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. Author manuscript; available in PMC: 2023 Jun 1.
Published in final edited form as: J Pediatr Surg. 2022 Feb 14;57(6):1018–1025. doi: 10.1016/j.jpedsurg.2022.01.063

Metastatic Human Hepatoblastoma Cells Exhibit Enhanced Tumorigenicity, Invasiveness and a Stem Cell-Like Phenotype

Raoud Marayati 1, Janet R Julson 1, Laura V Bownes 1, Colin H Quinn 1, Sara C Hutchins 2, Adele P Williams 1, Hooper R Markert 1, Andee M Beierle 1, Jerry E Stewart 1, Anita B Hjelmeland 3, Elizabeth Mroczek-Musulman 4, Elizabeth A Beierle 1,†
PMCID: PMC9119922  NIHMSID: NIHMS1780273  PMID: 35300860

Abstract

Background/Purpose:

Metastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis.

Materials/Methods:

Using a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3.

Results:

The HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin.

Conclusion:

This newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma.

Keywords: hepatoblastoma, metastases, stem cell, cisplatin

1. Introduction

Metastatic hepatoblastoma continues to pose a significant treatment challenge. Up to 20 % of hepatoblastoma patients have pulmonary metastasis at presentation, and the overall survival of these children may be as low as 25 % [1]. While patients with a primary tumor confined to the liver may be cured with surgical resection, treatment options for patients with metastatic disease rely on intensive chemotherapeutic regimens that carry serious toxicities and long-term effects.

Cancer cells must undergo a series of sequential events to metastasize and establish malignant secondary tumors. These events include survival in the circulation, extravasation through the vasculature, and invasion into normal tissue parenchyma [2, 3], but investigators have not fully defined these events in hepatoblastoma. Thus, there is a critical need for research efforts that will i) identify therapeutic targets, which might suppress one or more steps in the metastatic cascade, thereby preventing disease progression, and ii) lead to the use of a more specific and effective molecular-targeted therapies, alone, or in combination with current treatment regimens.

In the current study, we aimed to generate and characterize a metastatic hepatoblastoma cell line designed to investigate the molecular mechanism associated with hepatoblastoma metastasis. We showed that these metastatic hepatoblastoma cells exhibited enhanced tumorigenicity and invasiveness, were enriched for stem cell like cancer cells (SCLCCs), and were resistant to cisplatin chemotherapy. This novel cell line provides a tool to study the mechanisms responsible and potential therapeutics for hepatoblastoma metastasis.

2. Material and Methods

2.1. Cells and Cell Culture

The human long-term passage hepatoblastoma cell line, HuH6, was obtained from Thomas Pietschmann (Hannover, Germany) [4] and maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10 % fetal bovine serum (HyClone, GE Healthcare Life Sciences, Logan, UT), 1 μg/mL penicillin/streptomycin (Gibco, Carlsbad, CA), and 2 mmol/L L-glutamine (Thermo Fisher Scientific, Waltham, MA). We validated the HuH6 cell line within the past 12 months using short tandem repeat analysis (Genomics Core, University of Alabama at Birmingham (UAB), Birmingham, AL) and determined them free of mycoplasma. Cells were maintained at 37 °C and in a humidified atmosphere containing 5 % CO2.

The HuH6Luc cells were generously provided by the Hjelmeland laboratory and were established by stable transfection of HuH6 cells with the luciferase reporter cloned into the pCDH-CMV-MCS-EF1a-Puro lentiviral vector (System Biosciences, Palo Alto, CA). HuH6-Luc cells were cultured in the media described above with the addition of puromycin (1 μg/mL, Sigma-Aldrich, St. Louis, MO) to maintain selection. We validated the HuH6Luc cell line within the past 12 months using short tandem repeat analysis (Genomics Core, UAB) and determined them free of mycoplasma.

