Lipopolysaccharides Enhance Epithelial Hyperplasia and Tubular Adenoma in Intestine-Specific Expression of krasV12 in Transgenic Zebrafish

Intestinal carcinogenesis is a multistep process that begins with epithelial hyperplasia, followed by a transition to an adenoma and then to a carcinoma. Many etiological factors, including KRAS mutations and inflammation, have been implicated in oncogenesis. However, the potential synergistic effects between KRAS mutations and inflammation as well as the potential mechanisms by which they promote intestinal carcinogenesis remain unclear. Thus, the objective of this study was to investigate the synergistic effects of krasV12, lipopolysaccharides (LPS), and/or dextran sulfate sodium (DSS) on inflammation, tumor progression, and intestinal disorders using transgenic adults and larvae of zebrafish. Histopathology and pathological staining were used to examine the intestines of krasV12 transgenic zebrafish treated with LPS and/or DSS. LPS and/or DSS treatment enhanced intestinal inflammation in krasV12 transgenic larvae with concomitant increases in the number of neutrophils and macrophages in the intestines. The expression of krasV12, combined with LPS treatment, also enhanced epithelial hyperplasia and tubular adenoma, demonstrated by histopathological examinations and by increases in cell apoptosis, cell proliferation, and downstream signaling of phosphorylated AKT serine/threonine kinase 1 (AKT), extracellular-signal-regulated kinase (ERK), and histone. We also found that krasV12 expression, combined with LPS treatment, significantly enhanced changes in intestinal morphology, specifically (1) decreases in goblet cell number, goblet cell size, villi height, and intervilli space, as well as (2) increases in villi width and smooth muscle thickness. Moreover, krasV12 transgenic larvae cotreated with DSS and LPS exhibited exacerbated intestinal inflammation. Cotreatment with DSS and LPS in krasV12-expressing transgenic adult zebrafish also enhanced epithelial hyperplasia and tubular adenoma, compared with wild-type fish that received the same cotreatment. In conclusion, our data suggest that krasV12 expression, combined with LPS and/or DSS treatment, can enhance intestinal tumor progression by activating the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway and may provide a valuable in vivo platform to investigate tumor initiation and antitumor drugs for gastrointestinal cancers.


Introduction
Colorectal cancer (CRC) ranks third in terms of global cancer incidence. This disease causes more than 600,000 deaths every year, and the number of affected individuals continues to increase around the world [1][2][3]. In recent years, the incidence and mortality of CRC have also continued to rise rapidly. Between 2007 and 2016, the high mortality

Mifepristone and Chemical Treatments of Zebrafish
Each larva treatment group included 20 larvae, which were maintained in six-well plates. Each well contained 1X E3 medium and mifepristone (catalog number: M8046; Sigma-Aldrich, St. Louis, MO, USA), DSS (catalog number: D8906; Sigma-Aldrich, St. Louis, MO, USA), and/or LPS (catalog number: L4391; Sigma-Aldrich, St. Louis, MO, USA). For the DSS and/or LPS treatment groups, larvae were treated with 0.05% DSS and/or 40 ng/mL of LPS for 2 or 3 days postinduction (dpi). To induce kras V12 expression, larvae were also treated with 4 µM mifepristone for 2 or 3 dpi. Larvae were incubated at 28 • C, and mortality was determined daily. The 1X E3 medium, fresh mifepristone, and chemicals were treated every other day.
Each adult zebrafish treatment group was maintained in a 5 L tank at room temperature, and all zebrafish were fed normally. At 4 weeks postinduction (wpi), samples were collected to investigate long-term treatment effects. For the DSS and/or LPS treatment groups, 4-month-old zebrafish were treated with 0.00625% DSS and/or 40 ng/mL of LPS for 4 wpi. To induce kras V12 expression, zebrafish were exposed to 2 µM mifepristone at 4 wpi. The mortality of adult zebrafish was determined daily, and water, fresh mifepristone, and chemicals were treated every other day.

