The Role Of Tumor Plasiticity Into Endothelial-like Cells In Vasculogenic Mimicry Formation

Part 1 Tumor cells and bone marrow mesenchymal stem cells coordinate with each other in acquiring endothelial cell phenotype[Purpose]A co-culture system was used to investigate the interaction between mouse bone marrow mesenchymal stem cells (BMSCs) and B16 melanoma cells, observe phenotypic changes of tumor cells in the process of inducing BMSCs to differentiate into cells with endothelial phenotypic features as well as the role of BMSCs in inducing tumor cells to express endothelial markers. This is important for further understanding the degree and characteristics of differentiation of BMSCs into endothelial cells and its effect on the expression of endothelial markers in tumor cells in the contact co-culture microenvironment, and lays a foundation for identifying the origin of vascular endothelial cells.[Methods]1) Limb bone marrow from C57 mice was separated for primary culture. After purification by differential centrifugation, BMSCs were identified with stem cell phenotype with flow cytometry.2) A transwell co-culture model was established and immunofluorescence was used to observe the phenotypic changes of the BMSCs and melanoma cells in the process of co-culture. Transmission electron microscopy was used to confirm whether there were W-P corpuscles after BMSCs were induced. Cells and mediums were collected at various time regularly. ELISA was used to detect the change of the level of VEGF-a in medium and Western Blot was used to detect the time-dependent change of the level of VEGFR1, VEGFR2 and Factorâ…§in cell lysate.3) Immunofluorescence staining was performed to compare the change of cell phenotype and morphology between cells of non-contact co-culture and that of contact co-culture.[Results]1) Phenotype of cells separated from limb bone marrow from C57 mice after purified culture was identified as:CD44+/CD73+/CD90+/CD105+/CD166+/CD34-/ CD45-/CD133-, consistent with the phenotype of BMSCs.2) After co-culture with B16 melanoma cells for 72h, the expression of endothelial phenotype CD34,CD45,Factorâ…§,VEGFR and VEGFR-2 in mBMSCs was observed to be up-regulated gradually in a time-dependent manner, while the expression of CD4 and CD 105 was down-regulated. Flow cytometry showed that the percent of Factor VIII positive cells was 1.09% before induction and 30.17% after induction. Ultrastructure analysis found that Weibe-Palade bodies, which were specific for endothelial cells, could be observed under transmission electron microscope after co-culture for 72h.3) Proliferation rates of BMSCs and B16 cells from co-culture system increased significantly. The doubling time of mBMSCs in co-culture group was 25.02h, while that was 28.15h in control group. The doubling time of B16 cells in co-culture group was 26.30h, while that was 30.76h in control group.4) In non-contact co-culture system, mBMSCs can be induced by B16 cells to acquire endothelial cell phenotype. At the same time, B16 melanoma cells was observed to up-regulate the expression of VEGF-a, VEGFR-1, VEGFR-2 and Factor VIII. After directly co-culture (contact co-culture) of B16 cells and BMSCs for 72h, the percent of VEGFR1, VEGFR2, and Factorâ…§positive cells decreased significantly. B16 cells surrounding mBMSCs was observed.5) The level of VEGF-a in culture medium increased significantly with the extension of incubation time in co-culture system.[Conclusions]1) In the microenvironment of co-culture with the tumor cells, BMSCs can be induced by tumor cells to differentiate into endothelial cells and showed phenotype changes;2) Tumor cells can interact with BMSCs and showed up-regulation of endothelial cell markers;3) BMSCs and tumor cells can interact with each other to promote proliferation;4) The time-dependent increase of VEGF-a in culture medium was associated with appearance of endothelial markers of BMSCs after co-culture with tumor cells.Part 2 Clinicopathologic analysis----role of Twistl in vascular mimicry in human hepatocellular carcinoma[Purpose]Clinical specimens of human hepatocellular carcinoma of 97 patients with complete follow-up data were studied in this part to observe the existence of VM and its relationship with clinical prognosis and to analyze preliminarily the relationship between EMT-associated protein (characterizing protein E-cadherin and regulating protein Twistl, Twist2, Snail and Slug) and VM-related protein (CD31/PAS, VM-cadherin, MMPs) and its effect on clinical prognosis. EMT-related proteins that closely associated with VM were screened.