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

// 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 Akgun 1 , Marc Lutgehetmann 2 , Jan Dieckhoff 3 , Michael Kopke 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.