Characterization of in vivo chemoresistant human hepatocellular carcinoma cells with transendothelial differentiation capacities

Abstract

BackgroundChemotherapeutic 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. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model.MethodsSCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated 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. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel.ResultsHUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) 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 transdifferentiation 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. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression.ConclusionsChemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation 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.

MicroRNA‐760 Inhibits Doxorubicin Resistance in Hepatocellular Carcinoma through Regulating Notch1/Hes1‐PTEN/Akt Signaling Pathway

“…In HCC, Notch1 is elevated in HCC tissues, and Notch1‐mediated Notch signaling contributes to the growth and metastasis of HCC . Upregulation of Notch1 contributes to the acquired chemoresistance toward metronomic cyclophosphamide therapy . Overexpression of nuclear receptor binding protein 2, increases the chemosensitivity of HCC cells associated with inhibition of Notch1 expression .…”

Hes1: a key role in stemness, metastasis and multidrug resistance

“…55 Correspondingly, the Hes1 level is observed to be higher in chemically resistant, as well as cyclophosphamide-treated hepatocellular carcinoma. 56 There are several latent mechanisms by which cancer cells are able to promote transformations that increase drug resistance. Such instances include high level of Hes1 expression and enhancing STAT3 phosphorylation.…”

From Composition to Cure: A Systems Engineering Approach to Anticancer Drug Carriers

“…[157] In clinical trials, novel drug carriers will thus face the most difficult cancers to treat. It is important we pursue preclinical models that can better mimic the acquired drug resistance faced in human patients, by employing methods such as metronomic treatment of tumors in mice, [158] to induce drug resistance in vivo prior to the testing of drug carrier therapies. Furthermore, in clinical trials the performance of drug carriers will be compared against a standard of care, which may include combinations of multiple drugs or orthogonal methods of treatment.…”