Tissue Collection and Histopathology of Zebrafish Intestines
Control and transgenic zebrafish were euthanized at 5 months of age using 0.02% tricaine (catalog number: E10521; Sigma-Aldrich, St. Louis, MO, USA). Zebrafish intestines were then collected and fixed in 10% neutral buffered formalin solution (catalog number: HT501128; Sigma-Aldrich, St. Louis, MO, USA) overnight, embedded in paraffin, sectioned into 4 µm sections, and then mounted on poly-L-lysine-coated slides at different time points following mifepristone induction and chemical treatment. The slides were stored in slide boxes at room temperature.
Cytological analysis was also performed on the collected zebrafish intestines. After hematoxylin and eosin (H&E) staining was completed, intestinal histopathology was assessed via a single-blind evaluation of all samples. All intestine tissue evaluations were based on four consecutive sagittal serial sections, which composed the entire intestinal tract, anterior to posterior. Specifically, tissue samples were evaluated for epithelial hyperplasia, dysplasia, and tumors according to previously described diagnostic criteria [26,27].

Immunofluorescence (IF) and Periodic Acid-Schiff (PAS) Staining
The 4 µm zebrafish sections were dewaxed using histoclear (catalog number: H2779; Sigma-Aldrich, St. Louis, MO, USA) and hydrated in an ethanol gradient and Milli-Q water for 10 min, respectively. For antigen retrieval, endogenous peroxidase activity was blocked by heating the slides at 100 • C for 20 min in 10 mM citric acid buffer (catalog number: C9999; Sigma-Aldrich, St. Louis, MO, USA). This was followed by blocking with 3% H 2 O 2 for 15 min. The slides were then washed three times with 1X phosphate-buffered saline (catalog number: P3813; Sigma-Aldrich, St. Louis, MO, USA) with 0.1% Tween 20 (catalog number: 9005-64-5; Sigma-Aldrich, St. Louis, MO, USA) (PBST) for 5 min. Following this, slides were blocked again using 5% bovine serum albumin (BSA) (catalog number: A2153; Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min and then incubated with specific primary antibodies in a humidifying chamber at 4 • C overnight. After being washed with 1X PBST, the slides were incubated with conjugated fluorescent secondary antibodies and then incubated with 4 ,6-diamidino-2-phenylindole (DAPI) (catalog number: D9542 Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Finally, the slides were dehydrated, cleared, and mounted. To determine the specificity of primary antibodies for IF staining, we performed experiments using both appropriate positive (from a previously known positive case) and negative controls (slides not incubated with primary antibodies).
For PAS staining, tissues were also dewaxed using histoclear and hydrated in an ethanol gradient and Milli-Q water for 10 min, respectively. Following this, staining was performed using the Periodic Acid-Schiff Stain Kit (catalog number: 24200-1; Polysciences, Inc., Warrington, PA, USA) to detect goblet cells. Goblet cells were evaluated according to the number of villi. Finally, the slides were dehydrated, cleared, and mounted and then examined using light or fluorescent microscopy.

Statistical Analysis
Differences between experimental and control groups were analyzed by two-tailed Student's t-test and one-way ANOVA. Plot survival curves were derived using the Kaplan-Meier method, and log-rank tests were used to examine differences between experimental and control groups. p-Values of less than 0.05 were considered statistically significant.

Phenotype of Intestinal Tumors Induced by Sustained Expression of kras V12 with LPS Treatment in Transgenic Zebrafish
Results of previous studies demonstrated that treating adult-stage zebrafish with the colitogenic agent DSS can enhance intestinal tumorigenesis in kras V12 -expressing transgenic zebrafish [27]. To examine whether LPS could also enhance tumorigenesis in kras V12 adult transgenic zebrafish through the induction of inflammation, heterozygous kras V12 transgenic zebrafish were cotreated with 2 μM of mifepristone and 40 ng/mL of LPS for 4 weeks at 4 months postfertilization (mpf). We found no significant difference in body length between kras V12 transgenic zebrafish treated with LPS (kras+/LPS) and the control group ( Figure 2A). However, LPS treatment did lead to significantly reduced body

