Effect of Lipopolysaccharides on Liver Tumor Metastasis of twist1a/krasV12 Double Transgenic Zebrafish

The poor prognosis of patients diagnosed with hepatocellular carcinoma (HCC) is directly associated with the multi-step process of tumor metastasis. TWIST1, a basic helix-loop-helix (bHLH) transcription factor, is the most important epithelial-mesenchymal transition (EMT) gene involved in embryonic development, tumor progression, and metastasis. However, the role that TWIST1 gene plays in the process of liver tumor metastasis in vivo is still not well understood. Zebrafish can serve as a powerful model for cancer research. Thus, in this study, we crossed twist1a+ and kras+ transgenic zebrafish, which, respectively, express hepatocyte-specific mCherry and enhanced green fluorescent protein (EGFP); they also drive overexpression of their respective transcription factors. This was found to exacerbate the development of metastatic HCC. Fluorescence of mCherry and EGFP-labeled hepatocytes revealed that approximately 37.5% to 45.5% of the twist1a+/kras+ double transgenic zebrafish exhibited spontaneous tumor metastasis from the liver to the abdomen and tail areas, respectively. We also investigated the inflammatory effects of lipopolysaccharides (LPS) on the hepatocyte-specific co-expression of twist1a+ and kras+ in double transgenic zebrafish. Following LPS exposure, co-expression of twist1a+ and kras+ was found to increase tumor metastasis by 57.8%, likely due to crosstalk with the EMT pathway. Our results confirm that twist1a and kras are important mediators in the development of metastatic HCC. Taken together, our in-vivo model demonstrated that co-expression of twist1a+/kras+ in conjunction with exposure to LPS enhanced metastatic HCC offers a useful platform for the study of tumor initiation and metastasis in liver cancer.


Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and a major threat to human health [1][2][3]. Despite substantial progress in the treatment of HCC in recent years, 600,000 people still die from this disease annually, making it the third leading cause of cancer-related deaths worldwide [4,5]. Liver resection and transplantation are the most common HCC treatment methods; however, high recurrence and metastasis rates still lead to poor prognosis in HCC patients [6]. Therefore, early diagnosis and treatment of HCC are critical. A small number of genes have been linked to the occurrence and metastasis of HCC; however, elucidating the mechanisms which underlie liver tumor metastasis remains a pressing issue [7].
The significance of epithelial-mesenchymal transition (EMT) in the process of cancer metastasis has been explored previously. A large number of studies have found that EMT plays a key role in tumor invasion and metastasis, and TWIST1 has been identified as an important regulating factor in the EMT process. TWIST1 is a basic helix-loop-helix (bHLH) transcription factor and is also one of the most important EMT genes involved in embryonic development, tumor progression, and metastasis [8,9]. The fact that EMT has been shown to regulate various biological processes in tumors, including drug resistance, demonstrates its complexity [10]. TWIST1 negatively regulates E-cadherin and positively regulates Vimentin. Both of these proteins are important to EMT induction. Dysregulation of TWIST1 expression is associated with the E-cadherin-mediated loss of intercellular adhesion, activation of mesenchymal markers, and induction of cell motility [11]. Nonetheless, many aspects of EMT remain unclear and require further study. In particular, research is required to identify and understand EMT-related genes.
Abnormal expression of TWIST1 has been frequently observed in many types of cancers. The upregulation of TWIST1 in HCC cell lines promotes the proliferation, cell migration, invasion, and metastasis of cancer cells [7,[12][13][14]. Overexpression of TWIST1 is also associated with shorter overall survival in HCC patients [15]. Research into TWIST1 has broad applications and potential therapeutic value for HCC. However, the relationship between TWIST1 and the proto-oncogene K-RAS, which is a member of the RAS protein family and is mutated in a high percentage of human liver cancers, in HCC is unclear.
RAS proteins are a family of small molecular switches regulated by guanosine triphosphate, which can transmit signals from the cell membrane to the nucleus and activate a variety of signaling pathways involved in cell proliferation, transformation, and tumor progression. RAS family proteins include H-RAS, N-RAS, and K-RAS [16][17][18]. Many singlepoint mutations in RAS genes result in the constitutive activation of RAS with impaired GTPase activity, which leads to continuous stimulation of cell proliferation. The frequency of these gene mutations varies in different tumor types. In total, approximately 30% of human tumors have RAS gene mutations, and these mutations most commonly occur in the K-RAS gene [17]. For example, K-RAS mutations have been identified in 77% of human liver cancers, which is higher than the incidence of mutations in H-RAS and N-RAS in these cancers. The activation of RAS protein signals, which leads to the proliferation and transformation of hepatocytes, has also been observed in human HCC specimens [16,18,19].
Many microenvironmental inflammatory factors have been identified as potential therapeutic targets for HCC [20]. Aspirin, a non-steroidal anti-inflammatory drug, has been shown to reduce the risk of HCC and improve survival [21]. In addition, upregulation of Toll-like receptor (TLR) signaling, which is associated with inflammation-related cancers, has been found to play a key role in the prognosis of chronic and inflammatory diseases that lead to HCC [22]. Lipopolysaccharides (LPS), which are large molecules composed of lipids and polysaccharides that exist in the outer membrane of gram-negative bacteria, function by binding to toll-like receptor 4 (TLR4). In HCC, the cooperation of TLR4 and toll-like receptor 9 (TLR9) may activate the signal transducer and activator of transcription 3 (STAT3) [23][24][25][26]. Exposure to LPS leads to tumor growth and angiogenesis in HCC via the TLR4 receptor in vivo. The signaling which occurs following induction by LPS also promotes EMT in HCC [25,27,28].
The occurrence of tumors can be clearly divided into three independent stages: tumor initiation, progression, and metastasis [29,30]. In previous research, we characterized novel roles of twist1a and xmrk (an activated epidermal growth factor receptor (EGFR) homolog) in tumorigenesis and metastasis and proposed a new animal model for screening anti-tumor metastasis drugs [31][32][33]. However, no reports pertaining to the use of animal models in the study of how TWIST1 and K-RAS affect the initiation and maintenance of liver tumorigenesis have been published.
The use of zebrafish in the study of liver disease and HCC has recently become more widespread [34]. Thus, in the current study, we first investigated the potential relevance of twist1a and kras in liver tumors using a zebrafish model. We also explored the in-vivo mechanism which underlies the effects of LPS on liver tumors in kras or twist1a/kras transgenic zebrafish. Specifically, we were interested in how LPS promotes tumor progression and metastasis in these zebrafish. Results of this study helped to elucidate a new molecular mechanism of HCC and provided new insights pertaining to potential therapeutic targets against HCC.

