Study Of The Crosstalk Between Tumor-associated Macrophages And Tumor Cells And Its Effect On Chemoresistance And Prognosis After Neoadjuvant Chemotherapy In Gastric Cancer

As one of the most common malignant tumors in the digestive system,gastric cancer has the characteristics of low rate of early diagnosis,insidious symptoms and rapid progress,accounting for the fourth leading cause of cancer-related mortality worldwide.A large number of clinical and basic researches have achieved great progress in the diagnosis and treatment of gastric cancer,however,the 5-year survival rate of patients is still less than 50%,chemoresistance is one of the major contributing factors leading to poor prognosis of gastric cancer patients.The occurrence and progression of malignant tumor not only depend on the malignant biology of tumor cells,but also the regulation and support of tumour microenvironment(TME).Tumor-associated macrophages(TAMs)is the most abundant interstitial cell population,which role in TME depends on the phenotype of classic activation(M1 macrophages)or alternative activation(M2 macrophages)of macrophages.Several studies have found that M2 macrophages are related to the progression of malignant tumors,whose invasion is closely associated with the chemoresistance of several solid tumors.M2-type polarization of TAMs is induced by TME,including tumor cells.On the other hand,M2 macrophages effect the malignant biological properties of tumor in turn.Study on TME of gastric cancer only concentrated on the mechanism for the promotion of tumor rather than the crosstalk between gastric cancer cells and TAMs,especially the correlation between TAMs infiltrating in gastric cancer and prognosis in patients who recieved neoadjuvant chemotherapy.Maternal embryonic leucine zipper kinase(MELK)is a member of Ser/Thr protein kinase,which has been confirmed as the key driving gene for the occurrence and development of malignant tumor,involving in the proliferation,migration,invasion and chemoresistance of leiomyosarcoma of uterus,lung cancer,myeloma,liver cancer and cholangiocarcinoma.However,few study has reported its effect on the chemoresistance of gastric cancer.Therefore,exploring the crosstalk between gastric cancer cells and TAMs and the role of MELK in the progression of gastric cancer can put forth the novel targets and methods for the treatment of gastric cancer as well as the prognostic factors for neoadjuvant chemotherpay.Our study will be elaborated from the following four parts:Part I Crosstalk between tumor-associated macrophages and tumor cells promotes chemoresistance and enhences migration and invasion ability of gastric cancer cellsObjective:The purpose of this part is to establish chemoresistant gastric cancer cell line,constructing indirect co-culture model using parental or chemoresistant gastric cancer cell and TAMs to detect the effect of interaction between cells on the polarization of macrophages and the chemoresistance,the ability of migration and invasion of gastric cancer cells,and explore the mechanism involved.Methods:1.5-FU-resistent cell lines MKN45-R and HGC27-R were generated by repetitively exposing gastric cancer cells to increasing concentrations of 5-FU.Cell counting kit-8,western blot,wound-healing and transwell assay were used to evaluate the ability of chemoresistance,migration and invasion.2.TAMs from 5-FU-sensitive TME(MS)and 5-FU-resistant TME(MR)were obtained and harvested for experimental analysis.Real-time quantitative polymerase chain reaction(RT-qPCR)and flow cytometry(FCM)were used to compare the effect of parental and chemoresistant gastric cancer cell on the polarization of macrophages.3.MS and MR cells co-cultured with parental gastric cancer cells,then Cell counting kit-8(CCK-8),plate clone formation assay,FCM and Western blot(WB)were used to compare the effect of MS and MR cells on chemoresistance,migration and invasion of gastric cancer cells.4.RT-qPCR and enzyme linked immunosorbent assay(ELISA)were adopted to screen out the cytokines exerting mediate effect.5.FCM,transwell and WB were used to verify the mediate effect and the signal pathway involved.Results:1.MKN45-R and HGC27-R cells were significantly stronger in the ability of chemoresistance,migration and invasion than MKN45-S and HGC27-S cells.2.Co-culturing gastric cancer modulated macrophages to M2-like polarization,and chemoresistant cells were more effective in inducing M2 polarization.3.Co-culturing MR cells significantly promoted chemoresistance,migration and invasion of MKN45-S and HGC27-S cells.4.Macrophage-derived CXCL5 enhenced the ability of chemoresistance,migration and invasion via activating PI3K/AKT/mTOR signal pathway in gastric cancer cell,and recruited monocytes to be induce to be M2-like polarization TAMs.Conclusion:Interaction between TAMs and gastric cancer cells could induce macrophages polarization to M2 phenotype,which in turn further promoted the chemoresistance,migration and invasion of gastric cancer cells.TAMs-derived