Tumor-activated Neutrophils In Gastric Cancer Foster Immune Suppression And Disease Progression Through GM-CSF-PD-L1 Pathway

Gastric cancer(GC)is one of the leading causes of cancer death in less developed countries.The prognosis of GC is very poor,with a five-year survival less than 30%.Currently,the pathogenesis of GC is unclear.However,its occurrence,development,and prognosis are closely related to anti-tumor immunity in the tumor microenviroment.Tumor microenviroment is comprised of not only tumor cells,but also many other immune cells,including neutrophils.Neutrophils are prominent components of solid tumours and exhibit distinct phenotypes in different tumour microenvironments.However,in human GC,almost nothing is known about the phenotype,function,and associated clinical relevance of neutrophils.Objectives1.To clarify the distribution and phenotype of neutrophils in GC.2.To study the regulation of neutrophils in GC microenviroment.3.To elucidate the function and corresponding mechanisms of neutrophils in GC.4.To verify the function and corresponding mechanisms of neutrophils in vivo and to elucidate the clinical relevance of neutrophils in GC.Methods1.The distribution and phenotype of neutrophils in GCThe fresh gastrci tissue samples and blood samples were collected from the department of general surgery.Flowcytometry and immunhistochemistry were uesd to detect the distribution of neutrophils;flowcytometry was used to detect the p henotype of neutrophils.2.The regulation of neutrophils in GC microenviromentFreshly isolated peripheral neutrophils were stimulated with 20% tumor tissue culture supernatants(TTCS),40% TTCS,50% TTCS,80%TTCS or 50% non-tumor tissue culture supernatants(NTCS)for 16 h.The apoptosis of neutrophils were measured with annexin V apoptosis kit or TUNEL kit by flowcytometry.Freshly isolated peripheral neutrophils were stimulated with 50% TTCS or 50% NTCS for 12 h,the phenotype of neutrophils was detected by flowcytometry.The pro-inflammatory factors of gastric tissues were screened by microarray technology.Freshly isolated peripheral neutrophils were stimulated with different recombinant human cytokines(including GM-CSF,M-CSF,G-CSF,IFN-?,TGF-?,IL-1?,IL-6,IL-17 A,IL-23,IL-22,IL-10,etc)for 12 h,the phenotype of neutrophils was detected by flowcytometry.The protein of tumor tissues and non-tumor tissues were extracted.GM-CSF concentrations in tumor tissue,non-tumor tissue,TTCS and NTCS were measured by enzyme-linked immunosorbent assay(ELISA).The expression of EP-CAM and GM-CSF was examined by double immunofluorescence staining.Freshly isolated peripheral neutrophils were pre-treated with different signal pathway inhibitors(including AG490?FLLL32?BAY11-7082?SP600125?SB203580 or Wortmannin)for 2h,then sitmualted with TTCS or recombinant human GM-CSF for 12 h.The phenotype of neutrophils was detected by flowcytometry.3.The function and corresponding mechanisms of neutrophils in GC.Neutrophils from tumor tissue or non-tumor tissue were sorted by fluorescence activated cell sorter(FACS).In a 5-d coculture system,tumor-infiltrating neutrophils or non-tumor tissue-derived neutrophils were co-cultured with autologous bead-purified peripheral CD3+ T cells at 1:2 ratio,with or without anti-PD-L1 antibody.After 5-d incubation,the cells were harvested for intracellular cytokine staining.Freshly isolated peripheral neutrophils were stimulated with 50% TTCS or 50% NTCS for 12 h.TTCS-or NTCS-conditioned neutrophils were cocultured with CFSE labeled autologous or allogenic CD3+T cells,PD-1+T cells or PD-1-T cells for 5 days at 1:2 ratio,with or without anti-PD-L1 antibody.After 5-d incubation,the cells were harvested for intracellular cytokine staining.4.The function and corresponding mechanisms of neutrophils in vivo and the clinical relevance of neutrophils in GCNOD/SCID mice were injected subcutaneously with 106 SGC-7901 cells.TTCS-conditioned neutrophils(TCN)were obtained as above.Then,5×106 anti-CD3-and anti-CD28-stimulated polyclonal T cells were co-cultured with autologous peripheral neutrophils,or TCN at a 