Differential Transcriptomic Proles in Canine Intestinal Organoids Following Lipopolysaccharide Stimulation

Lipopolysaccharide (LPS) is associated with chronic intestinal inammation and promotes intestinal cancer progression in the gut. While the interplay between LPS and intestinal immune cells has been well characterized, little is known about LPS and intestinal epithelium interactions. In this study, we explored the differential effect of LPS on proliferation and the transcriptome in 3D enteroids/colonoids obtained from dogs with naturally occurring gastrointestinal (GI) diseases, such as Inammatory Bowel Disease (IBD) and GI mast cell tumor. The study objective was to analyze LPS-induced modulation of signaling pathways involving the intestinal epithelia and critical to colorectal cancer development in the context of IBD or a tumor microenvironment. While LPS incubation resulted in a pro-cancer gene expression pattern and stimulated proliferation of IBD enteroids and colonoids, down-regulation of several cancer-associated genes like CRYZL1, Gpatch4, SLC7A1, ATP13A2, and ZNF358 was also observed in tumor enteroids. Genes participating in porphyrin metabolism (CP), thiamine and purine metabolism (TAP2, EEF1A1), arachidonic acid, and glutathione metabolism (GPX1) exhibited a similar pattern of altered expression between IBD enteroids and IBD colonoids following LPS stimulation. In contrast, genes involved in anion transport, transcription and translation, apoptotic processes, and regulation of adaptive immune responses showed opposite expression patterns between IBD enteroids and colonoids following LPS treatment. In brief, the cross-talk between LPS/TLR4 signal transduction pathway and several metabolic pathways, such as fatty acid degradation and biosynthesis, and purine, thiamine, arachidonic acid, and glutathione metabolism, may be important in driving chronic intestinal inammation and intestinal carcinogenesis. biopsies biopsies rpm mixer/rocker chelation, cryptal release trituration and/or mild mild of nitrite production and cytokine synthesis in macrophages. Human hsp60 was found to synergize with IFN-γ in its proinammatory activity 109 . The inammatory response to LPS was evaluated in RGS2 −/− . It showed that it exhibited higher expression of TNF-α and phosphorylated p38 levels in cardiomyocytes. This study demonstrates that RGS2 plays a function in cardioprotection and anti-inammatory signaling via p38 110 . RGS2 inhibits G protein-coupled receptor signaling by increasing the rate of G protein deactivation or by decreasing G protein-effector interactions 111 . The KEGG 38 pathway analysis displayed that LPS induction modied the expression of thiamine, purine, and porphyrin metabolic pathway genes in tumor enteroids and IBD intestinal organoids but altered the expression of glycerophospholipid metabolism and arginine biosynthesis pathway genes in tumor enteroids. Purine metabolism is implicated in a variety of inammatory diseases, including IBD 112 . Purine metabolism regulates innate lymphoid cell function by balancing the levels of eATP and adenosine via the NTPDase enzyme and protects against intestinal injury 112 . As the co-enzyme of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, thiamine plays a key role in carbohydrate metabolism. Accumulation of LPS results in a decrease in thiamine content and transport 113 . When thiamine-degrading enzyme thiaminase was introduced to cell culture media containing thiamine, it inhibited the growth of breast cancer cells . In cancer cells, thiaminase reduced ATP levels signify its vital cell porphyrins


Introduction
Lipopolysaccharide (LPS) is the most effective cell wall-derived in ammatory toxin of Gram-negative bacteria with its inner component, lipid A, accountable for most of the toxin's in ammatory effects 1 . The intestinal lumen, a habitat for many trillions of commensal bacteria, is the primary LPS reservoir in the body 2 . During normal circumstances, the intestinal epithelium retains proper barrier function by promoting the transcellular movement of nutrients, water, and ions while reducing paracellular transport of bacteria or its products/toxins (including LPS) into the systemic circulation 3 . In intestinal permeability disorders, such as infection by pathogenic bacteria, and faulty detoxi cation of LPS, the defective tight junctions (TJ) barrier will permit enhanced paracellular ux of LPS and other phlogistic luminal antigens 4 . Interestingly, apical but not basolateral exposure to LPS, induces epithelial apoptosis through caspase-3 activation and stimulates disruption of tight junctional ZO-1, thus increasing epithelial permeability 5 . While transported in blood, LPS binds to either LPS binding protein (LBP) or plasma lipoproteins and induces systemic in ammation 4 . Basically, LPS-induced intestinal in ammation occurs through stimulation of toll-like receptor 4 (TLR4), which subsequently leads to recruitment of intracellular nuclear transcription factor-κB (NF-κB) followed by the release of chemokines and in ammatory cytokines including necrosis factor-alpha (TNF-α) 6 . TNF-α is accountable for various signaling events within cells, leading to necrosis or apoptosis, therefore playing a pivotal role in resistance to infection and cancers. Activation of the TLR-4 dependent FAK-MyD88-IRAK4 signaling pathway controls LPS-induced intestinal in ammation and tight junction permeability 7,8 .