[Methods]Resected specimens of 97 patients identified as hepatocellular carcinoma by pathologists with complete follow-up data were obtained from General Hospital and Cancer Institute and Hospital of Tianjin Medical University between February 2001 and December 2005 to analyze the medical records and clinical follow-up data; HE staining and CD31/PAS double staining were used to identify the existence of VM and the number of PAS/VM was counted; immunohistochemistry was used to detect EMT-related protein and VM-related protein and analyze their correlations between each other and the association with VM and clinical diagnosis with Statistical methods.[Results] 1) In 97 cases of HCC patients,18 cases (18/97,19%) had typical VM structures;2) Twistl expressed diffusely in the cytoplasm of HCC cells. In some cases, it expressed in the nucleus, or in the nucleus and cytoplasm simultaneously. There may be strong positive foci-like expression locally. The difference of nuclear expression of Twistl between two groups was significant. Results of Pearson test showed that there was a correlation between twist1-cyto,Twistl-nu and the number of VM-PAS-positive loop. The association between Twist2, Snail, Slug and VM was not strong;3) EMT-related characterizing protein VE-cadherin was correlated with VM as well as Twistl-cyto, Twistl-nu and Slug, but it was not correlated with Twist2 and Snail. MMP9 was correlated with VM and Twistl. MMP2 had no correlation with VM and Twistl in this study;4) Results of survival analysis showed that patients positive for Twistl-cyto, Twistl-nu, Snail, VE-cadherin, and MMP9 had a shorter survival and patients positive for E-cadherin had a longer survival. The expression of Slug and MMP2 had no significant correlation with the survival time.5) Results of Cox regression analysis showed that the expression of Twist1-Nu did the greatest contribution to the survival of patients.[Conclusions]1) VM was mainly existed in the poorly differentiated and high-malignant HCCs who belonged to solid and poorly differentiated type, suggesting that VM was associated with malignant biological behaviors of HCC;2) The nuclear expression of EMT regulatory protein Twistl had the most close association with VM and it affected the prognosis.3) The expression of Twistl was correlated with the expression of VE-cadherin and MMP9. Tumor cells may regulate the expression of VE-cadherin through the regulation of Twistl, thereby form tumor associated endothelial cells (TAEs) with characteristics of endothelial cells;4) Correlation between EMT regulatory proteins Twist2, Snail, Slug and VM formation were not observed.Part 3 Role of Twistl in enhancing tumor cell migration, motility and angiogenic remodeling[Purpose]HCC cell lines were used to study the effect of Twistl on migration, invasion and angiogenic remodeling of tumor cells in vitro to further identify the association between Twistl and remodeling of tumor cells and VM, and analyze the regulation of Twistl on the expression of VE-cadherin and the activity of MMPs.[Methods]HCC cell lines were selected. Twistl expression plasmid and shRNA plasmid were constructed. Cells were transfected to acquire up-regulation model and down-regulation model. Western Blot and RT-PCR were used to identify the transfection efficiency. Wound healing assay and transwell invasion assay were performed to evaluate the effect of Twistl on cell motility and invasion; three-dimensional matrigel culture was used to further analyze the effect of Twistl on angiogenic remodeling of tumor cells; chromatin immunoprecipitation assay and reporter gene assays were performed to investigate the regulation of Twistl on VE-cadherin transcription; zymography assays were performed to detect the effect of Twistl on the activity of MMP2 and MMP9.[Results]1) Expressions of twistl mRNA and protein in PLC, HepG2 and Huh-7 cell lines showed a low level, while those in Be17402 presented a high level;2) pcDNA3-Twistl can significantly up-regulate twistl expression in HepG2 and pGP-Twist1-shRNA can effectively reduce twistl expression in tumor cells. Along with the changes of Twistl expression, cell phenotypes changed correspondingly.3) Comparison between the up-regulated cell model in HepG2 and knockdown cell model in Be17402 suggested that Twist1 can enhance cell migration, motility and invasiveness. In three-dimensional culture, up-regulation of Twistl can promote HepG2 cells to form tubular structure, while down-regulation of Twistl can inhibit Be17402 from forming tubular structure.4) In three-dimensional culture, up-regulation of Twistl induced HepG2 cells to express VE-cadherin while down-regulation of Twistl inhibited expression of VE-cadherin in Be17402. Chromatin immunoprecipitation assay and reporter gene assays suggested that Twistl can combine with the promoter of VE-cadherin in a particular microenvironment and enhance its activity of transcription.5) Up-regulation of Twistl in HepG2 cells and Be17402 cells enhanced the activity of MMP2 and MMP9.