CXCL5 played a critical role in the cell-cell interaction and could recruit monocytes to form more M2-polarized macrophages in gastric cancer.Part Ⅱ Upregulated MELK promotes chemoresistance and induces TAMs polarization to M2 phenotype in gastric cancerObjective:The purpose of this part is to investigate the effect of MELK on chemoresistance of gastric cancer,and further explore the its effect on M2 polarization of TAMs and the mechanism involved.Methods:1.RT-qPCR and WB were used to detect and compare the expression of MELK in gastric epithelial cells GES-1,parental gastric cancer cells MKN45-S and HGC27-S,chemoresistant gastric cancer cells MKN45-R and HGC27-R.2.Gastric cancer tissues from neoadjuvant chemotherapy patients were collected and divided into chemotherapy sensitive group and chemotherapy resistant group according to chemotherapeutic efficacy.RT-qPCR assay compared the expression of MELK in normal gastric tissue,gastric cancer tissue from chemotherapy sensitive group and chemotherapy resistant group in mRNA level.IHC was used to further compare the expression of MELK in chemotherapy sensitive group and chemotherapy resistant group in protein level.3.Lentivirus infection was used to construct MELK overexpressing stable cell line(LV-MELK)in parental gastric cancer cells,and MELK suppressing stable cell line(sh-MELK)was constructed in chemoresistant gastric cancer cells.CCK-8,plate clone formation assay and FCM were used to investigate the effect of MELK on chemoresistance of gastric cancer.4.MELK overexpressing and suppressing stable cell lines co-culture with macrophages indirectly,and then the effect of MELK on M2 polarization of macrophages was explored.5.RT-qPCR and ELISA assay screened out the cytokines exerting mediate effect.FCW and WB assay were used to verify the mediate effect of CSF-1 and explore the signal pathway involved.6.The expression of MELK,CSF-1 and CD206 in gastric cancer tissue from neoadjuvant chemotherapy was detected by IHC,and the correlation with survival was further evaluated.Results:1.MELK was associated with the resistance of gastric cancer to chemotherapy.Compared with CES-1,the expression of MELK was higher in gastric cancer cells,especially in chemoresistant gastric cancer cells.The results of IHC showes that the expression of MELK in chemotherapy resistant group was much higher than in chemotherapy sensitive group and normal gastric tissue.2.Upregulated MELK could promote the chemoresistance of gastric cancer cells.Overexpressing MELK could significantly enhence the ability of parental gastric cancer cells in resistance to 5-FU and anti-apoptosis.On the contrary,suppressing MELK could reduce the ability of chemoresistant gastric cancer cells.3.Upregulated MELK could induce macrophages polarization to M2 phenotype.RT-qPCR and FCM assay found that MELK overexpressing stable cell line was more efficient in inducing M2 macrophages polarization through co-culture.In contrast,suppressing MELK in chemoresistant gastric cancer cells could reduce the ability.4.RT-qPCR and ELISA screened out that CSF-1 deriving from gastric cacer cells exerts mediate effect in gastric cancer cells inducing M2 macrophage polarization.FCM and WB assay further verified that CSF-1 exerts mediate effect via JAK2/STAT3 signal pathway.5.The expression of MELK in gastric cancer tissue from neoadjuvant chemotherapy correlated positively with CSF-1 and CD206.The OS and DFS were significantly shorter in patients with high expression of MELK,CSF-1 and CD206.Conclusion:Upregulated MELK could promote the chemoresistance of gastric cancer cells and induce M2 macrophages polarization through CSF-1 deriving from gastric cancer cells via activating JAK2/STAT3 signal pathway.The high expression of MELK,CSF-1 and CD206 was associated poor prognosis of gastric cancer patients recieving neoadjuvant chemotherapy.Part Ⅲ Infiltration of TAMs associated with chemotherapeutic efficacy and prognosis of gastric cancer after neoadjuvant chemotherapyObjective:The purpose of this part is to evaluate the correlation among the infiltration of TAMs with clinicopathologic characteristics and prognosis in gastric cancer after neoadjuvant chemotherapy and analyse correlation with the expression of CXCL5.Methods:1.Gastric cancer tissues from neoadjuvant chemotherapy patients were collected and divided into chemotherapy sensitive group and chemotherapy resistant group according to chemotherapeutic efficacy.IHC was used to detect the expression of CD68 for TAMs infiltration analysis and the differences between the two groups were compared.2.The clinicopathologic characteristics of patients were retrospectively collected.The correlation between the infiltration of TAMs and clinicopathologic characteristics was analyzed.IHC was used to detect the expression of CXCL5,the correlation between the infiltration of TAMs and the expression of CXCL5 was also analyzed.3.Cox regression analysis was used to explore the prognostic factors of gastric cancer.Kaplan-Meier method