Zebrafish Husbandry and Maintenance
All experimental protocols and procedures involving zebrafish were approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore and National Taiwan University. These experiments were also conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health. All zebrafish embryos and larvae were maintained in E3 medium. Adult zebrafish were maintained in a recirculating aquatic system at 28 • C with a 14-h light/10-h dark cycle in accordance with standard practice. Dry pellets (GEMMA Micro 150 and 300, Skretting Nutreco, Tooele, UT, USA) were fed to adult zebrafish twice a day at a designated amount of approximately 3% body mass and directly proportional to the density of zebrafish within the tank. The zebrafish were fed lab-grown brine shrimp or commercial fish feed three times per day [35,36].

RNA Isolation and Reverse Transcription PCR (RT-PCR)
Total RNA was extracted from primary liver tumors, metastatic liver tumors, and adjacent normal tissue of adult zebrafish using the RNeasy Mini Kit (Qiagen, Hilden, Germany); 1 µg RNA was then reverse transcribed into complementary DNA (cDNA) using the Quan-tiTect Whole Transcriptome Kit (Qiagen, Hilden, Germany). We amplified cDNA templates via polymerase chain reaction (PCR) using exTEN 2× PCR Master Mix (Axil Scientific, Singapore, Singapore). To assess liver markers, expression of fabp10a (Primers: forward, CCAGTGACAGAAATCCAGCA; reverse, GTTCTGCAGACCAGCTTTCC), tfa (Primers: forward, TGCAGAAAAAGCTGGTGATG; reverse, ACAGCATGAACTGGCACTTG), and actin (Primers: forward, CTCCATCATGAAGTGCGACGT; reverse, CAGACGGAGTATTTGCGCTCA) internal control in adult primary liver tumors, metastatic liver tumors, and adjacent normal tissue, we employed RT-PCR according to the following protocol: 1 µL of cDNA was amplified for 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. The cDNA was then incubated at 68 • C for an additional 7 min to allow for synthesis completion. The resulting PCR products were subjected to 1.0% agarose gel electrophoresis, in which actin was used as the internal control for the cDNA assay, in accordance with published primers and protocols [33,38,39].

