The Stemness Of Hepatocellular Carcinoma Involving Bile Duct Tumor Thrombi And CD90~+ HepG2/DC Fusion Cells Induced Immune Response Against Hepatocellular Carcinoma

Part Ⅰ The clinicalpathological features of and liver cancer stem cell marker expression in hepatocellular carcinoma involving bile duct tumor thrombiBackground Hepatocellular carcinoma(HCC)commonly invades portal vein,hepatic vein or inferior vena cava and form venous thrombi.HCC involving bile duct tumor thrombi(BDTT)is a rare event,which incidence is approximately 1.66%—13%.The clinicopathological feature of this kind of HCC is still not well classified.The BDTT is usually considered as the result of bile duct invasion and the symbol of late stage HCC.However,several research centers have reported that BDTT might occur at a very early HCC stage when the primary tumor was small or even undetectable.Therefore,HCC involving BDTT is reported that this kind of HCC might origin from liver stem cells,which locate at the Hering duct.In this study,in the purpose of illuminating the relationship between HCC involving BDTT and the liver stem cells,the clinicopathological feature and liver stem cell markers expression of 35 patients with HCC involving BDTT of943 HCC patients who underwent surgical treatment at our hospital were reviewed retrospectively.Objectives To illuminate the clinicopathological features of HCC patitients involving BDTT,guiding the diagnosis and treatment of this kind of HCC.To analyze the expression of liver cancer stem cell markers in the tissue of HCC patitients involving BDTT and explore the relationship betweent the liver cancer stem cell and this kind of HCCMethods 1.The clinicopathological data of 35 patients with HCC involving BDTT from January,2005 to December,2012 were reviewed retrospectively.2.The expression of liver stem cell marker: CD133,CD90,CK-19,Ep CAM,Ckit and VEGF on the tissue of 35 HCC patients and the control cases were analyzed by immunohistochemistry(IHC).3.The relation between the expression of liver stem cell markers and the clinicopathological feature was estimated,and Kaplan-Meier method was adopted for evaluating survival.Results 1.The results of univariate analysis showed that Child-Pugh classify,tumor size,micro-vessel invasion,liver cirrhosis and tumor differentiation might impact the prognosis,and multivariate analysis picked up the tumor differentiation as prognosis factor.2.The IHC results revealed that the expression of liver stem cell markers CD133,CD90,CK-19,Ep CAM,C-kit and VEGF in HCC involving BDTT were significantly higher than HCC patients without BDTT.And the pathologic differentiation degree of HCC involving BDTT was negatively correlated with the expression of liver stem cell markers.3.Kaplan-Meier survival analysis showed the high expression of liver stem markers in HCC involving BDTT resulted in poor prognosis(P < 0.001).Conclusions 1.HCC involving BDTT were high expressed of liver stem cell markers,and the expression was negatively correlated with the pathologic differentiation degree.2.The prognosis of HCC involving BDTT was significantly poor than HCC without BDTT.3.The high expression of liver stem markers in HCC involving BDTT reveal the close relationship between them,and implied the possibility of HCC involving BDTT originating from liver stem cells.Part Ⅱ CD90~+Hep G2/DC Fusion Cells Induced Immune Response against Hepatocellular CarcinomaBackground Hepatocellular carcinoma(HCC)is a common malignance worldwide with dismal prognosis.HCC is difficult for early diagnosis and no sensible to radiotherapy and chemotherapy.Furthermore,HCC is a kind of malignance easy to metastasis and recurrent.Despite continued advances in surgical treatment,the 5-year overall survival rate is still less than 15%.Consequently,new therapeutic strategies were urgently needed for improving the survival rate of HCC.According to recent studies,the relapse and metastasis of HCC may closely due to the cancer stem cells(CSCs),a small group of cells that are endowed with the ability to perpetuate themselves by self-renewal and then generate mature cells through differentiation.CSCs are responsible for tumor formation and progression because of these properties and the concept can explain the failure of conventional therapy.Therefore,targeting CSCs might be an effective