| BackgroundCholangiocarcinoma(CCA)is an intractable tumor originating from the epithelial cells in intrahepatic and extrahepatic bile ducts with poor prognosis.Surgical resection is the main curative treatment.However,most patients was diagnosed too late to resect or developed as metastatic disease.Moreover,lack of identifying biomarkers and efficient non-surgical therapy in current treatment caused non-second line therapy was demonstrated to improve long-term survival.Therefore,advanced understanding of molecular mechanisms of carcinogenesis is eagerly needed to develop novel therapies for the treatment of CCA.The costimulatory B7 family are cell-surface protein ligands,that can provide both stimulatory and inhibitory singal to regulation of T cell response.The B7 family comprises seven members,B7.1(CD80),B7.2(CD86),B7-DC(CD273,PD-L2),B7-H1(CD274,PD-L1),B7-H2(ICOS-L),B7-H3(CD276)and B7-H4(B7x,B7S1)(5).B7-H4,th e most recently identified member of the B7 family,is type I-transmembrane proteins via a glycosyl phosphate-dylinositol linkage with unidentified receptor.It has been shown that expression of B7-H4 is not detected in the majority of normal tissues,such as lung,colon,liver,kidney,pancreas,small bowel,breast,and uterus.However,several reports have recently demonstrated that the aberrant expression of B7-H4 protein is found in several tumour types,such as those of the breast,skin,lungs,colon,kidney,brain and ovarian.It is well known that the B7-H4 involved in the negative regulation of T cell-mediated immune responses in anti-tumor by inhibiting T cell activation,proliferation,cytokine production and cytotoxic activity.Furthermore,previous research reported that the expression level of B7-H4 is correlated with clinicopathological parameters and consider to be a prognostic maker in various tumors.However,the role of B7-H4 in tumor immunity and its correlation to different clinical outcomes are still controversial.In particular,expression of B7-H4 in CCA and its clinical significance have not been analyzed in detail.Further investigation of relationship between B7-H4 and CCA will be carried out and provide potential molecular targets for better methods of detection and treatment.Methods:1.Tissues were obtained from 137 patients who underwent surgery at the Southwest Hospital from 2005 to 2011.Patients who underwent pre-operative treatment,such as radiotherapy and/or chemotherapy,were excluded.A total of 110 cancerous samples and 28 lymph node metastatic samples from these patients were collected.In contrast,19 chronic inflammatory bile duct samples from patients with hepatolithiasis and 8 biliary adenoma samples were also collected from Southwest Hospital.The pathologic reports were reviewed.Expression of B7-H4 and TILs on paraffin clinical samples were evaluated by IHC.Correlations between B7-H4 expression and clinical features were analyzed.Correlation between tumor cell B7-H4 expression and densities of TILs were analyzed.2.A total of 110 CCA patients undergoing surgical resection between 2005 and 2011,in Southwest Hospital were included.Overall survival was calculated from the date of surgery until the date of death or last contact.Recurrence-free survival was computed from the date of surgery to the date of recurrence.Realtionship between B7-H4 and overall survival and recurrence were analyzed by using Kaplan-Meier and Multi-factor Cox models.3.Two human CCA cell lines,QBC939 and RBE,were cultured in present study.Cells were transfected with lentiviral vectors encoding short hairpin RNA targeting human B7-H4 for B7-H4 knockdown(shB7-H4)or a scrambled sh RNA as control(shControl).Impact of intervening B7-H4 expression on cell migration and invasion was evaluated by cell migration(wound healing)assay,cell invasion(transwell)assay respectively.4.Peripheral blood mononuclear cells(PBMCs)were isolated from 11 healthy donors.PBMCs were seeded into six-well culture plates containing 2ml RPMI-1640 and 10% FBS at a final concentration of 5–10x106cells/well.After 2 hours of incubation,non-adherent cells were removed by gentle washing with warm medium.The non-adherent cells(effector lymphocytes)were cryopreserved in FCS supplemented by10% DMSO.The resultant adherent cells containing DCs were cultured in medium supplemented with 500 U/ml recombinant human granulocyte-macrophage colony-stimulating factor(GM-CSF)and 1,000 U/ml recombinant human interleukin 4(IL-4)in37°C/5% CO2.Every 2 days,one-half of the medium was replaced by fresh medium containing a double concentration of GM-CSF and IL-4 as indicated above.On day 5,10 ng/ml of recombinant human tumor necrosis factor-α(TNF-α)was added to the medium to induce phenotypic and functional maturation of DCs.CCA cell were induced to apoptotic tumor