Induction of Transgene Expression and LPS Exposure in Transgenic Zebrafish
Each treatment group included 20 larvae that were incubated in 1× E3 medium and treated with 20 µg/mL Dox alone or with 20 µg/mL Dox + 40 ng/mL LPS (catalog number: L4391; Sigma-Aldrich, St. Louis, MO, USA) and then maintained in 6-well plates for 3 days. Adult zebrafish were treated with 10 µg/mL Dox alone or with 10 µg/mL Dox + 40 ng/mL LPS for 2 weeks. Following that, the double expression of twist1a+/kras+ in transgenic zebrafish was induced via treatment with 10 µg/mL Dox and 1 µg/mL 4-OHT in 5-L tanks. The twist1a+/kras+ treatment group was also exposed to 40 ng/mL LPS for 4 weeks. For these experiments, fresh water, Dox, 4-OHT, and LPS were changed every other day. The mortality of adult zebrafish was determined daily, and samples were collected to study long-term treatment effects.

Collection of Tissue, Paraffin Sectioning, and Histochemical Analysis
In accordance with published protocols [33,36,40], all zebrafish samples were collected following euthanization at 2-or 4-weeks post-induction (wpi). Liver tissues were then fixed and embedded in paraffin for histological analysis. Specifically, 5-µm sections were deparaffined, rehydrated, and examined using the EnVision™+ Dual Link System (Dako, Carpinteria, CA, USA) according to previous methodologies for immunohistochemistry (IHC)  , which were used to stain hepatic tissues of zebrafish at 4 • C overnight. After washing with 1× phosphate-buffered saline (PBS), peroxidase activity was detected by incubating tissue sections at room temperature with a universal secondary biotinylated antibody for 30 min and then adding Dako diaminobenzidine (DAB) substrate for development. Tissue sections were counterstained with Mayer's hematoxylin before being dehydrated, cleared, and mounted with slide covers. An Axio Imager Z2 microscope (Zeiss LSM 880, Goettingen, Germany) was used to visualize the sections. Images were analyzed with constant acquisition setting (microscope, magnification, light intensity, exposure time) using a 200× or 400× microscope objective. The results of histochemical analysis and larval measurement were evaluated by two independent senior scientists or pathologists in a single-blind manner to evaluate.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA), as previously described [36]. For all in-vivo experiments, a two-tailed unpaired Student's t-test or one-way analysis of variance (ANOVA) was applied to compare experimental and control groups. To determine the overall survival of zebrafish, survival rates were derived using the Kaplan-Meier method and log-rank test. Quantification of the IHC data using Image J software (NIH, Bethesda, MD, USA). Significance was set at a p-value of 0.05 or less.

Phenotype of twist1a+/kras+ Double Transgenic Zebrafish and Liver Tumor Metastasis Induced by Dox Treatment
For induction studies, 3 to 5 months of mpf adult zebrafish were used for treatment experiments. To comprehensively elucidate tumor progression and metastatic development in twist1a+/kras+ double transgenic zebrafish, experiments in this study employed twist1a+, kras+, and twist1a+/kras+ transgenic zebrafish, as well as their non-transgenic wild-type siblings. All zebrafish were treated with Dox and 4-OHT. Long-term treatment samples were collected and investigated at 2 and 4 weeks (Supplementary Figure S1A).
To study how twist1a+ and kras+ affected tumor growth and liver tumor metastasis over the long term, all zebrafish groups (twist1a+, kras+, twist1a+/kras+, and wild-type) were treated with 20 µg/mL Dox and 1µg/mL 4-OHT for 4 weeks. Compared with wild-type and twist1a+ control groups, an enlarged abdomen and obvious liver overgrowth were observed in both kras+ and twist1a+/kras+ zebrafish at 2 and 4 wpi. Hematoxylin and eosin (H&E) staining revealed that all liver tumors in the kras+ or twist1a+/kras+ zebrafish ranged from normal liver morphology to HCC and included the following classes: normal, hyperplasia (HP), hepatocellular adenoma (HCA), and HCC ( Figure 1A,G). Zebrafish hepatocellular neoplasms have similar histological characteristics to human hepatocellular neoplasms during the growth process. Therefore, the classification of zebrafish liver neoplasm types was based on the criteria for rodent hepatocellular neoplasms and established criteria as previously studied [41][42][43][44]. The classification criteria for liver neoplasm types are as follows: (1) The normal liver has a typical two-cell hepatic plate structure, uniform shape, size and clear boundaries of the cell. (2) The hyperplasia maintains hepatic plate arrangement with an increased prominent nucleus. (3) Unclear hepatic plates with clear cell boundary and relatively uniformed cell shape were found at HCA. (4) HCC was characterized by loss of cell boundaries and hepatic plate structure, increased mitotic cells and multiple nucleus. Furthermore, the body lengths of kras+ and twist1a+/kras+ transgenic zebrafish were significantly larger than those of the wild-type control; however, there was no significant difference in body weights among all groups ( Figure 1B,C). A significantly higher mortality was observed in kras+ or twist1a+/kras+ zebrafish ( Figure 1D). The representative images and percentages of kras+ and twist1a+/kras+ zebrafish exhibiting HCA or HCC development were as follows: HCA (2 wpi: 2/8; 1/11, respectively; 4 wpi: 3/8; 1/8, respectively) or HCC (2 wpi: 2/8; 3/11, respectively; 4 wpi: 3/8; 2/8, respectively). Some twist1a+/kras+ zebrafish also showed evidence of metastatic HCC at 2 and 4 wpi (2 wpi: 5/11; 4 wpi: 3/8) ( Figure 1E-G).

