Exacerbation of Liver Tumor Metastasis in twist1a+/xmrk+ Double Transgenic Zebrafish following Lipopolysaccharide or Dextran Sulphate Sodium Exposure

The poor prognosis for patients with hepatocellular carcinoma (HCC) is related directly to metastasis. The Twist1 gene encodes for a transcription factor essential to embryogenesis. It has also been shown to promote epithelial-to-mesenchymal transition (EMT), invasion, and metastasis; however, there is currently no in vivo evidence that Twist1 plays a role in the metastasis of liver tumors. Zebrafish are increasingly being used as an alternative cancer model. In the current study, an adult-stage zebrafish HCC model was used to examine the synergistic effects of twist1a and xmrk, a well characterized oncogene, during HCC metastasis. We also examined the effects of two inflammatory agents, lipopolysaccharides (LPS) and dextran sulfate sodium (DSS), on the hepatocyte-specific expression of transgenic twist1a and xmrk. The conditional overexpression of twist1a and xmrk was shown to promote liver tumor metastasis in zebrafish, resulting in increased apoptosis and cell proliferation as well as tumor maintenance and propagation independent of the inherent EMT-inducing activity of xmrk. Exposing twist1a+/xmrk+ transgenic zebrafish to LPS or DSS was shown to promote metastasis, indicating that the overexpression of twist1a and xmrk led to crosstalk between the signaling pathways involved in EMT. This study provides important evidence pertaining to the largely overlooked effects of signaling crosstalk between twist1a and xmrk in regulating HCC metastasis. Our results also suggest that the co-expression of twist1a/xmrk in conjunction with exposure to LPS or DSS enhances HCC metastasis, and provides a valuable in vivo platform by which to investigate tumor initiation and metastasis in the study of liver cancer.


Introduction
Hepatocellular carcinoma (HCC) is a common cause of death worldwide, particularly in Sub-Saharan Africa and Southeast Asia [1]. HCC is strongly associated with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, alcohol abuse, long-term exposure to aflatoxin B1, and metabolic diseases [2]. In recent years, researchers have observed increases in HCC incidence and HCC-related mortality [3]. Surgical resection and liver transplantation can achieve favorable treatment outcomes for patients with nonmetastatic HCC. These patients are also candidates for palliative local treatments, such as chemoembolization, radiofrequency ablation, and stereotactic radiotherapy [4]. However, for patients with advanced or metastatic HCC, palliative systemic therapy is the only option, and the survival benefits are limited [5]. As a result, HCC remains among the most deadly cancers, with a 5-year survival rate of only 5% [6].

Twist1a+ Transgenic Zebrafish, Diagram of Experimental Design, and Schedules of Specimen Collection from Long-Term Treatment
Following treatment with Dox, Twist1a transgene expression was demonstrated in F3 zebrafish by visualizing mCherry in the hepatocytes of seven dpf larva and five mpf adult zebrafish (Supplementary Figure S1A). To compare tumor growth and metastasis development between twist1a+ and twist1a+/xmrk+ double transgenic zebrafish, twist1a+/xmrk+ zebrafish were treated with Dox and 4-OHT or treated with Dox and 4-OHT exposed to LPS or DSS for up to 8 weeks. Samples were collected weekly for histological examination (Supplementary Figure S1B).

Expression of Liver Markers fabp10a and tfa in Primary and Metastatic Liver Tumors Tissues in twist1a+/xmrk+ Double Transgenic Zebrafish
After four weeks of treatment with 60 µg/mL Dox and 1 µg/mL 4-OHT, the immunofluorescence of twist1a+/xmrk+ transgenic zebrafish revealed evidence of tumor metastasis ( Figure 3A). Tissue samples were collected from the primary liver tumor and metastatic tumor as well as adjacent normal tissues to determine the origin of the metastases. We examined the expression of fabp10a and tfa RNA (two zebrafish liver markers) in the various tissues using a semiquantitative RT-PCR. Both zebrafish liver markers were expressed primarily in the primary and metastatic liver tumor tissue, thereby confirming that the origin of the metastatic tumors was indeed the liver ( Figure 3B). Neither fabp10a nor tfa RNA was observed in the adjacent normal tissues. Actin and a non-template sample, respectively, served as an internal control negative control.

