Colon Organoids Research Articles

Abstract 1215: Synthetic colibactins reveal the structural and biological mode-of-action of a microbial carcinogen

Abstract Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as “pks” encoding the biosynthesis of a secondary metabolite, colibactin, that putatively alkylates host DNA via nucleophilic cyclopropane ring addition. While evidence suggests colibactin’s genotoxicity promotes mutagenesis and cancer, it has been impossible to directly test this hypothesis due to compound instability and sparse production. Recently synthesized stable colibactin analogs facilitate the first studies investigating the compounds structural mode of action. We tested the genotoxic capacity of two compounds: colibactin 742 and an analogous molecule with modified proposed DNA-binding residues (colibactin 746), in human cell lines and colonic organoids. To assess transcriptional pathways associated with acute colibactin response, we compared transcriptional profiles in a normal human colonic cell line (FHC) after short-term exposure to colibactin 742 or 746 and pks+ K. pneumoniae or an isogenic ΔclbP mutant. To determine the mutagenic and transformative potential of colibactin 742, we exposed the human colonic epithelial cell line HCT 116 to 742 or 746 for 32-51 days followed by a 30-day recovery period. We assessed changes in gene expression by RNAseq and mutagenesis by whole-genome sequencing followed by mutational signature fitting. 742 but not 746 caused DNA damage in cell lines and human colonoids, quantified by γH2AX phosphorylation. 742 recapitulated transcriptomic responses to pks+ K. pneumoniae infection, and significantly upregulated pathways associated with p53 signaling, senescence, and IL-1 signaling, relative to 746 treated cells. Moreover, chronic cyclical exposure significantly increased expression of BRCA1-associated DNA repair pathways and genes involved in cross-link repair or replication-associated DNA damage response, consistent with colibactin’s putative cross-linking activity. Whole-genome sequencing revealed an increase in single-base substitutions (SBS) and indels (ID) in 742 treated cells, with a higher proportion of [T>N] SBS and 1-4 bp insertion/deletions in T-rich homopolymers, respectively. We found that 742-induced mutations could be attributed to the proposed colibactin signature (SBS88), reactive oxygen species (ROS; SBS17), or mismatch-repair deficiency (MMRd; SBS44). Colibactin 742 treatment caused an increase in H2DCFDA staining in intestinal epithelial cells, and an increase in transcriptomic pathways associated with peroxisome biogenesis or oxidative phosphorylation, supporting the theory that colibactin 742 treatment caused ROS-induced mutations. ID signatures primarily recapitulated pks-associated mutations. These findings provide the first direct evidence supporting colibactin’s proposed mode-of-action. Furthermore, our study suggests colibactin may indirectly cause mutations attributed to ROS or MMRd. Citation Format: Michael W. Dougherty, Rafael Valdés-Mas, Kevin M. Wernke, Raad Z. Gharaibeh, Alberto Riva, Ye Yang, Marcus Muehlbauer, Eran Elinav, Jens Puschhof, Seth B. Herzon, Christian Jobin. Synthetic colibactins reveal the structural and biological mode-of-action of a microbial carcinogen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1215.

Abstract 2574: An exon-skipping HDAC1 novel isoform promotes colorectal carcinogenesis

