Introduction
Primary liver cancer (PLC), including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), represents the third cause of cancer-related death [
1,
2]. Research has revealed that ICC and HCC share a monoclonal origin with bidirectional phenotype differentiation and may appear simultaneously [
3]. Combined HCC and ICC (cHCC-ICC) is a rare tumor accounting for 0.4–14.2% of all PLC [
4‐
7]. Since the diagnosis of cHCC-ICC relies on evidence of histological findings and patients who are not suitable for resection may be misdiagnosed with HCC or ICC alone, the actual incidence underestimates the current cHCC-ICC burden [
5]. Although curative surgical resection or liver transplantation is considered the mainstay of clinical practice, up to 80% of patients relapse within 5 years due to lymph node metastasis and vascular invasion [
8,
9]. Unfortunately, the 5-year survival in patients with unresectable cHCC-ICC does not exceed 30% due to inadequate response to current treatments [
10,
11]. Taking these findings into account when making treatment decisions, a more focused understanding of the molecular pathology and identification of potential therapeutic targets of cHCC-ICC thus are urgently needed.
Compared to xenograft tumor models, genetically engineered tumor models develop
de novo tumors that closely imitate the histopathological features of their human counterparts [
12]. Hydrodynamic tail vein injection (HDVI) and in situ electroporation (Epo) are two ways to transfer foreign plasmid DNA directly into hepatocytes. HDVI can create a pressurized blood force that redirects the blood flow directly into the liver, leading to plasmid DNA entering the intracellular compartment of hepatocytes [
13], while electroporation is an efficient way to introduce foreign genes into cultured cells and able to in situ transfer plasmids into hepatocytes [
14].
Recently, high-throughput genomic studies have revealed a series of driver genes contributing to HCC or ICC tumorigenesis [
15,
16].
c-Myc and
TP53 are two top frequently mutated genes in HCC patients. Recently, co-delivery of
c-Myc-encoding plasmid and CRISPR/Cas9-mediated
p53 knockout via HDVI successfully developed spontaneous HCC in mice [
17,
18]. In another study, Seehawer M et al. constructed a vector that co-expressed
Myc and
AKT1 to establish HCC in
p19Arf−/− mice via HDVI [
19]. Interestingly, the same vector led to ICC tumorigenesis by the approach of Epo, which could cause in situ necroptosis microenvironment, highlighting the hepatic microenvironment may contribute to lineage commitment during tumorigenesis [
19].
In the current study, we applied a Sleeping Beauty-dependent transposon plasmid co-expressing oncogenic Myc and AKT1 in combination with a CRISPR-Cas9 plasmid expressing single-guide RNA targeting p53 to compare their tumorigenic capacity via either HDVI- or Epo-dependent hepatocyte delivery. Notably, we found transfection of these plasmids by Epo led to the cHCC-ICC formation. Taking advantage of this novel spontaneous model, we proposed that LAMB1 may serve as a therapeutic target for cHCC-ICC.
Materials and methods
Vectors
A plasmid that co-expressing oncogenic Myc and AKT1 was a kind gift and has been constructed and described previously [
19]. The SB13 transposase-encoding vector was kindly provided by Dr Yue Zhao. pX330 backbone expressing sgRNA targeting p53 was obtained from Tyler Jacks (Addgene plasmid #59910).
Animal studies
Male 4 to 6-week-old C57BL/6 J mice were purchased from Charles River (Shanghai, China), and all the animals used in the study were fed in a specific pathogen-free facility. All animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhongshan Hospital, Fudan University.
Hydrodynamic tail vein injection and in situ electroporation
For hydrodynamic tail vein injection, 30 µg Myc + AKT1 co-expressing plasmid, 30 µg pX330 sg-p53 plasmid, and 10 µg SB13 transposase-encoding plasmid were prepared in 2 ml of sterile PBS and injected into a tail vein within 3–5 s per mouse. For in situ electroporation, 6-week-old wild-type C57BL/6 J mice were anesthetized, and the right lateral liver lobe was exposed after midline laparotomy. Plasmids described above were resolved in 50 µg sterile PBS and injected into the right lateral liver lobe using an insulin needle. In situ electroporation was performed with Squaure Wave Electroporator (Nepa Gene). The voltage and duration of electric pulse were 70 V and 75 ms, respectively. Two pulses were applied, and the interval was 500 ms.