Phenotype of Intestinal Tumors Induced by Sustained Expression of kras V12 with LPS Treatment in Transgenic Zebrafish
Results of previous studies demonstrated that treating adult-stage zebrafish with the colitogenic agent DSS can enhance intestinal tumorigenesis in kras V12 -expressing transgenic zebrafish [27]. To examine whether LPS could also enhance tumorigenesis in kras V12 adult transgenic zebrafish through the induction of inflammation, heterozygous kras V12 transgenic zebrafish were cotreated with 2 µM of mifepristone and 40 ng/mL of LPS for 4 weeks at 4 months postfertilization (mpf). We found no significant difference in body length between kras V12 transgenic zebrafish treated with LPS (kras+/LPS) and the control group ( Figure 2A). However, LPS treatment did lead to significantly reduced body weights in WT/LPS and kras+/LPS adult zebrafish compared with WT, WT/LPS, and kras+ zebrafish ( Figure 2B). Furthermore, by 4 wpi, four of the kras+ zebrafish and eight of the kras+/LPS zebrafish had died. During the same period, five WT/LPS and no WT control zebrafish died ( Figure 2C). At 4 wpi, we also evaluated the entire intestinal tract of all adult zebrafish for enteritis, epithelial hyperplasia, and the presence of tumors ( Figure 2D). Intestinal samples were collected from the WT, WT/LPS, kras+, and kras+/LPS groups, and histological examinations were then performed. While all WT zebrafish showed normal intestinal histology, LPS treatment respectively caused enteritis and hyperplasia in 55% and 35% of WT zebrafish. Conversely, kras+ zebrafish treated with mifepristone exhibited inflammation (10%), hyperplasia (30%), and tubular adenoma (30%). LPS treatment in kras+ zebrafish further increased the prevalence of abnormalities: 45% and 55% of adult kras+/LPS zebrafish respectively showed hyperplasia and tubular adenoma. These observations suggest that kras V12 expression combined with LPS treatment significantly enhanced intestinal tumors in adult zebrafish compared with untreated WT zebrafish, WT zebrafish treated with LPS, and kras+ zebrafish that did not undergo LPS treatment (Figure 2E).

Induction of kras V12 Expression with LPS Treatment Decreased the Number of Goblet Cells, Goblet Cell Size, Villi Height, and Intervilli Space and Increased Villi Width and Smooth Muscle Thickness in Fish Intestines
To examine the effects of LPS on the intestinal morphology of WT/LPS and kras+/LPS zebrafish, we examined the number and size of goblet cells; the height, intervilli space, and width of the villi; and the thickness of smooth muscles ( Figure 3A). In a 4-week induction experiment, histology analysis revealed that the number ( Figure 3B) and size (Figure 3C) of goblet cells were significantly reduced in WT/LPS and kras+LPS zebrafish compared with WT, WT/LPS, and kras+ zebrafish. Intestinal villi height ( Figure 3D) and intervilli space ( Figure 3F) were also significantly reduced in WT/LPS zebrafish compared with WT/WT/LPS and kras+ zebrafish. Conversely, intestinal villi width ( Figure 3E) and smooth muscle thickness ( Figure 3G) were significantly increased in WT/LPS and kras+/LPS zebrafish. These observations indicate that kras V12 expression combined with LPS treatment significantly enhanced changes in intestinal morphology. At 4 wpi, we also evaluated the entire intestinal tract of all adult zebrafish for enteritis, epithelial hyperplasia, and the presence of tumors ( Figure 2D). Intestinal samples were collected from the WT, WT/LPS, kras+, and kras+/LPS groups, and histological examinations were then performed. While all WT zebrafish showed normal intestinal histology, LPS treatment respectively caused enteritis and hyperplasia in 55% and 35% of WT zebrafish. Conversely, kras+ zebrafish treated with mifepristone exhibited inflammation (10%), hyperplasia (30%), and tubular adenoma (30%). LPS treatment in kras+ zebrafish further increased the prevalence of abnormalities: 45% and 55% of adult kras+/LPS zebrafish respectively showed hyperplasia and tubular adenoma. These observations suggest that kras V12 expression combined with LPS treatment significantly enhanced intestinal tumors in adult zebrafish compared with untreated WT zebrafish, WT zebrafish treated with LPS, and kras+ zebrafish that did not undergo LPS treatment ( Figure 2E).