Abstract Introduction: In a recent profiling for alternative splicing variants in patient-derived colorectal cancer (CRC) organoids by long-read SMRT sequencing, we identified a hitherto undescribed in-frame spliced transcript of HDAC1 with exon 7 skipping (HDAC1-ΔEx7). This study aims to explore the potential role of HDAC1-ΔEx7 in colorectal carcinogenesis. Methods: Quantitative PCR was performed to evaluate the expression of HDAC1 canonical and HDAC1-ΔEx7 isoforms in a cohort of CRC patient (n=103). Functional biology of HDAC1-ΔEx7 was studied in colon organoids and in vivo model. RNA sequencing was carried out for mechanistic investigation. Results: HDAC1-ΔEx7, but not the full-length HDAC1, showed significant upregulations in CRC tumors compared to matching adjacent non-tumoral tissue (p < 0.0001). Change in HDAC1-ΔEx7 expression was not observed in gastric, hepatocellular and esophageal cancer, suggesting HDAC1-ΔEx7 might be exclusive to CRC. The increased HDAC1-ΔEx7 expressions could draw significant associations with inferior patients’ survivals and likely represent an independent prognostication tumor biomarker. Our functional studies highlighted that HDAC1 full-length and HDAC1-∆Ex7 had distinct biological consequences in CRC. Overexpression of HDAC1-ΔEx7 could induce malignant features of non-tumoral colon organoids, including transforming the single layer-luminal non-tumoral organoids into cancerous organoids with compact and multi-layer cell cluster structure, and increasing the ability of organoids’ long-term propagation. In addition, HDAC1-ΔEx7 could trigger anchor-independent spheroid formation in CRC cell line and protect against stress induced cell death. Investigation of metastatic potential of HDAC1 isoforms in BALB/c nude mice indicated that HDAC1-ΔEx7, but not canonical, substantially increased lung metastatic burden with respect to both lesions size and numbers. Our mechanistic investigation highlighted a unique role of HDAC1-ΔEx7 in protecting CRC against ferroptosis induced cell death. Overexpression of HDAC1-ΔEx7 significantly scavenged the lipid ROS induced by glutamate and inhibited ferroptosis hallmark genes expression. Re-expression of HDAC1-ΔEx7 upon CRISPR-based knocked out of endogenous HDAC1 could robustly rescue CRC against ferroptosis induced from Erastin. Transcriptome revealed that HDAC1-ΔEx7 overcomes ferroptosis through regulating ion homeostasis. Conclusion: Our study identified a novel exon 7 skipping isoform of HDAC1 in CRC and showed its oncogenic role in promoting CRC carcinogenesis through inferring resistance to ferroptosis. Citation Format: Guangzheng Deng, Yujuan Dong, Zhongxu Zhu, Yue Guo, Xin Wang, Simon Ng, Nathalie Wong. An exon-skipping HDAC1 novel isoform promotes colorectal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2574.

Abstract 483: Hederacolchiside A1 inhibits autophagy via CTSC inhibition and inhibits growth in colon cancer

Abstract Background: Autophagy is a normal regulatory process that degrades non-functional or unnecessary cellular components. The degradation of components provides materials for the synthesis of cellular components and, during tumor development, may provide energy. Therefore, autophagy regulation may have potential therapeutic uses in oncology. The triterpenoid saponin hederacolchiside A1 (HA1) has potent antitumor effects; however, its role in autophagy is still unknown. Methods: In the present study, we used human colon cancer cells (SW480 and HT29) and mouse colon cancer cells (CT26) for the main experiments, and several additional cancer cell lines for other experiments. To assess the autophagy dysregulation caused by HA1, we evaluated the protein expressions of two autophagy markers (LC3 and SQSTM1) using Western blotting and the changes in organelles using LC3 puncta assay and transmission electron microscopy. We compared the effects of HA1 and other autophagy inhibitors. Additionally, we compared the HA1-induced decrease in cathepsin C (CTSC) expression in tumors and non-tumors using publicly available data. The effect of HA1 on tumor growth was evaluated in vitro using a three-dimensional spheroid model and patient-derived colon organoids, and in vivo based on tumor growth in a subcutaneous injection mouse model. Results: HA1 treatment resulted in marked vacuolization of colon and other cancer cells. Additionally, autophagy was induced by the accumulation of LC3-II and SQSTM1. These findings were supported by increased LC3 puncta and abnormal organelle morphology. Compared to treatment with other autophagy inhibitors, HA1 treatment resulted in the formation of distinct vacuoles and increased LC3 and SQSTM1, similar to chloroquine treatment. The publicly available data showed that the lysosomal protease CTSC was increased in several cancers, including colon cancer, compared to the normal tissues. Furthermore, HA1 decreased the CTSC expression and enzymatic activity independently of the proteasome degradation, resulting in autophagy dysregulation and, subsequently, autophagy flux inhibition. HA1 inhibited cell growth and induced cell cycle arrest in the two-dimensional culture, three-dimensional spheroids, patient-derived colon cancer organoids, and the in vivo experiments. Conclusion: HA1 inhibits cell growth and induces autophagy dysregulation. Therefore, HA1 may be a potential therapeutic agent in colon cancer. Citation Format: Solbi Kim, Eunji Kim, Minju Han, Youn Seo Oh, Sora Kang, Heung Jin Jeon, Hyo Jin Lee. Hederacolchiside A1 inhibits autophagy via CTSC inhibition and inhibits growth in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 483.

Selenoprotein W Ameliorates Experimental Colitis and Promotes Intestinal Epithelial Repair.