2.2. Generation of Metastatic Hepatoblastoma Cell Lines

The UAB Institutional Animal Care and Use Committee (IACUC-021420) approved all animal studies, and the studies conducted within institutional, national, and NIH guidelines. Animals were maintained in the specific pathogen-free facility with standard 12-hour light/dark cycles and access to chow and water ad libitum. Animals were humanely euthanized in their home cages with CO2 followed by cervical dislocation.

Metastatic hepatoblastoma cells were developed as depicted in Figure 1. HuH6Luc (2 × 106) cells in 100 μl of phosphate-buffered saline (PBS) were injected into the tail vein of 6-week-old female athymic nude mice (Charles River, Frederick, MD). Starting at 3 weeks following the initial injection and then weekly, the mice were administered d-luciferin substrate via peritoneal injection and imaged for bioluminescence using an IVIS® Lumina III with an EMCCD camera (PerkinElmer, Waltham, MA) to monitor for formation of lung metastasis. Animal weight was measured three times a week and animals were euthanized once they developed visible macrometastasis on imaging or reached IACUC parameters. Lungs were harvested, imaged ex vivo, and a portion fixed in 10 % formalin for histology. Grossly visible metastatic pulmonary lesions were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ) and plated into cell culture to derive the metastatic hepatoblastoma cell line, termed HLM_1. These cells were injected into the tail vein of a second animal and the same cycle continued as above to derive the metastatic hepatoblastoma cell lines HLM_2 and subsequently HLM_3. HLM_1, HLM_2, and HLM_3 cells were maintained under standard culture conditions and in the same media as described above with the addition of puromycin (1 μg/mL, Sigma-Aldrich).

Figure 1. Establishment of the metastatic hepatoblastoma cell lines.

Figure 1.

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(A) Using serial tail vein injections, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established the metastatic hepatoblastoma cell lines, HLM_1, HLM_2, and HLM_3. HuH6Luc (2 × 106) cells were injected into the tail vein of 6-week-old female athymic nude mice. Starting at 3 weeks, mice were imaged weekly for bioluminescence to detect formation of lung metastasis. Once metastasis detected, lungs were harvested, imaged ex vivo, with a portion fixed in formalin and another portion dissociated into single-cell suspension for culture to derive the metastatic hepatoblastoma cell line, HLM_1. When HLM_1 cells grew to confluence they were injected into the tail vein of a second animal and the same cycle continued to derive the metastatic hepatoblastoma cell lines HLM_2 and HLM_3. (B) To determine whether metastatic cell lines retained bioluminescence, cells (5 × 103) from each cell line were plated into 4 wells of a 96-well plate, allowed to attach overnight, then imaged for bioluminescence following addition of d-luciferin substrate using an IVIS® Lumina III in vivo imaging system. HLM_1Luc, HLM_2Luc, and HLM_3Luc cells maintained stable expression of luciferase comparable to the HuH6Luc parent cell line. The non-luciferase expressing HuH6 cell line, media only (first column, top 4 wells) and media with the addition of the selection agent, puromycin (first column, bottom 4 wells), were included as internal controls. (C) Cell morphology was compared between the parent and metastatic cell lines. Cells (1 × 105) were plated into 6-well plates and imaged after 24 hours to assess for morphology. Representative photomicrographs are shown. HLM_1, HLM_2, and HLM_3 cells did not differ in cell morphology from that of the HuH6 parent cell line. Scale bars represent 300 μm.

2.3. Cell Proliferation and Growth

Cell proliferation was measured using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). HLM_1, HLM_2, HLM_3, or the parent HuH6 cells (1 × 103 cells per well) were plated in 96-well plates. After 24 hours of incubation, 10 μL of CellTiter 96® reagent was added to each well and the absorbance read at 490 nm to detect the formazan product using a microplate reader (BioTek Gen5, BioTek, Winooski, VT). For cell growth over time, HuH6, HLM_1, HLM_2, or HLM_3 cells (5 × 104 cells per well) were plated in 12-well plates, incubated for 24, 48, 72, or 96 hours, stained with trypan blue (0.4 %, Gibco), and counted with a hemocytometer. Results of proliferation and cell growth from at least three biologic replicates were reported as mean fold change ± standard error of the mean (SEM).

