Colon Organoids Research Articles

Microbiota-derived I3A protects the intestine against radiation injury by activating AhR/IL-10/Wnt signaling and enhancing the abundance of probiotics

ABSTRACT The intestine is prone to radiation damage in patients undergoing radiotherapy for pelvic tumors. However, there are currently no effective drugs available for the prevention or treatment of radiation-induced enteropathy (RIE). In this study, we aimed at investigating the impact of indole-3-carboxaldehyde (I3A) derived from the intestinal microbiota on RIE. Intestinal organoids were isolated and cultivated for screening radioprotective tryptophan metabolites. A RIE model was established using 13 Gy whole-abdominal irradiation in male C57BL/6J mice. After oral administration of I3A, its radioprotective ability was assessed through the observation of survival rates, clinical scores, and pathological analysis. Intestinal stem cell survival and changes in the intestinal barrier were observed through immunofluorescence and immunohistochemistry. Subsequently, the radioprotective mechanisms of I3A was investigated through 16S rRNA and transcriptome sequencing, respectively. Finally, human colon cancer cells and organoids were cultured to assess the influence of I3A on tumor radiotherapy. I3A exhibited the most potent radioprotective effect on intestinal organoids. Oral administration of I3A treatment significantly increased the survival rate in irradiated mice, improved clinical and histological scores, mitigated mucosal damage, enhanced the proliferation and differentiation of Lgr5+ intestinal stem cells, and maintained intestinal barrier integrity. Furthermore, I3A enhanced the abundance of probiotics, and activated the AhR/IL-10/Wnt signaling pathway to promote intestinal epithelial proliferation. As a crucial tryptophan metabolite, I3A promotes intestinal epithelial cell proliferation through the AhR/IL-10/Wnt signaling pathway and upregulates the abundance of probiotics to treat RIE. Microbiota-derived I3A demonstrates potential clinical application value for the treatment of RIE.

Acinetobacter species secrete compounds that promote intestinal inflammation

Background: The gut microbiota is known to impact multiple aspects of human health, including immune modulation, nutrient uptake, cell metabolism, and defense against pathogens. The composition and diversity of the gut microbiota is known to be altered in the setting of inflammatory bowel disease (IBD). IBD, which encompasses the subsets Crohn’s Disease and Ulcerative Colitis, is a life-long condition characterized by chronic inflammation of the gastrointestinal tract. It has long been speculated that gut bacteria drive or exacerbate inflammation in IBD patients, but few studies have identified bacterial candidates. Analysis of 3,853 publicly available RNA-Seq samples revealed that Acinetobacter calcoaceticus was one of the top 10 highest elevated bacteria in Crohn's Disease patients. Acinetobacter are well-characterized by their antibiotic resistance, but little is known about its relationship to the intestine. We hypothesize that A. calcoaceticus stimulates intestinal inflammation and exacerbates colitis. Materials & Results: To identify if A. calcoaceticus could stimulate inflammation, we generated inside out mouse jejunal and colonic organoids and added live A. calcoaceticus bacteria to the cultures. As a positive control, we added another Acinetobacter species A. baumannii and as a negative control, we added E. coli Nissle. After 6 hrs, we observed significant expression of pro-inflammatory cytokines in response to A. calcoaceticus and A. baumannii, while E. coli Nissle did not generate the same responses. To further investigate the compounds responsible to inflammation, we grew our bacterial strains and exposed Caenorhabditis elegans to the bacterial secreted compounds. Similar to our organoid experiments, A. calcoaceticus and A.baumannii were lethal to worms, while E. coli had no effect on C. elegans viability. Next, we sought to address the effects of Acinetoacter on the gut in vivo. We oral gavaged adult BALB/c mice with A. calcoaceticus, A. baumannii and E. coli Nissle and found that in the setting of a complex gut microbiota, these bacteria alone did not stimulation inflammation. However, supplementation of anbitioics prior to introduction of our bacteria resulted in a loss of protection and enhanced inflammation. To model IBD, we administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) and induced colitis and then treated mice with our bacterial strains. We found that TNBS treated mice receiving A. calcoaceticus and A. baumannii lost more weight and had worse histological scores than mice treated with vehicle control and TNBS; indicating A. calcoaceticus worsens intestinal inflammation. Conclusions: These data show that A. calcoaceticus can promote intestinal inflammation and may be a contributing factor to the inflammation experienced by IBD patients. Funding Sources for this project include: 1T32GM132055-01, The Histochemical Society’s Cornerstone Grant and the Porter Physiology Development Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2018: The Role Of Interleukin 27 Receptor Signaling In Regulation Of Intestinal Barrier, Gut Microbiota And Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a cardiovascular disease characterized by accumulation of immune cells, inflammation, and degradation of medial layer of the abdominal aorta. Angiotensin II (Ang II) plays a key role in controlling blood pressure but is also able to regulate various immune and non-immune cells. While some studies suggested the role of Ang II in dysbiosis, the link between alteration of intestinal microbiota and AAA is largely unexplored. Cytokines are established regulators of inflammatory milieu in the aortic wall in AAA, but their distant contribution, for example via control of intestinal microbial homeostasis and cooperation with Ang II, has never been explored. Interleukin (IL)27 is a member of the IL6/IL12 superfamily that regulates various immune and non-immune cells. We previously showed that IL27R signaling cooperates with Ang II in the regulation of myelopoiesis thereby promoting AAA. Myeloid progenitor expansion and myeloid cell production driven by AngII are blunted in Apoe -/- Il27ra -/- mice. Here we found that Apoe -/- Il27ra -/- mice were also protected from gut dysbiosis and contain less pro-inflammatory bacterial genera in their colons. Unexpectedly, microbiota depletion accelerated AAA development in Apoe -/- Il27ra -/- mice, while Apoe -/- controls were protected from the disease. RNA seq analysis revealed upregulation of inflammatory pathways and downregulation in tight junctions and collagen in colons of Apoe -/- Il27ra -/- mice with ablated microbiota. Transcriptomic analysis of Apoe -/- colonic organoids stimulated with recIL27 shows upregulation of pathways linked to cell proliferation, cell cycle, and junction genes, indicating direct role of IL27. The analysis of the bone marrow revealed elevation of early myeloid progenitors which correlated with heightened circulating monocytes and neutrophils in Apoe -/- Il27ra -/- mice with ablated microbiota. Non-targeted metabolomics revealed microbiota and IL27R signaling-dependent alteration of serum metabolites likely mediating the effect on myeloid cells and AAA. Overall, our work uncovers novel role of IL27R signaling in the regulation of AAA, mechanistically connecting intestinal barrier and microbiota to the pathogenesis of this CVD.

