Experimental Research On Gene Expression And T Lymphocyte Immunosuppression Correlated With Hepatocellular Carcinoma Metastasis Of Rat Hepatic Stellate Cell

Hepatocellular carcinoma(HCC) is one of the malignant tumor of the highest incidence.The rapid development of cellular and molecular biology has deepened the understanding of the HCC formation mechanism.Currently,the most accepted hypothesis describes a step-by-step process of HCC in which external stimuli including hepatitis virus and other carcinogenic factors induce genetic or epigenetic alterations in mature hepatocytes resulting in the progression of normal cells through preneoplastic states into invasive cancers.However,none of the findings has turned into a breakthrough in the prevention and treatment for HCC.One reason is that the previous studies of HCC mostly focused on liver cells,and ignored the important role of mesenchymal cells in the malignant transformation.Gene and cell biology research for malignant tumor showed that interaction between parenchymal cells and stromal cells is important foundation of cancer formation and development.Mesenchymal cells include myofibroblasts,inflammatory cells,endothelial cells and so on, interacting with parenchymal cells by secreting cytokines,chemical factors, extracellular matrix(ECM) and direct cell-to-cell contiguity.The stroma cells are considered to be correlated with the angiogenesis,desmoplasia of the cancer and tumor immunity,and are therefore an important player in the progression,growth and spread of cancer.Studies about the role of tumor troma in the tumor development and progression are therefore becoming hot spot.In the liver,hepatoeytes represent the major cell type of the parenchyma,whereas mesenchymal compartment is composed of various cell types including Kuffper cells and hepatic stellate cells(HSC).HSC,also referred as Ito cells,lipocytes,or perisinusoidal cells,comprise 5%-8%of the total number of resident liver cells.They localized in Disse and characterized by their long processes by which they closely contact with hepatic sinusoid endothelial cells and hepatocytes.These provide the anatomical basis for the interaction between HSC and hepatocytes.Activation of HSC represents a final common pathway of the hepatic response to liver injury.In response to both acute and chronic liver injury,the normally quiescent HSC undergo a progressive activation and transdifferentiate into proliferative,expressingα-SMA, fibrogenic,proinflammatory and contractile myofibroblasts.Through increased secretion of cytokines,chemokines,growth factors,and extracellular matrix proteins and decreased degradation of extracellular matrix,activated HSC are responsible for modification,deposition,and accumulation of the majority of the excess extracellular matrix,resulting in liver fibrosis.Recently,studies suggested that it is possible that HSC play a great role in the progression and metastasis of HCC.Yet,the knowledge about the mechanism of activated HSC involving in the pathological event of HCC is currently limited.So far,a large body of knowledge has evolved regarding primary non-immunological functions of stellate cells including vitamin A homeostasis,liver fibrosis and regulation of hepatic blood flow.Profound studies extended the scope of stellate cell capacities and suggested a potential immune role in HSC.Accordingly, stellate cells were shown to express pattern recognition receptors including Toll-like receptors(TLRs),costimulatory molecules and cytokines required for modulation of immune responses.Most recent work demonstrated that hepatic stellate cells represented powerful antigen presenting cells(APC),which induce CD1-,MHC-Ⅰ-and MHC-Ⅱ-restricted T cell activation and mediate anti-infection immunity. Moreover,novel studies suggested a central role for stellate cells in retinoic acid mediated T cell differentiation.Activated HSC can also induce T cells apoptosis and assume the responsibility for immune equilibrium in liver.This overview takes you on a journey from non-immunological to immunological functions of stellate cells in the liver.At present,there are no related reports in what HSC immunological function is in HCC,and whether HSC immunological function is associated with the metastasis and recurrence of HCC.This subject is chiefly to investigate defferentially expression genes between induction-activated HSC(iHSC) and culture-activated HSC(aHSC).We confirmed that iHSC are different from aHSC in the biological function,which provided theretical foundation for studying relationships between iHSC immunosuppression and HCC metastasis.Subsequently,establishment of rat HCC lung metastatic model offered an experimental platform by which we can study correlation between intratumoral HSC(tHSC) immunology