Detection of fabp10a and tfa Expression in Primary and Metastatic Liver Tumors Tissues from twist1a+/kras+ Double Transgenic Zebrafish
To determine the expression of fabp10a and tfa at metastatic tumor cells, we collected primary liver tumors, metastatic HCC tissues, and adjacent normal muscle tissues on zebrafish body from twist1a+/kras+ transgenic zebrafish following treatment with 20 µg/mL Dox and 1 µg/mL 4-OHT. At 2 wpi, EGFP and mCherry fluorescence signal of twist1a+/kras+ zebrafish revealed evidence of metastatic HCC ( Figure 2A). To identify the expression of fabp10a and tfa at tumors, we semi-quantified the mRNA expression of two zebrafish liver markers (fabp10a and tfa) in primary liver tumors tissues, metastatic HCC tissues, and adjacent normal muscle tissues using semi-quantitative RT-PCR. We found that fabp10a and tfa genes were expressed in both primary tumors and metastatic HCC tissues, confirming that metastatic HCC may come from the liver. Note that mRNA expression of fabp10a and tfa was not observed in adjacent normal muscle tissues. Actin and non-template samples respectively served as positive and negative controls ( Figure 2B).  Student's t-test or oneway ANOVA were used to assess differences between variables: * p < 0.05, *** p < 0.001.

Detection of fabp10a and tfa Expression in Primary and Metastatic Liver Tumors Tissues from twist1a+/kras+ Double Transgenic Zebrafish
To determine the expression of fabp10a and tfa at metastatic tumor cells, we collected primary liver tumors, metastatic HCC tissues, and adjacent normal muscle tissues on zebrafish body from twist1a+/kras+ transgenic zebrafish following treatment with 20 μg/mL Dox and 1 μg/mL 4-OHT. At 2 wpi, EGFP and mCherry fluorescence signal of twist1a+/kras+ zebrafish revealed evidence of metastatic HCC (Figure 2A). To identify the expression of fabp10a and tfa at tumors, we semi-quantified the mRNA expression of two zebrafish liver markers (fabp10a and tfa) in primary liver tumors tissues, metastatic HCC tissues, and adjacent normal muscle tissues using semi-quantitative RT-PCR. We found that fabp10a and tfa genes were expressed in both primary tumors and metastatic HCC tissues, confirming that metastatic HCC may come from the liver. Note that mRNA expression of fabp10a and tfa was not observed in adjacent normal muscle tissues. Actin and non-template samples respectively served as positive and negative controls ( Figure 2B).

Co-Expression of twist1a/kras Significantly Increased Apoptosis, and twist1a Activated the EMT Pathway through E-Cadherin and Vimentin
To further compare the severity of liver tumorigenesis and metastasis between kras+ and twist1a+/kras+ zebrafish, the main hallmarks of cell proliferation and cell apoptosis, i.e., PCNA and caspase-3 staining, were examined in fish livers. After induction with 20 μg/mL Dox and 1 μg/mL 4-OHT of transgenic gene expression, kras+ and twist1a+/kras+ zebrafish showed a significant increase in the proliferation of liver cells and cell apoptosis