2.4. Motility Assays

For migration assays, 24-well culture plates (Corning Life Sciences, Corning, NY) were utilized as previously described [5]. Briefly, the bottom side of 8 μm Transwell® inserts were coated with collagen I (10 μg/mL, MP Biomedicals, Santa Ana, CA) overnight at 37 °C and then washed with PBS. For invasion assays, in addition to collagen on the bottom, the topside of the inserts was coated with 50 μL of Matrigel™ (1 mg/mL, BD Biosciences, San Jose, CA) overnight at 37 °C. For both migration and invasion, HuH6, HLM_1, HLM_2, or HLM_3 cells (3 × 104 cells) were plated onto the insert and allowed to migrate or invade for 24 hours. The inserts were fixed with 3 % paraformaldehyde and stained with 1 % crystal violet. Images of the inserts were obtained using a light microscope and the number of cells in seven random fields per insert were counted using ImageJ (https://imagej.nih.gov/ij). Migration and invasion were reported as mean fold change in number of cells migrating or invading ± SEM.

2.5. Anchorage-Independent Growth

Soft agar colony formation was used to assess for anchorage-independent growth as previously described [6]. Briefly, a 3 mL mixture of 1 % noble agar (BD Biosciences) and 2× culture media (in a 1:1 ratio) was poured into 60 × 15 mm petri dishes and allowed to cool. After solidification of the first layer, we added a second 1.5 mL layer containing the same ratio of agar and culture media, but also HuH6, HLM_1, HLM_2, or HLM_3 cells (1 × 104 cells per dish). We replaced 1 mL of culture media every 3–4 days. After 6 weeks, colonies were stained with crystal violet, images were taken using the Bio-Rad ChemiDoc™ MP Imager (Bio-Rad, Hercules, CA), and colony growth was quantified using ImageJ software. Colony count reported as mean fold change in number of colonies ± SEM.

2.6. In Vivo Formation of Lung Metastases

Having established that the HLM_3 cells proliferated more and had better motility than the HLM_1 or _HLM_2 cells, we completed the remaining studies using the HLM_3 cell line in comparison with the parent HuH6 cells.

For in vivo lung metastasis, tail vein injections of HuH6Luc or HLM_3Luc cells (2 × 106 cells in 100 μl of PBS) were performed (n=7 per group), and bioluminescence imaging was used to monitor the mice as described above. Animals were euthanized after 4 weeks and lungs were harvested, imaged ex vivo to quantify the number of metastatic lesions, and fixed for histologic studies.

2.7. Histology

Pulmonary metastasis were confirmed using hematoxylin and eosin (H&E) staining. Lungs were fixed in 10 % formalin, embedded in paraffin (FFPE), sectioned into 5 μm slices at 100 μm intervals through the entire lung of each animal, and stained with routine H&E. Sections at six levels from each lung were examined by a board-certified pediatric pathologist (E.M.M.), blinded to the treatment groups, to confirm the presence or absence of pulmonary metastasis.

To quantify the metastatic burden, H&E-stained lung sections at three different levels from each mouse were imaged using 10× objective of a light microscopy (Photometrics CoolSNAP HQ2 CCD camera (Tucson, AZ) attached to a Nikon Eclipse Ti microscope (Tokyo, Japan)). Metastatic burden was quantified using ImageJ software and by determining the total area of lung metastasis (in pixel squared). Results were reported as mean ± SEM.

2.8. CD133 Expression

Flow cytometry was used to evaluate the cell surface expression levels of CD133. HuH6 or HLM_3 cells (1 × 106) were labeled with allophycocyanin (APC)-conjugated mouse immunoglobulin G1 (IgG1) anti-human CD133/1 (clone AC133, Miltenyi Biotec, Waltham, MA) according to the manufacturer’s instructions. The percent of cells positive for APC was determined via flow cytometry using the Attune™ NxT Flow Cytometer (Invitrogen™, Thermo Fisher).