[Conclusions]1) Expression of Twist1 can promote cell migration, motility and invasiveness.2) Expression of Twistl can promote tubular remodeling in three-dimensional matrix through up-regulation of VE-cadherin;3) In the three-dimensional culture, Twistl combined with the promoter of VE-cadherin to promote the transcription of VE-cadherin;4) Expression of Twistl can enhance the activity of MMP2 and MMP9 in HCC cells;5) Twistl was the important molecular in the formation of VM by tumor cells via EMT. Part 4 Interaction between anti-apoptotic protein Bcl-2 and EMT regulatory protein Twistl[Purpose]We have reported that the VM was induced by hypoxia. Tumor cells formed LPPCN by hypoxia inducition and then obtain blood. LPPCN formation was associated with the anti-apoptosis protein Bcl-2. To investigate the upstream mechanism that initiates Twist1 expression, hypoxic micro-environment was simulated to evaluate time-dependent expression of anti-apoptotic protein Bcl-2 and Twistl. Yeast two hybrid assay, co-immunoprecipitation assay of overexpressing Twistl in eukaryotic cells and co-immunoprecipitation of endogenous protein were used to analyze direct interactions between Twistl and Bcl-2 to further identify the molecular mechanism of hypoxic microenvironment inducing tumor cells to remodel into vascular endothelial cells via EMT and enrich the theory of angiogenesis.[Methods]GasPak was used to simulate hypoxic tumor microenvironment. B16 cells were used as the model to study the time-dependent changes of the expression of Bcl-2 and Twistl; yeast two-hybrid assay was used to select the anti-apoptosis proteins those interacted with Twistl; co-immunoprecipitation was performed after Twistl was expressed in HepG2 cells to analyze the direct interaction between Twistl and Bcl-2. In hypoxia microenvironment, co-immunoprecipitation of endogenous protein was performed to analyze the direct interaction between Twistl and Bcl-2.[Results]1) After hypoxia for 30h, Twistl was significantly overexpressed in melanoma B16 cells, at the same time, Bcl-2 was overexpressed in a time-dependent manner with a peak expression between 24-36h followed by the decrease. Transcription factor Twistl, VEGFR2 and VE-cadherin were detected to be highly expressed when hypoxia was terminated at 30h.2) Detection of proteins those interacted with Twistl selected by Yeast two-hybrid assay suggested that there was Bcl-2 gene.3) After transfection with pcDNA3-Twistl for 48h, Bcl-2 and Twistl were detected to be overexpressed. Co-IP was used to precipitate Twist land Bcl-2 respectively, Bcl-2 and Twistl can be detected correspondingly.4) After hypoxia for 30h followed by re-oxygen for 24h, co-immunoprecipitation of endondogeneous Bcl-2 and Twistl can be detected.[Conclusions]1) Hypoxia can induce responsive overexpression of Bcl-2 and Twistl.The overexpression induced by hypoxia-reoxygen was greater than that by hypoxia.2) Yeast two-hybrid assay suggested that Twistl can interact with Bcl-2 directly.3) In tumor cells overexpressing Twistl, the direct interaction between Twistl and Bcl-2 can be detected; in hypoxia environment, the direct interaction between endogenous Twistl and Bcl-2 can be detected.4) Hypoxic and anti-apoptosis mechanism may induce tumor cells to undergo EMT and the overexpression of anti-apoptosis protein Bcl-2 may be one of initiators of EMT. Conclusions for whole manuscriptBased on the interaction between MSCs and tumor cells resulting in differentiation into endothelial cells, the relationship between EMT-related proteins and VM and the relationship between Bcl-2voverexpression and the formation of TAE in hypoxia, the possible mechanism of "LET" that could clarify the transformation from tumor cells to vascular endothelial cells was investigated. The contents were as followings:interactions between Bcl-2 and Twistl, time-dependent associations of appearance of tumor-associated endothelial cell markers, and molecular and functional evaluations of the corresponding tissues and cells. "LET" theory can better explain the biological behavior of tumor cells in particular microenvironment and further perfect the theory of tumor angiogenesis and EMT. At the same time, the theory provides a new idea and an access for basic research of tumor cells differentiation, provides new drug targets and information of treatment for transformational medicine research of tumor angiogenesis. It plays an important role in further understanding the biological behavior of tumor and in clinical diagnosis.