Induction of kras V12 Expression with LPS Treatment Decreased the Number of Goblet Cells, Goblet Cell Size, Villi Height, and Intervilli Space and Increased Villi Width and Smooth Muscle Thickness in Fish Intestines
To examine the effects of LPS on the intestinal morphology of WT/LPS and kras+/LPS zebrafish, we examined the number and size of goblet cells; the height, intervilli space, and width of the villi; and the thickness of smooth muscles ( Figure 3A). In a 4-week induction experiment, histology analysis revealed that the number ( Figure 3B Differences among the variables were assessed using Student's t-tests. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar: 100 µm.

Increases in Cell Apoptosis, Proliferation, and Downstream Signaling of Phosphorylated AKT and ERK Induced by Sustained Expression of kras V12 with LPS Treatment in Transgenic Zebrafish
We next aimed to clarify the effects of LPS treatment on intestinal cell apoptosis and proliferation in kras V12 -expressing zebrafish. For this, immunofluorescence staining was performed on intestine paraffin sections from WT, WT/LPS, kras+, and kras+/LPS zebrafish treated with 2 µM of mifepristone or 40 ng/mL of LPS. We found that kras V12expressing zebrafish treated with LPS showed significant increases in both caspase-3 ( Figure 4A,B) and PCNA-labeled cells ( Figure 4C,D) compared with WT/LPS and kras+ zebrafish. In addition, kras V12 expression combined with LPS treatment increased the expression of a mitotic marker of phosphorylated histone (p-histone) in intestinal epithelial cells compared with WT/LPS and kras+ zebrafish ( Figure S1). Our previous results revealed that the expression of kras V12 in zebrafish intestines significantly stimulated AKT and ERK activities, leading to the upregulation of the MAPK/ERK pathway (the MAPK/ERK pathway is the main downstream effector of RAS in the development of intestinal tumors) [27]. Furthermore, immunofluorescence staining for phosphorylated AKT (p-AKT) and ERK (p-ERK) revealed that compared with WT/LPS and kras+ zebrafish, kras V12 expression combined with LPS treatment significantly increased p-AKT levels ( Figure 5A

Cotreatment with DSS and LPS Enhanced Intestinal Inflammation in kras V12 Transgenic Zebrafish Larvae
We further tested potential synergistic effects on intestinal inflammation by cotreating WT/lyz+, kras+/lyz, WT/mpeg1+, and kras+/mpeg1+ zebrafish larvae from 4 to 6 or 7 dpf with 0.5% DSS and 40 ng/mL of LPS, in addition to treatment with 4 μM of mifepris-

Phenotype of Intestinal Tumors Induced by Sustained Expression of kras V12 with DSS and LPS Cotreatment in Transgenic Zebrafish
We further confirmed the cotreatment of DSS/LPS in kras V12 transgenic zebrafish at the adult stage. For this, 4 mpf heterozygous kras V12 zebrafish were cotreated with 2 μM of mifepristone and DSS/LPS (0.0625%; 40 ng/mL) for 4 weeks. No significant differences were found in terms of body length between kras+ zebrafish treated with DSS/LPS and control group fish ( Figure 7A). However, we observed significantly reduced body weights