The removal of circulating LPS (via amelioration of dysbiosis) facilitates the clinical recovery of in ammatory bowel disease (IBD), demonstrating its signi cant role in mediating chronic intestinal in ammation in IBD 2 . The role of LPS throughout the body has been well studied in the context of activation of immune cells (macrophages, dendritic cells, and T cells), where it causes pleiotropic in ammatory cytokine and chemokine secretion 9 . While LPS interactions with intestinal macrophages, dendritic cells, and T cells are well characterized 10,11 , few studies have investigated the direct interactions between LPS and the intestinal epithelium. Regardless of the signi cance of an impaired intestinal barrier in the development of intestinal in ammation in IBD, the effect of increased circulating levels of LPS on the intestinal epithelial barrier integrity remain mostly unknown. Concurrence of IBD among monozygotic vs. dizygotic twin pairs has validated the possible role of genetics in IBD development, with a frequency of 50-55% being detected 12 . Since the circulating LPS level is signi cantly elevated in IBD patients, a greater understanding of the role of LPS on intestinal gene expression modulating intestinal barrier function has important clinical implications.
As a trigger of in ammatory responses, LPS has been associated with cancer pathogenesis, including the development of gastrointestinal (GI) mast cell tumors and colorectal cancer (CRC) [13][14][15] . Different microbial products and TLR4 agonists have demonstrated the crucial role of TLR4 signaling in regulating tumor growth, survival, and progression in colonic, pancreatic, liver, and breast cancers 16 . The TLR4 signaling pathway has been shown to drive tumorigenesis and exhibit some antitumor effect on TLR4 activation 17,18 . Gene expression pro ling using microarray technology has been applied to identify physiologically and clinically signi cant subgroups of TNF-responsive tumors, elucidate the combinatorial and complex nature of cancers, 19,20 and advance our mechanistic understanding of oncogenesis.
The dog genome and its organization have been studied extensively in the last ten years. For understanding the biology and human diseases, dogs have emerged as a primary large animal model [21][22][23][24][25] . Of the large animal models used in translational GI research, the dog is particularly relevant since it presents similar gut physiology, dietary habits, and intestinal microbiota to humans 23,26 . Moreover, dogs spontaneously acquire severe chronic intestinal diseases such as IBD and CRC, which make them ideal animal models for translational GI research 25 . Our laboratory has successfully developed and characterized a canine 3D enteroids/colonoids model system to study intestinal biology during health and disease, and bridge the translational knowledge gap between mice and human 24,27 . Adult stem cell-derived intestinal organoids have previously been shown to retain their genetic and epigenetic phenotype in vitro after numerous passages 28 . We, therefore, hypothesized that a stromal mast cell tumor in the small intestine of a dog could in uence the phenotypic expression of the overlying epithelium, which would be retained in organoid culture. Mast cell tumors are known to secret histamine, proteases, prostaglandin D2, leukotrienes, heparin, as well as a variety of pro-in ammatory cytokines 29 , which could change the expression pro le of the epithelium overlying the tumor and priming it to be sensitized to LPS. This study aimed to broaden our understanding of LPS-induced regulation of signaling pathways in the intestinal epithelium and identify novel genes involved in in ammatory disease and colorectal cancer development.

