Carcinogenetic initiation contributed by EpCAM+ cancer cells in orthotopic HCC models of immunocompetent and athymic mice

Carcinogenetic initiation contributed by EpCAM+ cancer cells in orthotopic HCC models of immunocompetent and athymic mice

ABSTRACT

Purpose: Overexpression of epithelial cell adhesion molecule (EpCAM) correlates with poor prognosis, therapeutic failure and early tumor recurrence in hepatocellular carcinoma (HCC) patients. The tumor microenvironment dictates the fate of tumor-initiating cancer stem cells (CSCs); however, very limited studies were attempted to evaluate CSC tumorigenesis in the liver microenvironment. Here, we have systemically investigated the role of EpCAM+ cancer cells in tumor initiation in orthotopic HCC models.

Results: Control mice and the mice with bland steatosis failed to develop tumors. In the mice with steatohepatitis, EpCAM+ CSCs have shown significantly increased ability in terms of tumor initiation and growth, compared to that with EpCAM- non-CSCs inoculation (p < 0.005). For Hep3B inoculation, EpCAM-High group has shown significantly higher tumor growth compared with EpCAM-Low (p < 0.005). For HepG2 inoculation, both EpCAM-High and EpCAM-Low groups confirmed similar tumor incidence and growth.

Methods: Diet-induced compromised microenvironments were established to mimic clinical fatty liver and non-alcoholic steatohepatitis (NASH) patients and the tumorigenic capabilities of Hepa1-6 cells were evaluated. CSCs were enriched by spheroid culture and labeled with copGFP for EpCAM+ CSCs and with mCherry for non-CSCs. FACS-sorted cells were inoculated into left liver lobes, and tumor growth was monitored by high-frequency ultrasound. The subpopulations of Hep3B and HepG2 cells in terms of EpCAM-Low and EpCAM-High were evaluated in the orthotopic model of athymic mice.

Conclusions: NASH microenvironment promotes the EpCAM+ CSCs initiated tumorigenesis in immunocompetent mouse model. Differential EpCAM expression demonstrates distinct tumor biology in athymic mouse models.

INTRODUCTION

Hepatocellular carcinoma (HCC), a type of epithelial cancer, is the most common primary liver cancer (80–90%) in the United States [1]. HCC is the fifth most common cancer in men and seventh in women, and accounts for the third major cause of cancer-related deaths worldwide [2, 3]. HCC confers the highest death rate (~2.4%) among all cancers in United States [4]. A subset of cancer cells within the tumor microenvironment, defined as cancer stem cells (CSCs), contributes to aggressive tumor initiation, therapeutic treatment resistance, and tumor relapse in HCC patients [5].

The CSC concept has been elucidated in regard to tumor heterogeneity within primary HCC, and it helps to understand therapeutic resistance and early relapse [5–7]. Because of vast heterogeneity, no single marker can define the CSCs exclusively [8]. Identification of CSCs from human HCC tumors and human HCC cell lines has been performed using various CSC surface biomarkers, including CD90+, CD44+, epithelial cell adhesion molecule (EpCAM+), CD133+, OV6+, aldehyde dehydrogenase 1 (ALDH1+), and alpha-fetoprotein (AFP+) [5, 7, 9, 10]. EpCAM, one of the most characterized and well-accepted CSC markers, is associated with poor prognosis in HCC patients [10–14]. EpCAM is a target of Wnt/β-catenin signaling, and inhibiting Wnt/β-catenin signaling has been shown to destroy the EpCAM+ cells [13, 15]. There is no previous study to investigate EpCAM-expressing CSCs for their tumorigenic ability in a compromised liver microenvironment such as non-alcoholic fatty liver disease (NAFLD).

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“A diagnostic autoantibody signature for primary cutaneous melanoma” has the Altmetric score of 594. This article was published back in 2018 by Oncotarget and written by different experts from Hollywood Private Hospital, Edith Cowan University, Dermatology Specialist Group, St. John of God Hospital and The University of Western Australia. The introduction of the study mentions that “recent data shows that Australians are four times more likely to develop a cancer of the skin than any other type of cancer”, and shares an insight on melanoma that “is curable by surgical excision in the majority of cases, if detected at an early stage.”

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