Study On The Relationship Among Hepatic Stellate Cells, Their Associated Inflammatory Molecules And Metastasis/Recurrence In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a frequent malignancy accounting for90%of primary liver cancer and the third leading cause of cancer death worldwide. In China, it has already developed into the second highest cancer killer since the1990s. Typically, high mortality is involved in the lack of early diagnosis and therapeutic options. Indeed, atypical clinical manifestations of early HCC patients, the absence of specific markers for monitor and deficiencies of the present treatment strategies are limiting the further improvement of the prognosis. Even with curative resection which is supposed to be the most effective treatment in well-selected candidates, the5-year recurrence rate is still keeping more than60%. Therefore, it is of great urgency to find a way to accurately predict prognosis, reduce recurrence and metastasis after hepatectomy and perform an effective targeted intervention.Cumulative evidence indicates HCC is a clear example of inflammation-related cancer mainly due to exposure to hepatitis viral infection and cirrhosis. The chronic inflammatory status in HCC lead to an intricate tumor microenvironment which is a complex and dynamic network composed of a large variety of inflammatory/immune cells and mediators. Thus, to a large extent, HCC metastatic biologic behavior and poor prognosis may be determined and/or influenced by the local inflammatory status. Several lines of evidence suggest that during hepatocarcinogenesis, some inflammatory/immune cells can be activated and cooperate with each other to acquire more potent tumor-promoting activities and result in poorer prognosis. In this regards, these cells and some critical mediators in them therefore could provide us available target to constitute a promising therapeutic strategy for HCC. Notably, hepatic stellate cells (HSCs), myofibroblast-like inflammatory cells under activated state, display plastic phenotype and properties of fibrogenic cells. Recently, the role of HSCs in tumor microenvironment has gained a major interest. To reveal the mechanisms involved in cancer progression, various interpretations were raised. On one side, for both tumorigenesis and hepatic fibrogenesis, interventions with certain active signals in HSCs such as transforming growth factor (TGF)-β,hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) execute key roles in these pathological events. On the other side, upon activation directly induced by tumor cells, HSCs are conferred potency as a promoter in angiogenesis, invasiveness and metastasis through the molecular framework of tumor-stromal interaction. Moreover, it was reported that in tumor microenvironment, HSCs are perisinusoidal fibroblasts which transdifferentiate into cancer associated myofibroblasts (CAMFs) in response to paracrine factors secreted by cancer cells. Despite of the mechanisms of HSCs implicated in tumor progression on the mentioned findings, very little is still known about the nature and modulation of activated HSCs facilitating metastasis and recurrence in HCC.In this study, we first analyzed the gene expression profile of peritumoral HSCs by gene microarray, and also studied the relationship between the expression levels of several most characteristic HSCs phenotype/gene markers and the recurrence and metastasis of HCC using tissue microarray; then, based on the results of gene microarray of HSCs, an inflammatory factor TREM-1, also as probably a functional gene in HSCs, was selected as an object in our project to investigate the potential mechanisms that facilitate the migratory ability of tumor cells, moreover, the association of TREM-1and the prognosis of patients with HCC was set up; finally, given that IL-17receptors were expressed in HSCs (based on HSCs gene expression profile), the interaction between tumor-activated HSCs and IL-17+CD4+T cells was studied. Our data suggested the protumor power of different types of inflammatory/immune cells was probably involved in their crosstalk in HCC microenvironment.Part I Study on gene expression profile of tumor-activated hepatic stellate cells in immuno-microenvironmentThis study aims to investigate the phenotypic changes, density, location and differentially expressed genes of HSCs in HCC microenvironment based on the gene expression