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Marfels, Christian (2015): Characterization of in vivo chemoresistant human hepatocellular carcinoma cells with transendothelial differentiation capacities. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments, a better understanding of biochemical changes in the resistant tumors is needed. Therefore, especially in vivo models are very important tools to generate standardized cell-material, which can be examined by high throughput techniques. Thus, it should be possible to find new targets for therapy or even for diagnostic. This thesis focusses on the characterization of the in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft mouse model. First step of the work was the establishment of the xenograft mouse model. SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. After 10 weeks of response to the therapy, the tumor growth relapsed and tissue grew with very fast doubling time again, despite of ongoing treatment. This aggressive manner of growth under therapy could be also observed in a re-implantation study where the reisolated CPA chemoresistant HUH-REISO tumors grew without a lag phase, indicating an endogenous imprinted component. To evaluate this, tumors were examined by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Histochemical analysis of HUH-REISO tumors revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO in comparison to parental HUH-7 cells and in vivo passaged HUH-PAS cells (in vivo grown without chemotherapeutic CPA pressure). The pronounced network of host murine vascularization in parental HUH-7 tumors was completely substituted by a network of human and murine vessel-like structures in HUH-REISO tumors under therapy. In addition, cell lines of these tumors were analyzed in endothelial trans-differentiation studies on matrigel. In those studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. Such a trans-differentiation capacity requires a lineage of cells with pluripotent capacities like so called tumor stem cells. Indeed, I could show in a comparative study on stemness and plasticity markers that Thy-1, Oct-4, Sox-2 and Nanog were upregulated in resistant xenografts. Furthermore, under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression, which I could also show by qRT-PCR. Notch-1 raised in HUH-PAS under therapeutic pressure, meanwhile it was conversely regulated in comparison to Thy-1, Oct-4, Sox-2 and Nanog in HUH-REISO. In both groups Notch-3 was inducible by 2 times CPA treatment and fell back on base level after further four therapeutic cycles in HUH-REISO. To conclude all these finding: chemoresistance of HUH-REISO was not manifested under standard in vitro, but only under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of trans-differentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.