Transposon-based oncogene integration in Abcb4(Mdr2)-/- mice recapitulates high susceptibility to cholangiocarcinoma in primary sclerosing cholangitis

Abstract

Background & aims: Cholangiocarcinoma (CCA) is a dreaded complication of primary sclerosing cholangitis (PSC) that is difficult to diagnose and associated with high mortality. A lack of animal models of CCA recapitulating the hepatic microenvironment of sclerosing cholangitis has hindered the development of novel treatments. Herein, we sought to develop a mouse model of PSC-associated CCA.

Methods: Ten-week-old Mdr2^-/- mice with congenital PSC-like disease, and healthy wild-type littermates were subjected to either modified retrograde biliary instillation or hydrodynamic tail vein injection of a sleeping beauty transposon-transposase plasmid system with activated AKT (myr-AKT) and Yap (YapS127A) proto-oncogenes (SB AKT/YAP1). The role of TGFβ was interrogated via ALK5 inhibitor (SB-525334) administration. Tumor phenotype, burden and desmoplastic reaction were analyzed histologically and via RNA sequencing.

Results: While SB AKT/YAP1 plasmids administered via retrograde biliary injection caused tumors in Mdr2^-/-, only 26.67% (4/15) of these tumors were CCA. Alternatively, hydrodynamic tail vein injection of SB AKT/YAP1 resulted in robust tumorigenesis in all fibrotic Mdr2^-/- mice with high CCA burden compared to healthy mice. Tumors phenotypically resembled human CCA, expressed multiple CCA (but not hepatocellular carcinoma) markers, and exhibited a profound desmoplastic reaction. RNA sequencing analysis revealed profound transcriptional changes in CCA evolving in a PSC-like context, with specific alterations in multiple immune pathways. Pharmacological TGFβ inhibition led to enhanced immune cell tumor infiltration, reduced tumor burden and suppressed desmoplastic collagen accumulation compared to placebo.

Conclusion: We established a new high-fidelity cholangiocarcinoma model in mice, termed SB CCA.Mdr2^-/-, which recapitulates the increased susceptibility to CCA in the setting of biliary injury and fibrosis observed in PSC. Through transcriptomics and pharmacological studies, we show dysregulation of multiple immune pathways and TGFβ signaling as potential drivers of CCA in a PSC-like microenvironment.

Impact and implications: Animal models for primary sclerosing cholangitis (PSC)-related cholangiocarcinoma (PSC-CCA) are lacking. Thus, we have developed and characterized a new mouse model of PSC-CCA, termed SB CCA.Mdr2^-/-, which features reliable tumor induction on a PSC-like background of biliary injury and fibrosis. Global gene expression alterations were identified and standardized tools, including automated whole slide image analysis methodology for tumor burden and feature analysis, were established to enable systematic research into PSC-CCA biology and formal preclinical drug testing.

Keywords: ABCB4; TGFβ; cholangiocarcinoma; mouse model; primary sclerosing cholangitis.

Figure 1.. Liver tumorigenesis in wild-type and Mdr2−/− mice with hydrodynamic tail vein injection of AKT/YAP1 SB transposon-transposase complexes.

(A&B) Scheme of experimental design. Both female and male of WT and Mdr2−/− mice were subjected to HTVi of AKT/YAP1/SB (n = 4 – 10). (C&D) Development of liver tumors in healthy wildtype (WT) (left) and fibrotic Mdr2−/− (right) female and male mice 6 or 5 weeks after hydrodynamic transfection of AKT/YAP1 were revealed by gross examination of livers (upper panel) and immunohistochemistry staining of the cholangiocyte marker CK19 (lower panel). (E&F) Quantifications of tumor incidence, relative liver weight, total CK19+ tumor numbers in equally sized random samples from left, median and right lobes, and survival curves of WT and Mdr2−/− mice. Scale bar: 1 cm for gross images, 500 μm for magnification x20. P value was determined by Chi-square test for incidence, t test for relative liver weight and tumor number and log rank test for survival. HTVi, hydrodynamic tail vein injection; SB, sleeping beauty.

Figure 2.. Phenotypic characterization of the liver tumors in SB CCA.Mdr2−/− model.