Selenoprotein W (Selenow) is a ~9 kDa selenoprotein suggested to play a beneficial role in resolving inflammation. However, the underlying mechanisms are poorly understood. SELENOW expression in the human GI tract using ScRNAseq Gut Cell Atlas and Gene Expression Omnibus (GEO) databases revealed its expression in the small intestine and colonic epithelial, endothelial, mesenchymal, and stem cells and correlated with a protective effect in ulcerative colitis patients. Selenow KO mice treated with 4% dextran sodium sulfate (DSS) showed exacerbated acute colitis, with greater weight loss, shorter colons, and increased fecal occult blood compared to the WT counterparts. Selenow KO mice expressed higher colonic Tnfα, increased Tnfα+ macrophages in the colonic lamina propria, and exhibited loss in epithelial barrier integrity and decreased zonula occludens 1 (Zo-1) expression following DSS treatment. Expression of epithelial cellular adhesion marker (EpCam), yes-associated protein 1 (Yap1), and epidermal growth factor receptor (Egfr) were decreased along with CD24lo cycling epithelial cells in Selenow KO mice. Colonic lysates and organoids confirmed a crosstalk between Egfr and Yap1 that was regulated by Selenow. Overall, our findings suggest Selenow expression is key for efficient resolution of inflammation in experimental colitis that is mediated through the regulation of Egfr and Yap1.

A mesoporous polydopamine-derived nanomedicine for targeted and synergistic treatment of inflammatory bowel disease by pH-Responsive drug release and ROS scavenging.

Repurposing clinically approved drugs to construct novel nanomedicines is currently a very attractive therapeutic approach. Selective enrichment of anti-inflammatory drugs and reactive oxygen species (ROS) scavenging at the region of inflammation by stimuli-responsive oral nanomedicine is an effective strategy for the treatment of inflammatory bowel disease (IBD). This study reports a novel nanomedicine, which is based on the excellent drug loading and free radical scavenging ability of mesoporous polydopamine nanoparticles (MPDA NPs). By initiating polyacrylic acid（PAA）polymerization on its surface, a "core-shell" structure nano-carrier with pH response is constructed. Then, under alkaline conditions, using the π-π stacking and hydrophobic interaction between the anti-inflammatory drug sulfasalazine (SAP) and MPDA, the nanomedicines (PAA@MPDA-SAP NPs) loaded efficiently (928 μ g mg-1) of SAP was successfully formed. Our results reveal that PAA@MPDA-SAP NPs can pass through the upper digestive tract smoothly and finally accumulate in the inflamed colon. Through the synergistic effect of anti-inflammation and antioxidation, it can effectively reduce the expression of pro-inflammatory factors and enhance the intestinal mucosal barrier, and finally significantly alleviate the symptoms of colitis in mice. Furthermore, we confirmed that PAA@MPDA-SAP NPs have good biocompatibility and anti-inflammatory repair ability under inflammation induction through human colonic organoids. In summary, this work provides a theoretical basis for the development of nanomedicines for IBD therapy.

Obesogenic diet leads to luminal overproduction of the complex IV inhibitor H2 S and mitochondrial dysfunction in mouse colonocytes.

Obesity is characterized by systemic low-grade inflammation associated with disturbances of intestinal homeostasis and microbiota dysbiosis. Mitochondrial metabolism sustains epithelial homeostasis by providing energy to colonic epithelial cells (CEC) but can be altered by dietary modulations of the luminal environment. Our study aimed at evaluating whether the consumption of an obesogenic diet alters the mitochondrial function of CEC in mice. Mice were fed for 22 weeks with a 58% kcal fat diet (diet-induced obesity [DIO] group) or a 10% kcal fat diet (control diet, CTRL). Colonic crypts were isolated to assess mitochondrial function while colonic content was collected to characterize microbiota and metabolites. DIO mice developed obesity, intestinal hyperpermeability, and increased endotoxemia. Analysis of isolated colonic crypt bioenergetics revealed a mitochondrial dysfunction marked by decreased basal and maximal respirations and lower respiration linked to ATP production in DIO mice. Yet, CEC gene expression of mitochondrial respiration chain complexes and mitochondrial dynamics were not altered in DIO mice. In parallel, DIO mice displayed increased colonic bile acid concentrations, associated with higher abundance of Desulfovibrionaceae. Sulfide concentration was markedly increased in the colon content of DIO mice. Hence, chronic treatment of CTRL mouse colon organoids with sodium sulfide provoked mitochondrial dysfunction similar to that observed in vivo in DIO mice while acute exposure of isolated mitochondria from CEC of CTRL mice to sodium sulfide diminished complex IV activity. Our study provides new insights into colon mitochondrial dysfunction in obesity by revealing that increased sulfide production by DIO-induced dysbiosis impairs complex IV activity in mouse CEC.