Immunohistochemistry
Immunohistochemistry (IHC) was performed as previously described [
20]. In brief, the sections of tumors were incubated with the following antibodies: HNF4α (ab201460, Abcam), CK-19 (ab52625, Abcam), PCNA (2586, Cell Signaling Technology), p-AKT (Ser473, 4060, Cell Signaling Technology), p-ERK (Thr202/Tyr204, 4370, Cell Signaling Technology), p-NF-κB (Ser536, 3033, Cell Signaling Technology), β-catenin (8480, Cell Signaling Technology), TGF-β (21898-1-AP, Proteintech) and FGFR2 (13042-1-AP, Proteintech).
RNA-sequencing and analysis
RNA-sequencing for whole transcriptome analysis was performed using Illumina NovaSeq 6000 platform according to the manufacturer’s protocol. Three biological replicates were applied for RNA-seq, and all RNA samples passed quality control with 5–8 Gb and Q20 ≥ 90. Hierarchical clustering of RNA-seq was performed using R language with the ‘pheatmap’ package. A Euclidean method was used to calculate distance measurements, while the ‘complete’ method was utilized to calculate the dissimilarity values for hierarchical clustering. For the heatmap visualization, gene expression values were normalized.
Gene set enrichment analysis (GSEA) was performed using GSEA software version 4.1.0 with 1,000 permutations of the gene sets. The FPKM values from the RNA-seq were compared against the specific gene sets. Gene sets used in this study were downloaded from the MSigdb database (
http://software.broadinstitute.org/gsea/msigdb/index.jsp).
Statistics
The statistical analysis was performed using Prism Graphpad 7.0 software. Quantitative variables were analyzed by paired t-test. Kaplan-Meier analysis was used to compare OS between HDVI and Epo groups. Data were presented as mean ± standard deviation. p < 0.05 was considered statistically significant.
Discussion
cHCC-ICC is a rare type of PLC but has attracted increasing attention in recent years. Because the diagnosis of cHCC-ICC largely depends on histochemistry, the true incidence of cHCC-ICC is likely to be underestimated, making the knowledge and management of cHCC-ICC inaccessible. Thus, a tumor animal model that resembles clinical cHCC-ICC is essential for further investigation. In this study, we generated a spontaneous cHCC-ICC by induction of oncogenic Myc and AKT1 and loss of p53 through in situ electroporation. Evidence showed that this model shared similar transcriptome and oncogenic signaling pathways with human cHCC-ICC. Most importantly, we, for the first time, identified LAMB1 as a potential therapy target for cHCC-ICC.
Most solid tumors, especially HCC, develop in the context of chronic diseases and are composed of heterogeneous malignant cells. Importantly, the heterogeneous feature of tumor cells is a key reason for clinical drug resistance. Although traditional syngeneic or xenograft models are easy to perform, these models are unable to fully mimic specific human disease conditions and neglect the heterogeneous feature of tumor tissues. Thus, these common models have a limit to evaluating the drugs pre-clinically. It is well known that tumor cells are transformed from normal cells carrying oncogenic mutations, making it possible to induce PLC by genetic engineering. Accumulative studies have revealed specific mutations that lead to either HCC or ICC tumorigenesis. For example,
c-Myc,
CTNNB1 and
TP53 are among top mutated genes for HCC patients [
24]; while
KRAS, a powerful oncogene involved in glandular malignant, is able to induce ICC when specifically expressed in mouse hepatocytes [
25]. Currently, genetic engineered mouse model (GEMM) is an ideal tool for study HCC or ICC tumorigenesis and has clear genetic background resembling human disease, making it suitable for pre-clinical estimation. Unfortunately, few studies focus on cHCC-ICC as it’s not entirely clear how cHCC-ICC occurs. A recent study proved that an inflammatory tumor microenvironment directs lineage commitment of the PLC [
19]. Here, we used electroporation to induce a necroptotic liver microenvironment and established cHCC-ICC formation by combing
Myc and
AKT1 knockin with
p53 knockout. The morphological and genetic evidences proved successful induction of cHCC-ICC. Our work provides a simple GEMM for cHCC-ICC with direct clinical translational value. However, our study also has limitations. It remains elusive whether other gene combinations can lead to the cHCC-ICC formation in the same experimental setting. Moreover, our model does not exhibit the metastatic feature of cHCC-ICC, as evidenced by no lung metastatic lesion observed (data not shown). New gene combinations that can lead to metastasis should be tested in the future.