Co-Expression of twist1a/kras Significantly Increased Apoptosis, and twist1a Activated the EMT Pathway through E-Cadherin and Vimentin
To further compare the severity of liver tumorigenesis and metastasis between kras+ and twist1a+/kras+ zebrafish, the main hallmarks of cell proliferation and cell apoptosis, i.e., PCNA and caspase-3 staining, were examined in fish livers. After induction with 20 µg/mL Dox and 1 µg/mL 4-OHT of transgenic gene expression, kras+ and twist1a+/kras+ zebrafish showed a significant increase in the proliferation of liver cells and cell apoptosis compared with WT control zebrafish (Figure 3). At 4 wpi, we also found a dramatic increase in the percentage of twist1a+/kras+ zebrafish undergoing liver cell apoptosis compared with kras+ zebrafish (Figure 3A,B). Consistent with histological observations, the percentage of proliferating liver cells increased more rapidly in kras+ zebrafish and twist1a+/kras+ than in wild-type and twist1a+ control zebrafish from 2 and 4 wpi ( Figure 1F); however, at 4 wpi, the percentage of liver cells undergoing apoptosis was greater in twist1a+/kras+ zebrafish than in kras+ zebrafish ( Figure 3A,B). These observations were consistent with findings from our previous studies [32,36,45].
Biomedicines 2022, 10, 95 9 of 18 compared with WT control zebrafish (Figure 3). At 4 wpi, we also found a dramatic increase in the percentage of twist1a+/kras+ zebrafish undergoing liver cell apoptosis compared with kras+ zebrafish (Figure 3A,B). Consistent with histological observations, the percentage of proliferating liver cells increased more rapidly in kras+ zebrafish and twist1a+/kras+ than in wild-type and twist1a+ control zebrafish from 2 and 4 wpi ( Figure  1F); however, at 4 wpi, the percentage of liver cells undergoing apoptosis was greater in twist1a+/kras+ zebrafish than in kras+ zebrafish ( Figure 3A,B). These observations were consistent with findings from our previous studies [32,36,45]. In order to identify a potential mechanism and pathway involved in liver tumorigenesis or metastasis, we further compared the severity of liver tumor occurrence and metastasis between kras+ and twist1a+/kras+ zebrafish. For this, the main EMT hallmarks during cancer metastasis, E-cadherin and Vimentin, were examined by immunohistochemical staining of the liver. Immunohistochemical staining results revealed that, following induction with 20 μg/mL Dox and 1 μg/mL 4-OHT, kras+ zebrafish liver tissue had a decrease in E-cadherin and a corresponding increase in Vimentin at 4 wpi compared with wild-type zebrafish. However, the kras+ zebrafish liver tissue also showed an increase in E-cadherin and corresponding decrease in Vimentin compared with twist1a+/kras+ zebrafish ( Figure 4A). Quantification of the percentage of positive cells for E-cadherin and In order to identify a potential mechanism and pathway involved in liver tumorigenesis or metastasis, we further compared the severity of liver tumor occurrence and metastasis between kras+ and twist1a+/kras+ zebrafish. For this, the main EMT hallmarks during cancer metastasis, E-cadherin and Vimentin, were examined by immunohistochemical staining of the liver. Immunohistochemical staining results revealed that, following induction with 20 µg/mL Dox and 1 µg/mL 4-OHT, kras+ zebrafish liver tissue had a decrease in E-cadherin and a corresponding increase in Vimentin at 4 wpi compared with wild-type zebrafish. However, the kras+ zebrafish liver tissue also showed an increase in E-cadherin and corresponding decrease in Vimentin compared with twist1a+/kras+ zebrafish ( Figure 4A). Quantification of the percentage of positive cells for E-cadherin and Vimentin supported these findings ( Figure 4B). These observations suggest that co-expression of twist1a+ and kras+ could trigger crosstalk along the EMT pathway and could contribute to the liver tumorigenesis or metastasis in kras+ and twist1a+/kras+ zebrafish. These observations are also consistent with findings from our previous studies [33,40].
Vimentin supported these findings ( Figure 4B). These observations suggest that co-expression of twist1a+ and kras+ could trigger crosstalk along the EMT pathway and could contribute to the liver tumorigenesis or metastasis in kras+ and twist1a+/kras+ zebrafish. These observations are also consistent with findings from our previous studies [33,40].

