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. 2020 Oct 21;9:e53550. doi: 10.7554/eLife.53550

Figure 1. Development of peliosis-like regions after hepato-specific and focal Arid1a and Apc inactivation.

(a) Cre-loxP-generated hepatocyte-specific and inducible inactivation of Apc and/or Arid1a in 20% of hepatocytes after retro-orbital injection of infectious viral particles (ivp) of adenovirus encoding Cre recombinase (AdCre). The resulting mice are referred to as [Apc-Arid1a]ko-focal, [Apc]ko-focal, and [Arid1a]ko-focal. (b) Gross examination of mouse livers, 7 months after AdCre injection. Livers from [Apc-Arid1a]ko-focal mice had an irregular shape and a rough surface, with multiple dark red zones (indicated by arrows). (c) Incidence of hepatic lesions detected in WT (n = 10) and [Apc-Arid1a]ko-focal (n = 24) mice by ultrasonography. (d) Kaplan-Meier estimated survival curves of WT and [Apc-Arid1a]ko-focal mice over 15 months. n = 6 for each group. Inset: Liver of one mouse at necropsy (13 months after AdCre injection, representative of the three analyzed mice). (e) Hematoxylin Eosin (HE)-stained sections of mouse livers at 7 months post-injection. Large vascular spaces filled with blood cells were observed only in [Apc-Arid1a]ko-focal livers. Related data are found in Figure 1—figure supplements 1–4, and source data in ‘Figure 1—source data 1; Figure 1—figure supplement 1—source data 1; Figure 1—figure supplement 3—source data 1’.

Figure 1—source data 1. Emergence of peliosis (Figure 1c) and survival curve (Figure 1d).

Figure 1.

Figure 1—figure supplement 1. Focal inactivation of Apc and/or Arid1a genes in mouse liver.

Figure 1—figure supplement 1.

(a) Liver to Body weight (%) in mice; WT (n = 8), [Apc]ko-focal (n = 10), [Arid1a]ko-focal (n = 18), and [Apc-Arid1a]ko-focal (n = 19) mice. (b) RT-qPCR analysis of β-catenin-positive target genes from seven-month-old-mouse livers. WT (n = 5), [Apc]ko-TOTAL (n = 7), [Arid1a]ko-TOTAL (n = 12), and [Apc-Arid1a]ko-TOTAL (n = 10) mice. The data in (a,b) are expressed as the mean ± SEM and analyzed with one-way ANOVA. (c,d) Immunostainings against glutamine synthetase (Glul) and Arid1a after focal Apc and/or Arid1a loss. (c) Note the physiological staining of Glul in hepatocytes surrounding the centrolobular vein (cv). Focal activation of β-catenin signaling in single hepatocytes leads to an immunostaining of Glul, its hepatospecific target (red asterisk). Scale bars = 200 μm. (d) Immunofluorescence for Glul in hepatocytes without Arid1a nuclear fluorescence (white asterisk), shows the efficiency and specificity of the double Apc/Arid1a inactivation in [Apc-Arid1a]ko-focal livers (white asterisk). Scale bars = 100 μm.
Figure 1—figure supplement 1—source data 1. Liver to body weight ratios (Figure 1—figure supplements 1a) and expression of Glul and Axin2 mRNAs (Figure 1—figure supplements 1b).
Figure 1—figure supplement 2. Ultrasound features of livers from seven-month-old [Apc-Arid1a]ko-focal mice.

Figure 1—figure supplement 2.

(a) Echogenicity of peliotic areas within the [Apc-Arid1a]ko-focal liver (arrow), showing striking tissue modification. Scale bars = 2 cm. (b) Dynamic contrast-enhanced ultrasound using microbubble administration. Contrast-enhanced ultrasound imaging involves the injection of gas-filled micron-sized bubbles (microbubbles) that do not extravasate. This property makes them ideal contrast agents for imaging vascularity and blood perfusion. The protocol has been described in Snipstad et al., 2017. It revealed a decrease of hepatic vascular perfusion within echogenic areas compared to neighboring control tissue.
Figure 1—figure supplement 3. Blood vessel enrichment and angiogenesis in [Apc-Arid1a]ko-focal livers.

Figure 1—figure supplement 3.

(a) Peliotic areas appeared as abnormal tangles of irregularly shaped, leaky, small and large blood vessels filled with red blood cells, with multiple, mottled cyst-like spaces associated with sinusoidal dilatation and liver cell dropout in the livers of [Apc-Arid1a]ko-focal mice (1), relative to neighboring tissue (2). Scale bars = 200 μm (left panel) and 100 μm (right panel). (b) Immunofluorescence, at different magnifications, against β-catenin and Pecam1 of [Apc-Arid1a]ko-focal liver. Peliosis-like area, showing strong enrichment of blood vessels (1) compared to neighboring tissue (2). (c) RT-qPCR analysis of angiogenic factors from 7-month-old-mouse livers. WT (n = 5), [Apc]ko-TOTAL (n = 7), [Arid1a]ko-TOTAL (n = 12), and [Apc-Arid1a]ko-TOTAL (n = 10) mice. Data are presented as the mean + SEM and analyzed by one-way ANOVA.
Figure 1—figure supplement 3—source data 1. qPCR expression of angiogenic mRNAs (Figure 1—figure supplements 3c).
Figure 1—figure supplement 4. Hepatocarcinogenesis in β-catenin-activated and Arid1a-null context.

Figure 1—figure supplement 4.

(a–c) HCC incidence decrease in [Apc-Arid1a]ko-focal compared to [Apc]ko-focal mice. (a) Incidence of HCC was detected by ultrasound in [Apc]ko-focal (n = 13) and [Apc-Arid1a]ko-focal (n = 24) mice. (b) Two representative livers of ten-month-old [Apc]ko-focal and [Apc-Arid1a]ko-focal mouse livers presenting tumor. (c) Immunostaining of glutamine synthetase (Glul) and Arid1a in [Apc]ko-focal and [Apc-Arid1a]ko-focal liver sections. NT: non tumoral tissue; Tum: tumor; Scale bars = 200 μm. (d) EPO expression in human HCC depending on their CTNNB1 and ARID1A mutational status. Datasets in hepatocellular carcinoma were downloaded from the Cancer Genome Atlas (TCGA) data portal (http://tcga-data.nci.nih.gov). We extracted two types of molecular data including gene expression and somatic mutation using TCGA2STAT R package. Fours groups of tumors were clusterized based on mutation status: CTNNB1-mutated (n = 46), ARID1A-mutated (n = 11), CTNNB1/ARID1A-mutated (n = 5) and no CTNNB1 nor ARID1A mutations (n = 133). Analysis of variance and post-hoc tests were performed to test the association between EPO expression and mutation status.