Ethical animal use
The collection and analysis of intestinal biopsy samples from dogs with IBD and mast cell tumors were previously approved by the Iowa State University (ISU) Institutional Animal Care and Use Committee (IACUC-19-102; PI: Albert E. Jergens). All methods were performed in accordance with the relevant guidelines, and regulations of IACUC as required by U.S. federal regulations 30 . The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Crypt cell isolation and enrichment for enteroid and colonoid culture Ten to fteen endoscopic mucosal biopsies (using 2.8/3.2 mm forceps cup) of the ileum and colon were obtained from two dogs, one diagnosed with IBD (to derive colonoids) and another one with a mast cell tumor (to derive enteroids/colonoids). Epithelial crypts were isolated and enriched from intestinal biopsy samples, as reported previously 31 . Brie y, ileal and colonic biopsies were cut into small pieces (1-2 mm thickness) with a scalpel and were washed six times using the complete chelating solution (1X CCS). Pre-wetted pipette and conical tubes with 1% Bovine Serum Albumin (BSA) were used throughout the procedure to prevent the adherence of the crypt epithelia in the tubes and pipette, thereby minimizing loss of cryptal units 31 . Then, tissues were incubated with 1X CCS containing EDTA (20mM-30mM) for 45 minutes for colonic biopsies and 75 minutes for ileal biopsies at 4°C on a 20-degree, 24 rpm mixer/rocker (Fisher). After EDTA chelation, the cryptal epithelia release was augmented by trituration and/or mild shaking with a vortex. Additional trituration and/or mild shaking was performed after adding 2 mL of fetal bovine serum (FBS, Atlanta Biologicals) to maximize the number of isolated crypts. After tissue fragments settled to the bottom of the tube, the crypt suspension containing supernatant was transferred to a new conical tube then centrifuged at 150g, 4°C for 10 minutes. After centrifugation, the supernatant was removed, and the cell pellet was washed with 10 mL complete medium without an ISC growth factor (CMGF-) medium by repeating the centrifugation and decantation of the supernatant. Following crypt pellet washing, the cell pellet was re-suspended in 2 mL CMGF-medium, and the approximate number of crypt units isolated was enumerated using a hemocytometer 24 .
An estimated 50-100 crypts were seeded per well in 30 μL of Matrigel (Corning® Matrigel® Growth Factor Reduced [GFR] Basement Membrane Matrix) into a 24 well plate format and incubated at 37°C for 10 minutes 31 . 0.5 mL of complete medium with ISC growth factor (CMGF+) medium with the rho-associated kinase ROCK-I inhibitor Y-27632 (StemGent) and glycogen synthase kinase 3β inhibitor CHIR99021 (StemGent) was added to each well and placed in tissue culture incubator. The CMGF + medium with ROCK-I inhibitor and glycogen synthase kinase 3β inhibitor was used for the rst two days of ISC culture to enhance ISC survival and prevent apoptosis. After two days, the enteroid and colonoid cultures were replenished with CMGF + medium every two days for 6-7 days. These freshly isolated 3D enteroid and colonoid cultures were termed Passage-0 (P0).
For subsequent passaging, enteroids and colonoids in Matrigel media were mechanically disrupted using a 1 mL syringe with a 23g ¾" needle (trituration), collected into a 15 mL conical tube and washed with CMGF-medium, then pelleted by centrifugation at 4°C x 100 g for 10 minutes. The pelleted enteroids and colonoids were cultured as previously described and designated as P1. This process was repeated for additional cell culture passages.

LPS stimulation of canine enteroids and colonoids
After four passages, colonoids from a dog with IBD and enteroids and colonoids from a dog diagnosed with an intestinal mast cell tumor were grown on 18 wells of a 24 well culture plate. Among the 18 wells, 9 wells served as control cultures while 9 wells served as LPS treated cultures for these experiments. To investigate the in uence of a tumor microenvironment on the intestinal epithelium, we utilized enteroids from a canine intestinal mast cell tumor. In control enteroid/colonoid cultures, the culture wells were treated with only CMGF+ medium, whereas the LPS treatment group received CMGF+ medium added with LPS at a concentration of 5 µg/ml. A high dose of LPS (5 µg/ml) was used to mimic the high concentration of bacterial-derived luminal LPS 32 . After 48 hours 33 , photographs of culture wells containing the organoids were obtained using a phase-contrast microscope, and the culture media was collected and stored. All enteroids and colonoids from the separate 9 well groups were pooled and washed with PBS and then homogenized in TRIzol (Invitrogen™:TRIzol™ Reagent # 15596026) for microarray and quantitative real-time PCR (qPCR) analyses.
These samples were labeled separately (1-6) to avoid bias in interpretation and described below: AffylmGUI 35 was used to analyze the microarray data. The quality of the microarray chips was determined by NUSE and RNA degradation plots. Probe level data was converted into probe set expression data and normalized using GCRMA. Contrasts between treatment and control were made using the built-in linear model of affylmGUI.