2.9. Response to Cisplatin Therapy

To assess cell viability following cisplatin treatment, the alamarBlue® Cell Viability Assay (Thermo Fisher Scientific) was used. HuH6 or HLM_3 cells (5 × 103 cells per well) were plated in 96-well plates, allowed to attach overnight, and treated with cisplatin at increasing concentrations (0 to 100 μM). Following 72 hours of treatment, 10 μL of alamarBlue® reagent was added to each well and the absorbance was read at 562 nm (reduced reagent) and 595 nm (oxidized reagent) using a microplate reader (BioTek Gen5). Viability results from at least three biologic replicates were reported as mean fold change ± SEM.

2.10. Statistical Analysis

All experiments were performed with a minimum of three biologic replicates. Data were reported as mean ± SEM of separate experiments. Student’s t-test (two-sided) or analysis of variance (ANOVA) was used to compare means between groups as appropriate, with p ≤ 0.05 determined to be statistically significant.

3. Results

3.1. Metastatic Hepatoblastoma Cells Exhibited Enhanced Tumorigenicity In Vitro

Using a model of serial tail vein injections, non-invasive bioluminescence imaging, and dissociation of gross metastatic pulmonary lesions (Figure 1 A), we successfully established the metastatic hepatoblastoma cell lines, HLM_1, HLM_2, and HLM_3. These cell lines successfully propagated in culture, were frozen, banked, and thawed for successful regrowth in vitro and in vivo. The parent HuH6 and metastatic hepatoblastoma cells maintained stable expression of luciferase through serial passages through culture when evaluated using bioluminescence imaging (Figure 1 B). The metastatic hepatoblastoma cells exhibited comparable morphology to the parent hepatoblastoma cell line, HuH6 (Figure 1 C).

We first assessed proliferation using CellTiter 96® assay and found that HLM_1, HLM_2, and HLM_3 cells had significantly increased proliferation compared to the parent HuH6 cell line (1.15 ± 0.01, 1.33 ± 0.02, 1.63 ± 0.04 fold change HLM_1, 2, 3, respectively vs. HuH6, p≤0.001, Figure 2 A), which increased with each tail vein passage. Similarly, when we evaluated growth over time, we found with each subsequent tail vein passage an acceleration in growth compared to the parent HuH6 cell line (p≤0.05, Figure 2 B), suggesting an enhanced tumorigenicity in vitro.

Figure 2. Metastatic hepatoblastoma cells exhibited enhanced proliferation in vitro.

Figure 2.

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(A) CellTiter 96® assay measured proliferation. HuH6, HLM_1, HLM_2, and HLM_3 cells (1 × 103) were plated in 96-well plates. After 24 hours, 10 μL of CellTiter 96® reagent was added to each well and the absorbance read at 490 nm to detect the formazan product. HLM_1, HLM_2, and HLM_3 metastatic hepatoblastoma cells had significantly increased proliferation compared to the parent HuH6 cell line. (B) For cell growth over time, HuH6, HLM_1, HLM_2, or HLM_3 cells (5 × 104) were plated in 12-well plates, incubated for 24, 48, 72, or 96 hours, Cells were stained with trypan blue and counted to determine growth rates. HLM_1, HLM_2, and HLM_3 cells exhibited a significantly accelerated growth rate compared to HuH6 cells over the course of 96 hours. Data represent at least three biologic replicates and reported as mean ± SEM. * p≤0.05, *** p≤0.001