was used to analyze the prognosis and the log-rank test was used to compare the survival difference.Results:1.The number of TAMs infiltrated in chemotherapy resistant gastric cancer tissue was significantly higher than in chemotherapy sensitive gastric cancer tissue.2.Patients were dedided into high infiltration group and low infiltration group by the median of TAMs.The chemoresistant rate and lymph node metastasis rate in high infiltration group were higher than in low infiltration group,Depth of tumor infiltration and TNM staging were later.Age,gender,tumor size,tumor location,differentiated degree,lymphatic and venous invasion had no correlation with the infiltration of TAMs.3.The analysis of spearman rank correlation showed that the infiltration of TAMs had positive correaltion with the expression of CXCL5.4.Univariate Cox regression analysis showed that the infiltration of TAMs and the expression of CXCL5 were associated with the prognosis of gastric cancer.Kaplan-Meier method found that the OS of patients with high infiltration of TAMs and high expression of CXCL5 was respectively shorter than patients with low infiltration of TAMs and low expression of CXCL5.Multivariate Cox regression analysis showed that high infiltration of TAMs and high expression of CXCL5 were both independent risk factors for the prognosis of gastric cancer.Conclusion:The infiltration of TAMs was associated with the chemotherapeutic efficacy,depth of tumor infiltration and lymph node metastasis rate,and had positive correaltion with the expression of CXCL5.The high infiltration of TAMs and high expression of CXCL5 were both independent risk factors for the prognosis of gastric cancer after neoadjuvant chemotherapy.Part Ⅳ Comparison of the predictive value of pathological response at primary tumor and lymph node status after neoadjuvant chemotherapy in locally advanced gastric cancerObjective:This study aimed to investigate the prognostic value of pathological response at primary tumor and the lymph node status after receiving neoadjuvant chemotherapy.Methods:Data from 160 patients with loacally advanced gastric cancer treated with neoadjuvant chemotherapy followed by gastrectomy were retrospectively reviewed.Pathological evaluation after chemotherapy was based on the grade of pathological response of the primary tumor and the status of lymph node.Survival curves for overall survival(OS)and disease-free survival(DFS)were estimated via Kaplan-Meier method,and the log-rank test was used to compare survival difference.Univariate and multivariate analyses for prognostic factors were based on the Cox regression.Results:1.Among 160 selected cases,90 had pathological response(PR),while 70 had no pathological response(nPR)to chemotherapy.Smaller tumor size was presented in PR group,which also had lower level of signet ring cell features,compared to nPR group(all P<0.05).Based on the status of lymph nodes,nodal status(-)group showed smaller tumor size,lower depth of tumor invasion,better differentiated degree,lower level of signet ring cell features,lower rate of lymphatic and venous invasion and less advanced postoperative pathological stage(all P<0.05).2.Survival was equivalent between PR and nPR group(all P>0.05),while patients in NS-group had better DFS than in NS+group(HR 0.301,95%CI 0.194-0.468,P=0.002).3.The stratified analysis by the status of lymph node was further performed.For patients with pathological response,OS and DFS were better in patients with no lymph node metastasis than that with lymph node metastasis(OS:HR 0.346,95%CI 0.173-0.693,P=0.004;DFS:HR 0.312,95%CI 0.148-0.659,P=0.001).Similarly,for patients with no pathological response,the lymph node status showed remarkable prognostic significance in OS and DFS(OS:HR 0.439,95%CI 0.192-1.001,P=0.019;DFS:HR 0.320,95%CI 0.177-0.579,P<0.001).However,when stratified analysis was performed by pathological response,whether patients had lymph node metastasis or not,survival curves showed that pathological response was not related to OS and DFS(all P>0.05).4.Univariable Cox regression analysis of prognostic factors identified several potential predictors of OS and DFS.Clinical T stage,pathological lymph node status and ypTNM stage were associated with OS and DFS.Stepwise selection of variables and multivariable Cox regression analysis identified that pathological lymph node status(HR 1.756,95%CI 1.114-3.278,P=0.029)as being independent prognostic factors associated with OS,similar results were obtained regarding DFS(HR 1.901,95%CI 1.331-3.093,P=0.012).Conclusion:Lymph node status after neoadjuvant chemotherapy can serve as a potential independent prognostic factor for gastricc cancer patients treated with neoadjuvant chemotherapy followed by surgery,and may be more efficient than pathological response in primary tumor to some extent.The combined application of nodal status and pathological response could contribute to the clinical evaluation of neoadjuvant chemotherapy,as well as the prognosis of gastric cancer.