Co-Expression of twist1a and xmrk Significantly Increased Apoptosis and Cell Proliferation in the Hepatocyte Cells of Double Transgenic Zebrafish
The main hallmarks of tumorigenesis include cell cycle control and abnormal cell apoptosis [34]; therefore, we sought to determine whether the liver tumorigenesis and metastasis observed in the twist1a+/xmrk+ transgenic zebrafish were a consequence of aberrant cell cycle control and cell apoptosis. Caspase-3 staining for apoptotic cells and PCNA staining for proliferative cells were performed in zebrafish treated with 60 µg/mL Dox and 1 µg/mL 4-OHT ( Figure 4A,B). Overall, only a small number of apoptotic cells were observed in non-oncogenic livers in wild-type zebrafish. The number of apoptotic cells in the xmrk+ and twist1a+/xmrk+ transgenic zebrafish was significantly higher. In fact, the co-induction of twist1a and xmrk resulted in a 31% increase in the number of apoptotic cells ( Figure 4A,C). These findings are consistent with those of our previous research on HCC development in other oncogene transgenic zebrafish [31,35]. Remarkably, the percentage of apoptotic cells was higher in transgenic zebrafish with twist1a+/xmrk+ than in the xmrk+ induction group ( Figure 4C). Other studies have also reported that EGFR and Kras oncogenes can induce apoptosis through Ras signaling [36,37].
The number of proliferating cells was significantly higher in the xmrk+ and twist1a+/ xmrk+ transgenic zebrafish than in the wild-type control zebrafish. The number of proliferating cells was 27% higher in the twist1a+/xmrk+ transgenic zebrafish ( Figure 4B,D); however, the difference was less pronounced in the xmrk+ transgenic zebrafish ( Figure 4D). Note that the extent of apoptosis in individual twist1a+/xmrk+ transgenic zebrafish did not necessarily exceed that of individual xmrk+ transgenic zebrafish, which suggests that metastatic changes in liver tumors can be attributed primarily to cell proliferation. of liver tumor metastasis in twist1a+/xmrk+ transgenic zebrafish at 4 wpi. (F) Histological examination confirmed that xmrk+ and twist1a+/xmrk+ transgenic zebrafish developed HCC or metastatic HCC at 2 and 4 wpi, whereas normal liver histology was observed in all twist1a+ and wild-type siblings. Differences among variables were assessed using Student's t-tests or one-way ANOVA. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.  twist1a+/xmrk+ transgenic zebrafish than in the wild-type control zebrafish. The number of proliferating cells was 27% higher in the twist1a+/xmrk+ transgenic zebrafish ( Figure 4B and 4D); however, the difference was less pronounced in the xmrk+ transgenic zebrafish ( Figure 4D). Note that the extent of apoptosis in individual twist1a+/xmrk+ transgenic zebrafish did not necessarily exceed that of individual xmrk+ transgenic zebrafish, which suggests that metastatic changes in liver tumors can be attributed primarily to cell proliferation.

Twist1a Expression Activates EMT Pathway via E-cadherin and Vimentin
The expression of E-cadherin and vimentin are the main hallmarks of EMT during cancer metastasis [9,10]. We performed immunohistochemical staining for E-cadherin and vimentin to determine whether the liver tumor metastasis observed in the twist1a+/xmrk+ transgenic zebrafish was a consequence of aberrant metastasis in the liver ( Figure 5A,B). The twist1a+/xmrk+ transgenic zebrafish presented E-cadherin levels lower than those of the wild-type control or xmrk+ zebrafish with corresponding higher vimentin levels at 4 wpi. The quantification results revealed an 18% decrease in E-cadherin expression in the twist1a+/xmrk+ transgenic zebrafish ( Figure 5A,C) and a 17% increase in vimentin expression ( Figure 5B,D). This suggests that the co-expression of twist1a and xmrk triggers crosstalk along the EMT pathway and contributes to the liver tumor metastasis observed in this group of zebrafish.

