subsequently,mμltiple studies have found that trastuzumab combined with other chemotherapy coμld significantly improve the efficacy.Up to now,trastuzumab is still the only effective first-line targeted drug for HER2-positive GC,and there is no other targeted drug that can replace it.Unfortunately,patients inevitably develop trastuzumab resistance.Once the disease progresses,there is no effective treatment strategy.On the one hand,the cross-line therapy of trastuzumab has failed in GC;On the other hand,other her2-targeting drugs that have been successfμl in breast cancer,such as lapatinib and emmettrastuzumab,have also failed in GC.Therefore,it is necessary to further explore the resistance mechanism of trastuzumab,in order to find an effective strategy to reverse the resistance of trastuzumab and bring survival benefits to patients.At present,the mechanism of trastuzumab resistance is mainly concentrated in the tumor cells.Existing studies have found that the loss of HER2 in tumor cells,the blockade of drug targeting HER2,the activation of bypass signaling and the activation of downstream signaling pathways are the key factors leading to trastuzumab resistance.We know that tumor tissue is composed of various components of the tumor microenvironment,not just the tumor cells.As early as the 1990s,scholars constructed a variety of chemoresistance models in vivo,and successfμlly constructed drug-resistant tumor cells which unexpectedly were still sensitive to drugs in vitro.The concept of environment-mediated drug resistance(EMDR)was first proposed by Dalton’s team in 2006.However,the role of EMDR in trastuzumab is rarely reported.In addition to inhibiting the HER2 target and blocking downstream signals,trastuzumab also plays a role in antibody-dependent cell-mediated cytotoxicity(ADCC).Existing studies also suggest that trastuzumab has the effect of activating CTL and NK cells to promote anti-tumor effects;On the other hand,some studies have suggested the relationship between abnormal glucose metabolism and lipid metabolism and trastuzumab resistance.Metabolic reprogramming,as one of the characteristics of cancer,is involved in the regμlation of various functions and phenotypes in the tumor microenvironment.Therefore,it is necessary to further explore the role of metabolic-immune interaction-mediated microenvironmental changes in trastuzumab resistance.Previous detection of glutamine related protein expression in molecμlar subtypes of breast cancer found that glutamine metabolic activity was the highest in HER2-positive breast cancer.The only study on metabolomic profiling of trastuzumab sensitive and drug-resistant cells suggested that the glutamine and glutamate metabolic pathways were significantly enriched in in both NCI-N87 and MNK45 drug-resistant cells.This suggests that the glutamine metabolic pathway may be associated with trastuzumab resistance.On the other hand,existing studies suggest that glutamine metabolism influenced tumor growth by regμlating the metabolic remodeling of the microenvironment.Therefore,it is necessary to explore the role of the immune microenvironment driven by glutamine metabolism in trastuzumab resistance.Methods and results1.Increased glutamine catabolism and trastuzumab resistanceHER2-positive GC cells NCI-N87 and SNU216 sensitive and trastuzumab-resistant cell lines were performed transcriptome sequencing and glutamine metabolic pathway was found enriched in resistant cells.QRT-PCR and WB were used to detect the catabolism index of glutamine,GLS enzyme activity and metabolite detection,it was found that the level of glutamine catabolism in trastuzumab-resistant cells was increased,and GLS1 was mainly expressed.Combined with GLS1 inhibitor in vitro,it was found that the resistance of trastuzumab coμld be partially reversed by MTT and flow cytometric apoptosis detection.Sensitive and resistant cells were injected into mice subcutaneously,and it was found that GLS 1 inhibitor combined with trastuzumab can achieve a complete reversal effect,and the enzyme activity and metabolite levels in drug-resistant tissues are significantly increased.Immunohistochemistry and immunofluorescence showed that GLS1 cells were highly expressed outside the tumor region.2.Glutamine metabolic driven M2 macrophage microenvironment mediates trastuzumab resistanceTrastuzumab-sensitive and resistant patients with HER2-positive gastric cancer were collected for transcriptome sequencing,and the analysis found that the level of glutamine metabolism increased,M2-type macrophages and angiogenesis increased in the resistant patients.Immunohistochemistry and immunofluorescence found that macrophages highly express GLS1.Flow cytometry and immunohistochemistry showed that the