profile and phenotypic analysis. Also, this present project explored the valuable information on the prognosis and therapeutic targets of HBV related HCC in order to study the functional roles of stromal cells including HSCs during recurrence and metastasis of HCC in immuno-microenvironment.In this study we enrolled a random cohort containing224HBV related HCC patients following radical surgical resection in Zhongshan Hospital, Fudan University in2007. First, the expression of five most prominent phenotype markers of HSCs including α-SMA, glial fibrillary acidic protein (GFAP), Desmin, Vinculin and Vimentin were evaluated, and their association with metastasis and recurrence of HCC was studied; then, in vitro effect on phenotypic changes (e.g. GFAP, fibronectin, CD56and17R) of HSCs cell line LX-2induced by HCC conditioned medium was detected by flow cytometry; finally, HSCs and cancer associated myofibroblasts (CAMFs) were isolated respectively from normal, peritumoral human livers and cancer tissues to perform gene microarray analysis.The results showed that (1) Desmin and GFAP were both negatively expressed in all tissue sections. Vinculin and Vimentin were expressed ubiquitously on mesenchymal cells and parenchymal cells; both intratumoral and peritumoral a-SMA were expressed in mesenchymal cells, which showed irregular morphology and spindle or star-like shapes.(2) Peritumoral α-SMA was associated with tumor size (P=0.015), tumor differentiation (P=0.002) and TNM stage (P=0.028); both univariate and multivariate analysis verified that peritumoral α-SMA showed prognostic values for both time to recurrence (TTR) and overall survival (OS). Significantly, the predictive significance of peritumoral a-SMA was confirmed in early recurrence and AFP-normal subgroups (both P<0.05); no predictive value was found in peritumoral and intratumoral Desmin, GFAP, Vinculin and Vimentin as well as intratumoral α-SMA (P>0.05for OS and TTR).(3) The frequency of GFAP+HSCs was decreased when exposed to TCM from HCC cell lines (MHCC97L, HCCLM3and HCCLM6, P<0.05), other investigated biomarkers (fibronectin, CD56and IL-17R):showed no significance.(4) Compared with quiescent HSCs, peritumoral HSCs and intratumoral CAMFs expressed considerable up-and down-regulated genes associated with biological process, cellular component, molecular function and signaling pathways involved in fibrogenesis, inflammation and progress of cancer; by qRT-PCR, some identified genes confirmed the reliable results obtained in DNA microarray.These results suggested that HSCs are a kind of inflammatory cells constituting an integral part of immuno-microenvironment. Response to inflammatory stimulation in tumor microenvironment, heterogeneity of their phenotype and functional genes can result in a transdifferentiation process from quiescent state into activated cells or myofibroblast-like cells, and subsequently play protumor roles via inner crosstalk with distinct immune cells and tumor cells. The patients with more peritumoral activated HSCs have worse outcome, which revealed that HSCs had important positions in HCC metastasis and recurrence. Comprehensive evaluations of gene expression, density, distribution and so on could understand in depth the local immune status of HCC, also present rational biomarkers for HCC risk and promising therapeutic targets.Part II Study on the expression of TREM-1in hepatic stellate cells and the relationship with hepatocellular carcinoma prognosis This study aims to explore the link between inflammatory cells and prognosis of tumors through the research on TREM-1expressed in HSCs (based on HSCs gene expression profile, see part Ⅰ). Also, we investigated the mechanism that inflammatory cells dynamically reroute the direction from proinflammation response toward protumor in immuno-microenvironment via various inflammatory signals related cascade amplification response.Here, Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of soluble TREM-1(sTREM-1) in activated HSCs supernatant stimulated by LPS or HCC TCM, and in92preoperative and postoperative plasmas of patients with malignancy and/or benign liver tumor/disease, respectively. Expression levels of TREM-1were assessed by immunohistochemistry in tissue microarray from240patients with HCC (testing cohort and validation cohort). The association between locations and density of TREM-1-positive cells and HCC metastasis and recurrence was investigated by survival analysis.The results showed that (1) TREM-1expression was detected in both intratumoral areas and peritumoral tissues in most samples. Both cancer cells and hepatocytes showed cytoplasmic positive staining. Moreover, immunofluorescence and immunocytochemistry showed TREM-1was highly expressed in CAMFs and activated HSCs.