Exposure to DSS or LPS Induces Gut and Liver Inflammation in lyz:DsRed and mpeg1:mCherry Transgenic Zebrafish Larvae
Macrophages and neutrophils are the most abundant immune cells that infiltrate tumors, and both have been implicated in the development of HCC [38][39][40]. We evaluated the effects of elevated macrophage and neutrophil levels on HCC in the xmrk+ and twist1a+/xmrk+ transgenic zebrafish by determining whether inflammation could be induced by LPS or DSS in zebrafish larvae. This was achieved using Tg(lyz:DsRed, lyz+) and Tg(mpeg1:mCherry, mpeg1+) zebrafish to, respectively, measure the presence of neutrophils and macrophages. Four-day-old zebrafish larvae were treated with 40 ng/mL LPS or 0.5% DSS (i.e., lyz+/LPS, lyz+/DSS, mpeg1+/LPS, and mpeg1+/DSS) for 2 or 3 days. Lyz+ and mpeg1+ zebrafish larvae without exposure to inflammatory agents served as the controls. All the larvae in each group underwent imaging, whereupon the numbers of neutrophils and macrophages were quantified via immunofluorescence ( Figure 6A-D). The exposure to LPS or DSS significantly increased the number of neutrophils and macrophages in the Pharmaceuticals 2021, 14, 867 9 of 18 gut in the lyz+/LPS, lyz+/DSS, mpeg1+/LPS, and mpeg1+/DSS larvae, compared with the lyz+ and mpeg1+ control larvae. In the liver, significant increases in the number of neutrophils and macrophages were also observed following LPS exposure in the lyz+/LPS and mpeg1+/LPS zebrafish larvae; however, we did not observe any changes in the number of these immune cells following exposure to DSS ( Figure 6A,B).

Liver Tumor Phenotypes Induced by Sustained Expression of xmrk and Exposure to LPS or DSS in Transgenic Zebrafish Larvae and Adult Transgenic Zebrafish
We evaluated the effects of exposure to LPS or DSS on liver tumor progression following the short-term or long-term induction of xmrk in the xmrk+ transgenic zebrafish, which were treated with 20 µg/mL Dox alone or in conjunction with 20 µg/mL Dox and exposed to 40 ng/mL LPS or 0.5% DSS. Note that the xmrksiblings without Dox treatment served as the controls. Following short-term induction, liver size was significantly larger in the xmrk+, xmrk+/LPS, and xmrk+/DSS larvae than in the xmrkcontrol larvae ( Figure 7A,B).

Twist1a Expression Activates EMT Pathway via E-cadherin and Vimentin
The expression of E-cadherin and vimentin are the main hallmarks of EMT durin cancer metastasis [9,10]. We performed immunohistochemical staining for E-cadherin an vimentin to determine whether the liver tumor metastasis observed in the twist1a+/xmrk transgenic zebrafish was a consequence of aberrant metastasis in the liver ( Figure 5A,B The twist1a+/xmrk+ transgenic zebrafish presented E-cadherin levels lower than those o the wild-type control or xmrk+ zebrafish with corresponding higher vimentin levels at wpi. The quantification results revealed an 18% decrease in E-cadherin expression in th twist1a+/xmrk+ transgenic zebrafish ( Figure 5A,C) and a 17% increase in vimenti expression ( Figure 5B,D). This suggests that the co-expression of twist1a and xmrk trigger crosstalk along the EMT pathway and contributes to the liver tumor metastasis observe in this group of zebrafish.