Exposure to LPS Increased Liver Size in kras+ Transgenic Zebrafish Larvae
Before examining the long-term effects of LPS treatment, we examined the short-term effects of LPS treatment using kras+ transgenic larvae and non-transgenic wild-type sibling larvae. For this, four-day-old kras+ transgenic zebrafish larvae were treated with 20 μg/mL Dox alone or with 20 μg/mL Dox + 40 ng/mL LPS for 3 days. Wild-type (kras−) zebrafish larvae treated with 20 μg/mL Dox without exposure to LPS served as controls. All larvae in each group were imaged, and sizes of the livers were quantified ( Figure 5A). Exposure to LPS significantly increases liver size in kras+/LPS larvae, compared with kras+ and kras− control larvae ( Figure 5B). cells for all fields following staining at 4 wpi using ImageJ. Scale bar: 50 µm. Student's t-tests were used to assess differences between variables: * p < 0.05, ** p < 0.01, *** p < 0.001.

Exposure to LPS Increased Liver Size in kras+ Transgenic Zebrafish Larvae
Before examining the long-term effects of LPS treatment, we examined the short-term effects of LPS treatment using kras+ transgenic larvae and non-transgenic wild-type sibling larvae. For this, four-day-old kras+ transgenic zebrafish larvae were treated with 20 µg/mL Dox alone or with 20 µg/mL Dox + 40 ng/mL LPS for 3 days. Wild-type (kras−) zebrafish larvae treated with 20 µg/mL Dox without exposure to LPS served as controls. All larvae in each group were imaged, and sizes of the livers were quantified ( Figure 5A). Exposure to LPS significantly increases liver size in kras+/LPS larvae, compared with kras+ and kras− control larvae ( Figure 5B). dpi (white dotted frame: liver; black dots; green dots; pink dots: the number of zebrafish larvae, respectively). Scale bar: 200 μm. Student's t-tests were used to assess differences between variables: ** p < 0.01, *** p < 0.001.

Liver Tumor Phenotypes Induced by Sustained Expression of kras and Exposure to LPS in Adult Transgenic Zebrafish
After determining that short-term exposure to LPS can increase liver size, we next sought to evaluate the long-term effects of LPS exposure. For this, five-month-old adult kras+ transgenic zebrafish and their non-transgenic wild-type sibling zebrafish were treated with 10 μg/mL Dox alone or with 10 μg/mL Dox + 40 ng/mL LPS. Wild-type (kras−) adult zebrafish treated with 10 μg/mL Dox without exposure to LPS served as controls. The tumor status of all zebrafish in each group was examined at 2 wpi. H&E staining revealed that, following exposure to LPS, kras+/LPS zebrafish exhibited enlarged abdomens and obvious signs of liver overgrowth compared with kras+ and wild-type (kras−) control zebrafish ( Figure 6A). H&E staining also showed that liver tumors in both kras+ and kras+/LPS zebrafish ranged from normal liver morphology to HCC and included the following classes: normal, HP, HCA, and HCC ( Figure 6B). The classification of liver neoplasm types was based on established criteria as previously studied [41][42][43][44]. A significant increase in mortality was also observed in kras+ and kras+/LPS transgenic zebrafish compared with wild-type (kras−) control zebrafish ( Figure 6C). Histological analysis of kras+ zebrafish revealed normal, HP, HCA, and HCC (2 wpi: 4/20; 3/20; 3/20; 10/20, respectively), whereas kras+/LPS zebrafish presented more severe evidence of HCC (2 wpi: 15/15). All wild-type (kras−) control zebrafish exhibited normal liver morphology (2 wpi: 20/20) (Figure 6D). liver size in wild-type (kras−) control, kras+, and kras+/LPS zebrafish at 3 dpi (white dotted frame: liver; black dots; green dots; pink dots: the number of zebrafish larvae, respectively). Scale bar: 200 µm. Student's t-tests were used to assess differences between variables: ** p < 0.01, *** p < 0.001.

Liver Tumor Phenotypes Induced by Sustained Expression of kras and Exposure to LPS in Adult Transgenic Zebrafish
After determining that short-term exposure to LPS can increase liver size, we next sought to evaluate the long-term effects of LPS exposure. For this, five-month-old adult kras+ transgenic zebrafish and their non-transgenic wild-type sibling zebrafish were treated with 10 µg/mL Dox alone or with 10 µg/mL Dox + 40 ng/mL LPS. Wild-type (kras−) adult zebrafish treated with 10 µg/mL Dox without exposure to LPS served as controls. The tumor status of all zebrafish in each group was examined at 2 wpi. H&E staining revealed that, following exposure to LPS, kras+/LPS zebrafish exhibited enlarged abdomens and obvious signs of liver overgrowth compared with kras+ and wild-type (kras−) control zebrafish ( Figure 6A). H&E staining also showed that liver tumors in both kras+ and kras+/LPS zebrafish ranged from normal liver morphology to HCC and included the following classes: normal, HP, HCA, and HCC ( Figure 6B). The classification of liver neoplasm types was based on established criteria as previously studied [41][42][43][44]. A significant increase in mortality was also observed in kras+ and kras+/LPS transgenic zebrafish compared with wild-type (kras−) control zebrafish ( Figure 6C). Histological analysis of kras+ zebrafish revealed normal, HP, HCA, and HCC (2 wpi: 4/20; 3/20; 3/20; 10/20, respectively), whereas kras+/LPS zebrafish presented more severe evidence of HCC (2 wpi: 15/15). All wild-type (kras−) control zebrafish exhibited normal liver morphology (2 wpi: 20/20) ( Figure 6D). (D) Histological analysis shows that kras+ and kras+/LPS transgenic zebrafish developed HCC at 2 wpi. Scale bar: 50 or 200 μm. Student's t-test or one-way ANOVA were used to assess differences between variables: * p < 0.05, *** p < 0.001.