and HCC metastasis.Eventually,isolation and purification of HSC and T lyphocytes paved a way for exploring interaction between tHSC and T cell as well as studying potential mechanism of HSC immunosuppression and invasive and metastasis,thus provided experimental basis for in vivo and intervention study.The aim is to provide new immunotherapy target for HCC metastasis.Part one Gene different expression between culture-activated hepatic stellate cell and hepatocellular carcinoma cells induction-activated hepatic stellate cellThe main aim of this part was to study whether gene expression profiles are different between two kind of HSC in HCC and normal liver,and provide theoretical basis for follow-up investigation.We compared induced HSC by C5F cell lines conditioned medium with in vitro cultured HSC.Rat HSC were isolated from rat livers by perfusion of collagenase and pronase,followed by centrifugation over Nycodenz gradient.To prepare C5F conditioned medium induce HSC activation. 27100 gene expression was analyzed by cDNA microarray in quiescent, culture-activated and induction-activated HSC,and confirmed by real time polymerase chain reaction and Western blot analysis.Results demonstrated that 1967 genes were differentially expressed in iHSC and aHSC,including novel genes that encode proinflammatory mediators;cell adhesion moleculars;cell surface receptors; transduction molecules;and immunity factors.Some genes including Ccr1,Vcam1, Tagln,Colla1,Xlkd1 and Ccl24 were co-expressed in induction-activated and culture-activated HSC.Some genes including cystatin F,MMP-9,Jun,IgG-2a,Il7r, IAP1 and Igf1 were specifically expressed in induction-activated HSC.HCC cell induction-activated HSCs showed specific gene expression patterns,whereas culture activation only partially reproduced the gene expression changes observed during induced activation,suggesting that HCC cells can induce HSC activation. Induction-activated HSCs play a major role in HCC during invasion and metastasis. The above suggested that induce-activated HSC gene expression patterns are different from culture-activated HSC.Because culture activation does not properly regulate gene expression in HSC,in vivo activation should be considered the gold standard for the study of HSC biology.Part two Establishment of rat hepatocellular carcinoma lung metastasis model and primary study of intratumoral HSC immunityThe main aim of this part was to study whether T cell apoptosis appeared in hepatocellular carcinoma organization as well as correlation with HCC metastasis.We established rat hepatocellular carcinoma lung metastatic model.First,we injected rat HCC cell line McA-RH7777 1×10~6 into flank subcutaneously in 2 Buffalo rat.When subcutaneous tumor diameter is chalking to 1cm,they were cut into 1mm3 size and implanted rat liver left lobe(n=20).We observed hepatic tumor growth situation by B ultrasonic and MRI and drawn tumor growth curve.Randomly selected 1,2,3 and 4 weeks rats(n=3) and executed.Liver and lung tissue were dyed by HE,and lung metastasis focus amounts were counted.Normal liver and HCC tissue were detected by a-SMA and CD3 immunohistochemical staining or double staining,Tunel and CD3 double staining,then positive cells were counted(cell numbers/mm~2). Relationship in intratumoral HSC(tHSC),T cell apoptosis and lung metastasis was investigated by correlation analysis.Intratumoral HSC and spleen T lymphocytes were cocultured in vitro.Results showed that rat tumor formation rate was 100%,and the tumor in liver was clearly visible to one week by B ultrasonic localization, thereafter growing gradually.Metastatic tumor focus in lung occurred in all rats to four weeks by MRI detection.These are demonstrated by HE staining and pathology. We observed a-SMA expression around and within all HCC,and little in normal liver tissue.A-SMA content gradually increased with prolonged time.In normal liver, 1,2,3 and 4 week HCC,the number of a-SMA positive cells are 2.3±0.8,85±5.8, 151±12,239±14.7 and 321±16.9/mm~2 respectively(P＜0.05).Double CD3 and TUNEL stained firmed T cell apoptosis.The apoptotic T cells in HCC were more frequent than in normal liver and may spatially associated to tHSC.In normal liver,1, 2,3 and 4 week HCC,the number of apoptotic T cell are 1.5±0.5,43±2.4,82±3.1, 99±6.5 and 91±11/mm~2 respectively(P＜0.05).T cell apoptosis directly correlated to tHSC(r=0.711,P＜0.01),and lung metastasis directly correlated to T cell apoptosis in HCC(r=0.561,P＜0.05).To compare with activation T cells and quiescent HSC or quiescent T cells and tHSC cocultivation,T cell apoptosis was more frequently induced in cocultivation of T cell and tHSC(P＜0.05),moreover,tHSC was associated with T lymphocytes through direct contact.The above showed that T lymphocytes by permeating