Abstract 1909: Non-alcoholic steatohepatitis promotes EpCAM positive cancer stem cells mediated tumorigenesis in immunocompetent mouse model of hepatocellular carcinoma
Authors: Pandit Harshul , Li Yan , Cui Guozhen , Li Suping , Li Salina , Martin Robert C. 
Published: July 2017

Introduction: Non-alcoholic Steatohepatitis (NASH) is one of the major risk factors for cirrhosis and its progression to Hepatocellular Carcinoma (HCC). Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation and recurrence in tumor microenvironment. Understanding role of CSCs in tumorigenesis was limited to immunocompromised nude/SCID mouse models, and should be investigated in immunocompetent mouse model(s) representing clinical conditions. In this study, (1) we have investigated and confirmed CSCs properties in HCC cells lines, and (2) investigated tumor initiation capability of EpCAM positive CSCs in novel immunocompetent mouse model of HCC. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. To confirm CSCs, we analyzed CSC biomarkers (EpCAM,CD90,CD44,CD133) using flow-cytometry and Immunocytochemistry (ICC), and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance property of CSCs was studied using Doxorubicin (anthracycline antibiotic) and Sorafenib (multikinase inhibitor) by MTT assay. To study in vivo tumorigenesis, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells. Capability of EpCAM CSCs for tumor initiation was tested in 3 diet induced groups, i.e. control (10% dietary fat), high-fat (60% dietary fat), and NASH (60% dietary fat MCD diet with 0.1% methionine). 2 million FACS sorted EpCAM+/+ or EpCAM-/- cells were orthotopically injected into left liver lob. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression and significant higher chemoresistance compared with control HCC cells. Our in vivo findings confirmed that EpCAM CSCs required NASH microenvironment. In NASH group mice, EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p&lt;0.005). Histology analysis of NASH liver tissue confirmed lobular and portal inflammation, hepatocellular ballooning, and fibrosis. Control and high-fat diet group mice failed to develop tumors. Conclusions: We have developed a novel mouse model to study CSCs to overcome confounding limitations of immunocompromised mouse models for lacking functional immune system components. Our findings using immunocompetent mouse model suggest that, in HCC, (1) EpCAM+ CSCs cannot initiate tumor by itself within normal liver microenvironment in the presence of functional immune components, (2) NASH microenvironment promotes EpCAM+ CSCs mediated tumorigenesis while EpCAM-/- non-CSCs subset failed to develop tumor.

Citation Format: Harshul Pandit, Yan Li, Guozhen Cui, Suping Li, Salina Li, Robert C. Martin. Non-alcoholic steatohepatitis promotes EpCAM positive cancer stem cells mediated tumorigenesis in immunocompetent mouse model of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1909. doi:10.1158/1538-7445.AM2017-1909