MYO5B levels are linked to microRNAs and UNC45A in colorectal cancer

Background: Colorectal cancer (CRC) is currently the third most prevalent cancer worldwide and ranks as the second deadliest. Colon cancer is characterized by disruption in cellular structure and polarity, processes that are commonly regulated by cell traffcking. The molecular motor Myosin 5b (Myo5b) is a key component of intracellular traffcking in the intestinal epithelium. Myo5b moves along F-actin tracks to transport cargo to and from the apical membrane. When Myo5b function is impaired, it leads to significant changes in gastrointestinal epithelial cells, including hyper-proliferation, alterations in tight junction proteins, and reduced differentiation. For Myo5b to function properly it must be properly folded; a process regulated by the myosin chaperone Unc45a. Recent work demonstrates that Myo5b levels are decreased in gastric cancer and during the progression of colorectal tumors. However, the mechanism driving this altered Myo5b expression in colorectal cancer remains unknown. We hypothesize that microRNAs regulate the expression of Unc45a, which subsequently impacts Myo5b function, promoting colorectal cancer. Methods and Results: Analysis of high-throughput RNA-sequencing data from the Cancer Genome Atlas (TCGA) revealed hyper-methylation of the Myo5b gene in CRC tumors (n=286) compared to controls (n=41). Findings in gastric cancer have shown that hyper-methylation leads to the silencing of Myo5b gene expression. Consistent with these findings, we observe a significant reduction in Myo5b gene expression in CRC tumors. Immunostaining of CRC tumor arrays and analysis from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) demonstrated a corresponding decrease in Myo5b protein levels. For comparison, we examined Myo5b levels in normal human colonic organoids and the colon cancer cell lines T84,HT-29, HT29-MTX, HCT-8 and HCT-116. Using qPCR, we confirmed a significant reduction in Myo5b gene expression in cancer cells compared to human colonic organoids. We postulated that even if Myo5b was expressed in CRC, it might not function properly if Unc45a function was perturbed. Using the same databases, we found that while Unc45a gene expression levels remain unchanged, its protein levels were significantly reduced. Analysis of the miRMap data reveled multiple microRNAs that could target Unc45a. The top 10 Unc45a-targeting microRNAs were all found to be elevated in CRC compared to normal control tissue, suggesting that microRNAs may lead to reduced Unc45a protein levels. The functional importance of decreased Myo5b in CRC was underscored by the observation that CRC patients with lower levels of Myo5b had reduced survival compared to those CRC patients with higher levels of Myo5b. Additionally, the loss of Myo5b correlated with increased ribosome biogenesis, a key driver of tumor proliferation. Conclusions: These findings indicate that reduced Myo5b in CRC may be attributed to gene methylation and impaired protein folding by Unc45a. The loss of Myo5b holds significance, as it is associated with poorer prognosis and linked with cancer progression. National Diabetes and Digestive and Kidney Diseases Grant R43 DK109820, RO1 DK048370, KO1 DK121869, MUSC start up funds, Histochemical 2022 Cornerstone Grant, and NIDDK T32. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Histamine Receptors Are Altered in Colorectal Cancer