and infiltrating into HCC tissue may be induced apoptosis by tHSC, consequently associated with HCC metastasis.This model has high rates of tumor formation and survival,the activation consistency of HSC around and within HCC, and the histologic form similar to human HSC-T cells patterns,therefore is a reliable animal models that studied HSC immunological function in HCC as well as HCC metastasis correlation.Part three Experimental research of rat intratumoral HSC inhibit T lymphocyte functionA great deal of activated HSC in HCC tissue are associated with HCC metastasis. But little were known about intratumoral HSC(tHSC) immune response role in HCC. The main aim of this part was to further understand of tHSC immunosuppressive function and mechanism.To study tHSC inhibiting T lymphocyte function in vitro. The main methods are the isolation,culture and purification of HSC from Buffalo rat normal liver and HCC tissue.To detect tHSC surface molecules and gene expression by flow cytometry and fluorescence quantitative RT-PCR.To observe T cell proliferation and cytotoxic activity by ~3H thymine(~3H-TdR) incorporating and releasing experiment.To test IL-2,IL-10 and IFN-γexpression level in T cell by application of ELISA.TUNEL was used as T cell apoptosis detection method.To observe tHSC inhibition to T cell and then the influence of tumor invasion and movement ability by application of Transwell method.The above research results showed that quiescent HSC expressed many important surface molecules such as CD40,CD54,RT1A,RT1D,CD80 and expressed less B7-H1.Expression of CD40, CD54,RT1A,RT1D and CD80 in tHSC were downregulated,but B7-H1 upregulated (P＜0.05).Intratumoral HSC produced many immunosuppression related factors. Addition of the tHSC(but not quiescent) suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation and antigen-specific T cell killing activity in a dose-dependent manner.Addition of tHSC, compared with without addition,T cell apoptosis quantity increased significantly (P＜0.01).Addition of anti-B7-H1 antibodies decreased T cell apoptosis(P＜0.05). Addition of tHSC in activated T cells,compared to without addition,the movement and invasive ability of McA-RH7777 cells were significantly enhanced(P＜0.05). Addition of B7-H1 resistance antibodies,compared to without anti-B7-H1 antibody, weakened tumor cells movement and invasive ability(P＜0.05).Results indicated that high cytokine production by the T cells suggested that the inhibition was probably not a result of suppression of their activation.T-cell division was also found to be normal. The tHSC-induced T cell hyporesponsiveness was associated with enhanced T-cell apoptosis.Intratumoral HSC was associated with markedly enhanced expression of B7-H1.Blockade of B7-H1/PD-1 ligation significantly reduced tHSC immunomodulatory activity,suggesting an important role of B7-H1.In conclusion, the interactions between tHSCs and T cells may contribute to HCC recurrence and metastasis.The above in vitro study provide feasibility basis for contiguous in vivo experiment and interventional investigation.Conclusions1 Gene expression profiles of HCC cell induction-activated HSC are different from culture-activated HSC.HCC cell induction-activated HSC showed specific gene expression patterns.This study provided theoretical foundations for investigating biology of HSC in HCC.2 The established rat HCC lung metastatic models were verified with correlation between intratumoral HSC immunosuppression and HCC metastasis,which provides an experimental platform for further research on relationships between intratumoral HSC immunosuppression and HCC metastasis.3 Intratumoral HSC inhibit activated T lymphocyte function mainly through promoting T cell apoptosis,but not restraining T cell activation.The intratumoral HSC obviously improved the B7-H1 of expression.Blockade of B7-H1/PD-1 connection decreased intratumoral HSC immunosuppression activity,suggesting B7-H1 important role.Inhibition of T lymphocytes by intratumoral HSC may contribute to HCC recurrence and metastasis. Novelty1 To confirm that there are different gene expression patterns between HCC cell induction-activated HSC and culture-activated HSC first time.2 To prove that Morris HCC model can be used for studying relationships between intratumoral HSC immunosuppression and HCC metastasis first time.3 Preliminary proof suggest that intratumoral HSC promoted T lymphocytes apoptosis,then may contribute to HCC recurrence and metastasis first time.Potential merits for clinical application1 Intratumoral HSC was a potential therapeutic targets inhibiting HCC recurrence and metastasis.2 This study provided experimental basis for further research the mechanisms of intratumoral HSC immunosuppression and the invasion and metastasis of HCC.