Mechanosensitive extrusion of Enterovirus A71-infected cells from colonic organoids

Enterovirus A71 causes severe disease upon systemic infection, sometimes leading to life-threatening neurological dysfunction. However, in most cases infection is asymptomatic and limited to the gastrointestinal tract, where virus is amplified for transmission. Picornaviruses have previously been shown to exit infected cells via either cell lysis or secretion of vesicles. Here we report that entire Enterovirus A71-infected cells are specifically extruded from the apical surface of differentiated human colon organoids, as observed by confocal microscopy. Differential sensitivity to chemical and peptide inhibitors demonstrated that extrusion of virus-infected cells is dependent on force sensing via mechanosensitive ion channels rather than apoptotic cell death. When isolated and used as inoculum, intact virus-containing extruded cells can initiate new infections. In contrast, when mechanical force sensing is inhibited, large amounts of free virus are released. Thus, extrusion of live, virus-infected cells from intact epithelial tissue is likely to benefit both the integrity of host tissues and the protected spread of this faecal–oral pathogen within and between hosts.

Murine colon organoids as a novel model to study Trypanosoma cruzi infection and interactions with the intestinal epithelium.

Chagas disease (CD) is a life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 50,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne), but congenital and oral transmission have also been reported. The acute phase of CD presents mild symptoms but may develop into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In this immune-privileged reservoir, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models that approximate to the biological and structural complexity of this tissue. Therefore, to understand the role played by the intestinal tissue during transmission and chronic infection, physiological models resembling the organ complexity are needed. Here we addressed this issue by establishing and characterizing adult stem cell-derived colonoid infection models that are clinically relevant for CD. 3D and 2D systems of murine intestinal organoids infected with T. cruzi Dm28c (a highly virulent strain associated with oral outbreaks) were analyzed at different time points by confocal microscopy. T. cruzi was able to invade and replicate in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between organoids and cell lines (primate and intestinal human cell lines). So far, this represents the first evidence of the potential that these cellular systems offer for the study of host-pathogen interactions and the discovery of effective anti-chagasic drugs.

Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis.

Intestinal epithelial cells (IECs) are the first to encounter luminal microorganisms and actively participate in intestinal immunity. We reported that IECs express the β-glucan receptor Dectin-1, and respond to commensal fungi and β-glucans. In phagocytes, Dectin-1 mediates LC3-associated phagocytosis (LAP) utilizing autophagy components to process extracellular cargo. Dectin-1 can mediate phagocytosis of β-glucan-containing particles by non-phagocytic cells. We aimed to determine whether human IECs phagocytose β-glucan-containing fungal particles via LAP. Colonic (n=18) and ileal (n=4) organoids from individuals undergoing bowel resection were grown as monolayers. Fluorescent-dye conjugated zymosan (β-glucan particle), heat-killed- and UV inactivated C. albicans were applied to differentiated organoids and to human IEC lines. Confocal microscopy was used for live imaging and immuno-fluorescence. Quantification of phagocytosis was carried out with a fluorescence plate-reader. zymosan and C. albicans particles were phagocytosed by monolayers of human colonic and ileal organoids and IEC lines. LAP was identified by LC3 and Rubicon recruitment to phagosomes and lysosomal processing of internalized particles was demonstrated by co-localization with lysosomal dyes and LAMP2. Phagocytosis was significantly diminished by blockade of Dectin-1, actin polymerization and NAPDH oxidases. Our results show that human IECs sense luminal fungal particles and internalize them via LAP. This novel mechanism of luminal sampling suggests that IECs may contribute to the maintenance of mucosal tolerance towards commensal fungi.

A187 DIETARY FIBERS ELICIT GUT IMMUNE AND EPITHELIAL BARRIER MODIFYING EFFECTS IN INFLAMMATORY BOWEL DISEASES BASED ON FOOD SOURCE AND FIBER CHEMICAL FEATURES

