| Hepatocellular carcinoma (HCC) is one of the most common cancers and the second leading cause of cancer related death in China. Although surgical resection improves survival, the mortality rate remains high mainly because of frequent intrahepatic spread and extrahepatic metastasis.Consequently, elucidation of the molecular mechanism underlying HCC cell invasion is crucial to improve the clinical outcome of HCC therapy. Tumor microenvironment is the local pathological environment which has been shown to play critical roles in tumor invasion and metastasis and has become a focus in cancer research. Tumor associated macrophage (TAM), the main component of the inflammation cells that infiltrate most solid tumors in the tumor microenvironment, was primarily consisted of M2 tumor associated macrophages. In this study, we established the cell model of M2 tumor associated macrophage initially. Then the M2 tumor associated macrophages were cocultured with SMMC7721 cells in vitro to mimic the tumor microenvironment in the body using the three-dimensional cell culture apparatus BioLevitatorTM. The differentially expressed proteins by the three-dimensional cell coculture systems of M2 and SMMC7721 were screened by comparative analysis using quantitative proteomics technology of isobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrometry. The key differentially expressed protein molecules were chosen according to bioinformatics analysis combined with reports from previous studies. Interleukin 1 beta (IL-1 beta) is one of the key molecules and was further explored for its role and mechanism in HCC metastasis.The objective of this research is to elucidate the mechanism of HCC metastasis promoted by M2 tumor associated macrophages from the perspective of proteomics. The research may provide novel insight for targeted therapy of HCC metastasis in the future.Part One Establishment and identification of Ml and M2 tumor associated macrophage cell modelsMacrophage is one of the major components of the immune system which was differentiated from monocyte in the peripheral circulation and plays a crucial role in inherent immune response and adaptive immune response. It has been well established that macrophages can be divided into two subsets of macrophages with different phenotypes:one is classic activation of macrophage (Ml), another is alternative activation of macrophage (M2). The aim of this part was to establish the Ml and M2 tumor associated macrophage cell models for subsequent further research. Human monocytic cell line THP-1 was induced and differentiated into unactivation of macrophage (UaM) initially using phorbol myristate acetate (PMA). Then the UaMs were further induced into M1 tumor associated macrophage by lipopolysaccharides (LPS) and interferon gamma (IFN-y), and M2 tumor associated macrophage by IL-4 and IL-13, respectively. The cell morphology of M1 and M2 tumor associated macrophages was observed by inverted phase contrast microscope. The CD68, CD206, and CD 163 expressions of M1 and M2 tumor associated macrophages were determined by flow cytometry (FCM). The IL-10 and IL-12 secretion levels of M1 and M2 tumor associated macrophages were detected using enzyme linked immunosorbent assay (ELISA). The relative mRNA expressions of TNF-a, CCL3, Arg-1, iNOS, AMAC-1, and CCL22 of M1 and M2 tumor associated macrophages were detected using real time-polymerase chain reaction (RT-PCR). The results revealed that the IL-12, TNF-a, CCL3, and iNOS were highly expressed in M1 tumor associated macrophages, the IL-10, Arg-1, AMAC-1, CCL22, CD206, and CD 163 were highly expressed in M2 tumor associated macrophages, respectively. The results were consistent with reports from previous studies, suggesting that we have successfully established the cell models of M1 and M2 tumor associated macrophages.Part Two M2 tumor associated macrophage promotes HCC cell invasion and induces EMTEpithelial mesenchymal transition (EMT) is a critical step for cancer metastasis and has become a research focus in tumor metastasis. HCC is a malignance with high mortality. Metastasis is the main cause of the high mortality and is the major obstacle for the improvement of efficacy and survival in HCC treatment. Tumor microenvironment has been shown to play critical roles in tumor invasion and metastasis and has become a hot spot in cancer research. TAM is the main component of the inflammation cells in the tumor microenvironment which was primarily consisted of M2 tumor associated macrophages. In this part, we investigate the influence of the M2 tumor associated macrophage on invasion, migration, adhesion and EMT of the HCC cells and explore the effects of M2 tumor associated macrophage on HCC metastasis. Human monocytic cell line THP-1 was initially induced and differentiated to M2 tumor associated macrophage in vitro. Then the M2 tumor associated macrophage was co-cultured with Huh7 and SMMC7721 cells using transwell chambers to mimic the tumor microenvironment of HCC in the body. The effects of M2 tumor associated macrophage on invasion, migration, and adhesion of the HCC cells were assessed by transwell invasion assay, migration assay and adhesion assay, respectively. Real-time PCR was used to analyze the relative mRNA expressions of EMT related markers E-cadherin, N-cadherin, Vimentin and a-smooth muscle actin (α-SMA) of Huh7 and SMMC7721 cells. The results revealed that the Huh7 and SMMC7721 cells showed an obvious morphology change after co-cultured with M2 tumor associated macrophage. The cells lose the epithelial morphology and were converted to a spindle-like mesenchymal cell phenotype. The invasion and migration activities of Huh7 and SMMC7721 cells were increased while the adhesion activity of Huh7 and