cells(ATC)with 100ug/ml mitomycin for 24 hours as antigen,presented by DC and to induce specific CTL in vitro.On day 1,then non-adherent effector lymphocytes were co-cultured with ATC pulsed autologous DCs in a 6-well plate in the presence of 10 ng/ml recombinant human interleukin-7(IL-7).Half the medium was replaced with complete medium supplemented with 30 IU /ml recombinant human interleukin 10(IL-10)every 3 days.On day 7,lymphocytes were re-stimulated with ATC-pulsed autologous PBMCs in medium containing 10 ng/ml IL-7 and 20 U/ml IL-2.On day 10,after the fourth round of re-stimulation,cells were harvested and tested by CCA-specific CTLs assay.Knockdown of B7-H4 was performed in QBC939 and RBE cells by B7-H4-shRNA lentivirus transfection,respectively.By culturing with CCA-specific CTLs,the killing ability of CTL was analyzed by CTLs cytotoxic assay.Cytokine TGF-β,IL-6 concentration in the culture supernatant was tested by ELISA kit.Results:1.By IHC,we found that the expression of B7-H4 protein was detected in 54/110(49.0%)cancerous,18/28(64.2%)Lymph node metastatic and 4/19(21.1%)chronic inflammatory bile duct tissues,respectively.Positive staining of B7-H4 was detected in cancerous and lymph node metastatic samples,which was significantly higher than that observed in nontumorous tissues.Our data indicated that the high expression of B7-H4 was specific to the CCA tissues.B7-H4 expression in CCA tissues is significantly associated with tumor status(P = 0.025),lymph metastasis(P = 0.035)and UICC stage(P = 0.019)of CCA,but not with gender,age,tumor location and size.The patients with positive B7-H4 showed more aggressive pathological features,including higher tumor status,UICC stage and lymph node infiltration.As shown in Table 3,levels of B7-H4 expression on tumor cells were inversely correlated with the density of CD8+ T cells in tumor stroma(P = 0.0004),while they were not correlated with densities of CD8+ T cells in tumor nest(P = 0.776).In addition,there were no significant association between B7-H4 expression and CD4+ T cells in tumor stroma or nest(P = 0.567,P = 0.822)2.The median overall survival time of patients with negative and positive expression of B7-H4 were 19.5 and 12.6 months,respectively(P =0.015),and the median disease-free survival time of patients with negative B7-H4 expression was longer than that of patients with positive B7-H4 expression(16.7 vs.10.9 month,P P=0.046).Kaplan-Meier analysis showed that expression of B7-H4 is negative correlated with overall survival(log-rank p = 0.015)and recurrence(log-rank p= 0.046)in 110 cases.To validate this finding,the Multivariate Cox analysis was applied to the collective analysis of B7-H4 expression,gender,age,tumor location and TNM.The results showed that B7-H4 expression was an independent indicator of poor overall survival and early recurrence(HR = 1.786,95% CI = 1.110-2.872,p = 0.017;HR =2.062,95% CI = 1.160-3.665,p =0.014).Besides,tumor location was also a significant independent predictor of early recurrence(HR = 2.610,95% CI = 1.025-6.650,p = 0.044).In summary,our data suggested that aberrant expression of B7-H4 might be a risk factor and independent predictor of poor prognosis in CCA patients after surgical resection.3.Western blot analysis showed that B7-H4 was expressed in the QBC939 and RBE cells.Interestingly,the protein levels of B7-H4 were significantly higher in QBC939 cells than in RBE cells.Knockdown of B7-H4 was performed in QBC939 and RBE cells.By transwell assay,the number of invading cells was remarkably reduced by knockdown of B7-H4 in QBC939 and RBE cells.Consistently,cell migration was remarkably reduced by B7-H4 knockdown by wound-healing assay.Together,the data suggested an important role of B7-H4 in CCA cell invasion and metastasis.4.Knockdown of B7-H4 was performed in QBC939 and RBE cells by B7-H4-sh RNA lentivirus transfection,respectively.By culturing with CD8+ cytotoxic T cells,the killing ability of CD8+T was remarkably improved by knockdown of B7-H4 in both QBC939 and RBE cells.These dates indicated that knockdown of B7-H4 in tumor increase CD8+T–mediated cytotoxicity in vitro.The concentration of TGF-β,IL-6 in the culture supernatant was significantly higher than that of control.These dates indicated that knockdown of B7-H4 in tumor increase CD8+T–mediated cytotoxicity in vitro.Conclusions:In conclusion,our study showed that overexpression of B7-H4 associated with poor prognosis and promotes cell metastasis and invasion in CCA.We observed that aberrant expression of B7-H4 on CCA cell may greatly suppress the number and cytotoxicity of CD8+ T cells in tumor microenvironment.However,the cause of suppression of cytotoxicity may be that B7-H4 promotes secretion of TGF-β,IL-6. |
PDF Full Text Request