Background: Histamine is an endogenous amine that acts on a wide variety of cell types throughout the body. Its activity is mediated by four distinct receptors: HRH1, HRH2, HRH3 and HRH4. Each of these receptors induces a distinct signaling cascade that has diverse output. Histamine has been postulated to influence cancer progression, but little is known about the distribution of histamine receptors in the gut epithelium or its exact role in colorectal cancer. The aim of this study was to define the distribution of histamine receptors in the colonic epithelium in the setting of health and cancer. Methods & Results: We utilized publicly available single cell sequencing databases to examine the distribution of histamine receptors in the normal colon. We found that HRH1 was highly expressed in all colonic epithelial cells. Interestingly, HRH2, HRH3 and HRH4 were not expressed in any cell type in the colon. To confirm these findings, we grew human colonic organoids and found by qPCR analysis that HRH1 was the only receptor present in this epithelial specific system. In the setting of cancer, we found that HRH1 was elevated in colorectal cancer tumors compared to normal controls. Immunostaining of a tissue array confirmed that colon cancer tissue specimens had higher concentrations of immune associated HRH1; suggesting that immune cells were contributing to the higher levels of HRH1 observed in tumors. Single cell RNAseq data demonstrated that macrophages and monocytes had the highest expression of HRH1 among immune cells. Examination of immune infiltration and HRH1 expression in the Cancer Genome Atlas revealed a positive correlation between monocyte and macrophage HRH1 expression. Conclusions: These data suggest that HRH1 is the only receptor expressed in the colonic epithelium and that in cancer, infiltration of monocytes and macrophages harboring HRH1 elevate overall expression levels of this receptor in tumors. DDRC Pilot and Feasibility Award (Amy Engevik) Career Development Award (K01) (Amy Engevik) DK121869-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Sclareol protected against intestinal barrier dysfunction ameliorating Crohn's disease-like colitis via Nrf2/NF-B/MLCK signalling

BackgroundInflammation-induced intestinal barrier dysfunction is not only a pathological feature of Crohn's disease (CD) but also an important therapeutic target. Sclareol (SCL) is a nontoxic natural plant compound with anti-inflammatory effect, but its role in CD has not been established. MethodsIn vivo studies of mice with TNBS-induced colitis were carried out to evaluate the effects of SCL on CD-like colitis and intestinal barrier function. In vitro, a TNF-α-induced colonic organoid model was established to test the direct effect of SCL on inflammation-induced intestinal barrier injure and inflammatory response. The Nrf2/NF-κB/MLCK signalling was analysed to explore the mechanism of SCL. ResultsIn vivo, SCL largely alleviated the colitis in TNBS mice, as evidenced by improvements in the weight loss, colitis symptoms, endoscopic score, macroscopic histological score, and histological inflammation score. Moreover, SCL significantly improved intestinal barrier dysfunction, manifested as reduced intestinal permeability and decreased intestinal bacterial translocation in TNBS mice. Importantly, SCL antagonised the intestinal mucosal inflammation while protecting tight junctions in TNBS mice. In vitro, SCL largely depressed pro-inflammatory cytokines levels and improved intestinal epithelial permeability in a TNF-α-induced colonic organoid model. In the context of CD, the protective effects of SCL against inflammation and intestinal barrier damage are at least partially results from the Nrf2 signalling activation and the NF-κB/MLCK signalling inhibition. ConclusionsSCL improved intestinal barrier dysfunction and alleviated CD-like colitis, possibly through modulation of Nrf2/NF-κB/MLCK signalling. In view of SCL's safety profile, there is hope that it will be useful in the clinic.

Claudin-7 is essential for the maintenance of colonic stem cell homoeostasis via the modulation of Wnt/Notch signalling

Intestinal stem cells (ISCs) play a crucial role in the continuous self-renewal and recovery of the intestinal epithelium. In previous studies, we have revealed that the specific absence of Claudin-7 (Cldn-7) in intestinal epithelial cells (IECs) can lead to the development of spontaneous colitis. However, the mechanisms by which Cldn-7 maintains homeostasis in the colonic epithelium remain unclear. Therefore, in the present study, we used IEC- and ISC-specific Cldn-7 knockout mice to investigate the regulatory effects of Cldn-7 on colonic Lgr5+ stem cells in the mediation of colonic epithelial injury and repair under physiological and inflammatory conditions. Notably, our findings reveal that Cldn-7 deletion disrupts the self-renewal and differentiation of colonic stem cells alongside the formation of colonic organoids in vitro. Additionally, these Cldn-7 knockout models exhibited heightened susceptibility to experimental colitis, limited epithelial repair and regeneration, and increased differentiation toward the secretory lineage. Mechanistically, we also established that Cldn-7 facilitates the proliferation, differentiation, and organoid formation of Lgr5+ stem cells through the maintenance of Wnt and Notch signalling pathways in the colonic epithelium. Overall, our study provides new insights into the maintenance of ISC function and colonic epithelial homoeostasis.

Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Evaluation of EGFR and COX pathway inhibition in human colon organoids of serrated polyposis and other hereditary cancer syndromes.

Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.

Disruption of epithelium integrity by inflammation-associated fibroblasts through prostaglandin signaling.

Inflammation-associated fibroblasts (IAFs) are associated with progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial cells is unknown. Here, we developed an in vitro model whereby human colon fibroblasts are induced by specific cytokines and recapitulate key features of IAFs in vivo. When cocultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid expansion and barrier disruption due to swelling and rupture of individual epithelial cells. Colonoids cocultured with IAFs also show increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated by a paracrine pathway involving prostaglandin E2 and its receptor EP4, leading to protein kinase A -dependent activation of the cystic fibrosis transmembrane conductance regulator. EP4-specific chemical inhibitors effectively prevented IAF-induced colonoid swelling and restored normal proliferation and genome stability. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a therapeutic avenue to mitigate inflammation-associated epithelial injury.

ERdj5-mediated regulation of proliferation and differentiation in colon organoids under inflammatory stress

Background: Endoplasmic reticulum (ER) stress is resolved by ERdj5, which functions as a disulfide reductase. In a previous study, we confirmed that ERdj5 was not indispensable for the formation of intestinal epithelial cells in mice. However, studies evaluating the association of ERdj5 and growth factors are limited. In the present study, we observed changes in the proliferation of colon organoids cultured from intestinal crypts obtained from wild-type (WT) and ERdj5 knockout (KO) mice.Methods: We cultured colon organoids from WT and ERdj5 KO mice, and we then assessed differentiation- and proliferation-related markers after exposure to various stimuli, such as irradiation and the Toll-like receptor (TLR) 2 ligand Pam3CSK4.Results: Over 15 days of organoid culture, ERdj5 KO colon organoids demonstrated a similar increase in leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) expression as WT colon organoids. However, in response to Pam3CSK4-induced stress, the ERdj5 KO organoids exhibited decreased Lgr5 expression and increased C/EBP homologous protein (CHOP) levels. Moreover, after irradiation-induced stress, ERdj5 downregulated epithelial-related genes, including Lgr5, Villin 1, and Prominin 1.Conclusion: Inflammation through irradiation or TLR2-induced mild or severe ER stress in both groups, whereas ERdj5 deficiency eventually led to the downregulation of Lgr5. In summary, these findings suggest that colon organoids lack the ability to mount an effective defense against various inflammatory stimuli when there is a deficiency in the ERdj5-related ER stress resolution pathway.

Abstract 4780: TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death inthe United States and screening is the most effective method to reduce mortality. Chemoprevention, which aims to prevent CRC through eradication or prevention of precursor colonic adenomas, represents an alternative strategy to address this problem. The imipridone dordaviprone (ONC201) has been shown to induce apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. More recently, we have shown that ONC201 induces TRAIL and apoptosis in a dose-dependent manner and reduces adenoma formation in the Apcmin/+ CRC mouse model. We hypothesized that ONC201 would similarly enhance human colonic adenoma cell death through both TRAIL-dependent and TRAIL-independent mechanisms. We therefore evaluated the effects of ONC201 in two adenoma-derived organoid lines: one developed from an adenoma obtained from an individual with familial adenomatous polyposis (FAP) and the second from a sessile serrated adenoma (SSA). Methods: Sensitivity of the FAP and SSA organoid lines to ONC201 was assessed using CellTiter-Glo® luminescent cell viability assay. Organoids were plated in triplicates and treated with ONC201 (concentration ranging from 0 uM to 20 uM). Results were analyzed after 72 hours of incubation. Western blotting was performed on lysates generated from organoids treated with either 1.69 uM DMSO, 1.69 uM ONC201, or 3.38 uM ONC201 for FAP and 1.23 uM DMSO, 1.23 uM ONC201, or 2.46 uM ONC201 for SSA for 0, 24, or 48 hours. Co-culture experiments were performed with the adenoma-derived organoids (dyed using blue CMAC dye) and human NK-92MI cells (dyed with green CMFDA dye) treated with either 845 nM DMSO, 845 nM ONC201, or 1.69 uM ONC201 for FAP and 615 nM DMSO, 615 nM ONC201, or 1.23 uM ONC201 for SSA. Fluorescent images following administration of red live/dead dye were performed using DAPI, FITC, and cherry red channels on ImageXpress® at 0, 24, 48, and 72hours. Results: ONC201 demonstrated antineoplastic effects in adenoma-derived FAP and SSA organoids. Half maximal inhibitory concentrations (IC50) of ONC201 in FAP and SSA organoids were 1.69 uM and 1.23 uM, respectively. As was noted in rodent studies, the mechanism of action of ONC201 appears to be mediated through modulation of multiple pathways including the TRAIL pathway (TRAIL and DR5 were both upregulated) and the integrated stress response (ATF4 was upregulated). Co-culture with NK cells revealed an increase in NK-mediated organoid cell death. The ability of ONC201 to enhance NK-mediated killing is still being evaluated. Further analysis of the effects of ONC201 on stemness markers and on T-cell-mediated organoid cell killing are currently underway and may reveal other potential biomarkers and chemopreventive strategies for ONC201. Citation Format: Alexis J. Lannigan, Jasper Chan, Maximilian Schwermann, Lanlan Zhou, Varun V. Prabhu, Michael Dame, Dean Brenner, Wafik S. El-Deiry, Alexander G. Raufi. TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4780.

