| Objective:Gastric cancer is the fifth most common cancer in the world and the third leading cause of cancer-related deaths.In 2018,gastric cancer caused 782,685 deaths.Due to the lack of effective early diagnostic markers,most patients with gastric cancer have been shown to be advanced at the time of diagnosis.As one of the main methods of treating cancer,chemotherapy has been widely used in advanced gastric cancer.Traditional wisdom holds that chemotherapeutics kill tumor cells through direct cytostatic/cytotoxic effects.However,accumulating evidence indicates that chemotherapeutics can not only exert direct cytotoxic effects,but also affect the anti-tumor immune response.A large number of research results show that some chemotherapeutic drugs are immunogenic and can enhance the anti-tumor immune response by:(1)up-regulating MHC type I molecules,promoting antigen presentation and antigen recognition;(2)causing immunogenic cell death(ICD);(3)increase the number of effector T cells;(4)reduce immunosuppressive cells,including MDSC,Treg cells and tumor-associated macrophages(TAM);(5)regulate the secretion of cytokines.In recent years,many studies have paid attention to the immunosuppressive effects of some chemotherapeutics while paying attention to the immunostimulatory effects of chemotherapeutics.A number of studies have shown that chemotherapeutics reduce the anti-tumor immune response mainly in the following aspects:(1)reducing circulating immune cells;(2)inducing immunosuppressive monocytes;(3)upregulating programmed cell death on the surface of tumor cells Ligand 1(PD-L1)promotes tumor cell immune escape;(4)induces tumor cells to secrete extracellular vesicles(EV)to promote tumor metastasis.Among them,PD-L1 is an immuno-suppressive molecule expressed in a variety of tumor cells.By binding PD-L1 to PD-1 on the surface of activated T cells,tumor cells play a key role in evading the host's anti-tumor immune response.Several studies have shown that chemotherapeutics or chemotherapy can increase the expression of PD-L1 in tumor cells.For example,cisplatin can increase the expression of PD-L1,5-fluorouracil(5-FU)or FOLFOX in head and neck squamous cell carcinoma tumor cells.PD-L1 on the surface of colorectal cancer tumor cells,gemcitabine upregulated PD-L1 within 5 days before treatment,and paclitaxel upregulated PD-L1 expression in breast cancer cells.Exosome contains various biologically active molecules,plays a key role in intercellular communication,and can have an important effect on the physiological functions of cells.In addition,tumor cell-derived exosomes can transport their DNA,RNA,and proteins to the local or whole body,promoting tumor development and metastasis.A number of studies have shown that PD-L1 not only has a cell membrane surface type(Membrane PD-L1),but also has free PD-L1 released by cells to the outside of the cell.L1)and exosomal PD-L1(Exosomal PD-L1).In addition,the results of Mauro et al.Demonstrated that tumor cell-derived exosomes can carry PD-L1 to the lymph nodes,causing local and/or systemic immunosuppression,thereby promoting tumor progression[20].The published results of our research team show that Exosomal PD-L1 is more stable than Soluble PD-L1 and may have stronger immunosuppressive activity[21].As the core drug for chemotherapy of advanced gastric cancer,whether 5-FU can upregulate the expression of Exosomal PD-L1 in gastric cancer cells and thus cause immunosuppression in patients with advanced gastric cancer is unclear.The purpose of this study was to explore the effects and mechanisms of 5-FU on Exosomal PD-L1,and even on the immune status of patients with advanced gastric cancer through in vitro experiments and blood samples from clinical patients.In addition,the mechanism of 5-FU up-regulating PD-L1 was discussed in depth.The research results will provide scientific basis for understanding the immune regulating effects of 5-FU and improving clinical treatment.Methods:1.Western Blot was used to detect the expression of PD-L1,CD9,CD63,Cbl-b,STAT4,p-STAT4,STAT5a,p-STAT5 and GAPDH proteins.2.The expression levels of PD-L1,CD9,CD69 and CD25 on the surface of cells were detected by flow cytometry.3.Exosomes were isolated from the supernatant of gastric cancer cells by differential centrifugation,and were isolated from plasma samples of gastric cancer patients using Exosome Precipitation Solution.4.The expression levels of soluble PD-L1and Exosomal PD-L1 were detected by ELISA.5.The ultrastructure of Exosomes was observed under a transmission electron microscopy.6.Nanosight determines the diameter Size of Exosomes.7.The concentration of cytokines in plasma of patients was detected by Bio-Plex Pro Human Cytokine 8-plex Assay.8.Apoptosis in activated Jurkat T cell was detected by Flow cytometry.9.PBMCs were obtained from healthy donors by density gradient centrifugation.10.Lentiviral particles with short hairpin RNAs(shRNAs)targeting PD-L1 and Cbl-b genes were