1:1 ratio in the presence or absence of a neutrali zing antibody against human PD-L1 or a control Ig G antibody for 24 h,and were subsequently injected into the peritoneum in 100 ?l of buffered saline on day 10 after inoculation.Tumor size was measured every two days by two independent observers using callipers fitted with a vernier scale for 2w.Once the mice were sacrificed,the tumours were photographed,and were further fixed for immunohistochemicalstaining,and the spleens were dissociated into single cells for flow cytometry.Correlations between neutrophils and clinical parameters were assessed using the Pearson correlation analysis.Overall survival was defined as the interval between surgery and death.The known tumor-unrelated deaths(eg,accidental death)were excluded from the death record for this study.Cumulative survival time was caculated by Kaplan-Meier method,and survival time was measured in month;the log-rank test was applied to compare between 2 groups.Results1.The distribution and phenotype of neutrophils in GC1.1 Neutrophil percentage in peripheral blood of GC patients was higher than that in healthy donors.Moreover,the percentage of neutrophils in tumors was significantly higher than neutrophils in peritumoural or non-tumour tissues.As to the total number of neutrophils per million total cells in each tissue,similar observations were made.Furthermore,the accumulation of neutrophils in tumoral tissues was also confirmed by immunohistochemical staining.1.2 Compared to peripheral neutrophils,neutrophils in tissues expressed significantly high levels of neutrophil activation marker CD54 and immunosuppressvie molecule PD-L1.What's more,intratumoral neutrophils expressed significantly higher level of neutrophil activation marker CD54 and immunosuppressvie molecule PD-L1 than those expressed on peritumoral or non-tumor neutrophils.Significant correlations were found between intratumoral CD54+ neutrophils and PD-L1+ neutrophils,both in percentage and number.The co-exisitence of CD54 and PD-L1 on intratumoral neutrophils was verified by flowcytometry,indicating that tumor-infiltrating neutrophils exhibit an activated immunosuppressive phenotype.2.The regulation of neutrophils in GC microenviroment2.1 TTCS conditioned neutrophis exhibited a delayed onset of apoptosis in a dose-dependent manner when compared to NTCS conditioned neutrophils,which indicate that GC microenvironment prolonged lifespan of neutrophils to sustain their accumulation.2.2 TTCS-conditioned neutrophis expressed higher level of neutrophil activation marker CD54 and immunosuppressvie molecules PD-L1 than NTCS-or mediumconditioned neutrophis,implying that GC microenviroment plays important roles in activating nuetrophils and sustaining their immunosuppressiv e phenotype.2.3 Microarray screening of pro-inflammatory facors in gastric tumoral tissues showed than GM-CSF,M-CSF,G-CSF,IFN-?,TGF-?,IL-1?,IL-6,IL-17 A,IL-23,IL-22,IL-10,etc were among the most highly expressed cytokines.Then periph eral neutrophils were stimulated with these cytokines,respectively.The results showed that only GM-CSF could significantly up-regulate the expression of CD54 and PD-L1 on neutrophils.Moreover,blocking GM-CSF in TTCS significantly down-regulated PD-L1 expression,while provision of exogenous GM-CSF in NTCS significantly up-regulated the expression PD-L1 on neutrophils.ELISA results showed that tumoral tissues had higher level of GM-CSF than non-tumoral tissues and TTCS had higher level of GM-CSF than NTCS.There was clearly a positive correlation between PD-L1+ neutrophil infiltration and GM-CSF production within tumours.Immunoflourescence staining showed that GM-CSF most likely derived from EP-CAM+ tumor cells.Taken together,these results suggest the pivotal roles of tumor-derived GM-CSF in activating nuetrophils and sustaining their immunosuppressive phe notype.2.4 Blocking of JAK or STAT3 signalling pathway effectively suppressed CD54 and PD-L1 expression on TTCS or GM-CSF conditioned neutrophils either alone or in combination.Western blot(WB)showed that STAT3 was