Abstract Background Dietary fibers are not digested in the bowel; they are fermented by microbes, typically promoting gut health. However, IBD patients experience sensitivity to consumption of fibers. Our previous findings offered the first mechanistic evidence demonstrating that unfermented dietary β-fructans (inulin and oligofructose) can induce pro-inflammatory cytokines and altered epithelial barrier integrity in a subset of pediatric IBD colonic biopsies cultured ex vivo, and in the SYNERGY-1 (β-fructan) clinical study of adult remission UC patients. Fermentation of β-fructan by whole-microbiota intestinal washes from non-IBD or remission IBD patients (but not non-IBD microbes) reduced pro-inflammatory responses. Purpose Here we aimed to expand our findings to uncover the physiologically relevant gut immune and epithelial responses to over 50 unfermented and partially fermented dietary fibers (arabinoxylans, β-glucans, β-mannans, galatooligosaccharides, inulins, oligofructoses, pectins, raffinooligosaccharides, xyloglucans) sourced from commonly consumed fruits, grains, and vegetables to better understand which foods are safe for IBD patients, and in which disease state settings. Method Colonic biopsies cultured ex vivo, peripheral blood mononuclear cells (PBMCs), colonic organoids, and cell lines were incubated with individual dietary fibers or mixture of fibers extracted from commonly consumed fruits, grains, and vegetables. Epithelial barrier integrity (TEER, microscopy, FITC-dextran) and immune responses (cytokine secretion [ELISA/MSD] and expression [qPCR]) were assessed. Structural features of the different fibers (e.g., degree of polymerization, phenolic/phytic content, branching, sugar content) were measured by HPLC and gas chromatography and correlated to host cell responses. Result(s) Most significantly unfermented inulin, oligofructose, and arabinoxylan induced pro-inflammatory responses, particularly in myeloid cells. Pectin and galatooligosaccharides were either non-inflammatory or anti-inflammatory depending on the food source. The epithelial barrier response to select dietary fibers correlated more significantly with the chemical properties of the fibers; longer fibers (greater degree of polymerization; e.g., inulin) displayed improved barrier integrity while shorter dietary fibers with higher phenolic content displayed reduced barrier integrity. Fiber structural properties varied significantly between different fiber subtypes along with the same fiber subtype sourced from different foods. Conclusion(s) Our findings suggest that intolerance and avoidance of fibers in select IBD patients occurs in patients whose gut microbiota do not support fermentation of fibers resulting in increased presence of unfermented dietary fibers in the gut. Here we show which specific dietary fibers from specific food items can elicit gut barrier damage and inflammation in the gut dependent on fiber structural features, suggesting mechanisms underlying IBD patient avoidance of specific high-fiber foods. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; Weston Family Foundation, MMSF, NSERC, CRC Disclosure of Interest None Declared

The role of phosphatidylcholine 34:1 in the occurrence, development and treatment of ulcerative colitis.

Lipid homeostasis is considered to be related to intestinal metabolic balance, while its role in the pathogenesis and treatment of ulcerative colitis (UC) remains largely unexplored. The present study aimed to identify the target lipids related to the occurrence, development and treatment of UC by comparing the lipidomics of UC patients, mice and colonic organoids with the corresponding healthy controls. Here, multi-dimensional lipidomics based on LC-QTOF/MS, LC-MS/MS and iMScope systems were constructed and used to decipher the alteration of lipidomic profiles. The results indicated that UC patients and mice were often accompanied by dysregulation of lipid homeostasis, in which triglycerides and phosphatidylcholines were significantly reduced. Notably, phosphatidylcholine 34:1 (PC34:1) was characterized by high abundance and closely correlation with UC disease. Our results also revealed that down-regulation of PC synthase PCYT1α and Pemt caused by UC modeling was the main factor leading to the reduction of PC34:1, and exogenous PC34:1 could greatly enhance the fumarate level via inhibiting the transformation of glutamate to N-acetylglutamate, thus exerting an anti-UC effect. Collectively, our study not only supplies common technologies and strategies for exploring lipid metabolism in mammals, but also provides opportunities for the discovery of therapeutic agents and biomarkers of UC.

Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids.

There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during active inflammatory bowel disease (IBD), which is associated with an increased influx of immune cells into the gut mucosa. Therefore, studies on how chemokines are regulated in the intestinal epithelium may identify putative treatment targets in IBD. More recently, patient-derived ex vivo models such as intestinal organoids have facilitated molecular analysis of epithelial alterations in IBD patients own cells. Here, we describe refined experimental protocols and methods for the generation and maintenance of IBD patient-derived colonic organoids (colonoids) culture. We also give detailed description of medium, and supplements needed for colonoid establishment, growth, and differentiation, including production of Wnt-3A and Rspondin1 enriched media. Further, we present protocols for RNA and protein isolation from human colonoids, and subsequent gene expression analysis and Western blotting for e.g., signal transduction studies. We also describe how to process colonoids for chemokine protein expression analysis such as immunostaining, confocal imaging, and detection of secreted chemokines by e.g., enzyme-linked immunosorbent assay (ELISA). As proof of principle, we give examples of how the chemoattractant CCL20 can be regulated and expressed in colonoids derived from IBD-patients and healthy controls upon ligands-driven inflammation.

Understanding disruption of the gut barrier during inflammation: Should we abandon traditional epithelial cell lines and switch to intestinal organoids?