therapeutic strategy for HCC treatment.Dendritic cells(DCs)are the most important and most powerful antigen presenting cells in vivo.Multiple co-stimulate molecules were expressed on the surface of DC,which help present antigens to T lymphocyte and activate CD4+ and CD8+ CTLs.In previous studies,DCs were loaded with tumor-specific DNA,tumor total RNA or tumor antigen peptide to generate DC vaccine.Unfortunately,DC vaccines based on single antigen were limited on the unknown of most tumor antigens.Furthermore,DC vaccines loaded with DNA or RNA would lead to the cross immune response to the normal host cells.The strategy of DC fusing with the tumor cells would avoid the disadvantages above.DC would obtain all the tumor antigen message including the known and unknown one through the fusion process.The fusion cells present antigens to lymphocyte mainly through MHCⅠ molecules and activate CD8+ CTLs,which are the mainly effective cells to kill tumor cells in vivo.Recent study reported that the DC vaccine loaded with prostate stem cell antigen could induce specific immune response to prostate cancer stem cells.This finding proves the possibility of DC vaccine targeting tumor stem cells.However,there is no report about fusion DC vaccine targeting HCC stem cells so far.Previous studies have proved that the HCC cells with high expression of CD90 on the surface have a strong ability to form colony in vitro and form tumor nodule in vivo.Beside,studies reported that the serum-free suspension sphere culture method was able to efficiently enrich the stem cells in vitro.Therefore,CD90~+ HCC stem cells were enriched and fused with DC to generate DC vaccine in this study,and then the biological feature and immune function of the fusion cells were explored.Objectives In this study,CD90~+ HCC stem cells were fused with DC to estimate the potential immune response against HCC stem cells in vitro and in vivo models.Methods 1.Generation of DC and T cells Peripheral blood mononuclear cells(PBMCs)were isolated from HLA-A2+ healthy donors by Ficoll-Hypaque densitygradient centrifugation,and were incubated in six-well polystyrene plates at 37℃ in 5% CO2 for 2h in RPMI-1640.After that,non-adherent cells were removed and purified by nylon wool column,and subsequently cultured in RPMI-1640 complete medium with 20U/ml rh IL-2 as a source of autologous T cells.2.CSCs sorting,and enriching by suspension sphere culture CD90~+ Hep G2 cells sorting was performed by flow cytometry.Subsequently,the sorted cells were cultured by the method of suspension sphere culture for enrichment.Then the CD90~+ Hep G2 cells were identified in vivo and vitro.3.CD90~+Hep G2/DC fusion cell preparation and analysis of the biological features CD90~+Hep G2/DC fusion cells were induced by PEG and the fusion rates were estimate by flow cytometry.Then the expression of surface costimulate molecules were detected.Besides,the fluorescence decay of PKH26 was used to detect the capacity of CD90~+Hep G2-DC to stimulate T cells.And the IL-12p70 level was detected by ELISA.4.The anti-tumor capacity of CD90~+Hep G2-DC fusion cells The ability of CD90~+Hep G2-DCs induced CTLs against HCC in vitro was detected by LDH release assay and the ability of CD90~+Hep G2-DCs to inhibit Hep G2 cells induced tumor formation in nude mice was also estimated.The IFN-γ secretion of CD90~+Hep G2-DCs induced CTLs was analyzed by ELISPOT.Results 1.Immature or mature DCs could derive from PBMC by in vitro culture.2.The CD90~+Hep G2 purified from Hep G2 cell line have strong ability to form colony in vitro and tumor formation capacity in nude mice.3.The CD90~+Hep G2-DCs fusion cells expressed high level of surface costimulate molecules,including CD83、CD80、CD86、HLA-ABC、HLA-DR.These fusion cells effectively stimulate the proliferation of T cells and release IL-12p70.4.The amount of IFN-γ release CTLs induced by CD90~+Hep G2-DC fusion cells was increase.The CTLs targeted CD90~+Hep G2 most effectively and inhibited the tumor formation in nude mice.Conclusions 1.The strategy of CD90~+Hep G2 fusing DC was possible.2.CD90~+Hep G2-DCs could induce special CTLs targeting CD90~+Hep G2 effectively,and inhibit the tumor formation in nude mice.3.The ability of CD90~+Hep G2-DCs induced CTLs to inhibit tumor growth in nude mice might be associated to targeting to CD90~+HepG2.