3.2. Metastatic Hepatoblastoma Cells Exhibited a More Invasive Phenotype In Vitro

The enhanced ability to migrate, invade, and grow in an anchorage-independent manner facilitates the metastatic potential of cancer cells. Cell migration and invasion were assessed using modified Boyden chamber assays. HLM_1, HLM_2, and HLM_3 cells exhibited significantly increased migration and invasion compared to HuH6 cells (Figure 3 A, B). Anchorage-independent growth is another measure of cell motility and metastatic potential. We assessed anchorage-independent growth using soft agar assays. HLM_1, HLM_2, and HLM_3 cells exhibited increased fold change in anchorage-independent colony formation compared to HuH6 cells (5.2 ± 0.5, 15.4 ± 0.4, 35.6 ± 0.1 fold change HLM_1, 2, 3, respectively, compared to HuH6 cells, p≤0.001, Figure 3 C). These findings indicate that HLM_1, HLM_2, and HLM_3 cells exhibited a more invasive phenotype than the HuH6 parent cell line in vitro. Representative images of migration and invasion inserts and soft agar plates stained with crystal violet are shown below the graphs (Figure 3 A, B, C, scale bars 100 μm). When comparing each of the subsequent tail vein passages, the numbers of colonies significantly increased with each passage (204 ± 19 vs. 608 ± 17 colonies, HLM_1 vs. HLM_2, p≤0.001; 608 ± 17 vs. 1400 ± 3 colonies, HLM_2 vs. HLM_3, p≤0.001). These data suggest selection for more metastatic hepatoblastoma cell variants over each subsequent passage. We therefore chose to narrow our focus and employed the HLM_3 cell line for the remainder of this study.

Figure 3. Metastatic hepatoblastoma cells exhibited enhanced invasiveness in vitro.

Figure 3.

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(A) Migration and (B) invasion were assessed using modified Boyden chamber assays. HuH6 or HLM_1, HLM_2, or HLM_3 cells (3 × 104) were seeded into modified Boyden chambers. Inserts were coated on the bottom with chemoattractant collagen 1. A layer of Matrigel™ was added to the top of the insert for invasion studies. After 24 hours, photographs were taken with representatives shown (bottom panels) and migration and invasion from at least three biologic replicates reported as mean fold change in number of cells migrating or invading, respectively, ± SEM. Scale bars represent 100 μm. HLM_1, HLM_2, and HLM_3 cells exhibited a significant increase in (A) migration and (B) invasion compared to HuH6 cells. (C) Soft agar assays assessed anchorage-independent growth. Images of the soft agar dishes were taken and representatives shown (bottom panels). Anchorage-independent growth was quantified from at least three biologic replicates and reported as mean fold change in colony count ± SEM. HLM_1, HLM_2, and HLM_3 cells exhibited significantly increased colony formation compared to HuH6 cells. * p≤0.05, ** p≤0.01, *** p≤0.001

3.3. Metastatic Hepatoblastoma Cells Increased Formation of Lung Metastasis In Vivo

To establish whether the HLM_3 metastatic hepatoblastoma cells exhibited a more invasive phenotype in vivo, we evaluated their ability to form pulmonary metastasis following tail vein injections in athymic nude mice (n=7 per group). After 4 weeks, bioluminescence imaging of the lungs ex vivo revealed that 43 % of mice (3 of 7 animals, 5 metastatic lesions, white arrows) injected with the HLM_3 cells compared to 14 % of mice (1 of 7 animals, with a single metastatic lesion, white arrow) injected with HuH6 cells (Figure 4 A, B) had pulmonary metastasis. Pathologic examination of H&E staining of lung tissue confirmed the lesions to be hepatoblastoma pulmonary metastasis. Representative lung sections are shown (Figure 4 C) with the border between the metastatic lesions and normal lung tissue marked (dotted line). When histology sections from multiple lung levels were evaluated and microscopic metastasis quantified, animals injected with HLM_3 cells had a significantly greater metastatic burden compared with mice injected with HuH6 cells (p≤0.05, Figure 4 D).

Figure 4. Metastatic hepatoblastoma cells increased formation of lung metastasis in vivo.

Figure 4.