LPS Exposure Exacerbated Liver Tumor Metastasis as Well as Hepatocyte-Specific Expression of twist1a and kras in Double Transgenic Zebrafish
After our aforementioned research results demonstrated that induction of kras+ combined with LPS exposure increased liver size and the incidence of liver tumors, we next investigated how LPS exposure affected liver tumor metastasis in twist1a+/kras+ double transgenic, adult-stage zebrafish. For this, three-month-old twist1a+/kras+ zebrafish were treated with 10 μg/mL Dox and 1 μg/mL 4-OHT and exposed to 40 ng/mL LPS. Long-term treatment samples were collected and investigated at 4 weeks (Supplementary Figure  S1B). HCC and metastatic HCC were examined using H&E or immunofluorescence analysis ( Figure 7A,B). No significant differences in body lengths or body weights were found between twist1a+/kras+ and twist1a+/kras+/LPS groups ( Figure 7B,C), nor were there any differences in terms of mortality at twist1a+/kras+ and twist1a+/kras+/LPS groups (Figure (D) Histological analysis shows that kras+ and kras+/LPS transgenic zebrafish developed HCC at 2 wpi. Scale bar: 50 or 200 µm. Student's t-test or one-way ANOVA were used to assess differences between variables: * p < 0.05, *** p < 0.001.