Abstract 258: An organoid model for investigating Lynch syndrome tumorigenesis

Abstract Lynch syndrome (LS) is the most common inherited predisposition for developing colon cancer and is caused by germline variants in mismatch repair (MMR) genes. Still, the mechanism of tumorigenesis in these patients is unknown. An LS patient inherits one defective copy of an MMR gene. At some point in their lifetime, the remaining wild-type allele is lost in a cell rendering it entirely MMR deficient. MMR deficiency increases mutational rate and the chance of acquiring mutations in an oncogene or tumor suppressor that drive tumor initiation. However, loss of MMR in cells also leads to increased resistance to certain forms of DNA damage. Thus, our question is whether this phenotype imparts a selective advantage in colonic cells that may lead to clonal expansion of MMR-deficient cells as a first step towards tumorigenesis in LS patients. To test this, we are using stem cell-derived human colonic organoids and CRISPR gene editing to examine competition between MMR-deficient (MMRd) and MMR-proficient (MMRp) colonic cells over time. We have initially observed that MMRd stem cells are much more efficient at generating colonic organoids that grow larger and survive longer, consistent with our hypothesis of a selective advantage. To mimic the spontaneous loss of heterozygosity that occurs in LS patients’ colonic crypts, we are forming organoids from differentially labeled MMRd and MMRp stem cells. Live fluorescent imaging of individual organoids is used to quantify changes in cell population over time. Preliminary data indicate that MMRd cells can outgrow MMRp cells resulting in a shift in the ratio of green to red cells over a two-week time period while challenged with a DNA-damaging agent. These results suggest that loss of MMR function imparts an immediate selective advantage in colonic cells in response to DNA damage. Citation Format: Caroline Guild, Christopher Heinen, Samantha Nadeau. An organoid model for investigating Lynch syndrome tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 258.

Abstract 2170: Effects of folic acid supplementation on early stages of colitis-associated carcinogenesis

