Distribution,Functional Immunoregulation And Tumor-promoting Effects Of Mast Cells In Gastric Cancer

Gastric cancer?GC?,as a severe health problem,has been the sixth most common malignancies and the second leading cause of cancer death worldwide.Despite significant advances in prevention,diagnose,and therapeutic options and strategies in these years,many unanswered questions remain,particularly the pathogenesis of GC is not elaborated clearly.Nowadays,it is generally believed that the development and prognosis of GC are influenced by the cross-talk between tumors and host immune system.Mast cell is an important component in GC microenvironments.Some limited studies are mostly focused on the correlation between GC survival rate and mast cell infiltration by immunohistochemistry,and some on the relationships between the density of the infiltrating mast cells and local angiogenesis.However,the distribution,functional immunoregulation mechanism and tumor-promoting effects of mast cells in GC are presently unknown.Objective1.To investigate the infiltration of mast cells in gastric cancer and their mechanism.2.To clarity the regulatory mechanism on degranulation of mast cells and their effects on GC progression.3.To study the regulation of phenotype on mast cells in GC.4.To illuminate the immunosuppressive function and clinical significance of mast cells in GC.Methods1.The infiltration of mast cells in GC and their mechanismSingle cells were isolated from GC tissues?including intratumoral,marginal tissues,peritumoral and non-tumor tissue?,and infiltration of mast cells were detected by flow cytometry?FCM?.Besides,analysis the distribution of mast cells in GC tissues by immunohistochemistry.In GC,chemokine receptors on mast cells were screened.Mast cells were sorted from GC by FCM and performed by chemotaxis assay with neutralizing antibodies to elucidate the mechanism of mast cell infiltration.2.The regulatory mechanism on degranulation of mast cells and their effects on GC progressionHuman umbilical cord blood derived mast cells?hCBMC?were cultured with tumor tissue culture supernatantas?TTCS?and autologous non-tumor tissue culture supernatantas?NTCS?for 1 h to detect the degranulation of mast cells by measuring?-hexosaminidase release.The pro-inflammatory cytokines in tumor tissues were screened by protein microarray.And then,different human recombinant cytokines were added into LAD2 cells?a human mast cells cell line?for 1 h to stimulate?-hexosaminidase release.The concentration of ADM and?-hexosaminidase in tumor or non-tumor tissue were analysed by ELISA.The expression of ADM and RAPM2 in GC were detected by immunofluorescence staining.ADM receptor antagonist AMA?1?M?was added into TTCS/mast cell co-culture system to inhibit mast cell degranulation by blocking ADM-ADMR interaction.For signaling pathway study,LAD2 cells were pretreated with different signaling pathway inhibitors?10?M?for 2 h,and then were stimulated with TTCS for 1 h for detecting mast cell degranulation.The expression of AKT and p-AKT in LAD2 cells exposed to TTCS?10 min?with or without AMA were analyzed by western blot.In vitro,GC cell lines were cultured with different mast cell culture supernatants for24 h or 72 h,which proliferation or apoptosis was analyzed by CCK-8 or annexin V and deoxyuridine triphosphate nucleotides?dUTP?detection respectively.In vivo,establishing gastric tumor bearing mice model and mast cells?BMMCs or LAD2 cells?were injected into the tumor cavity with or without cromolyn?10 mg/kg?by intraperitoneal injection.Tumor growth was recorded every 2 days.The proliferating cell nuclear antigen?PCNA?expression in transplant tumors of mice was compared by immunohistochemistry staining.The production of IL-17A from hCBMC exposed to autologous NTCS,TTCS with or without cromolyn was analyzed by ELISA.In vitro,GC cell lines were stimulated with the culture supernatants from TTCS-conditioned hCBMCs?referred as TTCS-hCBMCs?plus control IgG or IL-17A neutralizing antibodies?20?g/ml?for 24 h or 72 h,which proliferation or apoptosis was analyzed by CCK-8 or annexin V and deoxyuridine triphosphate nucleotides?dUTP?detection respectively.In vivo,establishing gastric tumor bearing mice model and mast cells(BMMCs from WT or IL-17A^-/-mice or LAD2 cells)were injected into the tumor cavity.In some cases,control IgG or IL-17A neutralizing antibodies?20?g per mouse?were injected into the peritoneum every 2 days after the mast cell injection.Tumor growth was recorded every 2 days.The proliferating cell nuclear antigen?PCNA?expression in transplant tumors of mice was compared by immunohistochemistry staining.3.The regulation of phenotype on mast cells in GCSingle cells were isolated from GC tissues and the phenotype of mast cells infiltrated in GC was analyzed by FCM.Besides,the expression of PD-L1 on mast