Since pooled data was used without replication, there were no replicates, and Empirical Bayes statistics could not be computed. MA plots were generated in R, where M is the log 2 ratio of probe intensity, and A is the average probe intensity in the contrast ( Table-S10; Probe IDs and their associated gene information are available: https://www.ebi.ac.uk/arrayexpress/ les/A-AFFY-149/A-AFFY-149.adf.txt). The MA plot shows a cone shape where log 2 fold change M decreases with average probe intensity A. Probes that were on the edge of this cone shape are of potential interest as they fall outside the bulk of the probes for a given intensity. Therefore, to identify probes of interest, the hexbin library in R was used to identify probes that fall in low-density regions of the MA plot, which corresponds to the edges of the MA plot 36 . This method was chosen to select probes without bias and for reproducibility. One advantage of this approach is that the probes that are most different in a comparison of samples will be identi ed. However, one point of caution is that regardless of the M value magnitude for all sample comparisons, there will still be a relatively small number of probes identi ed (~500). KEGG ids from these probes were explored using GenomeNet Kegg pathway search.

BLAST search and KEGG pathway analyses
Blast2GO was used to determine the function and localization of differentially expressed genes (DEGs) 37 . It is a widely used annotation platform that uses homology searches to associate sequence with Gene Ontology (GO) terms and other functional annotations. Blast2GO generated gene ontology annotations were determined for the three sub-trees of GO, (a) biological process, (b) molecular function (c) cellular component. The KEGG (Kyoto Encyclopedia of Genes and Genomes) 38,39 pathway analyses were performed by Blast2GO.

LPS stimulates higher intestinal epithelial cell proliferation
LPS at a concentration of 5 µg/ml caused an increase in the size and numbers of canine enteroids and colonoids, which were apparent under phase-contrast microscopy ( Fig. 1a). Although all enteroids and colonoids showed increased proliferation following LPS stimulation, the magnitude of proliferation was higher in tumor enteroids, followed by IBD enteroids and IBD colonoids. To con rm the magnitude of increased proliferation of enteroids and colonoids after LPS stimulation, qPCR was used to evaluate mRNA expression of Ki-67, a nuclear proliferation marker 40 . LPS stimulation caused a 4-fold increase in Ki-67 mRNA expression in tumor enteroids and a 3.4-fold increase in IBD enteroids, whereas LPS caused a 1.7-fold increase in IBD colonoids (Fig. 1b).
Microarray analysis reveals differential expression of genes stimulated by LPS in IBD intestinal organoids and tumor enteroids In this study, we used GeneChip® Canine Genome 2.0 Array that contains more than 42,800 Canis familiaris probe sets to evaluate gene expression 41 . These probe sets targeted more than 18,000 C. familiaris mRNA/EST-based transcripts and more than 20,000 non-redundant predicted genes for more comprehensive coverage 41 . We performed multiple comparisons between samples to determine the effect of LPS treatment, anatomical location, and the in vivo microenvironment (tumor/IBD) on mRNA expression pro les of organoids following LPS stimulation. The highest number of highly differentially expressed mRNAs (684) was found in LPS-treated IBD colonoids vs. Control IBD colonoids (Table 1). Among this total number of highly differentially expressed mRNAs, 48% were upregulated, and 52% were down-regulated (Table 1). Although similar numbers of differentially expressed mRNAs (677) were found between LPS-treated tumor enteroids vs. control tumor enteroids, 56% were upregulated, and 44% were downregulated.
Notably, the Blast2GO analysis revealed numerous genes involved in various biological processes, and these genes were signi cantly affected by LPS-induced in ammation, in ammatory bowel disease, and carcinogenesis ( Fig. 3- Fig. S2-9, Table S1-S10). Changes in genes associated with cellular metabolic processes, stress response, cell death, and immune response were primarily overrepresented in the biological process category; intracellular organelle, extracellular region, and plasma membrane were overrepresented in the cellular component category; and transferase activity, one-carbon group transfer, and metal ion binding were overrepresented in the molecular function category (Fig. S1-S10).
Genes showing similar expression patterns between IBD enteroids and colonoids following LPS stimulation The pattern of gene expression was compared between LPS-stimulated IBD enteroids and colonoids. We identi ed 34 genes across these two groups with similar expression patterns ( Fig. 6 & Table 2-3). Following LPS stimulation, 13 of these 34 genes were elevated in both IBD enteroids and colonoids, whereas 21 were downregulated. These 34 genes may represent the intestinal epithelium signature LPS responsive gene network, regardless of the intestinal regions.
Genes upregulated in IBD enteroids and down-regulated in IBD colonoids (and vice versa) following LPS stimulation Interestingly, 18 genes were found to be altered in the opposite direction ( Fig. 5 & 7, & Table-2-3 & S9). These 18 genes were elevated in LPS-treated IBD enteroids; however, these same 18 genes were down-regulated in LPStreated colonoids. These ndings imply that a distinct gene signature might be utilized to differentiate the intestinal region in response to LPS stimulation.
