EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features

Abstract

Background & aims: Cancer progression/metastases and embryonic development share many properties including cellular plasticity, dynamic cell motility, and integral interaction with the microenvironment. We hypothesized that the heterogeneous nature of hepatocellular carcinoma (HCC), in part, may be owing to the presence of hepatic cancer cells with stem/progenitor features.

Methods: Gene expression profiling and immunohistochemistry analyses were used to analyze 235 tumor specimens derived from 2 recently identified HCC subtypes (EpCAM(+) alpha-fetoprotein [AFP(+)] HCC and EpCAM(-) AFP(-) HCC). These subtypes differed in their expression of AFP, a molecule produced in the developing embryo, and EpCAM, a cell surface hepatic stem cell marker. Fluorescence-activated cell sorting was used to isolate EpCAM(+) HCC cells, which were tested for hepatic stem/progenitor cell properties.

Results: Gene expression and pathway analyses revealed that the EpCAM(+) AFP(+) HCC subtype had features of hepatic stem/progenitor cells. Indeed, the fluorescence-activated cell sorting-isolated EpCAM(+) HCC cells displayed hepatic cancer stem cell-like traits including the abilities to self-renew and differentiate. Moreover, these cells were capable of initiating highly invasive HCC in nonobese diabetic, severe combined immunodeficient mice. Activation of Wnt/beta-catenin signaling enriched the EpCAM(+) cell population, whereas RNA interference-based blockage of EpCAM, a Wnt/beta-catenin signaling target, attenuated the activities of these cells.

Conclusions: Taken together, our results suggest that HCC growth and invasiveness is dictated by a subset of EpCAM(+) cells, opening a new avenue for HCC cancer cell eradication by targeting Wnt/beta-catenin signaling components such as EpCAM.

Figure 1

HpSC-HCC represents a subset of invasive HCCs with cancer stem cells (CSC) Features. (A) Hierarchical cluster analysis based on 793 HpSC-HCC-coregulated genes in 156 HCC cases. Each cell in the matrix represents the expression level of a gene in an individual sample. Red and green cells depict high and low expression levels, respectively, as indicated by the scale bar. (B) Pathway analysis of HpSC-HCC-coregulated genes. The top ten canonical signaling pathways activated in cluster-A (upper panel) or cluster-B (lower panel) with statistical significance (P < 0.01) are shown. (C) Expression patterns of well-known HpSC and MH markers in each HCC subtype as analyzed by microarray. (D) Kaplan-Meier survival analysis of the cases used for array analysis. (E) Frequency of macroscopic and microscopic portal vein invasion in HpSC-HCC and MH-HCC used for IHC. (F) Representative images of EpCAM, AFP, and CK19 staining in HpSC-HCC samples analyzed by IHC and IF. EpCAM staining illustrates heterogeneous expression of EpCAM in HpSC-HCC (left panel). EpCAM⁺ cells were disseminated in the invasive border (left panel black arrows) with expression of AFP (right top panel) and CK19 (right bottom panel).

Figure 2

Characterization of hepatic stem cell marker expression in HCC cell lines. (A) IF analysis of six HCC cell lines (EpCAM⁺ AFP⁺ cell lines; HuH1, HuH7, and Hep3B, EpCAM⁻ AFP⁻ cell lines; SK-Hep-1, HLE, and HLF) stained with anti-EpCAM, anti-CD133, anti-CD90, anti-CK19, anti-Vimentin, anti-Hep-Par1, and anti-β-catenin antibodies. (B) FACS analysis of six HCC cell lines stained with anti-EpCAM, anti-CD133, and anti-CD90 antibodies.

Figure 3

Characterization of EpCAM⁺ and EpCAM⁻ cells in HuH7 cells. (A) FACS analysis of EpCAM⁺ and EpCAM⁻ cells day 1 after cell sorting. (B) IF analysis of cells stained with anti-EpCAM, anti-AFP, anti-CK19, or anti-β-catenin antibodies. (C) qRT-PCR analysis of EpCAM⁺ and EpCAM⁻ HuH7 cells (left upper panel) or hepatic stem cells (HpSC) and mature hepatocytes (MH) (left lower panel). Experiments were performed in triplicate. Hierarchical cluster analysis of HpSC, MH, EpCAM⁺ and EpCAM⁻ HCC cells using a panel of genes expressed in human embryonic stem cells (right panel). Gene expression was measured in quadruplicate. (D) Representative photographs of the plates containing colonies derived from 2,000 EpCAM⁺ or EpCAM⁻ HuH7 cells (upper panel). Colony formation experiments were performed in triplicate (mean ± SD) (middle panel). Cell invasiveness of EpCAM⁺ and EpCAM⁻ cells using the Matrigel invasion assay (lower panel). (E) Flow cytometer analysis of EpCAM⁺ and EpCAM⁻ HuH7 cells stained with anti-EpCAM at day 1 and 14 after cell sorting. (F) Percent of sorted EpCAM⁺ and EpCAM⁻ cells after culturing with various times as analyzed by IF. Numbers of EpCAM⁺ and EpCAM⁻ cells were counted in three independent areas of Chamber Slides at day 1, 3, 7, and 15 after cell sorting. The average percentages of EpCAM⁺ or ⁻ cells are depicted as red or blue, respectively.

