Background
Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the second leading cause of cancer mortality [
1]. HCCs’ prognosis is poor due to chemo and radiotherapy resistance [
2,
3]. Consequently, there is a serious need for new treatment options. Small animal models are often the only available means of testing the safety, potency, and efficacy of new anticancer agents prior to clinical trials. Several types of mouse models of HCC are available, depending on tumor-inducing mechanisms. For drug development, the most frequently used models are xenograft models. They are easy to obtain and follow up, with a rapid growth rate, but their relevance is limited, as the resemblance between xenograft tumors and human HCC is rather poor [
4]. Additionally, there are significant differences in tumor growth inhibition between HCC cell lines [
5].
Spontaneous developing tumor models (genetically engineered mice) are more relevant as tumors occur through a multistep process of hepatocarcinogenesis. The natural history is respected (hepatocyte proliferation, dysplasia, neoplasia) and HCCs result from the cooperation and dependency between oncogenes, growth factors and viral genes [
5]. Genetically induced tumor models are generally used to investigate carcinogenic pathways, but very few attempts were made to use them for pharmacological testing.
A hepatocyte-specific
Trim24-null mutant mouse line was used in this study. TRIM24 is a ligand-dependent nuclear receptor co-regulator interacting with Retinoic Acid Receptors (RARs). It was shown to function as a potent liver-specific tumor suppressor by attenuating RAR alpha mediated transcription. Indeed, in genetically engineered mice with silencing of
Trim24 gene, an aberrant activation of RAR alpha, leads to sequentially development of hepatocyte alteration, preneoplastic lesions, and HCC [
6,
7]. In order to perform pharmacological testing in this spontaneously developing HCC model, it was necessary to assess the presence and to ensure the follow-up of intra-abdominal tumor growth without animal sacrifice.
Micro-computed tomography (micro-CT) for small animal imaging has been increasingly used over the last decade [
8]. Liver imaging has been obtained with the enhancement of a radiological agent either through an ApoE receptor-mediated mechanism or a mechanism of nanoparticles uptake by the reticuloendothelial system [
9]. Both types of contrast agents resulted in variable quality of liver imaging depending on dose, imaging timing, subjacent liver pathology [
10], and even mouse strain [
11].
In this study, two main objectives are addressed: first, to provide a reliable imaging method for spontaneously developing liver tumors, and secondly, to evaluate the response to a new anticancer drug, namely myo-inositol trispyrophosphate (ITPP) [
12‐
14] in the hepatocyte-specific
Trim24-null mutant mouse model.
Methods
Animal models
Hepatocyte-specific Trim24-null mice (Trim24L2/L2Alb-Cre)
A hepatocyte-specific
Trim24-null mouse in mixed C57BL/6 x129/Sv mouse strain was used as a spontaneous developing HCC model. As previously described [
6],
Trim24-null mice as well as hepatocyte-specific
Trim24
L2/L2Alb-Cre [
15] develop sequentially abnormal hypertrophic hepatocytes with enlarged nuclei and increased DNA content and ploidy (3 months of age), clear-cell foci of altered hepatocytes (7 months), adenomas (from 9 months), and invasive hepatocarcinomas (from 12 months) giving rise to lung metastases.
Wild type mice were 2 months old male littermates of the Trim24
L2/L2Alb-Cre mice.
Partial hepatectomy
Based on the principle of partial hepatectomy (PH) as promoter of liver carcinogenesis [
16], a two-third partial hepatectomy was performed in
Trim24
L2/L2Alb-Cre males at the age of 2 months. Surgery was performed under isoflurane/oxygen general anesthesia (induction at 3 %, maintaining 1.5 %). A right subcostal laparotomy was performed. A single silk thread was used for suture of the left and medial lobes of the liver (constituting approximately 70 % of the total liver mass) along with the gallbladder. Gallbladder, left and medial lobes were resected, followed by careful hemostasis. The abdominal wall was closed with Vicryl 3/0 separate sutures in two layers (muscular layer and skin). Animals were allowed to recover on a 37 °C warm plate.
The early and medium-term effects of PH were evaluated in five groups of 10 mice (5 wild-types and 5 Trim24
L2/L2Alb-Cre). Mice were sacrificed at 2 months of age, and postoperatively at 48 h, 72 h, 5 days, and 15 days respectively after PH. The long-term effect of PH was evaluated in a group of 6 Trim24
L2/L2Alb-Cre mice, which underwent a PH at the age of 2 months and were sacrificed postoperatively at 5 months. Livers were harvested for analysis under isoflurane/oxygen general anesthesia prior to euthanasia.
HCC xenograft model
This model was obtained as previously described, by means of an orthotopic injection of a suspension of 2 × 10
6 Hep 55.1C cells in C57BL/6 J mice [
17].