Abstract Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by persistent mucosal inflammation extending throughout the colon. Due to compromised intestinal surface integrity and the prolonged use of medication, individuals with UC often develop a folate deficiency, requiring folic acid (FA) supplementation. Prior findings from this group indicate that FA supplementation, at clinically relevant levels, promotes UC-associated dysplasia. Supplementation with FA resulted in an increased multiplicity of dysplasia and a significant elevation in the expression of several inflammatory genes (e.g. IL-6, Cox-2, Iκκ-β) within the colonic mucosa of mice with UC. Genome-wide expression profiling of normal and dysplastic colonic epithelial cells from mice treated with different doses of FA revealed differentially expressed genes were enriched for members of the ERK and NF-κB pathways. Subsequent in vitro analyses, utilizing isogenic RKO colon adenocarcinoma cells exposed to various doses of FA, demonstrated the downregulation of ERK and p-ERK in wild type (WT) p53+/+ cells, while an increase in expression was observed in p53−/− cells at the protein level. The goal of this study is to investigate the role of the MAPK/ERK pathway in the development of UC-associated neoplasms. An in vitro model that faithfully reproduces the inflamed microenvironment in the context of WT vs. mutant p53, the putative gatekeeper of UC-associated carcinogenesis, is needed to enhance our understanding of early changes in the colon that are induced by FA and drive tumor formation. Colon organoids are being generated by isolating crypts from WT p53+/+ and mutant p53+/515A mice. These organoids are then co-cultured with mouse macrophages (RAW264.7 cells) in inserts to stimulate crosstalk between the macrophages and colonic epithelial cells, as in the inflamed colon. Analyses demonstrate that the macrophages express pivotal inflammatory cytokines characteristic of UC, including IL-1β, IL-10, TNF-α, and IL-6, irrespective of the presence or absence of LPS stimulation. Co-cultured organoids and macrophages will be exposed to different concentrations of FA for various lengths of time, and the impact of treatment on the levels of ERK, p-ERK, and p65 will be assessed. The ability of p-ERK to mediate the transcriptional activation of NF-κB and its downstream effector IL-6 will also be evaluated. Subsequent investigations employing this innovative experimental model will reveal the mechanisms by which FA supplementation promotes the progression of UC-associated dysplasia. Given the potential risks associated with FA supplementation in individuals with UC, there is an urgent need to translate these results to a clinical setting. (Supported by CA262551 and the Timothy and Aurora Hughes Cancer Research Fund). Citation Format: Ariane Rocha Bartolomeu, Wen-Chi Chang, Lisa Vanderveer, Kristen N. Harvey, Mitchell Cheung, Harry S. Cooper, Margie Lee Clapper. Effects of folic acid supplementation on early stages of colitis-associated carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2170.

Abstract 6844: Discovery of a novel sialic acid O-acetyltransferase in colon tissue

Abstract Sialic acids have critical functions relating to host-pathogenic interactions and the immune microenvironment. Subsequent post-translational O-acetyl modifications add an extra layer of control by modulating many of these interactions in both normal and transformed colorectal cells. The processes through which this modification is added to sialic acids are not completely understood. To date only one gene, CASD1, has been identified to encode a sialic acid O-acetyltransferase in human cells. We used whole-genome sequencing on normal human colon tissue to identify a haplotype strongly associated with colorectal sialic acid O-acetylation, as determined by histopathological mPAS staining. Only SNP’s of a single candidate gene within the haplotype correlated perfectly in a 91-sample validation set. CRISPR-Cas9 editing of this gene in 2D cell line and 3D normal colon organoid models yielded changes to modified sialic acid levels. These findings suggest that this candidate gene functions as a novel sialic acid O-acetyltransferase in colorectal tissue and could have implications as a diagnostic tool for patients with inflammatory diseases of the bowel or as an indicator for therapeutic targets in cancer. Citation Format: Bum Seok Lee, Ashley Cook, Maged Zeineldin, Surojit Sur, Laura Dobbyn, Maria Popoli, Sana Khalili, Bert Vogelstein, Nickolas Papadopoulos, Shibin Zhou, Chetan Bettegowda, Phillip Buckhaults, Tatianna Larman, Kathy Gabrielson, Kenneth W. Kinzler, Nicolas Wyhs. Discovery of a novel sialic acid O-acetyltransferase in colon tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6844.

Abstract 7552: Inhibition of histone methyltransferase EZH2 for immune-interception of colorectal cancer in Lynch syndrome

Abstract Introduction: Colorectal pre-cancers in Lynch Syndrome (LS) display a distinct immune profile thus presenting a unique opportunity to develop novel immune-interception approaches to halt carcinogenesis. Epigenetic modulation by EZH2 of immune-related genes has been implicated in many cancers including colorectal but it has yet to be explored in LS carcinogenesis. The main objective of this study is to test the effect of EZH2 inhibition on immune cell profiling, colorectal carcinogenesis, gene expression, and histone methylation, using ex vivo organoids and a LS mouse model. Methods: A mouse model of LS colorectal carcinogenesis (Villin-Cre;Msh2fl/fl;TgfβRII-KI; herein referred to as VCMsh2THu) and ex vivo colonic organoids were utilized to assess the effects of the EZH2 inhibitor GSK503. Mice were treated for 9-weeks with GSK503 with downstream studies exploring polyp multiplicity, changes to the immune landscape, and epigenetic analyses to derive mechanistic insights into EZH2 regulation in LS. Results: GSK503 significantly altered transcriptomic changes in VCMsh2THu organoids with tissue-specific tropism for dysplastic organoids as compared to organoids prepared from adjacent normal mucosa. GSK503 significantly increased CD4+and CD8+ T cells in splenocytes and colonic mucosa of treated mice compared to controls in vitro and in vivo. A preventive-dose (200μg/kg) of GSK503 for 9 weeks significantly reduced adenoma multiplicity in the large intestine of VCMsh2THu mice with significant changes in the immune landscape of treated mice compared to controls. The data suggests GSK503 significantly enriched both total and activated cytotoxic T-lymphocytes CD8+/CD137+), stromal macrophages (CD68+), and activated helper T-lymphocytes (CD4+/CD134+) in the large intestine of VCMsh2THu mice. Transcriptome and molecular analyses using IHC, Western blots, and computational tools showed activation of immune and apoptotic markers with concomitant reduction of H3K27 methylation levels in colonic crypts. ChIPseq analyses revealed decreased levels of H3K27me3 and H3K4me1 (enhancer), a slight increased levels of H3K27ac (enhancer) marks with no changes in levels of H3K4me3 in treated mice, indicating that EZH2 inhibition yields global reprograming of poised promoters and active enhancers in the LS mouse genome. Conclusions: Collectively, our findings reveal that GSK503 has a promising potential as an immuno-interception modality establishing the framework for launching phase I clinical trials with EZH2 inhibitors for LS carriers. Citation Format: Fahriye Duzagac, Charles M. Bowen, Laura Reyes-Uribe, Mahira Zaheer, Jacklyn Thompson, Nan Deng, Melissa Taggart, Abhinav Jain, Winfried Edelmann, Krishna M. Sinha, Eduardo Vilar. Inhibition of histone methyltransferase EZH2 for immune-interception of colorectal cancer in Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7552.