While genes involved in nitrogen metabolism, drug metabolism, bisphenol degradation (CA1), arginine and proline metabolism (ANPEP), primary bile acid biosynthesis, taurine and hypotaurine metabolism (BAAT) responded in IBD intestinal organoids and tumor enteroids, LPS treatment modulated expression of ACSL5. ACSL5 is involved in the degradation and biosynthesis of fatty acids, as well as the purine and thiamine metabolism pathways (Fig. S10 & Table-4). FAR1 was strongly expressed in untreated tumor enteroids and was found to be involved in wax production, as demonstrated by KEGG pathway studies (Table-4). Following LPS stimulation, genes involved in porphyrin metabolism (CP), thiamine and purine metabolism (TAP2, EEF1A1), arachidonic acid, and glutathione metabolism (GPX1) expressed similarly in IBD enteroids and colonoids (Table-4).

EggNOG Annotation
Functional gene annotation was carried out against the eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) database 39,42 . In order to focus on potentially functional activities of the genes in response to LPS stimulation in IBD enteroids and colonoids, and characterize their evolutionary signi cance, we extracted and grouped genes in four general categories: (A) information storage and processing genes (representing 37.93% of all functional genes identi ed), including Transcription, translation, ribosomal structure and biogenesis and RNA processing and modi cation, (B) cellular processes and signaling (representing 34.48% of all functional genes identi ed) including intracellular tra cking, secretion, and vesicular transport, Signal transduction mechanisms, posttranslational modi cation, protein turnover, chaperones and extracellular structures (C) metabolism (representing 10.34%) including secondary metabolites biosynthesis, transport and catabolism, Inorganic ion transport and metabolism, and energy production and conversion and (D) genes poorly characterized or with unknown functions (representing 10.34% of all functional genes identi ed in both IBD enteroids and colonoids) (Supplemental Information-1). Out of 29 identi ed genes stimulated in both IBD enteroids and colonoids, 29 genes were 10.74% orthologous in Vertebrata/ Chordata/ Mammalia/ Bilateria/ Metazoa/ Opisthokonta. In comparison, 22 genes were 8.15% orthologous in Carnivora, 6 genes were 2.22% orthologous among Euarchontoglires, and 5 genes were 1.85% orthologous among Rodentia as well identi ed (Supplemental Information-1) 39,42 .

Discussion
In this study, we generated enteroids and colonoids from dogs diagnosed with IBD or intestinal mast cell tumor. The goal of these experiments was to better understand how intestinal epithelial cells respond to LPS stimulation under different pathological conditions. We further investigated differences between organoids derived from various intestinal compartments (small intestine vs. colon) in IBD dogs following LPS stimulation. Transcriptomic analyses using microarray 43 were used to identify differentially expressed genes in enteroids and colonoids derived from diseased dogs. These data are the rst to characterize regional speci c transcriptomic changes in intestinal epithelial cells of dogs with IBD and intestinal cancer in response to ex vivo LPS stimulation.
In the present study, tumor enteroids had the highest proliferation indices (based on Ki-67 estimates), followed by IBD enteroids and IBD colonoids, while all enteroids and colonoids showed an increase in proliferation following LPS stimulation. Additionally, we observed that organoid proliferation varied between enteroids and colonoids from the IBD dog, with enteroids exhibiting greater proliferation than colonoids. It is possible that LPS-induced enhanced proliferation in tumor enteroids was caused by increased expression of genes involved in cell cycle process like centromere protein F (CENPF) and ap endonuclease GEN homolog 1 (GEN1) as well as a receptor protein tyrosine kinase (STYK1). CENP-F is a centromere-kinetochore complex-associated protein, and its expression is increased in tumors 44 and it serves as a marker for cell proliferation in human malignancies 45 50 . Additionally, the receptor STYK1 is required for cell proliferation 51 . STYK1's oncogenic potential has been studied widely in gallbladder cancer (GBC) and was reported to be largely dependent on the PI3K/AKT pathway. STYK1's tumor-stimulating activity was abolished by the AKT-speci c inhibitor MK2206, as well as by STYK1 gene silencing 51 . A crucial NF-κB regulator, Sam68 (KHDRBS1), was also observed to be elevated in LPS-treated tumor enteroids, along with the genes GEN1, KRIT1, CENPF, and STYK1. Sam68 (KHDRBS1) exhibited prognostic signi cance in various malignancies and was elevated in cancer cell lines 52,53 . Additionally, the genome-wide study identi ed Sam68 (KHDRBS1) co-expression with cancer-related genes 52,53 . Our results suggest the possible roles of GEN1, KRIT1, CENPF, STYK1, and Sam68/KHDRBS1 in promoting cancer cell proliferation, and these ndings may provide a potential therapeutic target to control mast cell malignancy.