Disruption of the intestinal epithelial barrier is a hallmark of mucosal inflammation. It increases exposure of the immune system to luminal microbes, triggering a perpetuating inflammatory response. For several decades, the inflammatory stimuli-induced breakdown of the human gut barrier was studied in vitro by using colon cancer derived epithelial cell lines. While providing a wealth of important data, these cell lines do not completely mimic the morphology and function of normal human intestinal epithelial cells (IEC) due to cancer-related chromosomal abnormalities and oncogenic mutations. The development of human intestinal organoids provided a physiologically-relevant experimental platform to study homeostatic regulation and disease-dependent dysfunctions of the intestinal epithelial barrier. There is need to align and integrate the emerging data obtained with intestinal organoids and classical studies that utilized colon cancer cell lines. This review discusses the utilization of human intestinal organoids to dissect the roles and mechanisms of gut barrier disruption during mucosal inflammation. We summarize available data generated with two major types of organoids derived from either intestinal crypts or induced pluripotent stem cells and compare them to the results of earlier studies with conventional cell lines. We identify research areas where the complementary use of colon cancer-derived cell lines and organoids advance our understanding of epithelial barrier dysfunctions in the inflamed gut and identify unique questions that could be addressed only by using the intestinal organoid platforms.

Aryl Hydrocarbon Receptor (AhR) Signaling in Colonic Cells and Tumors.

The aryl hydrocarbon receptor (AhR) is overexpressed in many tumor types and exhibits tumor-specific tumor promoter and tumor suppressor-like activity. In colon cancer, most but not all studies suggest that the AhR exhibits tumor suppressor activity which is enhanced by AhR ligands acting as agonists. Our studies investigated the role of the AhR in colon tumorigenesis using wild-type and AhR-knockout mice, the inflammation model of colon tumorigenesis using mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) and APCS580/+; KrasG12D/+ mice all of which form intestinal tumors. The effects of tissue-specific AhR loss in the intestine of the tumor-forming mice on colonic stem cells, organoid-initiating capacity, colon tumor formation and mechanisms of AhR-mediated effects were investigated. Loss of AhR enhanced stem cell and tumor growth and in the AOM/DSS model AhR-dependent suppression of FOXM1 and downstream genes was important for AhR-dependent anticancer activity. Furthermore, the effectiveness of interleukin-22 (IL22) in colonic epithelial cells was also dependent on AhR expression. IL22 induced phosphorylation of STAT3, inhibited colonic organoid growth, promoted colonic cell proliferation in vivo and enhanced DNA repair in AOM/DSS-induced tumors. In this mouse model, the AhR suppressed SOCS3 expression and enhanced IL22-mediated activation of STAT3, whereas the loss of the AhR increased levels of SOCS3 which in turn inhibited IL22-induced STAT3 activation. In the APCS580/+; KrasG12D/+ mouse model, the loss of the AhR enhanced Wnt signaling and colon carcinogenesis. Results in both mouse models of colon carcinogenesis were complemented by single cell transcriptomics on colonic intestinal crypts which also showed that AhR deletion promoted expression of FOXM1-regulated genes in multiple colonic cell subtypes. These results support the role of the AhR as a tumor suppressor-like gene in the colon.

TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype

Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Influence of Butyrate on Impaired Gene Expression in Colon from Patients with High Blood Pressure.

Hypertension (HTN) is associated with gut dysbiosis and the depletion of butyrate-producing bacteria in animal models and people. Furthermore, fecal material transfer from donor hypertensive patients increases blood pressure in normotensive recipient animals and ameliorates HTN-associated pathophysiology. These observations have implications in the impaired interactions between the gut and gut microbiota in HTN. Although this concept is supported in animal models, little is known about human HTN. Therefore, our objective for this study was to compare gene expression with transcriptomics and its potential to influence microbiota in subjects with normal and high blood pressure (HBP). Colon samples from reference subjects with normal blood pressure (REF) and HBP were used for RNA-seq to analyze their transcriptomes. We observed the significant downregulation of gene sets governing immune responses (e.g., SGK1 and OASL), gut epithelial function (e.g., KRT20 and SLC9A3R1), gut microbiota (e.g., PPARG and CIDEC) and genes associated with cardiovascular and gut diseases (e.g., PLAUR and NLN) in HBP subjects; the expression of genes within these pathways correlated with blood pressure. Potential drug targets in the gut epithelium were identified using the Drug Gene International Database for possible use in HTN. They include peroxisome proliferator-activated receptor gamma (PPRG), active serum/glucocorticoid regulated kinase 1 (SGK1) and 3 beta-hydroxysteroid isomerase type II inhibitor (HSD3B). Finally, butyrate, a microbiota-derived short-chain fatty acid, restored the disrupted expression of certain functional genes in colonic organoids from HBP subjects. Patients with HBP exhibit a unique transcriptome that could underlie impaired gut-microbiota interactions. Targeting these interactions could provide a promising new therapeutic intervention for hypertension management.