Exposure to DSS or LPS Induces Gut and Liver Inflammation in lyz:DsRed and mpeg1:mCherry Transgenic Zebrafish Larvae
Macrophages and neutrophils are the most abundant immune cells that infiltrat tumors, and both have been implicated in the development of HCC [38][39][40]. We evaluate the effects of elevated macrophage and neutrophil levels on HCC in the xmrk+ an twist1a+/xmrk+ transgenic zebrafish by determining whether inflammation could b induced by LPS or DSS in zebrafish larvae. This was achieved using Tg(lyz:DsRed, lyz+ and Tg(mpeg1:mCherry, mpeg1+) zebrafish to, respectively, measure the presence o neutrophils and macrophages. Four-day-old zebrafish larvae were treated with 40 ng/m LPS or 0.5% DSS (i.e., lyz+/LPS, lyz+/DSS, mpeg1+/LPS, and mpeg1+/DSS) for 2 or 3 day Following long-term induction, the xmrk+ transgenic zebrafish were treated with 20 µg/mL Dox alone or 20 µg/mL Dox and 40 ng/mL LPS or 0.00625% DSS. Note that the xmrksiblings without Dox treatment served as the controls. At 2 wpi, samples were collected for the assessment of tumor status. The xmrk+, xmrk+/LPS, and xmrk+/DSS transgenic zebrafish exhibited enlarged abdomens (compared with the xmrkcontrol group) and obvious signs of liver overgrowth. A H&E examination revealed that following xmrk induction, the liver phenotype progressed from predominantly normal to HCC ( Figure 7C). A significant increase in mortality was also observed in the xmrk+/LPS and xmrk+/DSS transgenic zebrafish, compared with the xmrk+ transgenic zebrafish and the xmrkcontrols ( Figure 7D). Histological analysis of the xmrk+, xmrk+/LPS, and xmrk+/DSS transgenic zebrafish revealed a combination of the normal liver phenotype (4/17, 23.53%; 5/18, 27.78%; and 0/9, 0%, respectively), hyperplasia (3/17, 17.65%; 2/18, 11.11%; and 1/9, 11.11%, respectively), and hepatocellular carcinoma (10/17, 58.82%; 11/18, 61.11%; and 8/9, 88.89%, respectively), whereas the xmrkcontrols all presented the normal liver phenotype (19/19, 100%). The HCC status was more severe in the xmrk+/DSS than in the xmrk+ or xmrk+/LPS transgenic zebrafish ( Figure 7E). Lyz+ and mpeg1+ zebrafish larvae without exposure to inflammatory agents served as the controls. All the larvae in each group underwent imaging, whereupon the numbers of neutrophils and macrophages were quantified via immunofluorescence ( Figure 6A-D). The exposure to LPS or DSS significantly increased the number of neutrophils and macrophages in the gut in the lyz+/LPS, lyz+/DSS, mpeg1+/LPS, and mpeg1+/DSS larvae, compared with the lyz+ and mpeg1+ control larvae. In the liver, significant increases in the number of neutrophils and macrophages were also observed following LPS exposure in the lyz+/LPS and mpeg1+/LPS zebrafish larvae; however, we did not observe any changes in the number of these immune cells following exposure to DSS ( Figure 6A,B).

Liver Tumor Phenotypes Induced by Sustained Expression of xmrk and exposure to LPS or DSS in Transgenic Zebrafish Larvae and Adult Transgenic Zebrafish
We evaluated the effects of exposure to LPS or DSS on liver tumor progression following the short-term or long-term induction of xmrk in the xmrk+ transgenic zebrafish, which were treated with 20 μg/mL Dox alone or in conjunction with 20 μg/mL Dox and exposed to 40 ng/mL LPS or 0.5% DSS. Note that the xmrk-siblings without Dox treatment served as the controls. Following short-term induction, liver size was significantly larger in the xmrk+, xmrk+/LPS, and xmrk+/DSS larvae than in the xmrk-control larvae ( Figure  7A,B).
Following long-term induction, the xmrk+ transgenic zebrafish were treated with 20 μg/mL Dox alone or 20 μg/mL Dox and 40 ng/mL LPS or 0.00625% DSS. Note that the Figure 6. Increases in the numbers of neutrophils and macrophage in the gut and liver of lyz+/LPS, lyz+/DSS, mpeg1+/LPS, and mpeg1+/DSS zebrafish larvae exposed to LPS or DSS. Quantification (via fluorescence) of the number of cells in the gut and liver of zebrafish larvae testing positive for (A) neutrophils (lyz+) or (B) macrophages (mpeg1+) accompanied by representative fluorescence images of (C) neutrophils and (D) macrophages. Differences among the variables were assessed using Student's t-tests. Statistical significance: *** p < 0.001. Scale bar: 25 µm.