Figure 4

Spheroid formation of EpCAM⁺ HuH1 HCC cells. A representative phase contrast image of an HCC spheroid derived from an EpCAM⁺ cell (scale bar = 100 μm) (A) and total numbers of spheroids from 1,000 sorted cells (B) are shown. Experiments were performed in triplicate and data are shown as mean ± SD. (C) Representative confocal images of an HCC spheroid co-stained with anti-EpCAM, anti-AFP and DAPI (scale bar = 50 μm). (D) A 3-D image of an HCC spheroid co-stained with anti-EpCAM, anti-AFP and DAPI (scale bar = 50 μm) reconstructed from confocal images using surface rendering. (E) FACS analysis of EpCAM⁺ cells cultured as spheroids cells (red) or attached cells (blue) for 14 days after cell sorting. (F) Confocal images of an HCC spheroid co-stained with anti-PCNA, anti-AFP and DAPI (scale bar = 50 μm).

Figure 5

Tumorigenic and invasive potential of EpCAM⁺ HCC cells. (A) Representative NOD/SCID mice (upper panel) with subcutaneous tumors (lower panel) from EpCAM⁺ (black arrows) or EpCAM⁻ (white arrows) HuH1 cells. (B) Tumorigenicity of 200 sorted HuH1 cells. (C) Histological analysis of EpCAM⁺ HuH1-derived xenografts. H&E staining of a subcutaneous tumor (left upper panel) with capsular invasion (left lower panel) and muscular invasion (right lower panel) and IF of the tumor stained with anti-EpCAM, anti-AFP, and DAPI (right upper panel) (scale bar = 50 μm). (D) Tumorigenicity of 1,000 sorted cells derived from an EpCAM⁺ HuH1 xenograft. Data are generated from 10 mice in each group. (E) Representative NOD/SCID mice (left panel) with subcutaneous tumors from CD133⁺ (black arrows) or CD133⁻ (white arrows) (mid panel) and EpCAM⁺ (black arrows) or EpCAM⁻ (white arrows) (right panel) HuH1 cells. (F) Tumorigenicity of 1,000 HuH1 cells sorted by anti-EpCAM (left panel) or anti-CD133 (right panel) antibodies.

Figure 6

Wnt/β-catenin signaling augments EpCAM⁺ HCC cells. (A) Flow cytometer analysis of HuH1, HuH7, and HLF cells treated with 2μM of BIO (orange) or MeBIO (green) for 10 days and stained with anti-EpCAM, anti-CD133 and anti-CD90 antibodies. (B) TOP-FLASH luciferase assays of HuH7 cells treated with 2μM of BIO (red) or MeBIO (green). (C) Flow cytometer analysis of HuH7 cells cultured in the normal media (DMEM supplemented with 10% FBS) or Wnt10B conditioned media (details are described in the Materials and Methods). Cells were cultured in each medium for 2 weeks. (D) Representative phase contrast images (left panel, scale bar = 100 μm) or IF images (right panel, scale bar = 50 μm) of HuH7 cells treated with 2μM of BIO or MeBIO for 14 days. (E) qRT-PCR analysis of representative HpSC-HCC related genes in HuH7 cells treated with 2μM of BIO or MeBIO for 14 days. (F) Spheroid formation assay of HuH7 cells treated with 2μM of BIO or MeBIO for 14 days (mean ± SD).

Figure 7

EpCAM blockage inhibits the tumorigenic and invasive capacity of EpCAM⁺ HCC cells. (A) Enrichment of EpCAM⁺ cells after 5-FU treatment. HuH1 cells were without (green) or treated with 2 μg/ml of 5FU for 3 days and analyzed by FACS using anti-EpCAM and anti-CD133 antibodies. (B) Spheroid formation of HuH1 cells treated with 2 μg/ml of 5FU (red) for 3 days. (C) FACS analysis of HuH1 cells treated with a control siRNA (orange) or EpCAM-specific siRNA (green) at day 3 after transfection. Spheroid formation (D) or invasive capacity (E) of EpCAM⁺ HuH1 cells transfected with a control siRNA or EpCAM-specific siRNA. Experiments were performed in triplicate and the data are shown as mean ± SD. (F) Inhibition of tumor formation in vivo by EpCAM gene silencing. EpCAM⁺ HuH1 cells were transfected with siRNA oligos and 1,000 cells were injected 24 hours after transfection.