P072 Circadian misalignment by simulated night shifts decreases colonoid barrier integrity in Ulcerative Colitis

Abstract Background Circadian rhythms orchestrate multiple biological processes with 24-hour oscillations that regulate diverse bodily functions. The central clock is in the suprachiasmatic nucleus and orchestrates peripheral circadian clocks in every organ system including the gastrointestinal tract (GIT). Previous work has shown that circadian misalignment in Ulcerative Colitis (UC) is associated with increased colonic permeability, and colonic organoids recapitulate the host circadian phenotype. The goals of this study were to examine whether circadian misalignment would disrupt the colonic barrier integrity in UC. Methods 5 subjects, 2 UC and 3 Healthy Controls (HC), were recruited into the study. All UC subjects were inactive (partial Mayo Score ≤ 1) and stable medications with no flares for the last 3 months. For two weeks prior to entry into the circadian lab all subjects were on a prescribed regular sleep schedule. Subjects were then kept in the circadian lab for 6 days with strict control of their light/dark cycle (Figure 1). All subjects then underwent two unprepped flexible sigmoidoscopies at: (1) baseline with circadian alignment and (2) after three days of simulated night shifts with circadian misalignment. Colonic organoids generated created from the colon tissue biopsies grown in Matrigel and barrier integrity was assessed in apical-out colonoids by FITC-Dextran (4 kDa) Net Fluorescence was estimated by ImageJ. Results Colonic organoids at baseline had significantly decreased ZO-1 in UC versus HC, Figure 2. At baseline there was an increase in UC colonic organoid barrier integrity as measured by net fluorescence (density/area). In UC vs HC barrier integrity was 855 ± 203.8 vs 333.1 ± 52.9, p<0.05. After three days of simulated night shifts, barrier integrity significantly increased in both groups at was 1100 ± 342 vs 656 ± 197.9,p <0.5 (Figure 3). After 24 hours of treatment with cytokines, barrier integrity in HC, UC, misaligned HC, and misaligned UC was 2227 ± 376, 2030 ± 264, 1787 ± 402, and 1506 ± 332, (Figure 4). Conclusion Circadian misalignment by alternation of light:dark cycles (simulated night shift) causes misalignment of host central clock and impacts circadian regulated biological process like tight junction proteins which decreases colonic barrier integrity. This study highlights the importance of studying environmental factors that impact circadian timing in UC including night shift work which may be a significant risk factor for a disease flare. Further in vivo and in vitro studies are ongoing to determine the mechanisms through which circadian misalignment impacts colonic barrier integrity in UC (NCT05180279).

P031 CKD-506, a new histone deacetylase 6 inhibitor, suppresses immune cells and restores intestinal epithelial function

Abstract Background Inflammatory bowel disease (IBD) represents a group of chronic immune-mediated diseases of the gastrointestinal tract, characterized by recurrent inflammation and consequential damage of the gastrointestinal tract. Histone deacetylases (HDACs) are a family of mostly ubiquitous enzymes that remove acetyl groups from lysines on histone proteins to regulate gene transcription. HDAC6, which is localized to the cytoplasm, appears to be a promising candidate in IBD treatment. The purpose of the current study was to examine the anti-inflammatory effects of CKD-506, a novel histone deacetylase 6 (HDAC6) inhibitor, on human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells and to explore the relationship between CKD-506 and gut epithelial barrier function. Methods Lipopolysaccharide (LPS)-stimulated human PBMCs from inflammatory bowel disease (IBD) patients were treated with CKD-506, and tumor necrosis factor-alpha (TNF-α) expression was measured using enzyme-linked immunosorbent assay. The proliferation of CD4+ T cells from IBD patients was evaluated using flow cytometric analysis. The effects of CKD-506 on gut barrier function in a cell line and colon organoids, with examinations of mRNA production, goblet cell differentiation, and E-cadherin recovery, were investigated using quantitative reverse transcription PCR, immunofluorescence, and FITC-dextran permeability assay. Results TNF-α secretion, a pivotal pro-inflammatory mediator in IBD, of LPS-triggered PBMCs was markedly decreased by CKD-506 treatment in a dose-dependent manner and to a greater extent than by tofacitinib or tubastatin A treatment. E-cadherin mRNA expression and goblet cell differentiation increased significantly and dose-dependently in HT-29 cells in response to CKD-506, and inhibition of E-cadherin loss after TNF-α stimulation was significantly reduced both in HT-29 cells and gut organoids. Caco-2 cells treated with CKD-506 showed a significant reduction of barrier permeability in a dose-dependent manner. Conclusion The present study demonstrated that CKD-506 has anti-inflammatory effects on PBMCs and CD4+ T cells and improved gut barrier function, indicating its potential as a promising and competent candidate for small-molecule medicine for IBD treatment.