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(A) Tail vein injections of HuH6Luc or HLM_3Luc cells (2 × 106) with stable expression of the luciferase reporter (superscript Luc) were performed (n=7 per group). At 4 weeks, mice were euthanized and the lungs imaged for bioluminescence ex vivo. (B) Pulmonary metastases were observed in 43 % of mice (3 out of 7 mice, white arrows) injected with the HLM_3Luc cells compared to 14 % of mice (1 out of 7, white arrow) injected with the parent HuH6Luc cells (NS, p=0.2). (C) H&E staining of lung tissue confirmed the presence of pulmonary metastasis. Representative H&E stained lung sections from both groups are shown with the metastatic lesions marked (dotted line). (D) H&E-stained lung sections at three different levels from each mouse were imaged using light microscopy. Metastatic burden was quantified using ImageJ software and by determining the total area of lung metastasis (in pixel squared). Results from each group were reported as mean ± SEM. Tail vein injections of HLM_3Luc cells resulted in an increased metastatic burden compared to those mice injected with HuH6 Luc cells. ** p≤0.01

3.4. Metastatic Hepatoblastoma Cells Exhibited a Stem Cell-Like Phenotype

SCLCCs, a subpopulation of cancer cells with stem-cell like properties, are thought to contribute to cancer metastasis [7]. To determine whether the HLM_3 metastatic hepatoblastoma cells exhibited a more stem-cell like phenotype than the parent HuH6 cell line, we evaluated two characteristics of hepatoblastoma SCLCCs: i) cell surface expression of CD133, and ii) mRNA abundance of common stem cell markers.

The HLM_3 cell line was enriched for hepatoblastoma SCLCCs as evidenced by the increase in i) cell surface expression of CD133 (57 ± 6 % of HLM_3 vs. 31 ± 1.6 % of HuH6 cells positive for CD133, p≤0.01, Figure 5 A) and ii) mRNA abundance of the common stemness markers Oct4, Nanog, Sox2, and nestin (2.6, 2.7, 2.9, and 2.1 fold higher in HLM_3, respectively, p≤0.05, Figure 5 B). Representative contour plots are shown below the graphs in Figure 5 A. A negative control consisting of cells with no antibody staining was included for each flow cytometry assay (Figure 5 A, bottom left panel).

Figure 5. Metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were resistant to cisplatin.

Figure 5.

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(A) CD133 cell surface expression was determined using flow cytometry. HLM_3 cells had significantly increased CD133 expression compared to HuH6 cells. A negative control consisting of HuH6 or HLM_3 cells with no antibody staining was included for each flow cytometry assay. Representative contour plots for each cell line along with a negative staining control (for HuH6) are shown below the graph. (B) Quantitative real-time PCR was used to examine the mRNA abundance of Oct4, Nanog, Sox2, and nestin. Gene expression was normalized to β-actin and calculated as fold change to HuH6 cells using the ΔΔCt method. HLM_3 cells had increased mRNA abundance of stem cell markers compared to HuH6 cells. (C) HuH6 or HLM_3 cells (5 × 103) were plated in 96-well plates and treated with cisplatin at increasing concentrations (0 to 100 μM). Following 72 hours of treatment, 10 μL of alamarBlue® reagent was added to each well and the absorbance read. Viability of HuH6 cells was significantly decreased compared to HLM_3 cells, beginning at 2.5 μM concentration of cisplatin. There was no significant change in HLM_3 viability at even the highest concentration of cisplatin (100 μM). Data represent at least three biologic replicates and reported as mean ± standard error of the mean. * p≤0.05, ** p≤0.01, *** p≤0.001.

3.5. Metastatic Hepatoblastoma Cells were Resistant to Cisplatin

Since cisplatin chemotherapy is the standard of care in hepatoblastoma treatment, and SCLCCs are often chemoresistant, we sought to evaluate the sensitivity of the HLM_3 metastatic hepatoblastoma cells to cisplatin. HuH6 or HLM_3 cells were treated with cisplatin at increasing concentrations (0 to 100 μM) for 72 hours and their viability assessed using an alamarBlue® assay. The HLM_3 metastatic hepatoblastoma cells were more resistant to cisplatin compared to the parent HuH6 cell line as evidenced by their continued viability at high concentrations of cisplatin and their statistically significant increased viability compared to HuH6 cells beginning at cisplatin concentrations of 2.5 μM (Figure 5 C).