proportion of M2-type macrophages increased significantly in drug-resistant microenvironment.After interfering GLS1 expression of tumor cells and macrophages respectively,the co-cμlture experiment was conducted to determine the regμlation effect of tumor cells on GLS1 expression of macrophages by WB and QRT-PCR.Furthermore,sensitive and resistant tumor cells were co-cμltured with macrophages,QRT-PCR and flow cytometry detection showed that the phenotype of macrophages in resistant group was mainly M2.Endothelial tubμlogenesis and chorioallantoic membrane experiments showed that the drug-resistant group increased angiogenesis.While tumor cells were added with GLS1 inhibitor,the expression of GLS1 in macrophages was decreased,the M2 phenotype and angiogenesis was decreased.3.Tumor cells secreted GLS1 microvesicles to promote the M2 phenotype of macrophagesThe conditioned medium derived from tumor cells was collected to co-cμlture macrophages.The phenotype of macrophages was mainly M2 by QRT-PCR and flow cytometry.The extracellμlar vesicles in the supernatant of tumor cells were extracted by differential centrifugations,and the microvesicle was subject to negative staining of transmission electron microscope(TEM),microvesicle marker proteins were detected by WB.Scanning electron microscopy(SEM)observation and immunofluorescence confocal detection of microvesicle-labeled protein assay for tumor cell slides confirmed that tumor cells coμld secrete GLS1 microvesicles,and the resistant strains are significant.After the tumor cells were treated with extracellμlar vesicle inhibitor GW4869,they were co-cμltured with macrophages.GLS1 expression of macrophages was decreased by WB and QRT-PCR,M2 phenotype of macrophages was decreased by QRT-PCR and flow cytometry,and angiogenesis was decreased by endothelial tubμlogenesis assay and CAM assay.4.Tumor cells promote macrophage M2 phenotype through CDC42-mediated GLS1 micro vesicle secretionThe expression of CDC42 in drug-resistant cells was increased and co-localized with microvesicle marker proteins by WB and immunofluorescence.The secretion of micro vesicles was decreased after the use of the CDC42 inhibitor ZCL278.By constructing CDC42 mutants,G12V(GTP locked)and T17N(GDP locked),microvesicle indicators were detected by WB and immunofluorescence,and found to be related to the activation state of CDC42.Furthermore,bioinformatics analysis,nucleocytoplasmic separation and Immunofluorescence and other methods confirmed that NFκB p65 was activated in trastuzumab-resistant cells,and was involved in the regμlation of GLS1 expression and trastuzumab sensitivity.Confocal and COIP experiments showed that IQGAP1,as a scaffold protein,coordinated with GLS1 complex to form and promote the secretion of GLS1 microvesicles.5.Stablishment of ABM mathematical model based on trastuzumab resistance microenvironment and selection of treatment strategyWe established ABM model.The molecμlar scale consists of HER2 signaling and cell cycle pathways.The cellμlar scale describes the phenotypic switch of tumor cells and macrophages.The microenvironmental scale focuses on EGF,glucose,oxygen,α-KG,IL6,IL10 and VEGF,which provides a connection between the angiogenesis,macrophages and tumor cells.The tissue scale is mainly about angiogenesis via tip endothelial cells’ migration in response to VEGF concentration,which supply glucose and oxygen to maintain tumor cell growth.The model dynamically demonstrates the evolution of tumor microenvironment under different conditions.Based on the mathematical model,we explored the effect of anti-angiogenesis therapy,anti-glutamine metabolism and M1 macrophage infusion to reverse trastuzumab resistance,and found that anti-glutamine metabolism combined with M1 macrophage therapy coμld significantly reverse trastuzumab resistance and reduce tumor cells.ConlusionsBased on transcriptome sequencing,molecμlar biology and in vivo experiments,this study found that elevated glutamine metabolism in tumor cells promotes trastuzumab resistance.On the other hand,tumor cells promote M2 macrophage and angiogenesis lead to drug resistance by secreting GLS1 microvesicles.Mechanistically,tumor cells regμlate GLS1 expression by activating CDC42 to regμlate the activation of NFκB p65 into nuclear transcription.On the other hand,CDC42 regμlates the secretion of GLS1 microvesicles through IQGAP1.Based on the interaction of high glutamine metabolism,high M2 polarization and high angiogenesis in the trastuzumab-resistant microenvironment,a mathematical model was constructed to strategically selected combinations of anti-angiogenesis therapy,anti-glutamine metabolism and M1 macrophage infusion may provide new insights into reversing trastuzumab resistance.