| Hepatocellular carcinoma (HCC) is one of the most common cancer in the world, and the third leading cause of cancer-related death worldwide, accounting for at least610,000deaths per year, nearly half of them in China. Despite tremendous achievements of cellular and molecular biology in HCC have been made during past decades, the prognosis of HCC remains dismal. Although surgery achieves the best outcome in well-selected candidates, the5-year recurrence rate remains as high as60-70%after HCC resection. At the same time, about80%of HCC cases are associated with chronic hepatitis (type B or C hepatitis) and cirrhosis in China, and most of them lost the chance to accept the currative operation. Although the systematic therapies including chemotherapy and targeted therapy were investigated deeply and applied widespread in the patients with late stage, the tumors always progress after several months treatment remains a major obstacle for more therapeutic efficacy.The novel high throughput analysis technologies, such as genomics, protemics and tissue microarray, greatly facilitate the studies on the progression and metastasis of tumor. However, none of the findings has turned into a breakthrough in the prevention and treatment for HCC. The vast majority of previous studies focused solely on malignant cells themselves, and ignored the important role of stromal cells in the malignant transformation. The invason and metastasis of HCC are complicated procedures with multi-step and multi-factor, and also depened on the interaction between malignant cells and stromal cells. 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 initiation and development of cancer. HSC, also referred as Ito cells, lipocytes, or perisinusoidal cells, comprise13%of the total number of liver cells. They localized in Disse and characterized by their long processes by which they closely embrace hepatocytes and contact with sinusoidal endothelial. The activation of hepatic stellate cells (HSC) is recognized as a central event in the development of hepatic fibrosis and lastly, cirrhosis. A three-step process of "inflammation-fibrosis/cirrhosis-HCC" is believed to be involved in hepatocarcinogenesis, the activation of hepatic stellate cells (HSCs) may serve as an important mediator in the process of inflammation-fibrosis-carcinoma axis, by increasing secretion of cytokines, chemokines, growth factors, and extracellular matrix proteins and decreasing degradation of extracellular matrix. Moreover, in HCC the stroma is infiltrated by activated HSC/myofibroblasts. Activated HSCs release paracrine factors that may promote hepatocarcinogenesis through unknown mechanisms including release of survival signals. Clinical evidences disclosed that peritumoral HSCs were related to more early HCC recurrences.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. Recently, studies suggested that it is possible that HSC play a great role in the progression of HCC, even in tumor metastasis, but the related mechanism remains unclear.Our research group confirmed that: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. Intratumoral HSC inhibit activated T lymphocyte function mainly through promoting T cell apoptosis, and then promote tumor cells and movement ability in vitro.In this study, we take induction-activated HSCs (iHSC) and McA-RH-7777hepatoma cells to co-culture, and confirm that iHSC alter expression genes and promote the growth and invation of McA-RH-7777hepatoma cells in vitro through the mechanism of paracrine secretion. iHSCs also enhance growth and invation of the subcutaneouly hepatoma in rat with the alteration of immune microenviroment. Subsequently, we investigate the interaction between iHSCs and T lyphocytes or DCs in vitro to explore potenial mechanism of HSC immunosuppression in HCC microenviroment, the results show iHSCs induce T cell apoptosis, but not promote DCs apoptosis. Eventually, we attempt to improve the immune microenviroment in liver by infusion DCs through portal vein to control the development and metastasis of HCC, and provided experimental basis and intervention study in vivo. The aim is to explore the novel immunotherapy for reccurence and metastsis of HCC Part1Rat hepatic stellate cells alter the gene expression profiles and promote the growth, migration, invation of hepatocellular carcinoma cells in vitroThe aim of this part was to investigate the induction-activated HSCs (iHSC) with tumor-CM alter the gene expression profiles and promote the growth, migration, of hepatocellular carcinoma cells in vitro through the mechanisms including release of paracrine