Micro-CT-scan
A small animal micro-scanner X (micro CAT II - Imtek/Siemens Medical Solutions, Malvern, PA) was used. The micro CAT II scanner has a single X-ray source and detector technology, and it provides reconstructed images with a voxel size of 119x119x119μm. The technical parameters employed were the following: exposure time: 300 milliseconds, X-ray voltage: 80.0 kVp, and anode current: 500 μA. Micro-CT-scan imaging was performed under isoflurane general anesthesia and with respiratory gating. Images were captured after peak expiration, resulting in a mean scanning time of 15 min. Radiological contrast agents Fenestra™ LC and Fenestra™ VC (ART Advanced Research Technologies Inc., Montreal, Canada) consist in polyiodinated lipids, which are selectively taken up by hepatocytes via an ApoE receptor-mediated pathway. Contrast agent injection was performed in conscious mice, placed in a restraining tube and light heated. In order to prevent volume overload and liver toxicity (see Additional file
1), contrast agent dosages, route and timing of administration were established as follows: Fenestra™ LC (10 μl/g) intraperitoneally, 6 h prior to imaging and Fenestra™ VC (10 μl/g) intravenously in the tail vein, 15 min prior to imaging.
Micro-CT-scan images exported by the Amira™ 3D software were processed with 3D-VPM software (IRCAD, Strasbourg, France) for 3D reconstruction and to compute tumor volumes.
To evaluate micro-CT-scan accuracy, a group of 28 Trim24
L2/L2Alb-Cre mice aged between 3 and 25 months were imaged by means of micro-CT-scan, and then dissected and histologically examined.
HCC follow-up under ITPP treatment - study design
A group of 23 hepatocyte-specific
Trim24-null mice was considered for this study of HCC follow-up under ITPP treatment (ITPP was a gift from Jean Marie Lehn, ISIS, Strasbourg, France). All mice had partial hepatectomy at 2 months of age and three successive micro-CT assessments, at 5, 12 and 15–16 months of age. Then, ITPP treatment was started in 12 mice using an intraperitoneal injection of 3 mg/g/week over 10 months, and 11 untreated mice were considered as controls. All 23 mice were imaged by means of micro-CT scan at 3 and 9 months after treatment/survey initiation. 3D reconstructions of tumor and liver were performed: tumor volumes were calculated, allowing for the measurement of tumor doubling time (TDT) [
18] and growth rate.
Follow-up was performed until mice were terminally ill (cachexia, bad grooming, major weakness) or reached 25 months of age, when the eight surviving mice (5 in the ITPP group and three in the control group) were sacrificed. Dissection, macroscopic measurements and sampling for histology and molecular analysis were performed. Liver and tumor measurements at dissection were corroborated with the last micro-CT-scan rendering.
Survival curve was constructed over 25 months of age (average lifespan of wild-type mice).
Animal experiments were approved by the local ethics committee and were performed according to the revised European Community directive (2010/63/EU, September 24, 2010) for the protection of animals used for scientific purposes.
Gross examination, histology, and molecular biology methods
Gross examination was performed on livers harvested under isoflurane anesthesia. Livers were weighed first. If present, the number of tumors, their measurements and their localization were recorded. Tumor volume was calculated as D1 x D2 X D3 x π/6, where D1, D2 and D3 are tumor diameters.
Histology was performed on tissue samples, fixed in buffered formaldehyde for 24 h, dehydrated and paraffin-embedded. Standard 5-μm slides were obtained and stained by routine Hematoxylin - Eosin (HE) staining. Slide analysis was performed using the AxioVision 4.6 software (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).
Ki67 labeling was used for evaluation of proliferation rate and TUNEL assays (Terminal deoxynucleotidyl transferase dUTP nick end labeling) for apoptosis assessment, according to standard procedures. The percentage of Ki67-positive cells and TUNEL-positive cells was determined from five randomly chosen fields/section and three sections/liver for each animal.
Molecular biology
Trim24 genotyping was performed on genomic DNA obtained from tail biopsy sampled on 10-day-old mice, by means of a standard PCR technique, using Chromo 4 Thermocycler (Bio-rad, Marnes-la-Coquette, France). Primers sequences are available in Additional file
2.
Gene expression analysis in tumor and normal liver samples was performed using the
RT q-PCR technique. Tissue samples were homogenized in a FastPrep®-24 Instrument (MP Biomedicals Inc., lllkirch, France) in Lysis Matrix B tubes (MP Biomedicals, Inc.) containing lysis buffer (Sigma-Aldrich, Saint Quentin Fallavier, France). RNAs were then purified with the mammalian GenElute Gel Extraction Kit (Sigma-Aldrich), according to the manufacturer’s recommendations. RNAs (3 μg) were used as template for reverse transcription with random hexamer and anchored oligo dT, in the presence of 200 units of reverse transcriptase (MP Biomedicals, Inc.). Resulting cDNAs were analyzed using the RT qPCR on a Chromo 4 Cycler, using QuanTitect MasterMix (Qiagen, Courtaboeuf, France) and the primers corresponding to genes of interest (see Additional file
2). Results were analyzed with the Opticom 3 software (Bio-rad). Expressions of genes of interest were normalized by housekeeping gene HPRT and represented as tumor/liver ratio.