predominantly phosphorylated in TTCS conditioned neutrophils,and this phosphorylation was abolished when blocking GM-CSF in TTCS.These results imply that JAK-STAT3 signalling pathway is crucial for neutrophil activation and PD-L1 induction.3.The function and corresponding mechanisms of neutrophils in GC3.1 The co-localization of neutrohils and CD3+ T cells in tumor tissues suggest that these neutrophils may promote tumor progression by impairing T cell immunity.3.2 Neutrophil/T-cell co-cultures showed that tumor-infiltrating neutrophils were superior to non-tumour-derived neutrophils in inhibiting T cell proliferation and interferon-?(IFN-?)production in a PD-L1 dependent manner.3.3 TTCS conditioned neutrophils significantly inhibited autologous or allogenic T cell proliferation and IFN-? production in a PD-L1 dependent manner.3.4 TTCS conditioned neutrophils significantly inhibited autologous or allogenic PD-1+ Tcell proliferation and IFN-? production in a PD-L1 dependent manner.No such phenomena were found when refer to PD-1-T cells.Moreover,significant negative correlations were found between the percentage and number of PD-1+ T cells and PD-L1+ neutrophils in human GC tissues.4.The function and corresponding mechanisms of neutrophils in vivo and the clinical relevance of neutrophils in GC4.1 In vivo model showed that mice without T cell transfusions,mice treated with T cells plus TCN or control Ig G-treated TCN,showed tumour growth and disease progression.However,mice treated with T cells plus PD-L1 blocking antibody-treated TCN showed reduced tumour volumes and disease progression at each measurement time point from day 18.Moreover,mice treated with T cells plus PD-L1 blocking antibody-treated TCN,also showed an increased CD3+ T cell infiltration and IFN-? production from T cells,and a decreased tumour cell proliferati on,compared with the mice treated with T cells plus TCN or control Ig G-treated TCN.These findings suggest that tumour-activated neutrophils suppress T cell immunity in vivo dependent on PD-L1 and thereby contribute to tumour growth and GC progression.4.2 An increased neutrophil percentage was correlated with increased tumor size,advanced lymphatic invasion and tumor stage.The patients were divided into two groups based on the median neutrophil percentage level(5.24%),the 44-month survival rate was significantly lower for those within the higher neutrophil percentage group.Similar results were obtained when the patient cohort was stratified based on neutrophil number.Moreover,when patients were divided into two groups based on the median PD-L1+ neutrophil percentage level(24%),the 20-month survival rate was significantly lower for those within higher PD-L1+ neutrophil percentage group.Similar results were obtained when the patient cohort was stratified based on PD-L1+ neutrophil number.Conclusions1.Neutrophils accumulated in the tumor tissues of GC patients and exhibited an activated immunosuppressive phenotype.2.GC microenviroment prolonged the life span of tumor-infiltrating neutrophils;tumor cells derived GM-CSF activated neutrophils and up-regulated the expression of PD-L1 on neutrophils through JAK-STAT3 signalling pathway.3.Tumor-activated neutrophils inhibit T cells function through PD-L1-PD-1 pathway,thus suppressed tumor immunity and promote GC progression.4.Neutrophil percentage or number increased with tumor progression.Both neutrophil percentage and number predicted reduced overall survival.5.PD-L1+ neutrophil percentage or number increased with tumor progression.Both PD-L1+ neutrophil percentage and number predicted reduced overall survival.SignificanceOur in vitro and in vivo data together provide a model of immunosuppression involving GM-CSF-PD-L1 on neutrophils in GC.Our findings further suggest several possible therapeutic targets,including GM-CSF,neutrophils and PD-L1.Given the apparent relationship between neutrophils levels and the advanced progression observed in the studied GC patients,thought should be given to the use of these PD-L1+ neutrophils as novel diagnostic biomarkers for GC.