SMMC7721 cells was decreased. The relative mRNA expression of epithelial marker E-cadherin was down-regulated, while the relative mRNA expressions of mesenchymal markers N-cadherin, Vimentin and α-SMA were up-regulated as compared with the untreated cells. The results suggested that the M2 tumor associated macrophage promotes HCC cell invasion through inducing EMT.Part Three Establishment of three-dimensional culture systems of M2 tumor associated macrophage and HCC cells and quantitative analysis of secretome of the three-dimensional cell culture systemsSecretome was consisted of secreted proteins from the cells and tissues. Secretomics was to quantitatively analyze the secreted proteins using proteomics technology. It was reported that tumor microenvironment of the solid tumors consisted of malignant tumor cells and stromal cells.It has been suggested that the stromal cells in the tumor microenvironment of the solid tumors are highly dynamic and flexible and play a critical role in tumor progression, and the secreted proteins from the stromal cells were shown to act as a "bridge" in the interaction between the stromal cells and the tumor cells. TAM is the main component of the stromal cells in the tumor microenvironment which are primarily consisted of M2 tumor associated macrophages. In this part, we first established the cell model of M2 tumor associated macrophage. Then the M2 tumor associated macrophages were co-cultured with the SMMC7721 cells in vitro to mimic the tumor microenvironment in the body using the three-dimensional cell culture apparatus BioLevitatorTM. The differentially expressed proteins in the three-dimensional cell coculture systems of M2 and SMMC7721 were screened by comparative analysis using quantitative proteomics technology of iTRAQ combined with mass spectrometry. The key differentially expressed protein molecules were chosen according to bioinformatics analysis combined with reports from previous studies, and the expression levels of the key molecules were validated using western blot analysis. The objective of this part was to provide theoretical basis and experimental evidence for further elucidating the molecular mechanism of HCC metastasis promoted by M2 tumor associated macrophages in the tumor microenvironment.Part Four The effects and mechanism of macrophage-derived interleukin 1 beta on HCC metastasisHCC is a typical inflammation-related cancer with chronic inflammation and immunomodulatory disorder which was mainly caused by hepatitis virus. Recently, more and more studies have been performed to investigate the effects of inflammation on carcinogenesis and the relationship between chronic inflammation and cancer metastasis had also been widely investigated. Interleukin 1 beta (IL-1 beta), mainly produced by macrophages and monocytes, plays a key role in inflammatory response and immune regulation. In this part, the exogenous recombinant human IL-1 beta was used to induce SMMC7721 cells. The effects of IL-1 beta on invasion, migration, and adhesion of the SMMC7721 cells were assessed by transwell invasion assay, migration assay and adhesion assay, respectively. The relative mRNA expressions of EMT markers of the SMMC7721 cells were analyzed by real-time PCR. The protein expressions of EMT-related marker Snail and signaling factors NF-κB, IκBα, and P-IκBα in the NF-κB pathway were determined by western blot analysis. The results revealed that the SMMC7721 cells showed an obvious morphology change after induced by IL-1 beta. The cells lose the epithelial morphology and were converted to a spindle-like mesenchymal cell phenotype. The invasion and migration activities of the SMMC7721 cells were increased while the adhesion activity was decreased. The relative mRNA expression of the epithelial marker E-cadherin was down-regulated, while the relative mRNA expressions of the mesenchymal markers N-cadherin, Vimentin, Snail, Slug, ZEB1, and ZEB2 were up-regulated as compared with the untreated cells. The protein expressions of NF-κB、P-IκBα, and Snail were up-regulated while the protein expression of IκBα was down-regulated in a time-dependent manner. In addition, the introduction of the inhibitor BAY 11-7082 of the NF-κB pathway attenuates even reverses the above effect of IL-1 beta on SMMC7721 cells.The results suggest that IL-1 beta facilitates hepatocellular carcinoma metastasis through inducing EMT via NF-κB/Snail signaling. The promotion of IL-1 beta on HCC metastasis can be reversed by blocking the NF-κB pathway. The IL-1 beta may be a potential therapeutic target for HCC metastasis in the future.Conclusions1. We have successfully established the cell models of Ml and M2 tumor associated macrophages.2. M2 tumor associated macrophage promotes invasion and metastasis of HCC cells through inducing EMT.3. We have successfully established the three-dimensional cell culture systems of M2, SMMC7721, and M2+SMMC7721 in vitro by mimicing the tumor microenvironment in the body using three-dimensional cell culture apparatus BioLevitatorTM. The differentially expressed protein molecules by the three-dimensional cell culture systems of M2, SMMC7721, and M2+SMMC7721 were successfully screened by comparative analysis using quantitative proteomics technology of iTRAQ combined with mass spectrometry. IL-1 beta was found to be one of the key molecules in the differentially expressed proteins of the M2+SMMC7721 co-culture system which has been shown closely associated with tumor metastasis.4. IL-1 beta promotes hepatocellular carcinoma metastasis through inducing epithelial-mesenchymal transition (EMT) via NF-KB/Snail signaling. IL-1 beta is a potential therapeutic target for HCC metastasis in the future. |
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