Exposure to LPS or DSS Exacerbated Liver Tumor Metastasis in Hepatocyte-Specific Expression of twist1a+/xmrk+ Double Transgenic Zebrafish
Our findings revealed that the simultaneous induction of xmrk+ and twist1a+ under exposure to inflammatory agents can enhance liver tumorigenesis and metastasis. Thus, we examined the effects on liver tumor metastasis in twist1a+/xmrk+ transgenic zebrafish exposed to LPS or DSS in the adult stage. At 4 mpf, the twist1a+/xmrk+ zebrafish were treated with 20 µg/mL Dox and 1 µg/mL 4-OHT and exposed to 40 ng/mL LPS or 0.00625% DSS for 4 weeks. The immunofluorescence analysis revealed evidence of tumor metastasis ( Figure 8A,B). In terms of mortality, a substantial number of zebrafish in all the groups began to succumb from approximately 10 dpi. No significant difference in mortality was observed between the twist1a+/xmrk+ transgenic zebrafish exposed to LPS and the corresponding unexposed twist1a+/xmrk+ control zebrafish ( Figure 8C). Nonetheless, mortality was significantly higher among the twist1a+/xmrk+ zebrafish exposed to DSS than among the unexposed twist1a+/xmrk+ controls ( Figure 8D). Furthermore, we observed that at 4 wpi, the incidence of liver tumor metastasis was significantly higher in the twist1a+/xmrk+/LPS and twist1a+/xmrk+/DSS zebrafish (6/9, 66.67%; and 23/26, 88.46%, respectively) than in the two unexposed twist1a+/xmrk+ control groups (3/8, 37.50%; and 15/44, 34.10%, respectively) ( Figure 8E,F). zebrafish developed HCC at 2 wpi, compared to normal liver histology in xmrk-siblings. Differences among the variables were assessed using Student's t-tests or one-way ANOVA. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Discussion
HCC involves a multi-stage alteration in gene expression involving cell proliferation, invasion, and metastasis. Recent advances in surgical techniques have led to significant improvements in local tumor control; however, the prognosis of patients with metastatic HCC remains poor [41]. The dysregulation of human Twist1 has been reported in HCC and other cancers, and research suggests that Twist1 plays an important role in promoting the invasion and metastasis of HCC and intrahepatic cholangiocarcinoma [42,43]. Moreover, in a mouse model of metastatic breast tumor, twist1 has been identified among the most up-regulated genes [26].
Zebrafish is an excellent model by which to investigate the mechanisms underlying metastasis in human cancers [44]. It can also serve as a tool for the screening of therapeutic drugs [2,30,31,45,46]. In the current study, we utilized transgenic zebrafish in the larvae and adult stages to elucidate the processes of liver tumorigenesis and metastasis. We hypothesized that there are additional EMT-related genes (e.g., twist1a) that are independent of the primary xmrk oncogene, but that can have cumulative effects on tumor progression. In this study, we performed long-term tumor induction for up to 8 weeks (Figures 1 and 2). The hepatocyte-specific overexpression of twist1a and xmrk in the liver of zebrafish was shown to accelerate apoptosis ( Figure 4A,C) and cell proliferation ( Figure 4B,D), with concomitant hepatocyte transformations by 4 wpi, including liver overgrowth, HP, HCC, and metastatic HCC (Figures 2 and 3).
The overexpression of twist1a was associated with a reduction in the expression of E-cadherin ( Figure 5A,C) and an increase in the expression of vimentin ( Figure 5B,D), both of which are involved in the promotion of EMT. One clinical study reported that Twist1 was indicative of tumor cell EMT and endothelium-dependent angiogenesis in HCC [47], suggesting that the activation of the twist1a gene could be mediated via regulation of the EMT pathway. By contrast, Twist1 has been shown to prevent oncogene-induced apoptosis and/or senescence, while decreasing Ras and Myc expression via the repression of the p16 and/or the p19ARF/p53 pathways. The Twist1 was shown to be essential to the initiation and maintenance of p53-deficient cancer stem cells in a KRas D12 p53-deficient mouse model. That study identified both p53-dependent and p53-independent roles for twist1 in tumor initiation, proliferation, apoptosis, and propagation [12]. In mice, the impaired expression of E-cadherin was found to promote hepatocellular carcinogenesis [48], and the dysregulation of E-cadherin was also identified in a number of transgenic mouse models of liver cancer [49]. In HCC patients, a decrease in the expression of E-cadherin is indicative of poor prognosis [50]. Moreover, the accelerated autophagic degradation of E-Cadherin via sirtuin 6, a protein deacetylase to promote EMT in HCC [51]. In in vitro and in vivo studies of breast cancer, the repression of E-cadherin expression occurs involving the direct binding of Twist1 to the E-cadherin promoter, such that the downregulation of E-cadherin attenuates cell-cell adhesion and enhances migration and invasion [26].
Vimentin plays important roles in regulating the migration of many cell types [52]. Impaired cell migration has been demonstrated through the recirculation of endocytic cell adhesion receptors to the plasma membrane by vimentin as well as through the disruption of vimentin function [53]. Previous studies have reported the overexpression of vimentin in cases of breast cancer and HCC metastasis [54,55]. The overexpression of vimentin has also been shown to increase integrin traffic, migration, and invasion in a vimentin-negative MCF7 breast cancer cell line [53,56]. In a number of triple-negative breast cancers, vimentin expression has been identified as a marker of basal-like breast cancer cells associated with poor prognosis [57]. Serum vimentin can serve as a surrogate marker for small HCC tumors [58]. Furthermore, vimentin is a potential therapeutic target in a sorafenib-resistant HepG2 cell line [59]. In the current study, we observed that twist1a not only maintains but in fact accelerates xmrk-induced liver tumorigenesis and metastasis. This finding is consistent with previous studies on other types of cancers, such as those of the skin, bone, and lung [12,60,61].
In the tumor microenvironment, macrophages and neutrophils are the most abundant immune cells that infiltrate tumors [38,39]. As such, macrophages and neutrophils also play a key tumor support role in HCC [40]. Zebrafish models have been used to study the effects of LPS or DSS on inflammation [62,63]. In the current study, we confirmed the following: (1) LPS or DSS can induce inflammation by recruiting neutrophils and macrophages ( Figure 6); (2) The effects of LPS or DSS exposure are enhanced when combined with xmrk expression (Figure 7); and (3) These effects stimulate the immune system, resulting in accelerated tumor metastasis in twist1a+/xmrk+ double transgenic zebrafish (Figure 8). In the adult-stage twist1a+/xmrk+ zebrafish, the hepatocyte-specific co-expression of twist1a and xmrk coupled with LPS ( Figure 8A,E) or DSS ( Figure 8B,F) exposure led to a higher incidence of metastatic HCC. This suggests that twist1a, xmrk, LPS, and DSS can interact with the immune system and thereby participate in the development of tumor metastasis. This provides strong support of assertions indicating a relationship between chronic inflammation and tumor metastasis.