used to establish stably transfected gastric cancer cell lines.11.STAT5a and Cbl-b si RNAs were transiently transfected into gastric cancer cells using Lipofectamine 2000 reagent.12.RT-PCR was used to detect the expression of miR-940.13.The binding of Cbl-b and STAT5a was detected by co-immunoprecipitation technique.14.RNA sequencing data of gastric cancer patients in TCGA-STAD and GSE62254 datasets were downloaded from TCGA website and GEO website for correlation analysis.15.Statistical processing:each experiment was repeated three times independently,and the experimental data were expressed as mean+standard deviation.The t-test,Spearman rank correlation test,one-way ANOVA or two-way ANOVA involved in the article were all carried out using SPSS 16.0 statistical software,which confirmed that P<0.05 had statistical significance.Results:1.5-FU upregulated the expression of PD-L1,membrane PD-L1 and soluble PD-L1 in gastric cancer cells in a dose-and time-dependent manner.The PD-L1 high-expression gastric cancer cell lines MKN74 and PD-L1 low-expression gastric cancer cell lines MGC803 were treated with different concentrations of 5-FU(0,0.1,1.0?g/ml),and the expression levels of different forms of PD-L1 were detected.Western blot showed that the expression of PD-L1 in MKN74 and MGC803 was significantly increased after 5-FU treatment compared with control group.Flow cytometry results showed that the expression of membrane PD-L1 was significantly increased in MKN74 and MGC803 after 5-FU treatment.ELISA results showed that the expression of soluble PD-L1 was significantly increased in MKN74 and MGC803 after5-FU treatment.These results suggest that 5-FU can upregulate the expression of PD-L1in gastric cancer cells.2.5-FU upregulates Exosomal PD-L1 derived from gastric cancer cells.The exosomes derived from MKN74 were isolated by differential centrifugation,and the bilayer membrane structure was observed under transmission electron microscopy.The vesicle diameter was measured by Nanosight.The positive detection of exosome markers CD9 and CD63 by flow cytometry and Western blot confirmed that the extracted vesicles were indeed exosomes.5-FU(0,0.1,1.0?g/ml)were used to treat gastric cancer cells MKN74 for 48 h and 72 h,respectively.Exosomal PD-L1 level was detected by Western blot.Results showed that MKN74-derived exosomal PD-L1 was significantly increased after 5-FU treatment compared with control group.These results suggest that 5-FU upregulates exosomal PD-L1 derived from gastric cancer cells.3.Exosomal PD-L1 PromotionJurkat T cell apoptosis.MKN74/shPD-L1 cells stably knocked out of PD-L1 and control MKN74/NC cells were constructed,and exosomes derived from MKN74/shPD-L1 and control MKN74/NC cells were extracted,respectively,to act on activated Jurkat T cells,and the apoptosis in activated Jurket T cells was detected by flow cytometry.The results showed that MKN74/NC cell-derived exosomes induced Jurkat T cell apoptosis significantly higher than MKN74/shPD-L1-derived exosomes.These results suggest that exosomal PD-L1promotes Jurkat T cells apoptosis.4.Exosomal PD-L1 significantly inhibited the early activation marker CD69 of T cells,but not the late activation marker CD25.MKN74/shPD-L1 and MKN74/NC cell-derived exosomes from control group were used to act on activated lymphocytes in PBMC,and the changes of lymphocyte activity were detected by flow cytometry.The results showed that MKN74/NC cell-derived exosome-treated group down-regulated the early activation marker CD69 of T cells significantly more than MKN74/shPD-L1cell-derived exosome-treated group compared with untreated activated PBMC.The downregulation of CD69 by MKN74/NC cell-derived exosomes was partially reversed after the addition of the PD-1 inhibitor.In addition,the results showed that MKN74/shPD-L1 and MKN74/NC cell-derived exosome-treated groups downregulated the late activation marker CD25 of T cells less significantly than untreated activated PBMCs.These results suggest that exosomal PD-L1 significantly inhibits the early activation marker CD69 of T cells,but not the late activation marker CD25 of T cells.5.Fluorouracil upregulates Exosomal PD-L1 in peripheral blood of patients with gastric cancer,but it is not time-dependent.We obtained blood samples at baseline and after different chemotherapy cycles from 21 patients with stage III-IV gastric cancer who underwent single-agent multi-cycle chemotherapy with fluoropyrimidine.Exosomes in plasma samples were extracted with an exosomal isolation kit and exosomal PD-L1 level was detected by ELISA.Results showed that exosomal PD-L1 was upregulated after 2and 4 cycles of fluorouracil chemotherapy compared to baseline levels(N=21,P=0.009;N=10,P=0.047).Only 3 patients received 6 cycles of chemotherapy,and exosomal PD-L1 upregulated after 6 cycles of chemotherapy(N=3,P=0.023)compared with baseline levels.These results suggest that fluorouracil upregulates exosomal PD-L1 in peripheral blood of