(2) Expression levels of sTREM-1, IL-6and TNF-a were increased in supernatants of activated HSCs co-cultured with HCC cells or normal hepatocytes (P<0.001). In a time-dependent manner, not dose-independent, the levels of sTREM-1from activated HSCs/myofibroblasts were all detected to elevate at30minutes induced by LPS (10μg/ml-lng/ml), and peaked at1-2hours.(3) Migratory ability was greatly changed in both types of HCC cells after incubation with activated HSCs/CAMFs-CM and TREM-1agonist or inhibitor.(4) Expression levels of sTREM-1, IL-6and TNF-a were up-regulated in plasmas of patients with HCC compared to benign liver tumors/diseases (P<0.005).(5) In testing cohort, peritumoral TREM-1expression level was found to be associated with other several variables including vascular invasion (P<0.001), tumor size (P=0.001) and high TNM stage (P<0.001); on univariate analysis of our data, high density of peritumoral a-SMA and TREM-1were associated with bad OS (both P<0.001) and TTR (both P<0.001); significant factors were used for further multivariate analysis. Increased peritumoral a-SMA and TREM-1can predict death (P=0.009and P=0.008, respectively) and elevated risks of recurrence (P=0.015and P=0.005, respectively); high peritumoral TREM-1level was more likely to suffer from tumor early recurrences (univariate analysis p<0.001and multivariate analysis p=0.006, respectively) rather than late recurrences; similarly, high density of peritumoral TREM-1significantly correlated with poor OS (P<0.001) and TTR (P=0.002) in validation cohort.Taken together, these data revealed that mesenchymal cells such as HSCs were pivotal components in the tumor immuno-microenvironment and closely related with HCC metastasis and recurrence. Through the effects of inflammatory signals or pathways, they amplify the inflammatory response in the tumor microenvironment after a series of cascade reaction and further strengthen the malignant biological behavior of tumor, and therefore lead to the invasion and metastasis of cancer cells. As an inflammatory signal, TREM-1is related with the malignant behavior of tumor and provides us a rational biomarker and a novel anti-inflammatory therapy target to make a judgment on prognosis and therapeutic effect after hepatectomy in HCC.Part Ⅲ study on the association among IL-17/IL-17RE, hepatic stellate cells and hepatocellular carcinoma prognosisThe purpose of this study is to explore the intercellular communication between the different inflammatory stromal cells (HSCs and Thl7) in the tumor microenvironment and their synergistic effect to promote tumor metastasis and recurrence, in order to further study the regulation between various inflammatory cells and tumor cells.We have already demonstrated several IL-17receptors (e.g. IL-17RA, RB and RE) expressed in HSCs based on the data of HSC gene expression profile. Therefore, IL-17receptors were selected as objects in this study. We designed and set up a set of tissue microarray containing a random cohort of300HCC patients after curative hepatectomy in Zhongshan Hospital, Fudan University. The expression and prognostic value of IL-17and IL-17R (A-E) were examined by immunohistochemistry. Six Th17associated cytokines (IL-6,-9,-17,-22,-17R and TNF-a) in serum (n=111) were quantified using enzyme-linked immunosorbent assays. Phenotypic features of IL-17+CD4+T cells were determined by flow cytometry analysis.The results showed that (1) IL-17receptor family members were focal, scattered and diffuse on various liver cells and cancer cells, which showed a variety of staining patterns; the localization of IL-17RA was very similar to that of IL-17RB, and the expression patterns of them in tissues were diffuse; moreover, IL-17RD and IL-17RE were located in similar staining patterns in mesenchymal cells besides parenchymal cells.