factors. Rat HSCs were isolated from rat livers by perfusion of collagenase and pronase, followed by centrifugation over percoll density gradient. We prepared rat McA-RH7777hepatoma cells conditioned medium (CM) and induced HSCs activation in vitro. The iHSCs were collected and co-cultured with McA-RH7777cells in transwell systems. We compared the gene expression profiles of co-cultured McA-RH7777cells with the control McA-RH7777cells.28,728gene expression was analyzed by cDNA microarray, and confirmed by real time polymerase chain reaction and Western blot analysis. We prepared iHSC CM, and investigated wether iHSC CM promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through the experiments, such as CCK-8(cell count kit-8), scratch repair, transwell invation. The cytokines of supernant in the co-cultured McA-RH7777cells and the control McA-RH7777cells were assayed by ELISA and compared.Results demonstrated that573genes were differentially expressed in the co-cultured McA-RH7777cells and control McA-RH7777cells with more than2-fold up/down-regulated (among them,432genes were up-regulated and141genes were down-regulated), including cell surface receptors, metabolic process, cell adhesion moleculars, signal transduction molecules, chemokines and immunity factors. By the release of paracrine factors from iHSC, some genes in the co-cultured McA-RH7777cells were up regulated such as Tapbp, Ccl2, Cxcll, Cxcl10, Junb, Igfl, Statl, Irfl, Irf7, Irf9and Csfl, and some genes including4-Sep, Cck, Pdgfra, Itgae, Cd36were specifically down-regulated. iHSC also promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through release of paracrine factors. Compared with the control McA-RH7777cells, some cytokines of supernant were increased in the co-cultured McA-RH7777cells, such as HGF, IL-6, MMP-2, MMP-9. In conclusion, iHSCs can alter the gene expression profiles and promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through the release of paracrine factors. The above suggested that iHSC play a major role in HCC during growth, and metastasis.Part2Rat hepatic stellate cells promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment in vivoThe aim of this part was to investigate whether HSCs promoted the growth, invasion of hepatocellular carcinoma, involved the evolution of tumor immune microenviroment in vivo. McA-RH7777hepatoma cells (3×106cells) were injected into flank subcutaneously in Buffalo rats. After3weeks, the long diameter of subcutaneous tumor was about2cm, the tumor tissures were cut into1mm3size and implanted into rat liver left lobe (n=25). Hepatic tumor growth was observed by CT scan.At the time of1,2,3,4weeks after implantation, the rats (n=5) were randomly selected to sacrifice, liver and lung tissue were dyed by HE. Early stage (1week after implantation) and late stage (4week after implantation) hepatoma tissue were detected by a-SMA, CD3, CD68, and S-100immunohistochemical staining. Then positive cells were counted and compared in the early and late stage hepatoma tissue. Experiment of hypodermic tumorigenicity:Buffalo rats were divided into2groups (n=6), and injected subcutaneously with a cell suspension of0.2ml containing either:(A, control group)2×106McA-RH7777cells,(B, exprimental group) a mixture of2×106McA-RH7777cells and1×106induction-activated HSC. The tumor growth and immune microenviroment were detected and compared in the2groups. Experiment of tumorigenicity in liver:Buffalo rats were divided into2groups (n=6), and injected into each rat liver left lobe with a cell suspension of0.2ml containing either:(A, control group)2×106McA-RH7777cells,(B, exprimental group) a mixture of2×106McA-RH7777cells and1×106induction-activated HSC. The tumor growth and lung metastasis were detected and compared in the2groups. Results showed that rat tumor formation rate was100%. The tumors in liver were clearly visible at2week by CT scan, and the lung metastases were detected at4weeks and obviuosly at5weeks by CT scan. The rate of lung metastasis formation was also100%. We observed a-SMA expression within all HCC. Compared with the early stage HCC, the a-SMA content (intratumal HSC, tHSC) and macrophages increased while the dendritic cells (DCs) and T lymphocytes decreased in the late stage HCC. The induction-activated HSCs promote growth and invasion of hypodermic hepatoma in Bufallo rats with more HSCs, macrophages and less DCs, T lymphocytes in the tumor immune microenviroment. The mixture of McA-RH7777cells and induction-activated HSC innoculation in liver didn’t promote the gowth or metastasis of hepatoma, compared to the simple