patients with gastric cancer,but it is not time dependent.6.Fluorouracil inhibits immunostimulatory cytokines IL-2,IFN-?,GM-CSF,TNF-?and IL-6 after 2 cycles of chemotherapy,but the inhibition is not obvious after 4 and6 cycles of chemotherapy.Bio-Plex Pro Human Cytokine 8-plex Assay detected the levels of 8 cytokines in 21 patients at baseline and after different cycles of chemotherapy.The results showed that after 2 cycles of chemotherapy,immunostimulatory cytokines IL-2,IFN-?,GM-CSF,TNF-?and IL-6 significantly reduced(P=0.009,P=0.014,P=0.014,P=0.004,P=0.031),no significant changes in immunostimulatory cytokines IL-4,IL-8 and the immunosuppressive cytokine IL-10.In addition,compared with baseline levels,there were no significant changes of all eight cytokines after 4 and 6cycles of chemotherapy.These results suggest that fluorouracil inhibits immunostimulatory cytokines IL-2,IFN-?,GM-CSF,TNF-?,and IL-6 after 2 cycles of chemotherapy,but does not show significant inhibition after 4 and 6 cycles of chemotherapy.7.Circulating exosomal PD-L1 amounts were elevated obviously in non-responders,while the absolute counts of T cell subgroups and the levels of eight cytokines did not change significantly.Query the evaluation of fluorouracil therapeutic effect of 21patients based on imageology from the Hospital Information System(HIS)of the First Affiliated Hospital of China Medical University.According to the objective response rate,among 21 gastric cancer patients,those showing treatment efficacy as complete response(CR)or partial response(PR)were considered responders.Meanwhile,patients whose treatment efficacy was stable disease(SD)or progressive disease(PD),were considered non-responders.After collating and analyzing,we observed that circulating exosomal PD-L1 amounts were elevated obviously in non-responders while only slightly increasing or even decreasing in responders(P=0.018).Meanwhile,the absolute counts of CD4~+and CD8~+T cells were decreased more significantly in non-responders compared with responders,although no statistically significant differences were observed(P=0.1025 and P=0.6689 respectively).Additionally,the changes of cytokines showed no differences between responders and non-responders.8.5-FU upregulates the expression of STAT5a thus promotes PD-L1 expression.Western blot detected the expression of STAT5a and its phosphorylation level after treatment of MKN74 and MGC803 with 1.0?g/ml 5-FU for 6 h and 12 h.The results showed that compared with the control group,STAT5a and its phosphorylation level were significantly increased.Western blot was used to detect the changes of PD-L1expression level after STAT5a was knocked down in MKN74 and MGC803 with small interfering RNA(siRNA).The results showed that compared with the control group,MKN74/siSTAT5a and MGC803/siSTAT5a cells reduced PD-L1 expression.These results suggest that 5-FU promotes PD-L1 expression through upregulation and activation of STAT5a.9.5-FU promotes STAT5a and PD-L1 expression by inhibiting Cbl-b.The results of co-immunoprecipitation experiments show that Cbl-b can directly bind to STAT5a.Cbl-b of MGC803 was silenced with siRNA,and then treated with 1.0?g/ml 5-FU for 72 h.The expression of Cbl-b,STAT5a and PD-L1 was detected by Western blot.The results showed that 5-FU treatment of MGC803 downregulated Cbl-b expression,while silencing Cbl-b increased 5-FU-induced up-regulation of STAT5a and PD-L1.These results indicate that Cbl-b can directly bind to STAT5a and inhibit its expression by ubiquitination of STAT5a.5-FU upregulates STAT5a and PD-L1 expression by inhibiting Cbl-b.10.5-FU inhibits Cbl-b expression by upregulating miR-940 thus promotes STAT5a and PD-L1 expression.The results of Blast comparative analysis showed that miR-940binding sites were present in the 3'UTR of the Cbl-b transcript.RT-PCR analysis showed that the relative level of miR-940 was significantly increased in MGC803 cells following5-FU treatment(P<0.01).Western blot results showed that Cbl-b expression decreased after MGC803 cells were transfected with miR-940 mimics.Next,MGC803 cells were transfected with mimics of miR-940 or negative control followed by 5-FU treatment.Western blot results showed that transfected mi R-940 mimics further decreased 5-FU induced Cbl-b expression,whereas p-STAT5 and PD-L1 were increased.These results indicate that 5-FU inhibits Cbl-b expression by up-regulating miR-940,and promotes up-regulation of STAT5a and PD-L1 expression.Conclusion:1.5-FU upregulates the expression of Exosomal PD-L1 in a time-and dose-dependent manner.2.Exosomal PD-L1 derived from gastric cancer cells promotes T cell apoptosis and inhibits T cell activation.3.Immunosuppressive effect of Fluorouracil is mainly through increasing circulating blood Exosomal PD-L1,accompanied by immune-activating cytokine suppression.4.5-FU can upregulate PD-L1 expression through the miR-940/Cbl-b/STAT5a axis. |
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