Isolation of RNA and Reverse-Transcription-PCR (RT-PCR)
Total RNA was isolated from the primary liver tumor, metastatic liver tumors, and adjacent normal tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA (1 µg) was then reverse transcribed into cDNA using the QuantiTect Whole Transcriptome Kit (Qiagen, Hilden, Germany). Following reverse transcription, cDNA templates were amplified via polymerase chain reaction (PCR) using exTEN 2X PCR Master Mix (Axil Scientific, Singapore). The primer sequences of the liver markers and internal control used for RT-PCR were as follows: fabp10a (Forward: CCAGTGACAGAAATCCAGCA; Reverse: GTTCTGCAGACCAGCTTTCC), tfa (Forward: TGCAGAAAAAGCTGGTGATG; Reverse: ACAGCATGAACTGGCACTTG), and actin (Forward: CTCCATCATGAAGTGCGACGT; Reverse: CAGACGGAGTATTTGCGCTCA). In the RT-PCR reaction, 1 µL of cDNA was amplified using the following protocol: 1 cycle at 95 • C for 5 min, followed by 35 cycles at 95 • C for 10 s, 58 • C for 30 s, and 68 • C for 1 min, eventually followed by incubation at 68 • C for an additional 7 min to allow for synthesis completion. Assaying the cDNA involved subjecting the PCR product to 1.0% agarose gel electrophoresis, using actin as an internal control.