Phenotype of Intestinal Tumors Induced by Sustained Expression of kras V12 with DSS and LPS Cotreatment in Transgenic Zebrafish
We further confirmed the cotreatment of DSS/LPS in kras V12 transgenic zebrafish at the adult stage. For this, 4 mpf heterozygous kras V12 zebrafish were cotreated with 2 µM of mifepristone and DSS/LPS (0.0625%; 40 ng/mL) for 4 weeks. No significant differences were found in terms of body length between kras+ zebrafish treated with DSS/LPS and control group fish ( Figure 7A). However, we observed significantly reduced body weights in adult WT/DSS/LPS and kras+/DSS/LPS zebrafish compared with WT or WT/DSS/LPS zebrafish ( Figure 7B). to inflammation, hyperplasia, and tubular adenoma in 9.1%, 27.3%, and 27.3% of these zebrafish, respectively. DSS/LPS treatment in kras+ zebrafish further increased the prevalence of abnormalities, whereby 45.5% and 54.5% of zebrafish respectively exhibited hyperplasia and tubular adenoma. These observations suggest that kras V12 expression combined with DSS/LPS treatment significantly increased the prevalence of intestinal tumors in adult zebrafish compared with WT zebrafish treated with DSS/LPS ( Figure 7E). We also confirmed the WT or kras+ cotreatment of DSS/LPS at the adult stage of zebrafish compared with WT/LPS or kras+/LPS. Results from WT/DSS/LPS and kras+/DSS/LPS transgenic zebrafish were not significantly different from those of WT/LPS and kras+/LPS control groups ( Figure S3).  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. Scale bar: 50 μm.

Discussion
The development of CRC is a multistep process that involves the progression of normal epithelium to an adenoma and then to an adenocarcinoma, where the adenocarcinoma may eventually metastasize to different organs [35]. The development a genetic model of colorectal tumorigenesis was introduced in 1990. In this model, APC, KRAS, TP53, and DCC were proposed as genes that promote CRC progression [36]. Since the introduction of this model, many potential molecular mechanisms of CRC have been investigated. There is a consensus that CRC development is related to LPS-induced systemic inflammation, and these events alter the signal transduction of TLR4, NF-κB, and transforming growth factor beta 1 (TGF-β1) pathways [17,37]. In mice, LPS has been found to 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. Scale bar: 50 µm. By 4 wpi, 5 of the WT/DSS/LPS or kras+ zebrafish from each group and 13 of the kras+/DSS/LPS zebrafish had died, whereas no WT zebrafish died during the same pe-riod ( Figure 7C). At 4 wpi, we evaluated the entire intestinal tract of adult zebrafish for enteritis, epithelial hyperplasia, and the presence of tumors ( Figure 7D). For this, intestinal samples were collected from WT, WT/DSS/LPS, kras+, and kras+/DSS/LPS groups, and histological examinations were then performed. While all WT zebrafish showed normal intestinal histology, DSS/LPS treatment resulted in 55% and 35% of WT zebrafish respectively developing enteritis and hyperplasia. In kras+ zebrafish, mifepristone treatment led to inflammation, hyperplasia, and tubular adenoma in 9.1%, 27.3%, and 27.3% of these zebrafish, respectively. DSS/LPS treatment in kras+ zebrafish further increased the prevalence of abnormalities, whereby 45.5% and 54.5% of zebrafish respectively exhibited hyperplasia and tubular adenoma. These observations suggest that kras V12 expression combined with DSS/LPS treatment significantly increased the prevalence of intestinal tumors in adult zebrafish compared with WT zebrafish treated with DSS/LPS ( Figure 7E). We also confirmed the WT or kras+ cotreatment of DSS/LPS at the adult stage of zebrafish compared with WT/LPS or kras+/LPS. Results from WT/DSS/LPS and kras+/DSS/LPS transgenic zebrafish were not significantly different from those of WT/LPS and kras+/LPS control groups ( Figure S3).

