Surgical resection and radiofrequency ablation initiate cancer in cytokeratin-19+- liver cells deficient for p53 and Rb

The long term prognosis of liver cancer patients remains unsatisfactory because of cancer recurrence after surgical interventions, particularly in patients with viral infections. Since hepatitis B and C viral proteins lead to inactivation of the tumor suppressors p53 and Retinoblastoma (Rb), we hypothesize that surgery in the context of p53/Rb inactivation initiate de novo tumorigenesis. We, therefore, generated transgenic mice with hepatocyte and cholangiocyte/liver progenitor cell (LPC)-specific deletion of p53 and Rb, by interbreeding conditional p53/Rb knockout mice with either Albumin-cre or Cytokeratin-19-cre transgenic mice. We show that liver cancer develops at the necrotic injury site after surgical resection or radiofrequency ablation in p53/Rb deficient livers. Cancer initiation occurs as a result of specific migration, expansion and transformation of cytokeratin-19+-liver (CK-19+) cells. At the injury site migrating CK-19+ cells formed small bile ducts and adjacent cells strongly expressed the transforming growth factor β (TGFβ). Isolated cytokeratin-19+ cells deficient for p53/Rb were resistant against hypoxia and TGFβ-mediated growth inhibition. CK-19+ specific deletion of p53/Rb verified that carcinomas at the injury site originates from cholangiocytes or liver progenitor cells. These findings suggest that human liver patients with hepatitis B and C viral infection or with mutations for p53 and Rb are at high risk to develop tumors at the surgical intervention site.


Liver organoids
Organoids isolated from bile duct fragments of wild type (WT) and p53/Rb deficient liver were cultured as described before [1]. To investigate proliferation of WT vs p53/Rb deficient liver organoids, 5-ethynyl-2′-deoxyuridine (Edu) (Invitrogen, A10044) was incorporated for 2 hours at 37°C from day 2 until day 5 of culture. To investigate the effect of TGF-β on liver organoid proliferation, organoids were incubated for 24 hours with TGF-β (R&D systems, 7666-MB-005) at concentration of 0, 5 and 10 ng/ml respectively. Then organoids were pulsed with Edu for 2 hours. For hypoxia experiments, organoids were incubated for 24 hours in humidified chamber at 37°C, 5% CO 2 and 2.5% O 2 in Don Whitley H35 hypoxystation (Don Whitley Scientific, UK). Organoids cultured under normoxia were maintained in a standard cell culture incubator. For oxidative stress experiments, organoids were incubated with or without 1.25 μM N-acetylcysteine (Sigma Aldrich) and 1mM Hydrogen peroxide (Merck Millipore) for 24 hours .

EDU-incorporation assays and trypan blue analysis
After 18hours, organoids cultured under normoxia, hypoxia and oxidative stress experiments were incubated with EdU for 6hours, and then organoids were analyzed for trypan blue exclusion using TC20 automated cell counter (Bio-rad). For EdU analysis, cells were fixed with 4% paraformaldehyde and processed in a click reaction with 5 μM Alexa Fluor 488 azide (Life Technologies, A10266) according to manufacturer's instruction. Nuclei were stained with DAPI (Sigma-Aldrich, D9542). The percentage of EdU positive cells was analyzed using automated image acquisition and data analysis with the Cellomics ArrayScan VTI HCS Reader (Thermo Scientific) and confocal SP II microscope (Leica Microsystems)

Flow cytometry analysis
Organoids were trypsinized and fixed with ice cold 70% ethanol in PBS and stored in the fridge. Prior to FACS analysis, nuclei were stained with Propidium iodide as described previously [2] and analyzed with FACS Calibur and CellQuest software (BD Bioscience). Histograms overlay were created using FlowJo software.

Histological image acquisition and processing
Haematoxylin and eosin as well as immunohistochemistry images were acquired using DP25 camera, Labsens soft imaging version 1.1 and Olympus BX45 microscope (Olympus, Zoeterwoude, The Netherlands). Immunofluorescence images were acquired using Leica DFC 425c camera and Leica Application Suite version 4 (Leica Microsystems B.V. Rijswijk, The Netherlands, and BX60 microscope (Olympus, Zoeterwoude, The Netherlands). Images were processed using Adobe photoshop cs6/CC and exported to Adobe illustrator cs6/CC (Adobe Systems, Inc., San Jose, California).

Pathological analysis of liver tissues
Pathological analysis was performed by a board-certified veterinary pathologist (A.d.B.) and a human pathologist (T.R.). Liver tumors were classified according to the nomenclature and diagnostic criteria for hepatobiliary lesions in rats and mice [3].

Genotype analysis
Genotyping on livers and liver tumors was performed using allele-specific primers. The

Statistics
Survival curve was drawn using SPSS statistical package and statistical difference was calculated using SPSS log rank test. Statistical test used in table is student's t-test; statistical test for bar graphs was computed using Mann-Whitney U test, mean, standard deviation and standard error of the mean were calculated using Microsoft excel (2010) ®.