Induction of Transgene Expression using Doxycycline and 4-Hydroxytamoxifen
Transgenic larvae were screened for fluorescence at 5 days post-fertilization (dpf) using a fluorescence stereo microscope (SMZ18, Nikon, Japan). The larvae were sorted according to whether EGFP and/or mCherry fluorescence was detected. The induction study was conducted on adult fish (3 to 4 months post-fertilization; mpf) in 5-L tanks with fresh water replenished every other day. Doxycycline (Dox, Sigma-Aldrich, St Louis, MO, USA) was used for the induction of xmrk, and 4-Hydroxytamoxifen (4-OHT, Sigma-Aldrich, St Louis, MO, USA) was used for the induction of twist1a. Long-term liver tumor metastasis induction involved treating twist1a+, xmrk+, and twist1a+/xmrk+ transgenic zebrafish as well as their wild-type siblings using 30 (low dose) or 60 (high dose) µg/mL Dox and 0.5 or 1 µg/mL 4-OHT for 2, 4, 5, 6, or 8 weeks to maintain tumor growth and induce metastasis.

Induction of Transgene Expression, and Chemical Exposure to Transgenic Zebrafish
We sought to determine whether inflammatory agents DSS (Catalog number: D8906; Sigma-Aldrich, St. Louis, MO, USA) or LPS (Catalog number: L4391; Sigma-Aldrich, St. Louis, MO, USA) could cause inflammation in 4-day-old lyz:DsRed and mpeg1:mCherry transgenic zebrafish larvae. Each exposure group included 30 larvae maintained in 6-well plates containing 1 × E3 medium and 0.05% DSS or 40 ng/mL LPS for a period of 2 or 3 days.
The double expression of twist1a/xmrk was induced via exposure to LPS or DSS in 5-L tanks at room temperature. Each treatment group was treated using 60 µg/mL Dox and 1µg/mL 4-OHT with LPS 40 ng/mL or DSS 0.00625%. The double expression of twist1a/xmrk was induced via exposure to 60 µg/mL Dox with 1 µg/mL 4-OHT for 4 wpi.
In this set of experiments, all larvae were maintained in 6-well plates containing 1 × E3 medium. After reaching the adult stage, the zebrafish were maintained in 5-L tanks at room temperature. Fresh water, Dox, 4-OHT, LPS, and DSS were replenished every other day. Samples were collected to investigate long-term treatment effects, and the mortality of adult zebrafish was estimated daily.

Collection of Tissue and Immunohistochemistry Staining
Tissue samples were collected from zebrafish following euthanization at 2, 4, 5, 6, or 8 wpi. Liver tissues were fixed and embedded in paraffin for histological and immunohistochemistry analysis, as previously described [45,46]. The 5-mmicrometer sections were deparaffined, rehydrated, and then treated with 3% H 2 O 2 to block endogenous peroxidase activity, followed by heating in 10 mM citrate buffer at 100 • C for 20 min for antigen retrieval. Immunohistochemical analysis was performed with EnVision™+ Dual Link System . After washing with 1× PBS, slides were washed in 1x PBS with 0.1% Tween 20, developed with Dako DAB staining buffer, counterstained with hematoxylin before being dehydrated, cleared, and mounted with slide covers for evaluation using an Axio Imager Z2 microscope (Zeiss, Carl Zeiss Meditec AG, Germany).

Statistical Analysis
All statistical analysis in this study involved comparisons between experimental and control groups using one-way analysis of variance (ANOVA) and a two-tailed unpaired Student's t-tests. Kaplan-Meier survival curves and log-rank tests were performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) as previously described [45,46]. p-values of 0.05 or less were considered statistically significant.

Conclusions
In conclusion, our results identify Twist1 as an effective target gene against human HCC metastasis. This study provides the first in vivo demonstration that twist1a plays a critical role in both the maintenance and acceleration of xmrk-induced liver tumor metastasis in adult-stage zebrafish. We generated a novel autochthonous transgenic zebrafish model to demonstrate that twist1a and xmrk overexpression cooperates with inflammatory agents to accelerate the onset of tumor metastasis.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/ph14090867/s1, Figure S1: Fluorescent images of twist1a+ transgenic zebrafish and a timeline of experimental design with the schedule used for specimen collection; Figure  Funding: This work was supported by grants from Ministry of Education of Singapore (R154000B88112 and R154000B70114) at Singapore, and National Taiwan University Hospital (UN109-062) at Taiwan.