Discussion
The development of CRC is a multistep process that involves the progression of normal epithelium to an adenoma and then to an adenocarcinoma, where the adenocarcinoma may eventually metastasize to different organs [35]. The development a genetic model of colorectal tumorigenesis was introduced in 1990. In this model, APC, KRAS, TP53, and DCC were proposed as genes that promote CRC progression [36]. Since the introduction of this model, many potential molecular mechanisms of CRC have been investigated. There is a consensus that CRC development is related to LPS-induced systemic inflammation, and these events alter the signal transduction of TLR4, NF-κB, and transforming growth factor beta 1 (TGF-β1) pathways [17,37]. In mice, LPS has been found to contribute to tumor progression and hepatic metastasis of colon cancer cells [17,38,39]. Furthermore, the DSSinduced colitis model is widely used because it has many similarities with human ulcerative colitis [40]. Furthermore, a huge advantage of transgenic zebrafish can be an exceptional platform for mimicking human intestinal disorder and establishing vertebrate models for drug screening. For intestinal disease research, high tumor incidence and convenient chemical treatment make the inducible transgenic zebrafish a reasonable platform for intestinal inflammation and tumor progression [23,27]. However, no previously published studies have reported that kras V12 expression combined with LPS treatment can induce intestinal tumors in zebrafish. This is also the first study to report that LPS and/or DSS treatment promotes intestinal tumor progression in kras V12 -expressing transgenic zebrafish.
Tumor-associated macrophages and tumor-associated neutrophils are among the most abundant immune cells in the tumor microenvironment. In CRC, they play pivotal tumorsupporting roles [41,42]. In this study, a zebrafish model was used to study the effects of LPS and/or DSS treatment on intestinal inflammation in kras V12 transgenic zebrafish larvae. Specifically, we were interested in (1) the effects that kras V12 expression has on neutrophils and macrophages when combined with LPS and/or DSS treatment and (2) how these effects stimulate the immune system ( Figure 1). We previously found that treating kras V12 zebrafish larvae with LPS led to significant increases in neutrophil count and neutrophil density in the liver. These increases in neutrophils further led to an enlargement in liver size [43]. In adult zebrafish, the intestine-specific expression of kras V12 with LPS treatment also led to an increase in hyperplasia and tubular adenoma ( Figure 2D,E). In addition, intestinal morphology ( Figure 3A) revealed that goblet cell number, goblet cell size, villi height, intervilli space, villi width, and smooth muscle thickness ( Figure 3B-G) were also significantly altered in these kras V12 zebrafish. Goblet cells in intestinal epithelium produce and secrete mucins (predominantly MUC2), which enter the intestinal lumen to form a mucus layer [44]. The mucus and mucins of goblet cells and intestinal epithelial cells compose the first line of defense for the gastrointestinal tract and interact with the immune system [45]. In clinical CRC samples from mice, SCF/c-KIT signaling has been shown to promote mucus secretion in colonic goblet cells as well as the development of mucinous colorectal adenocarcinoma [46]. CRC tumors and cell lines are characterized by an increased expression of goblet cell marker genes, and there is an association between the proportion of goblet cells in a tumor and the probability that the tumor is assigned as consensus molecular subtype 3 (CMS3) (CMS3 is a tumor subtype that is mutually exclusive from mucinous adenocarcinoma pathologies [47]). Changes in mucin gene expression and mucin glycan structure can occur in intestinal cancers, which leads to cancer progression [48]. Our results indicated that kras V12 and/or LPS can critically interact with the immune system and may be involved in the development of intestinal carcinogenesis.
We analyzed intestinal tumor formation using histological and immunochemical methods. Immunochemical data showed an increase in active caspase-3 ( Figure 4A,B), PCNA expression ( Figure 4C,D), and downstream signaling of phosphorylated AKT ( Figure 5A,B) in kras+/LPS zebrafish compared with kras+ zebrafish, which suggests that LPS is associated with intestinal tumor formation. LPS has been reported to cause rapid apoptosis and death in intestinal epithelial cells as well as loss of epithelial integrity, which is contingent on apoptosis functioning normally [49][50][51]. LPS treatment has also been found to increase (1) AKT phosphorylation at residue Ser473 and (2) increase liver metastasis of HT-29 cells, both in vitro and in vivo [17]. Our data indicate that kras V12 and LPS can be linked through the AKT pathway. The AKT pathway has roles in apoptosis [52], cell proliferation, cell migration [53], angiogenesis [54], and metabolism [55]. Thus, this kras/LPS/AKT link may be the mechanism that underlies the development of intestinal carcinogenesis in kras V12 transgenic zebrafish.
Recently, inflammation has been found to increase the risk of CRC [56][57][58]. DSS has been shown to induce inflammation of the colonic mucosa and has a tumor-promoting effect in mouse and zebrafish models [27,59]. Moreover, a DSS-induced inflammatory bowel disease (IBD)-like enterocolitis model has also been established in zebrafish [60]. Inflammatory stimuli induced by DSS treatment following initiation with AOM carcinogens are effective at rapidly inducing inflammation and intestinal tumors [61]. In mice, high-fat diets can also promote colon tumors associated with AOM/DSS-induced colitis [62]. In addition, DSS treatment has been found to initiate the development of small intestinal polyps or tumors at 2% and 4%, respectively, in mouse models [63,64]. Therefore, we further investigated whether cotreatment with DSS/LPS can exacerbate intestinal inflammation associated with neutrophils or macrophages in kras V12 transgenic zebrafish larvae. In kras V12 zebrafish larvae, DSS/LPS treatment led to significant increases in both of neutrophil and macrophage numbers in the intestines ( Figure 6). In addition, LPS/DSS cotreatment significantly enhanced the increase in neutrophils and macrophages in the intestines in kras+/lyz+/LPS and kras+/lyz+/DSS as well as in kras+/mpeg1+/LPS and kras+/mpeg1+/DSS zebrafish during the larval stage ( Figure S2). For adult zebrafish, kras V12 expression combined with DSS/LPS cotreatment led to an increase in the prevalence of hyperplasia and tubular adenoma compared with WT/DSS/LPS adult zebrafish ( Figure 7D,E). Therefore, the current research also strongly supports a relationship between chronic inflammation and intestinal tumorigenesis. However, we did not observe significant differences in intestinal tumorigenesis between WT/LPS and WT/LPS/DSS or between kras+/LPS and kras+/DSS/LPS adult-stage zebrafish ( Figure S3). These results indicate that extending the treatment time may be necessary to strengthen the state of chronic inflammation [65,66].