cell in GC was detected by immunofluorescence staining.Mast cells?hCBMCs?were exposed to 50%autologous TTCS or NTCS for 24 h,or exposed to 20%,40%,80%TTCS for 24 h or exposed to 50%TTCS for 6,12,24 h,and the expression of PD-L1 on hCBMC was analyzed by FCM.Mast cells?hCBMCs?were stimulated with different human recombinant cytokines?100 ng/ml?for 24 h and the expression of PD-L1 on hCBMC was analyzed by FCM.TNF-?concentration between autologous tumor and non-tumor tissues or between autologous TTCS and NTCS was detected by ELISA.Mast cells?hCBMCs?were exposed to 50%TTCS with or without anti-TNF-?antibody for 24 h and the expression of PD-L1 on hCBMC was analyzed by FCM.Mast cells were pre-treated with signaling pathways inhibitors and then exposed to TTCS for 24 h,the phenotype of mast cells was analyzed by FCM.Mast cells?LAD2 cells?were exposed to autologous TTCS,NTCS,and TTCS with anti-TNF-??20?g/ml?or LAD2 cells pretreated with or without BAY 11-7082?an I?B?inhibitor?were exposed to TTCS,and then the expression of PD-L1,p65 and p-p65 were analyzed by western blot.4.The immunosuppressive function and clinical significance of mast cells in GCMast cells were sorted from non-tumor or tumor tissues by FCM and co-cultured with CFSE?carboxyfluorescein succinimidyl amino ester?-labeled peripheral CD3⁺T cells from autologous GC patients with or without anti-PD-L1?20?g/ml?for 5 days.The cells were collected for intracellular staining and the supernatants were harvested for ELISA.In vivo,gastric tumor bearing mice model was established.CD3⁺T cells were co-cultured with TCM with anti-PD-L1?20?g/ml?or a control IgG?20?g/ml?for 5 days,and were injected into the peritoneum on day 7 after mice inoculation.Tumor growth was recorded every 2 days.PCNA expression in transplant tumors was analyzed by immunohistochemistry staining.The transplant tumor protein was extracted and the concentration of IFN-?,perforin and granzyme B was detected by ELISA.According to the infiltration degree of mast cells in GC,patients were divided into high or low mast cell group.Overall and disease free survival was calculated by the Kaplan-Meier method and measured in months;two groups were compared by log-rank test.Results1.The infiltration of mast cells in GC and their mechanismThe infiltration of mast cells was higher in intratumoral tissues than marginal,peritumoral,and non-tumor tissues by FCM and immunohistochemistry staining.The majority of tumor-infiltrating mast cells expressed CXCR4,while,mast cells in peritumoral or non-tumor tissues showed lower CXCR4 expression.The concentrations of CXCL12 were significantly increased in tumor tissue or TTCS.TTCS could induce more tumor-infiltrating mast cell migration than NTCS by mast cell chemotaxis assay,and this effect could be blocked by CXCL12 and/or CXCR4neutralizing antibodies.These data indicate that mast cells can be recruited into GC microenvironment through CXCL12-CXCR4 axis.2.The regulatory mechanism on degranulation of mast cells and their effects on GC progressionCompared to NTCS,TTCS significantly induced mast cell degranulation.Mast cells were stimulated with human recombinant cytokine highly-expressed in GC,and found ADM remarkably induced mast cell degranulation in a dose-dependent manner.The induction of mast cell degranulation by TTCS could be blocked by adding ADM receptor antagonist AMA into TTCS/mast cell co-culture system.In signaling pathways study,blocking the signal transduction of PI3K with Wortmannin effectively suppressed the degranulation of mast cells.AKT was predominantly phosphorylated in mast cells after treatment with TTCS in a time-dependent manner,and AKT phosphorylation could be abolished by blocking tumor derived-ADM.These data show that tumor-derived ADM plays an important role in mast cell degranulation by activation of PI3K-AKT signaling pathway in GC.In vitro,GC cells were stimulated with the culture supernatants from TTCS-condit-ioned hCBMCs.This potently induced GC cells proliferation and inhibited apoptosis compared to the culture supernatants from NTCS-conditioned hCBMC.In vivo,mast cells were injected with or without degranulation inhibitor cromolyn into our established gastric tumor bearing mice model.Compared to mice injected with mast cells,mice injected with mast cells plus cromolyn showed decreased tumor volumes and tumor cell proliferation.GC cells were stimulated with different recombinant cytokines and found that only IL-17A remarkably induced GC cells proliferation.And mast cell-derived IL-17A could be blocked by inhibition of mast cell degranulation with cromolyn.In vitro,the proliferation of GC cells was inhibited and apoptosis of GC cells was promoted efficiently when added IL-17A neutralizing antibodies in the culture supernatants from TTCS-hCBMC.In vivo,gastric tumor bearing mice model was