4. Discussion

Despite advances in the care of hepatoblastoma patients, survival in those presenting with metastatic disease remains as low as 25 % [1]. This poor survival is likely due to the systemic nature of metastatic disease and a more complex tumor biology driving metastasis, including expression of aggressive gene signatures leading to vascular invasion, metastatic spread, and the resistance of disseminated cancer cells to existing anti-cancer therapies. Current treatment protocols for metastatic hepatoblastoma heavily rely on treatment plans generated over twenty years ago and frequently do not incorporate targeted therapies. The ability to test such novel therapies is limited due to the inaccessibility of cell lines and mouse models [8, 9]. The majority of hepatoblastoma models described in the literature are derived from patients with favorable histology primary hepatoblastoma tumors, thereby lacking the diversity of high-risk subtypes and distant metastasis.

There are only a handful of reports describing the occurrence of metastasis in the context of hepatoblastoma models. Mokkapati et al. reported lung metastases in a genetically-engineered murine model (2 out of 20 mice), however the mice phenotypically expressed both hepatocellular carcinoma (HCC) and hepatoblastoma, and it was not clear which of the cell types gave rise to the metastases [10]. In other studies utilizing intra-splenic injections of HuH6 cells, extra-hepatic tumor was detected in 15 % of cases (2 out of 13 mice) but was localized to the peritoneum and ovaries, and no pulmonary metastasis were detected [11], which is not consistent with the clinical picture of metastatic hepatoblastoma. One promising approach to modeling hepatoblastoma has been the development of patient-derived xenografts (PDX). Bissig-Choisat et al. were the first to observe lung metastasis in 3 out of their 6 established hepatoblastoma PDXs that were engrafted in the murine liver capsule [12]. Only a single PDX derived from a human hepatoblastoma lung metastasis has been described in the literature [13]. PDX models are limited by several factors including the inability to propagate well in culture and their inaccessibility for high-throughput drug testing [14]. To our knowledge, there is no known long-term passage cell line derived from metastatic hepatoblastoma. In the current study, we established a novel human hepatoblastoma metastatic cell line that maintains its features in serial culture and forms metastasis in animals. We employed the HuH6 human hepatoblastoma cell line, which remains the only commercially available long-term passage cell line whose gene expression signature is consistent with hepatoblastoma [15, 16] to create the HLM_3 metastatic cell line. We acknowledge solely using long-term stable cell lines for cancer research has limitations, since these cells are cultured under contrived conditions that may lead to a loss of genetic and phenotypic characteristics that more closely represent the human condition.

SCLCCs may be responsible for both metastatic initiation and progression. Growing evidence supports a hierarchical model for metastasis [17], in which pre-existing subpopulations of stem-like cells act as a source for migratory and invasive cells in the primary tumors that initiate metastasis. Research has suggested that although many tumor cells may enter the bloodstream, only SCLCCs survive and produce metastases at distant sites [18–20]. We demonstrated that the HLM_3 metastatic cells exhibited enhanced tumorigenicity and invasiveness in vitro and in vivo, which was associated with an increased SCLCC phenotype compared to the parent HuH6 cells. This finding was consistent with other investigators who found metastatic tumors to have an undifferentiated molecular signature and were comprised of a greater proportion of SCLCCs than primary tumors [21]. Lawson et al. isolated metastatic cells from PDX models of human breast cancer and, utilizing single-cell analysis, revealed a stem-cell program [17]. In colon cancer, highly metastatic cell populations contained more SCLCCs compared to primary colon cancer cells, and had increased expression levels of Sox2, Nanog, and Oct4 [22].