Discussion
Recent evidence points to an increasing incidence of HCC among men in countries such as Japan, Italy, France, Switzerland, the United Kingdom, and the United States [46][47][48]. The process from clinical diagnosis to treatment primarily involves medical imaging, surgery, regional tumor treatment, and biotherapy. Although medical research has progressed remarkably with regard to HCC, effective treatments for patients are still lacking. Due to tumor recurrence and metastasis, the relative 5-year survival rate of liver cancer patients remains low [47,48], highlighting that further investigations into the mechanisms governing liver tumor metastasis should remain a top priority.
TWIST1 is involved in biological processes required for normal growth and development and regulates the expression of many specific genes [49]. However, TWIST1 has known roles in the carcinogenesis of tumor cells. For example, TWIST1 plays an important role in the vascular invasion and lung metastasis of tumor cells [11]. Primary tumor cells undergo EMT and, in the process of tumor metastasis, travel through the circulatory system to distant organs. The occurrence of EMT in tumor cells and the stimulation of tumor metastasis are promoted by TWIST1. In addition, TWIST1 inhibits apoptosis and senescence and promotes the immortalization of cells [50]. However, the mechanisms by which TWIST1 affect the metastasis of tumor cells remains unclear, although mechanisms are believed to differ depending on tumor type. One previous study that employed a Kras-induced lung cancer transgenic mouse model found that Twist1 inhibited cell senescence, thereby accelerating and maintaining the tumorigenic effects of mutant Kras genes [51]. In the present study, our results showed that twist1a+/kras+ double transgenic zebrafish developed spontaneous metastatic tumour. The mortality of twist1a+/kras+ zebrafish was also significantly higher compared to that of kras+ zebrafish (Figure 1) despite the significant increase in liver cell apoptosis in twist1a+/kras+ zebrafish. However, the percentage of apoptotic liver cells was exacerbated in twist1a+/kras+ zebrafish compared with kras+ zebrafish (Figure 3). These results are also consistent with the previous results of twist1a+/xmrk+ zebrafish compared with xmrk+ zebrafish [33]. Thus, the increase in apoptosis of twist1a during the liver tumor metastasis at kras+ or xmrk+ zebrafish is different from the decrease in apoptosis found in rhabdomyosarcoma [52], which means that it has multiple functions. These observations suggest that cell apoptosis or cell proliferation were key factors in the tumorigenesis or metastasis of liver tumors in kras+ or twist1a+/kras+ zebrafish, which is consistent with findings from our previous study on twist1a+/xmrk+ transgenic zebrafish [33].
TWIST1 acts as an EMT regulator and promotes tumor progression through distinct mechanisms. The upregulation of TWIST1 in HCC cell lines promotes cell proliferation and migration [12]. TWIST1 also regulates downstream genes such as E-cadherin and vimentin to promote EMT [53], wherein E-cadherin is the first confirmed gene target of TWIST1expression. TWIST1 inhibits E-cadherin expression by binding to the E-cadherin promoter, resulting in the downregulation of E-cadherin and consequent attenuation of cell-cell adhesion as well as the enhancement of cell migration and invasion. An increase in angiogenesis related to tumor progression is also promoted by TWIST1, which acts by increasing the production of vascular endothelial growth factors [54]. Moreover, knockout of TWIST1 has been found to significantly reduce the number of Vimentin-positive breast tumor cells, which indicates that Twist1 expression is positively associated with Vimentin expression. In specific mouse models, TWIST1 has also been shown to promote EMT in HCC by regulating vimentin via cullin2 circular RNA [12,55]. The results indicate that E-cadherin and Vimentin proteins are significantly increased or decreased, respectively, in kras+ zebrafish compared with twist1a+/kras+ zebrafish (Figure 4), which is consistent with findings from our previous studies [33,40].
The tumor microenvironment, which is composed of stromal cells, endothelial cells, immune cells, inflammatory cells, cytokines, and extracellular matrix, plays a major role in the initiation and development of HCC [56]. LPS induces inflammation in zebrafish by activating (1) TLR4/myeloid differentiation primary response 88 (MyD88)/nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) as well as (2) TLR4/MyD88/mitogenactivated protein kinases (MAPKs) signaling pathways [57]. LPS is a ligand for TLR4 and functions by mediating specific effects of bacterial products. TLR4 is also expressed in different types of cells in the liver, including tumor, hepatic stellate, and kupffer cells [58][59][60]. In a mouse model of HCC, LPS was found to promote angiogenesis by stimulating the activation of hepatic stellate cells via the TLR4 pathway [27]. LPS has also been found to be related to the up-regulation of matrix metalloproteinases (MMPs) expression and activation by other pro-inflammatory signals, and zebrafish is a very suitable in vivo model for studying the regulation and activation of MMPs [61][62][63]. The upregulation of vascular endothelial growth factor (VEGF) expression by LPS has also been shown to induce angiogenesis in HCC cells through a STAT3-dependent pathway both in vitro and in vivo [25]. Another previous study found that LPS induced hepatic stellate cells to secrete a variety of pro-angiogenic factors, including VEGF, platelet-derived growth factor (PDGF), and angiopoietin-1 (Ang-1) [27]. Our results, obtained after exposing zebrafish larvae ( Figure 5) and adult-stage zebrafish ( Figure 6) to LPS, are consistent with those of previous studies that employed zebrafish models of kras-induced liver or gut tumors (i.e., these studies also reported that LPS treatment accelerated tumor progression) [32,33,36,64]. In addition, co-expression of twist1a+/kras+ in zebrafish that had been exposed to LPS was found to exacerbate metastasis compared with twist1a+/kras+ zebrafish that had not been exposed to LPS, indicating that LPS could activate liver tumor progression and metastasis in kras mutants in vivo by cooperating with the twist1a gene. On the other hand, we also noticed that the twist1a+/kras+ group has a higher survival tendency than the twist1a+/kras+/LPS group. However, there is no statistically significant difference. We speculate that expanding the number of zebrafish will reduce this trend (Figure 7). Together, these results reveal the twist1a+ and kras+ genes have a cooperative relationship in chronic inflammation, which may contribute to interactions within the immune system that exacerbate the development of tumor metastasis.

Conclusions
In conclusion, our results indicate that TWIST1 may be an effective target gene in treating HCC metastasis. This is the first in-vivo demonstration that twist1a plays an important role in the maintenance and acceleration of liver tumor metastasis in kras+ adultstage zebrafish. We also determined that the inflammatory agent LPS plays a significant role in twist1a+/kras+ double transgenic zebrafish, which could exacerbate HCC metastasis.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10010095/s1, Figure S1: Experimental design and long-term treatment samples were collected weekly for investigation.