While LPS treatment did not affect the expression of genes involved in proliferation and malignancy, such as GEN1, KRIT1, CENPF, STYK1, and Sam68/KHDRBS1, it did inhibit numerous genes implicated in tumor growth. LPS treatment, for example, reduced the expression of the CRYZL1, Gpatch4, SLC7A1, ATP13A2, and ZNF358 genes.
While CRYZL1 was previously detected in circulating tumor cells in the blood of female cancer patients 54 , GPATCH4 was identi ed in melanoma patient sera and was revealed to be increased in hepatocellular carcinoma 55 . Similarly, the SLC7A1/CAT1 arginine transporter plays a critical function in colorectal cancer by increasing arginine metabolism 56 . After LPS treatment, another important gene, ATP13A2, was downregulated in tumor enteroids. ATP13A2 regulates autophagy, as demonstrated by ATP13A2 knockdown decreasing cellular autophagy levels, reversing ATP13A2-induced stemness in colon cancer cells with the autophagy inhibitor ba lomycin A1 57 , and reduction of the volume of colon cancer xenografts in mice treated with ATP13A2 siRNA 57 . While all of these pieces of evidence point to ATP13A2 and autophagy, studies also reveal a link between the level of ATP13A2 expression and the survival rate of colon cancer patients. Colon cancer patients with elevated ATP13A2 expression display shorter overall survival than those with low ATP13A2 57 . While proliferation-promoting genes such as GEN1, KRIT1, CENPF, STYK1, and Sam68/KHDRBS1 were elevated in LPS-treated tumor enteroids, ZNF139 expression was decreased. ZNF139 increases proliferation and prevents apoptosis by increasing Survivin, x-IAP, and Bcl-2 expression and decreasing Caspase-3 and Bax expression 58 . ZNF139 is substantially expressed in gastric cancer and is used as a prognostic marker in this disease 59 .
Interestingly, while LPS treatment increased proliferation and expression of the GEN1, KRIT1, CENPF, STYK1, and Sam68/KHDRBS1 genes, it decreased the expression of cancer-associated CRYZL1, Gpatch4, SLC7A1, ATP13A2, and ZNF358 genes 47 Studies have shown a connection between LPS stimulation and NO production and the Ceruloplasmin (CP) activity 87 . Without changing iNOS expression levels, CP enhances LPS-activated iNOS activity. An unknown Cp receptor activates this intracellular signaling that cross-talks with the response stimulated by LPS 88 . Members of the S100 family, S100A2 and S100A16, were also induced by LPS stimulation and IBD; similar induction of S100A2 expression induction and secretion has been reported in the previous studies 89 . S100A2 is believed to be a functional component in the immune response 89,90 due to its increased expression in LPS-stimulated immune cells. S100A2 has also been linked to cancer through regulating downstream of the BRCA1/ΔNp63 signaling axis 91 .
Furthermore, our current research shows associations between S100 proteins and IBD, with LPS and IBD both stimulating S100A16 activity. Our present study, along with earlier reports 92 points to the potential use of S100 markers for diagnostic purposes, speci cally S100A2 in cancer and S100A16 in IBD. Use of S100 A proteins in canine feces as biomarkers of in ammatory activity has been previously reported 93 .
The transmembrane ion pump Na+/K+-ATPase (ATP1A1) has been linked to nuclear factor kappa B (NFκB) signaling 94 , a signal associated with the LPS induced immune response. While α2Na+/K+-ATPase haploinsu ciency was reported to regulate LPS-induced immune responses negatively, we observed that ATP1A1 was suppressed in both IBD enteroids and colonoids treated with LPS in the present study. Nonetheless, our demonstration of the inhibited expression of ATP1A1 in IBD intestinal organoids identi es it as a promising candidate for further analysis.
The GI tract microbial ecology varies according to its microbial diversity and anatomic location. Oxygen tension is a primary determinate of microbial numbers and complexity 95 . The upper GI tract, stomach, and small intestine have a lower pH, shorter transit time, and reduced bacterial population. However, the colon harbors the greatest diversity of bacteria, as it has a low cell turnover rate, a low redox potential, and a longer transit time 96 . As a result of the LPS reaction, the gene expression pro le of enteroids and colonoids from IBD dogs also alters. Eighteen genes were increased in IBD enteroids treated with LPS but were downregulated in colonoids treated with LPS. These 18 unique signature genes might be used to differentiate the intestinal regions in response to LPS stimulation.