Few definitive stem cell markers are available for the investigation of hepatoblastoma SCLCCs. Stafman et al. identified CD133 as one such marker, revealing that CD133+ cells exhibited greater ability to form tumorspheres in non-adherent conditions and had high tumorigenic potential in animals compared to CD133− cells [23]. In the current study, we utilized CD133 cell surface marker expression as well as mRNA abundance of the stemness markers Oct4, Nanog, Sox2, and nestin to demonstrate that HLM_3 cells are enriched for hepatoblastoma SCLCCs.

Since SCLCCs are resistant to a variety of therapeutic interventions [7], partly because of enhanced survival mechanisms [24], metastatic cells may also be resistant to therapy. Um et al. reported that highly metastatic colon cancer cell lines were resistant to anticancer drugs and ionizing radiation [25]. Similarly, uveal melanoma cells derived from a metastatic liver lesion contained SCLCCs that were able to survive cisplatin chemotherapy compared to cells derived from primary tumors [26]. This finding was evident in the HLM_3 metastatic hepatoblastoma cells, which were more resistant to cisplatin chemotherapy than the parent HuH6 cells. Cisplatin, a DNA cross-linking agent, is commonly used to treat patients with advanced or metastatic hepatoblastoma [27]. However, with chemoresistance being a significant limitation to effective treatment of many hepatoblastoma patients, it is essential to develop effective ways to study these chemoresistant metastatic cells to enable the development of innovative interventions.

A limitation of this study, which is inherent to the model used to generate the metastatic hepatoblastoma cells, is the utilization of Luc-tagged cells. Bioluminescence imaging of luciferase-labeled cells [28] has proven to be a powerful methodology allowing for repeated and non-invasive tracking and quantification of cells in intact small animals [29]. However, the expression and production of the luciferase enzyme may not be stable over time and across different culture conditions [30]. The bioluminescence application may not be sensitive enough to detect small microscopic metastatic lesions [31]. In addition, loss of reporter signal has been reported. For example, Baklaushev et al. described the development of natural or induced immune responses against the reporter that may restrict both the invasiveness and the metastatic potential of some reporter-labelled tumor cells, although those studies involved the use of immunocompetent hosts [32]. The cells in the current study were monitored for the continued expression of the reporter and found to retain expression through multiple passages in culture. Histologic examination confirmed the pulmonary lesions identified by bioluminescence were metastasis and identified microscopic metastases that were not detected by bioluminescence.

5. Conclusions

Using serial tail vein injections of cells from hepatoblastoma pulmonary metastasis, we have successfully established a metastatic human hepatoblastoma cell line that demonstrated enhanced tumorigenicity and invasiveness, both in vitro and in vivo. Furthermore, the metastatic hepatoblastoma cells were enriched for SCLCCs and were more resistant to cisplatin. Future studies utilizing these cell lines will enable us to identify novel targets and design innovative therapeutic strategies for the treatment of children with metastatic hepatoblastoma.

Acknowledgements

The authors wish to thank Vidya Sagar Hanumanthu and the UAB Comprehensive Flow Cytometry Core (supported by NIH P30 AR048311 and NIH P30 AI27667), the Small Animal Imaging Facility (supported by the O’Neal Cancer Center Preclinical Imaging Shared Facility P30CA013148), the IVIS Lumina III S10 instrumentation grant 1S10OD021697.

Funding

This project was made possible by funding from the National Cancer Institute of the National Institutes of Health under award numbers T32 CA229102 (RM, JRJ, and LVB), T32 CA183926 (APW), 5T32GM008361 (CHQ), P30 AR048311 and P30 AI027767 to the UAB Flow Cytometry Core, and P30CA013148 to the UAB Small Animal Imaging Facility. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Other funding sources include Cannonball Kids cancer (EAB), Starr Fund-Vince Lombardi Cancer Foundation (EAB), and the Society of University Surgeons (RM).

Footnotes

Declaration of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

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