established.Compared to mice injected with LAD2cells plus control IgG or mast cells from WT mice,mice injected with LAD2 plus IL-17A neutralizing antibodies or mast cells from IL-17A^-/-mice showed decreased tumor volumes and tumor cell proliferation.These findings suggest that mast cell-derived IL-17A promote GC cell proliferation and contribute to tumor progression in vitro and in vivo.3.The regulation of phenotype on mast cells in GCImmunosuppressive molecule PD-L1 expressed higher on intratumoral mast cells than that on peritumoral and non-tumor mast cells.Besides,TTCS could increase PD-L1expression on mast cells than NTCS and up-regulated PD-L1 expression in both time-dependent and dose-dependent manners.The concentrations of TNF-?were higher expression in tumor tissues or TTSC than that in non-tumor tissues or NTCS.The induction of PD-L1 on mast cells by TTCS could be blocked by anti-TNF-?,indicating that tumor-derived TNF-?plays an essential role in mast cell PD-L1 induction.In signaling pathway study,blocking the signal transduction of NF-?B with BAY11-7082 could suppress PD-L1 expression on TTCS-conditioned mast cells by FCM.PD-L1 expression or p65 phosphorylation was abolished when blocking NF-?B signaling pathway on mast cells or neutralizing TNF-?in TTCS by western blot,suggesting that tumor-derived TNF-?activates NF-?B pathway to induce PD-L1 expression on mast cells.4.The immunosuppressive function and clinical significance of mast cells in GCThe infiltration of mast cells and T cells in GC was the significant negative correlations and mast cells and T cells were contacting in GC by immunofluorescence staining,suggesting that mast cells may influence the function of T cells.In vitro,mast cells sorted from GC tissues were co-cultured with T cells with or without anti-PD-L1 showed tumor-infiltrating mast cells had a stronger inhibition on T cell proliferation and IFN-?secretion than non-tumor-derived mast cells and neutralizing PD-L1could weaken the inhibition of tumor-infiltrating mast cells.In vivo,gastric tumor bearing mice model was established.T cells were co-cultured with TTCS-conditioned mast cells?TCM?with anti-PD-L1 or a control IgG and injected into the peritoneum.Mice injected with T cells plus control IgG-treated TCM showed a promotion of tumor growth and disease progression,while,mice injected with T cells plus PD-L1 neutralizing antibody-treated TCM showed an inhibition of tumor growth and disease onging.Moreover,mice injected with T cells plus PD-L1 neutralizing antibody-treated TCM also showed decreased tumor cell proliferation by immunohistochemistry staining.These data indicate that tumor-associated mast cells suppress T cell function in vitro and in vivo depending on PD-L1,which lead to tumor growth and GC progression.Analyzing the infiltration of mast cell in GC and patients clinical information,a significant positive association between the percentage of mast cells and advanced clinical features of GC.Besides,according to the infiltration of mast cells in GC,patients were divided into high or low mast cell group.Overall and disease free survival rates were significantly lower for those within the higher mast cell percentage group.Conclusion1.Patients with GC show a significantly higher mast cell infiltration in tumors and tumor-infiltrating mast cells accumulate in tumors by CXCL12-CXCR4 axis.2.Tumor-derived ADM induced mast cell degranulation via PI3K-AKT signaling pathway,which effectively promotes the proliferation and inhibits the apoptosis of GC cells in vitro and contributes to the growth and progression of GC tumors in vivo,and the effect could be reversed by blocking IL-17A production from these mast cells.3.Intratumoral mast cells express higher PD-L1,which was induced by tumor-derived TNF-?though activating NF-?B signaling pathway.4.The tumor-infiltrating and tumor-conditioned mast cells effectively suppress normal T-cell immunity through PD-L1 in vitro,and tumor-conditioned mast cells contribute to the suppression of T-cell immunity and the promotion of GC growth in vivo.The effect can be reversed by blocking PD-L1 on these mast cells.5.Increased intratumoral mast cells are positive association with advanced clinical features of GC and associated with poor survival of GC patients.SignificanceOur results illuminate novel protumorigenic and immunosuppressive roles of mast cells in GC,suggesting that tumor-infiltrating mast cells may become a helpful clinical prognostic marker in the future.We clarify functional evidence for these mast cells degranulation to GC progression and identify TNF-?-PD-L1 immunosuppressive pathway in inhibiting T cell immunity.Overall,blocking these pathological mast cells that infiltrate tumors or the secreting protumorigenic mediators from mast cells may be a useful therapeutic strategy for preventing GC progress.