(A) Representative Hematoxylin-Eosin (H&E) and connective tissue (picrosirius red) staining of the tumor lesions in the Mdr2−/− mice. Low magnification images (x50, scale bar 200 μm) and x200 blow-up (scale bar 50 μm). All tumors were defined as intrahepatic cholangiocarcinoma (CCA) characterized by atypical cells forming tubular structures with pronounced desmoplastic reaction. (B) Tumors are uniformly positive for markers of active cell replication (Ki67), cholangiocyte lineage marker CK19, Sox9, and negative for hepatocellular markers HNF4α, glutamine synthetase (GS), AFP, CPS1, and Glypican 3 (GPC3). Tumors were positive for mucin by Alcian Blue stain and myofibroblast marker α-smooth muscle actin (α-SMA), poorly vascularized (low endothelial cell marker CD31) and demonstrated TGFβ signaling activation as evidenced by robust phospho-SMAD2 expression. Original magnification, x200. Tumor (T) border is demarcated by a punctate line. Scale bar: 50 μm.

Figure 3.. Transcriptional signatures associated with CCA tumors arising in the settings of biliary injury and fibrosis in SB CCA.Mdr2−/− model.

Bulk RNA sequencing of total liver RNA of tumor-bearing healthy livers (WT CCA, n=6) and fibrotic Mdr2−/− livers (Mdr2−/− CCA, n=6) was performed and differentially regulated CCA-associated genes identified in comparison to respective non-tumor bearing control livers transduced with empty SB transposon-transposase complexes (WT and Mdr2−/−, respectively, n=6). (A) Venn diagrams representing the overlap between fibrotic (CCA.Mdr2−/−) and non-fibrotic (CCA.WT) CCA-associated differentially expressed genes (DEGs, ∣log2FoldChange∣ > 1, Padjust < 0.05) identified as induced (top) or repressed (bottom) relative to the corresponding control livers. (B) Heatmap showing the same DEGs. The left columns 1-2 shows up- (red) and down-regulated (blue) genes; the right part displays the z-score for expression of each gene across all samples within identical comparison. (C) Gene Set Enrichment Analysis (GSEA) with the MSigDB gene sets C2 demonstrate that class 1 and 2 human cholangiocarcinoma signatures are enriched in CCA.Mdr2−/− tumors. (D) Hallmark gene set analysis pathways revealed 4 common pathways enriched in CCA arising in fibrotic Mdr2−/− and healthy WT liver (upper panel) as well as several inflammation and immunity related pathways that exhibited dramatically different regulation in fibrotic CCA (lower panel). (E) Volcano plot representing genes of major cytokine families (IL1, IL6, IL10, TNF, IFN and TGFb), corresponding receptors, and av integrins based on their fold change and P value in tumors when compared with the control liver tissues. The genes with fold change >2 are highlighted in red, the genes with fold change between 1 and 2, and P value < 0.05 are highlighted in blue.

Figure 4.. TGFβ signaling functionally drives tumor progression in SB CCA.Mdr2−/− model.

(A) Scheme of experimental design for the study on administration of ALK5 inhibitor (n=8 for female, n=4-6 for male). (B) Effective inhibition of intracellular TGFβ signaling as evidenced by markedly diminished phospho-SMAD2 staining upon ALK5 antagonism. Magnification x200, scale bar 50 μm. (C&D) Representative CK19 stainings show marked inhibition of CCA growth upon ALK5 inhibition in mice of both sexes. (E&F) Relative liver weight and CCA burden assessed morphometrically via total CK19⁺ tumor numbers (x20). Scale bar: 500 μm. (G) Correlation of ductular reaction (CK19+ cell number) and fibrosis (% collagen area, PSR) with tumor burden in SB CCA.Mdr2−/− model (left) with representative images (middle). Magnification x100, scale bar 100 μm. Morphometric quantification of non-tumor CK19 and PSR staining upon ALK5 inhibition. P value was calculated via t test, and Pearson’s correlation to analyse association of tumor burden to nontumor ductular reaction and fibrosis.

Figure 5.. AI-powered automated OncoNest analysis of the tumor burden in SB CCA.Mdr2−/− model and therapeutic effect of ALK5 inhibition.

(A) Representative images illustrating the learning process to allow the automated identification of CK19-positive tumors regions. (B) Area-normalized tumor number (count per mm², or tumor burden), tumor area ratio (%), and Tumor Composite Score were automated identified as shown in the charts. P value was determined by t test.

Figure 6.. Quantitative and architectural changes in intratumoral desmoplastic reaction in SB CCA.Mdr2−/− model upon TGFβ inhibition.

(A) Representative images of desmoplastic tumor tissue (picrosirius red, original magnification x100, scale bar: 100 μm). (B) Representative images (FibroNest^™) to show identification of assembled collagen fibers (white) and fine collagen fibers (pink) based on morphometric quantification (x100, scale bar: 100 μm). (C) Fibrosis per tumor in the whole section was quantified for assemble collagen area, fine collagen area, total collagen area. P value was determined by t test. CAR, collagen area ratio.