The differential expression of TOMM20 and eIF3F between IBD enteroids and colonoids may be a reason why IBD enteroids with higher TOMM20 expression and lower eIF3F expression proliferate more than colonoids. Mitochondrial protein, translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20), promotes proliferation and resistance to apoptosis and serves as a marker of mitophagy activity 97 . Reduced TOMM20 expression in response to LPS treatment implies that LPS activates mitophagy 98 , resulting in decreased proliferation in IBD colonoids (as observed in the current study). In tumor cells, enhanced eIF3F expression inhibits translation, cell growth, cell proliferation and induces apoptosis, whereas knockdown of eIF3f inhibits apoptosis, displaying the role of eIF3f as an essential negative regulator of cell growth and proliferation 99,100 . Furthermore, the expression of FAM168A(TCRP1) in IBD enteroids implies that IBD enteroids are more protected than colonoids against LPS stimulation. FAM168A operates via the PI3K/AKT/NFKB signaling pathway and has previously been shown to protect cells against apoptosis 101 .
Several genes that participated in RNA metabolism, protein synthesis, import, protein complex assembly and proteolysis, anion transport, adaptive immune response, and apoptosis were also differentially expressed in LPStreated IBD colonoids and enteroids. For instance, LPS treatment enhanced the expression of SF3A1, S100P, CRIP1, ANXA1, and RGS2 in IBD colonoids. The presence of SF3A and SF3B is required for a robust innate immune response to LPS and other TLR agonists 102 . SF3A1, a member of the SF3A complex, regulates LPS-induced IL-6 by primarily inhibiting its production 102 . Similarly, S100 proteins are known to be secreted in response to TLR-4 activation. S100 proteins also in uence proliferation, differentiation, and apoptosis, in addition to in ammation 90 . Earlier reports and our recent observation of enhanced cysteine-rich intestinal protein 1 (CRIP1) expression in response to LPS suggest that CRIP may play a role in immune cell activation or differentiation 103 . While CRIP1 is abundant in the intestine 104 , it is abnormally expressed in certain types of tumor 103 . CRIP1 inhibits the expression of Fas and proteins involved in Fas-mediated apoptosis 103 . LPS activates the AnxA1 gene signi cantly, and in the absence of AnxA1, LPS induces a dysregulated cellular and cytokine response with a high degree of leukocyte adhesion. The protective role of AnxA1 was demonstrated in AnxA1-de cient mice. In AnxA1-de cient mice, LPS induced a toxic response manifested by organ injury and lethality, restored by exogenous administration of AnxA1 105,106 .
In contrast, following LPS treatment, GTF2A2 expression was increased in IBD enteroids. GTF2A2 is required for NF-kB signaling in LPS-induced TNFα responsive module 107 . Additionally, LPS promoted HSPD1 and Cyr61 expression in IBD enteroids. Cyr61 is known to be activated by LPS and may have pleiotropic responses to LPS 108 . Human hsp60 directly promotes nitrite production and cytokine synthesis in macrophages. Human hsp60 was found to synergize with IFN-γ in its proin ammatory activity 109 . The in ammatory response to LPS was evaluated in RGS2 −/− . It showed that it exhibited higher expression of TNF-α and phosphorylated p38 levels in cardiomyocytes. This study demonstrates that RGS2 plays a function in cardioprotection and anti-in ammatory signaling via p38 110 . RGS2 inhibits G protein-coupled receptor signaling by increasing the rate of G protein deactivation or by decreasing G protein-effector interactions 111 .
The KEGG 38 pathway analysis displayed that LPS induction modi ed the expression of thiamine, purine, and porphyrin metabolic pathway genes in tumor enteroids and IBD intestinal organoids but altered the expression of glycerophospholipid metabolism and arginine biosynthesis pathway genes in tumor enteroids. Purine metabolism is implicated in a variety of in ammatory diseases, including IBD 112 . Purine metabolism regulates innate lymphoid cell function by balancing the levels of eATP and adenosine via the NTPDase enzyme and protects against intestinal injury 112 . As the co-enzyme of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, thiamine plays a key role in carbohydrate metabolism. Accumulation of LPS results in a decrease in thiamine content and transport 113 . When thiamine-degrading enzyme thiaminase was introduced to cell culture media containing thiamine, it inhibited the growth of breast cancer cells 114 . In cancer cells, thiaminase reduced ATP levels signify its vital role in cancer cell bioenergetics 114,115 . LPS-induced TNF-α production in mice was inhibited by porphyrins suggesting its inhibitory effects in TNF-α cytokine production 116,117 .
