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Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model

2017; Impact Journals LLC; Volume: 8; Issue: 70 Linguagem: Inglês

10.18632/oncotarget.23312

1949-2553

Michael Thomaschewski, Kristoffer Riecken, Ludmilla Unrau, Tassilo Volz, Kerstin Cornils, Harald Ittrich, Denise Heim, Henning Wege, Ercan Akgün, Marc Lütgehetmann, Jan Dieckhoff, Michael Köpke, Maura Dandri, Daniel Benten, Boris Fehse,

RNA Interference and Gene Delivery

// Michael Thomaschewski 1 , Kristoffer Riecken 1 , Ludmilla Unrau 1 , Tassilo Volz 2 , Kerstin Cornils 1 , Harald Ittrich 3 , Denise Heim 2 , Henning Wege 2 , Ercan Akgün 1 , Marc Lütgehetmann 2 , Jan Dieckhoff 3 , Michael Köpke 2 , Maura Dandri 2 , Daniel Benten 2, 4, * and Boris Fehse 1, * 1 Research Department of Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center (UMC) Hamburg-Eppendorf, Hamburg, Germany 2 Department of Medicine, Gastroenterology and Hepatology, UMC Hamburg-Eppendorf, Hamburg, Germany 3 Diagnostic and Interventional Radiology, UMC Hamburg-Eppendorf, Hamburg, Germany 4 Department of Gastroenterology, Helios Klinikum Duisburg, Duisburg, Germany * These authors have contributed equally to this work Correspondence to: Boris Fehse, email: fehse@uke.de Daniel Benten, email: d.benten@helios-gesundheit.de Keywords: HCC; insertional mutagenesis; RGB marking; LeGO vectors; hTERT Received: September 15, 2017 Accepted: December 04, 2017 Published: December 14, 2017 ABSTRACT We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.