Abstract 2796: Colibactin mutation signatures are associated with younger age of onset in colorectal cancer

Abstract Escherichia coli (E. coli) strains carrying the polyketide synthase (pks) island produce the genotoxin colibactin associated with pathogenesis of colorectal cancer (CRC). Colibactin binds directly to DNA, causing alkylating damage leading to double stranded DNA breaks. Colibactin mutagenesis results in mutations in hexameric A-T rich DNA motifs that can be identified by whole genome sequencing (WGS) as mutation signatures SBS28 and SBS88 (SBS-pks). Since WGS is not routinely used clinically, we developed an approach to specifically identify SBS-pks using a clinical targeted exon capture assay, MSK-IMPACT. In an institutional pan-cancer clinical cohort of 78,905 tumors we identified 15,967 samples with > 20 mutations detected by MSK-IMPACT. We identified 150/1,884 samples with microsatellite stable CRC with >10% mutations attributable to SBS-pks. Examining clinicopathologic and genomic features of SBS-pks+ compared to SBS-pks- CRC tumors, we found that SBS-pks+ signatures are significantly enriched in younger age of onset compared to SBS-pks- CRC patients (median age 55 vs median age 59, p= 0.0028). SBS-pks+ CRC exhibit similar driver mutations and tumor mutational burden (TMB) compared to SBS-pks- CRC but are associated with increased fraction of genome altered (FGA). To mechanistically investigate colibactin-associated damage we developed a novel long-term human colon organoid-microbe co-culture model system that takes advantage of reversal of polarity in suspension culture. WGS of human CRC organoids co-cultured for three months with pks+ and delta-pks E. coli NC101 confirmed induction of pks+ specific SBS-pks signatures, validating the model. Leveraging this model and a biobank of normal colon organoids from donors aged </> 50 years, we are investigating the molecular mechanisms underlying the association of SBS-pks with younger age of onset CRC. Citation Format: Stefanie Gerstberger, Melissa Lumish, Saskia Hartner, Farheen Shah, Seongmin Choi, Anisha Luthra, Qingwen Jiang, Hyung Jun Woo, Ahmed Mahmoud, Henry Walch, Simran Asawa, Mark Donoghue, Andrea Cercek, Rona Yaeger, Andrew McPherson, Francisco Sanchez-Vega, Karuna Ganesh. Colibactin mutation signatures are associated with younger age of onset in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2796.

Abstract 7305: Chemoprevention of colorectal cancer: Role of trace minerals in conjunction with calcium