Statistical analysis
Data are expressed as mean ± standard deviation. Comparative analysis was performed using a two-way analysis of variance and unpaired t test with Welch correction (p < 0.05 was considered statistically significant). Linear regression equations, confidence intervals, and log-rank tests were performed using the GraphPad InStat statistics software (GraphPad Software, Inc., La Jolla, CA). Correlation between data series was evaluated using Pearson or Spearmen correlation coefficient as appropriate. The determination coefficient R2 < 0.5 was interpreted as an absence of correlation, and values close to one as a strong correlation.
Discussion
The major interest of transgenic mouse models of HCC is that tumor development reflects the clinical reality: pathological liver background, natural tumor - liver interactions, and well-vascularized tumors. This in vivo animal study valorized the genetic knowledge on the Trim24 gene, which had enabled the engineering of a HCC mouse model - Trim24
L2/L2Alb-Cre. In practice, to test an anticancer agent in a genetically induced model presents reputable disadvantages: long tumor developing time, unpredictability of tumor development, and non-homogeneity of the model. In order to challenge these shortcomings, a step-by-step methodology was described in this study.
First, evidence was provided that PH resulted in prolonged cell-cycling, with significantly high proliferation rate, beyond 15 days after PH and resulting in earlier development of HCC at long term follow-up. Based on this finding, PH was systematically performed in all animals, in order to boost the hepatocarcinogenesis process.
In order to assess the presence and to ensure the follow-up of HCC in
Trim24
L2/L2Alb-Cre mice, a contrast-enhanced micro-CT-scan imaging protocol was created. Several issues needed to be addressed: understanding of the particular rendering of the spontaneously developed tumors, coping with repeated contrast agent injection on the pathological liver background and with toxicity of the contrast agent. Data from the literature are mainly based on graft model imaging, in which the high resolution of the micro-CT-scan, along with specifically developed contrast agents [
9,
10,
19,
20] allowed to detect liver tumors starting with a 300 μm diameter [
9,
10], to calculate in vivo tumor volume [
21], and to monitor disease progression [
22,
23]. Monitoring of drug/therapy efficacy using micro-CT-scan was shown to be feasible in orthotropic models [
24,
25] and in a pilot study in three transgenic ASV-B mice [
10]. In these studies, HCCs were visualized as non-enhanced areas on the background of the normal, contrast-enhanced liver parenchyma. These renderings are consistent with our team’s experience in xenograft tumor models (Fig.
3a). However, they were not confirmed for spontaneously developing tumors in
Trim24
L2/L2Alb-Cre mice, in which the rendering of HCC was much more polymorphic (i.e., hypodense, isodense or hyperdense nodules and diffuse mass).
In the present study, a contrast agent injection protocol was described, consisting in an intraperitoneal contrast agent injection 6 h before imaging and vascular agent injection 15 min prior to imaging. This protocol allowed for the optimal visualization of spontaneously developing liver tumors, with low toxicity, adapted to the pathological liver background. Diagnosis of liver malignancy was achieved with an accuracy of 82.1 %, comparable to that obtained in prospective clinical trials [
26].
Finally, ITPP was pharmacologically tested in
Trim24
L2/L2Alb-Cre spontaneous HCC model. ITPP is a synthetic allosteric effector of hemoglobin, which increases the oxygen-releasing capacity of red blood cells leading to the suppression of hypoxia-inducible factors and to the down-regulation of hypoxia-inducible genes. Consequently, tumor growth is markedly affected in hypoxic tumors [
12]. ITPP proved its anticancer efficacy in graft models of melanoma and breast cancer [
12], colon cancer [
13], pancreatic cancer [
14], and hepatoma [
25]. In the present setting, the non-invasive imaging approach allowed to collect comprehensive data on tumor growth rate, survival and molecular findings with minimal animal sacrifice.
Tumor growth could be objectified before the ITPP treatment was started, and at the 3 and 9 months follow-up. Significant progression of tumor burden was equally demonstrated in the control group as well as in the ITPP group with no difference between groups. However, when further analyzing TDT, a mild acceleration of tumor growth (decrease of TDT) at 3 months of ITPP treatment was observed, with return to initial growth rate at 9 months of follow-up. Tumor growth acceleration seemed to be in contradiction with previous data [
25]. It was nevertheless consistent with molecular findings, which could not demonstrate HIF1a and VEGF suppression at the end of the follow-up period. These findings were probably the consequence of the more important vascularization of tumors, as compared to graft models. In this genetically induced model, in which hepatocytes are continuously cell cycling, hypoxia might be of minimum importance in the neoplastic development, and subsequently the efficacy of the treatment on tumor burden is of less importance. In return, in the ITPP group, tumors were better tolerated, mice continuing to survive despite very large tumors and a low volume of functional livers. In the ITPP group, treatment seemed to protect against the onset of new HCCs, as suggested by higher levels of p53 in non-tumor livers. This could account for the long-term effects of the product, with a significant prolongation of survival, which favored the ITPP treatment.
Acknowledgements
The authors would like to thank Guy Temporal for editorial assistance in proofreading the manuscript.