LPS treatment also altered expression of arachidonic acid and glutathione metabolism pathway genes in IBD enteroids and colonoids. The arachidonic acid (AA) pathway is implicated in a variety of in ammatory diseases 118, 119 . The glutathione (GSH) pathway is a critical metabolic integrator in T cell-mediated in ammatory responses 120 . Metabolism of AA results in reactive oxygen species (ROS) generation 121 . GSH and its metabolic enzymes protect tissues from oxidative damage 83,84,122-127 . By interacting in both the AA and GSH pathways, GPX1 is a critical determinant of AA effects 121 . Fatty acyl-CoA reductase 1 (FAR1) expression was greater in IBD enteroids than tumor enteroids. In mammals, fatty alcohol synthesis is accomplished by two highly expressed FAR isozymes 128 . FAR1 is a potential tumor suppressor, and its increased expression has been associated with improved survival rates in colorectal and breast cancer patients 129 . The enzymes of wax biosynthesis and its association with cancer have been previously reported 128,130 .
As in ammation is a prominent regulator of drug-metabolizing enzymes 131 , DEGs involved in drug metabolism were identi ed in IBD intestinal organoids and tumor enteroids. Additionally, LPS treatment altered Acyl-CoA synthetase 5 (ACSL5) expression in the intestine and tumor enteroids. ACSL5 is required for the de novo synthesis of lipids and fatty acid degradation, and its role in in ammation and cancer development has been reported.
ACSL5 interacts with proapoptotic molecules and suppresses proliferation 132 . Following LPS treatment, genes involved in glutathione metabolism and arginine biosynthesis pathways were signi cantly altered in serum, whereas genes involved in the bile acid biosynthesis pathway were signi cantly changed in numerous rat tissues 133 . We also observed alterations in the expression of genes involved in these metabolic pathways in IBD intestinal organoids stimulated with LPS. Similarly, intestinal in ammation affects multiple metabolic pathways 134 , as evidenced in IBD enteroids and colonoids that express DEGs from primary metabolic process.
In conclusion, the current study provides new and comprehensive data describing how LPS induces differential gene expression in intestinal organoids derived from dogs with chronic intestinal in ammation and small intestinal cancer. The cross-talk between LPS/TLR4 signal transduction pathway and other metabolic pathways like fatty acid degradation and biosynthesis, purine, thiamine, arachidonic acid, and glutathione metabolism demonstrates an important role for LPS in chronic in ammation and carcinogenesis. Additionally, we observed contradictory effects of LPS. While it induced the proliferation and expression of several tumor-associated genes, it decreased the expression of other cancer-associated genes in tumor enteroids, including CRYZL1, Gpatch4, SLC7A1, ATP13A2, and ZNF358. In summary, this study may pave the way for novel approaches to developing anti-in ammatory and anticancer therapeutics.   LPS stimulates higher proliferation.
(a) Enteroids and colonoids from dogs with IBD and intestinal mast cell tumor, 48 hours after LPS stimulation.
Representative phase-contrast images of enteroids and colonoids after LPS stimulation. The Control group received complete growth medium whereas the LPS group received LPS 5 µg/ml in complete growth medium.
(b) Expression of Ki-67 in enteroids and colonoids from dogs with IBD and intestinal mast cell tumor, 48 hours after LPS stimulation as measured by qPCR. The Control group received complete growth medium, whereas the LPS group received LPS 5 µg/ml in complete growth medium. GAPDH was used to normalize. Ent-IBD-C: control IBD enteroids; Ent-IBD-T: IBD enteroids following LPS stimulation; Col-IBD-C: control IBD colonoids; Col-IBD-T: IBD colonoids following LPS stimulation; Ent-Tum-C: control tumor enteroids; Ent-Tum-T: tumor enteroids following LPS stimulation.   Table-S1A-C, and the detailed information of genes is presented in Table-S1D. Gene Ontology (GO) analysis of 27 DEGs in common/same direction between IBD enteroids and colonoids following LPS stimulation. The pie graphs show DEGs annotated for (a) biological process, (b) molecular function, and (c) cellular component categories. The numbers in parentheses indicate the percentage of total genes in each functional category. The summary of genes in each functional category is represented in Table-S8A-C, and the detailed information of genes is presented in Table-S8D.  Table-S9A-C, and the detailed information of genes is presented in Table-S9D.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.