(2) Peritumoral IL-17RE density had relationship with vascular invasion (P=0.004) and late TNM stage (P=0.012). None of clinicopathologic variables was found to be associated with expression levels of intratumoral IL-17RE and IL-17. IL-17RE density was significantly associated with TTR and OR in both peritumoral and intratumoral tissues (all P<0.001), and intratumoral IL-17density was an independent prognostic factor in this HCC cohort. By ROC analysis, combination of intratumoral IL-17RE and IL-17densities showed higher predictive accuracy on outcome of HCC patients than them alone. Combination of intratumoral IL-17RE and IL-17densities were found to be more likely to suffer from tumor early and late recurrences by univariate and multivariate analysis.(3) Expression levels of IL-6,-22,-17R and TNF-α were increased in serum of patients with HCC compared to hemangiomas patients (P<0.001). At postoperative5days, all of their expression levels were decreased (P<0.001).(4) Conditioned medium of peritumoral activated human HSCs induced expansion of circulating of IL17+CD4+and CD3+T cells significantly (P<0.01). There was no difference of primary peripheral IL-17+CD4+and CD3+T cells without stimulation between the groups of HCC patients and hemangiomas patients (P>0.05).Collectively, these data suggested that in tumor microenvironment, cytokines or receptors in different inflammatory cells built a bridge for intercellular communication. The synergistic effects between the distinct types of inflammatory cells may change the homeostasis of the tumor microenvironment and dynamically regulate the development of tumors. As the roles of IL-17and IL-17RE in the process of HCC metastasis and recurrence, they could provide useful predictors for triaging at-risk patients with poor prognosis of HCC following resection and also possible therapeutic targets against this disease.Conclusions1In gene expression profile, compared with quiescent HSCs, peritumoral HSCs and intratumoral CAMFs expressed considerable up-and down-regulated genes associated with biological process, cellular component, molecular function and signaling pathways involved in fibrogenesis, inflammation and progress of cancer;2Peritumoral α-SMA showed prognostic values for both TTR and OS. Significantly, the predictive significance of peritumoral α-SMA was confirmed in early recurrence and AFP-normal subgroups;3TREM-1expression was detected in cancer cells, hepatocytes and HSCs. Increased peritumoral α-SMA and TREM-1can predict death and elevated risks of recurrence; high peritumoral TREM-1level was more likely to suffer from tumor early recurrences rather than late recurrences. 4IL-17RE density was significantly associated with TTR and OR in both peritumoral and intratumoral tissues. Intratumoral IL-17density was an independent prognostic factor in this HCC cohort. Combination of intratumoral IL-17RE and IL-17densities were found to be more likely to suffer from tumor early and late recurrences by univariate and multivariate analysis.The novelty of this study1For the first time, we analyzed and reported the gene expression profile of peritumoral activated HSC and intratumoral CAMFs, and found considerable changed functional genes. By study on these genes, we could understand in depth the local immune status of HCC, also present rational biomarkers for HCC risk and promising therapeutic targets.2For the first time, we demonstrated that the expression of TREM-1was not only limited in myeloid cells, but also in hepatocytes, liver cancer cells and HSCs. The expression level of peritumoral TREM-1is an independent predictive indictor, showing its potential values as a rational biomarker and novel anti-inflammatory therapy target to make a judgment on prognosis and therapeutic effect after hepatectomy in HCC.3For the first time, we demonstrated that the prognostic ability of peritumoral and intratumoral IL-17RE in patients with HCC following resection, also found interaction of inflammatory cells promoted their proliferation.The potential application of this project1The density of various inflammatory/immune cells (HSCs and Th17) in tumor microenvironment is closely related with metastasis and recurrence of HCC after resection, which is beneficial in biological indicators for HCC risk monitor and promising therapeutic targets.2Numerous changed genes could be found from the analysis of the gene expression profiles of inflammatory/immune cells in HCC microenvironment. By mining and integration of these genes, a set of HCC risk prediction model could be created and provide guide for individualized treatment.