McA-RH7777cells. The above showed that the rat orthotope hepatoma model has high rates of tumor formation and lung metastasis. During the progression of hepatoma, the immune microenviroment changes and evolves with infiltrated tHSC increase and DCs, T lymphocytes decrease. Rat HSC promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment in vivo.Part3Preliminary experimental research on the mechanism by which rat activated hepatic stellate cells regulated the immunity microenviroment in hepatocellular carcinomaActivated HSC in HCC tissue are associated with immunosuppressive microenviroment and early recurrence. But little were known about HSC immune interaction with immunocytes in HCC. The aim of this part was to investigate iHSC immunosuppressive function in immune microenviroment and mechanism, and whether iHSCs effected T lymphocytes or DCs in vitro.We prepared rat McA-RH7777hepatoma cells conditioned medium (CM) and induced HSCs activation in vitro. T lymphocytes and DCs were isolated, purified and cultured respectively. The iHSCs were collected and co-cultured with actived-T lymphocytes (aT) or DCs respectively. To assay iHSC inhibition to aT cytotoxic activity by LDH releasing experiment. TUNEL was used to detect T cell apoptosis induced by iHSC. To observe iHSC inhibition to aT and then the influence of tumor invasion ability by application of Transwell method. Flow cytometry was used to detect DC apoptosis induced by iHSC. Experiment of adoptive immunity of DCs in liver:Buffalo rats were divided into2groups (n=6), the rats in control group were implanted into each rat liver left lobe with a1mm3size tumor tissue, the rats in intervention group were infused with DCs loaded with tumor antigen through hepatic vein and then implanted with tumor tissue. The tumor growth, lung metastasis and immunocytes in microenviroment were detected and compared in the2groups. Results showed that activated T cell apoptosis quantity increased significantly by iHSC induction in vitro while DC apoptosis quantity didn’t increase. Addition of iHSC induced activated T cells apoptosis, compared to without addition, the invasive ability of McA-RH7777hepatoma cells were significantly enhanced (P<0.05) in Transwell systems. Compared to the control group, tumor weight and lung metastases were decreased and local immune state were improved (P<0.05) in the intervention group which accepted adoptive immunity of DCs. In summary, iHSC promotes HCC invasion and regulates immune microenviroment through the mechanism of which iHSC induced activated T cell apoptosis, not DC apoptosis.Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of hepatoma, and improves local immune state in vivo. The above in vivo study provide feasibility basis for therapy of HCC.Conclusions1. Induction activated HSCs can alter the gene expression profiles and promote the growth, migration, invasion of hepatocellular carcinoma cells in vitro through the release of paracrine factors. The above suggested that iHSC play a major role in HCC during growth, and metastasis.2. The rat orthotope hepatoma model has high rates of tumor formation and lung metastasis. During the progression of hepatoma, the immune microenviroment changes and evolves with infiltrated tHSC increase and DCs, T lymphocytes decrease. Rat HSC promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment. in vivo.3. Induction activated HSC promotes HCC invasion and regulates immune microenviroment through the mechanism of which iHSC induced activated T cell apoptosis, not DC apoptosis.Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of hepatoma, and improves local immune state in vivo. The above in vivo study provide feasibility basis for therapy of HCC.Novelty of this study 1For the first time, we confirm that induction activated HSCs can alter the gene expression profiles and promote the growth, migration, invasion of hepatocellular carcinoma cells through the release of paracrine factors.2To confirm that induction activated HSC regulates HCC immune microenviroment, and iHSC does not enhance DC apoptosis. Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of HCC, and improves local immune state in vivo.The potential application of this study1Induction ctivated HSC in HCC microenviroment was a potential biomark of HCC characteristics. This study provided experimental basis for further research the mechanisms of intratumoral HSC immunosuppression in HCC immune microenviroment.2HSC was a potential treatment target inhibiting HCC recurrence and metastasis, especially for HCC associated with fibrosis/cirrhosis. |
PDF Full Text Request