Conclusions
In conclusion, our results (based on zebrafish treated with LPS alone or cotreated with LPS and DSS) provide evidence that LPS and/or DSS exacerbates the development and progression of intestinal tumors in kras V12 transgenic zebrafish. These findings are an extension of our previous data [27], which showed that DSS increased kras V12 -induced intestinal tumors in zebrafish. The current study demonstrated that kras V12 expression combined with LPS and/or DSS treatment also significantly exacerbated the development and progression of intestinal tumors, and this is achieved through the modulation of the PI3K-AKT signaling pathway. Therefore, further research is necessary to explore the effects of other inflammatory agents on CRC progression in humans.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/biomedicines9080974/s1: Figure S1: Expression of kras V12 with LPS treatment enhanced the increase in p-Histone in intestinal epithelial cells, Figure S2: LPS/DSS co-treatment significantly enhanced the increase number in neutrophils and macrophages in the intestine during the larval stage in kras+/lyz+/LPS and kras+/lyz+/DSS as well as in kras+/mpeg1+/LPS and kras+/mpeg1+/DSS zebrafish, Figure S3: No significant synergistic effects on intestinal tumorigenesis between WT/LPS and WT/LPS/DSS or between kras+/LPS and kras+ with DSS/LPS adult stage zebrafish, Table S1: Information of antibodies used in this study.