Abstract Background: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. CRC usually develops from adenomas (CRC precursors) after a long latency period. Prevention is crucial in reducing CRC incidence and progression to cancer. Among several prevention options, calcium has been a widely considered chemo-preventative agent. Aims: Calcium plays a vital role in the regulation of epithelial cell differentiation, which is essential for epithelial growth control. Epidemiological studies have demonstrated that adequate calcium intake is crucial for protection against colonic polyp formation and colorectal cancer. However, recent interventional trials with calcium have had only modest and mixed results, and the question is how to improve efficacy. One potential strategy is to use calcium in combination with additional minerals and trace elements. Results: Our preclinical studies employing monolayer cultures with CRC cell lines and colon organoids cultures from colon polyps have shown that a calcium-rich mineral combination derived from fossilized red algae (Aquamin) is more effective than calcium alone in inducing differentiation, suppressing growth and upregulation of calcium-sensing receptor. When colonic adenomas in culture were subjected to differential proteomic analysis, Aquamin upregulated several proteins involved in proliferation suppression, DNA mismatch repair and inducing differentiation, and apoptosis. Two long-term mouse studies have demonstrated that Aquamin is more effective in suppressing polyp & CRC incidence compared to calcium alone. Based on these findings, we conducted a 90-day FDA-approved trial (a pilot study with ten subjects per arm) comparing the two interventions in human subjects at risk for CRC. Results showed that Aquamin treatment reduced proliferation and increased cell differentiation observed by immunohistochemistry. Proteomic analysis also revealed that the minerals upregulated several proteins related to growth suppression, inducing differentiation and those contributing to cell-cell and cell-matrix adhesion functions and downregulated proteins involved in proliferation and nucleic acid metabolism. Conclusion: These results provide convincing evidence that a multi-mineral combination derived from red algae can provide benefits in preventing CRC that are not seen with calcium alone. Further studies, such as a polyp-prevention trial in human subjects, are warranted to advance these CRC prevention efforts. Citation Format: Isabelle Harber, Shannon McClintock, James Varani, Muhammad N. Aslam. Chemoprevention of colorectal cancer: Role of trace minerals in conjunction with calcium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7305.

Abstract 1168: Anti-CSF1R antibody monotherapy inhibits minimal residual disease in a novel immunocompetent murine colorectal cancer metastasis model

Abstract Background: The high mortality rate of colorectal cancer (CRC) is primarily due to metastatic disease. Microscopic minimal residual disease (MRD) detected in patients by circulating tumor DNA (ctDNA) may remain radiographically undetectable and persist following therapeutic intervention, resulting in recurrence. Preclinical models are urgently needed to elucidate the mechanisms whereby MRD escapes anti-tumor immune surveillance. We have developed and characterized a preclinical liver MRD model that recapitulates immune response, with the goal to discover novel strategies to improve outcomes for patients with ctDNA+ MRD. Methods: We generated genetically engineered mouse colonic organoids CDX2 CRE; Rosa26 LSL-CAS9-GFP with APC and TP53 mutation and performed intrasplenic injection in syngeneic C57BL/6 mice to recapitulate microsatellite stable CRC metastasis to the liver. Mice were monitored for radiographically detectable disease using MRI. Tumors were resected at various timepoints, with comparison between metastases at various sizes of development (dichotomized as micro- and macro-metastases). We performed multiplex immunofluorescence staining, single cell RNA seq, and spatial transcriptomic analysis to delineate the immune compartments of micro- and macro-metastasis in untreated mice. After identifying colony stimulating factor -1 receptor (CSF1R) as a target, we performed a vehicle controlled anti-CSF1R monotherapy study for 4 weeks. Treatment began at either 7 days before metastasis is radiographically detectable to mimic treatment of MRD, or 21 days post tumor engraftment. Results: Immune exclusion was confirmed through intratumoral CD45+ leukocyte densities, with transition to an immune excluded phenotype occurring above 0.5mm in size (defined as macro-metastases). In our preclinical model, immunosuppressive populations of macrophages (IBA1+CD163+) dominated in the micro-metastases. This observation was confirmed in 13 patients with paired micro- and macro-metastases to the liver (p=0.0065). Strikingly, anti-CSF1R treatment in the murine model eradicated IBA1+CD163+ macrophages (p<0.0001) and induced complete remission of liver micro-metastasis (0/7 mice with residual disease), in contrast to limited efficacy for macro-metastases (5/5 mice with progressed radiographically disease). Additionally, a long-term study confirmed 100% survival (5/5 mice) and no evidence of disease via MRI at 6 months upon anti-CSF1R treatment compared to vehicle (0/5 mice). Conclusions: We established a reproducible preclinical model of MRD with similar immunopathological features of MRD in patients. Our data further identified infiltration of immunosuppressive macrophage populations as an early determinant of MRD progression and indicated CSF1R as a potential new clinical therapeutic target for ctDNA+-defined MRD patients. Citation Format: Alaa M. Mohamed, Kyung Serk Cho, Melinda Soeung, Amanda Anderson, Jumanah Alshenaifi, Ganiraju Manyam, Oscar Villareal, Jennifer Davis, Will Norton, Sisi Gao, Christopher Bristow, Federica Carbone, Stefania Napolitano, Oluwadara Coker, Isha Khanduri, Justin Huang, Dipen M. Maru, Mauro Di Pilato, Ryan Sun, Linghua Wang, David G. Menter, Giannicola Genovese, Giulio Draetta, Natalie Fowlkes, Scott Kopetz. Anti-CSF1R antibody monotherapy inhibits minimal residual disease in a novel immunocompetent murine colorectal cancer metastasis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1168.