Whether cHCC-ICC is a unique or a subtype of HCC or ICC has long been controversial. cHCC-ICC can be further divided into three subtypes according to Allen and Lisa’s criteria that are separate type (HCC and ICC components physically separated), combined type (HCC and ICC component in the same tumor with clear boundaries), and mixed type (HCC and ICC component in the same tumor with no boundaries) [
26]. Histologically, we found that
MAPEpo tumors exhibited features with a combined type of cHCC-ICC. Recently, Xue et al. [
3] comprehensively analyzed a total of 133 cHCC-ICC cases and revealed that combined type of cHCC-ICC acquired intense ICC-like landscapes, including high expression of
KRT19 but a lower expression of HCC markers (including
AFP and
GPC3), which is also supported by our transcriptome data (Fig.
2A). Compared with
MAPHDVI tumors,
MAPEpo tumors tended to express markers of biliary differentiation, especially for
Krt19,
Krt7,
Nes,
Tgfb2, and
Jag1. Consistently,
Afp expression is much higher in
MAPHDVI tumors than in
MAPEpo tumors. These data suggested that
MAPEpo tumors might exhibit more ICC-like characteristics.
Despite apparent molecular discrepancy among subtypes of cHCC-ICC, they all have poorer prognosis and more invasive features than HCC and are similar to ICC [
27]. Interactions between cell adhesion or migration and ECM are vital factors that mediate tumor metastasis. In our study, we identified 73 overlapping genes that are highly upregulated in cHCC-ICC. Bioinformatic analysis indicated that these genes were mainly involved in ECM bioprocesses and cell adhesion. These findings further reinforced the invasive feature of cHCC-ICC.
Laminins, a family of extracellular matrix glycoproteins, are among the predominant component of ECM [
28]. Evidence reported that laminins participated in tumor metastasis by promoting cell adhesion and migration, and their receptors expressed on the tumor cell surface [
29‐
31]. We screened these 73 genes and identified a main differential gene
Lamb1, a member of the laminin family. Taking advantage of several data sets reported previously, we established that
Lamb1 was upregulated in cHCC-ICC compared to HCC, but its expression seemed comparable with ICC. It has been reported that LAMB1 overexpressed in several types of tumors and correlated with tumor metastasis and poor prognosis [
32‐
34]. The prognosis value of LAMB1 was further screened tin TCGA database. The results suggested that LAMB1 is elevated in a series of tumors, but LAMB1 is negatively correlated with both OS and DFS for LIHC, CHOL, and COAD (Additional file
1: Fig. S1). Most importantly, KEGG and GSEA analysis showed that LAMB1 played a pivotal role in pro-metastatic processes, including focal adhesion, ECM-receptor interaction, and cellular junction in LIHC, CHOL, and COAD, highlighting that LAMB1 might also be a potential treating target for cHCC-ICC (Additional file
1: Fig. S2). Since clinical transcriptome data of patients with cHCC-ICC remains very limited, we were able only to verify the expression of
Lamb1 in current published data; further larger-scale studies should be performed to assess its clinical value. Summarily, the present study established a human-resembling cHCC-ICC model via in situ electroporation. This novel preclinical model can be used to investigate the molecular feature of cHCC-ICC.
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