DOP68 Autophagy induction in the intestinal epithelium protects against inflammation-induced barrier loss in vivo.

Abstract Background Loss of the paracellular tight junction (TJ) barrier and defective autophagy in the gut are pathogenic factors for inflammatory bowel disease (IBD). Earlier, we demonstrated the role of autophagy in the degradation of the pore-forming claudin-2 protein and the resultant TJ barrier enhancement. Here, we investigated the role of autophagy in the regulation of the TJ barrier in vivo and in Crohn’s disease (CD) patient-derived tissues and patient-derived organoids. Methods Wild type and autophagy-deficient Atg7-/- mice were given intraperitoneal rapamycin injection to induce autophagy along with LPS or TNF-α to induce inflammation. Surgically resected tissues from CD patients were incubated with rapamycin and the paracellular barrier was measured by Ussing chambers. Autophagy was induced in human colonic organoids by incubation with either rapamycin or by nutrient starvation. Results Intraperitoneal rapamycin injection in WT mice successfully induced autophagy as evidenced through the degradation of the autophagy substrate p62/SQSTM. Rapamycin injection increased the colonic occludin levels and enhanced the paracellular TJ barrier as evidenced by the decreasing paracellular flux of inulin. Rapamycin prevented LPS and TNF-a induced colonic paracellular barrier loss and reduction in occludin levels in WT mice. Rapamycin reduced the LPS and TNF-a-induced increase in occludin’s association with caveolin-1 thereby preventing its caveolae-mediated endocytosis and degradation. The protective effect of rapamycin was dependent on autophagy. Conditional knockout of Atg7 (cAtg7) in mouse colon decreased baseline occludin levels and increased inulin flux in mouse colon. Furthermore, rapamycin failed to prevent the LPS and TNF-a-induced colonic barrier loss in the cAtg7 mice. Additionally, these mice lacking the ability to undergo autophagy in the colon were more susceptible to Dextran Sodium Sulphate (DSS)-induced colitis. In normal and inflamed colonic tissues from CD patients, incubation with rapamycin induced autophagy, evidenced through the reduction in p62/SQSTM1 protein levels, increased occludin levels and enhanced the paracellular TJ barrier. Autophagy induction by rapamycin or nutrient starvation also increased occludin levels and its membrane localization in patient-derived organoids. Conclusion Our data show that rapamycin successfully induced autophagy in WT mice’s colon and protected these mice from the barrier disruptive effect of both LPS and TNF-α, indicating the importance of autophagy in regulating the intestinal TJ barrier. Our data also highlights the application of autophagy induction as a potential therapeutic approach in the treatment of IBD.

Genomic and epigenomic responses to aspirin in human colonic organoids.

Aspirin (ASA) is a proven chemoprotective agent for colorectal cancer, though mechanisms underlying these effects are incompletely understood. Human organoids are an ideal system to study genomic and epigenomic host-environment interactions. We use human colonic organoids to profile ASA responses on genome-wide gene expression and chromatin accessibility. Human colonic organoids from one individual were cultured and treated in triplicate with 3 mM ASA or vehicle control (DMSO) for 24 h. Gene expression and chromatin accessibility were measured using RNA- and ATAC-sequencing, respectively. Differentially expressed genes were analyzed using DESeq2. Top genes were validated by qPCR. Gene set enrichment was performed by SetRank. Differentially accessible peaks were analyzed using DiffBind and edgeR. Peak annotation and differential transcription factor motifs were determined by HOMER and diffTF. The results showed robust transcriptional responses to ASA with significant enrichment for fatty acid oxidation and peroxisome proliferator-activated receptor (PPAR) signaling that were validated in independent organoid lines. A large number of differentially accessible chromatin regions were found in response to ASA with significant enrichment for Fos, Jun, and Hnf transcription factor motifs. Integrated analysis of epigenomic and genomic treatment responses highlighted gene regions that could mediate ASA's specific effects in the colon including those involved in chemoprotection and/or toxicity. Assessment of chromatin accessibility and transcriptional responses to ASA yielded new observations about genome-wide effects in the colon facilitated by application of human colonic organoids. This framework can be applied to study colonic ASA responses between individuals and populations in future studies.