Mouse Colonic Organoids Research Articles

The glucocorticoid receptor potentiates aldosterone-induced transcription by the mineralocorticoid receptor

The glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) have distinct, yet overlapping physiological and pathophysiological functions. There are indications that both receptors interact functionally and physically, but the precise role of this interdependence is poorly understood. Here, we analyzed the impact of GR coexpression on MR genome-wide transcriptional responses and chromatin binding upon activation by aldosterone and glucocorticoids, both physiological ligands of this receptor. Transcriptional responses of MR in the absence of GR result in fewer regulated genes. In contrast, coexpression of GR potentiates MR-mediated transcription, particularly in response to aldosterone, both in cell lines and in the more physiologically relevant model of mouse colon organoids. MR chromatin binding is altered by GR coexpression in a locus- and ligand-specific way. Single-molecule tracking of MR suggests that the presence of GR contributes to productive binding of MR/aldosterone complexes to chromatin. Together, our data indicate that coexpression of GR potentiates aldosterone-mediated MR transcriptional activity, even in the absence of glucocorticoids.

Molecular mechanism of BMP signal control by Twisted gastrulation

Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.

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.

Tissue-location-specific transcription programs drive tumor dependencies in colon cancer

Cancers of the same tissue-type but in anatomically distinct locations exhibit different molecular dependencies for tumorigenesis. Proximal and distal colon cancers exemplify such characteristics, with BRAFV600E predominantly occurring in proximal colon cancers along with increased DNA methylation phenotype. Using mouse colon organoids, here we show that proximal and distal colon stem cells have distinct transcriptional programs that regulate stemness and differentiation. We identify that the homeobox transcription factor, CDX2, which is silenced by DNA methylation in proximal colon cancers, is a key mediator of the differential transcriptional programs. Cdx2-mediated proximal colon-specific transcriptional program concurrently is tumor suppressive, and Cdx2 loss sufficiently creates permissive state for BRAFV600E-driven transformation. Human proximal colon cancers with CDX2 downregulation showed similar transcriptional program as in mouse proximal organoids with Cdx2 loss. Developmental transcription factors, such as CDX2, are thus critical in maintaining tissue-location specific transcriptional programs that create tissue-type origin specific dependencies for tumor development.

PVA enema ameliorates DSS-induced acute colitis in mice

BackgroundUlcerative colitis (UC) represents a clinically challenging condition characterized by persistent damage to the colonic epithelial mucosa as the principal pathological feature. Polyvinyl alcohol (PVA) solution, primarily composed of glue, is a biodegradable polymer material that has found utility in the medical field. This research endeavors to investigate the therapeutic potential of PVA water solution in ameliorating UC in mice.MethodsUC was induced in 48 C57BL/6 mice by administering 2.5% DSS in their diet for 6 days. Mice were treated with different concentrations of PVA (0.1 mg/ml PVA, 0.3 mg/ml PVA, 1 mg/ml PVA, 3 mg/ml PVA, 10 mg/ml PVA) enemas (n = 6). Disease Activity Index (DAI) and histologic score were evaluated for inflammation degree. Furthermore, mouse colon organoids were cultured, which were used to assess the effects of PVA on expansion in vitro.ResultsPVA aqueous solutions (1 mg/ml and 3 mg/ml) were able to alleviate the DAI in mice. By DAY 6, there was a significant 3/5-fold decrease in DAI within the 1 mg/ml PVA group (p = 0.02). Histopathology scores demonstrated improvements, while the levels of inflammatory factors in the intestinal mucosal tissue were reduced. Additionally, it was confirmed that PVA could promote the expansion of colonic organoids in vitro.ConclusionsIn summary, our investigation has yielded findings indicating that PVA holds the potential to ameliorate symptoms associated with colitis in murine subjects afflicted by DSS-induced colitis, primarily through its facilitation of intestinal stem cell expansion. This study might provide a new candidate for the clinical treatment of ulcerative colitis.

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.

Sotetsuflavone ameliorates Crohn's disease-like colitis by inhibiting M1 macrophage-induced intestinal barrier damage via JNK and MAPK signalling

ObjectivesIntestinal inflammation and intestinal barrier dysfunction are two important pathological changes in Crohn's disease (CD). Sotetsuflavone (SF) is a natural monomeric herbal compound with anti-inflammatory and cytoprotective effects that is mostly nontoxic. The effect of SF on CD-like spontaneous colitis was investigated in this study. MethodsIl-10−/− mice were used as a CD model and were administered different doses of SF. Lipopolysaccharide (LPS) plus IFN-γ-induced macrophages (RAW264.7) and a coculture system (RAW264.7 and organoids) were used in vitro. The protective effects of SF against CD-like colitis and macrophage differentiation and the mechanisms were evaluated. ResultsSF treatment markedly improved spontaneous colitis in the CD model, as shown by the following evidence: reductions in the DAI, macroscopic scores (3.63 ± 1.30), colonic tissue inflammatory scores (2 ± 0.76) and proinflammatory factor levels and the attenuation of colon shortening (8 ± 0.93 cm) and weight loss (1.75 ± 1.83 g). Decreased intestinal permeability and intestinal bacterial translocation rates provided evidence of the protective effect of SF on intestinal barrier function. We also found that SF suppressed M1 macrophage-induced inflammatory responses. In the coculture system of mouse colonic organoids and RAW264.7 cells, SF significantly ameliorated M1 macrophage-induced intestinal epithelial damage. In addition, SF inhibited JNK and MAPK (p38) signalling in both Il-10−/− mice and LPS plus IFN-γ-induced macrophages (RAW264.7). ConclusionsThe protective effects of SF against CD-like colitis may be achieved partially by inhibiting M1 macrophage-induced intestinal barrier damage via JNK and p38 signalling. SF may have therapeutic potential for treating CD, especially considering its safety.

Ruscogenins Improve CD-Like Enteritis by Inhibiting Apoptosis of Intestinal Epithelial Cells and Activating Nrf2/NQO1 Pathway.

Interaction of intestinal barrier dysfunction and intestinal inflammation promotes the progression of Crohn's disease (CD). A more recent study has suggested that ruscogenins (RUS) can exert anti-inflammatory effects through activation of the Nrf2/NQO1 pathway. The current study is aimed at determining the functionalization of RUS on CD-like colitis. Wild-type (WT) mice induced with trinitrobenzene sulfonic acid (TNBS) exhibit a significant inflammation in their colon and are hence widely used for CD models. In the current study, the mice were treated with the Nrf-2 antagonist (ML385) or ruscogenin (RUS) whereas normal WT mice were kept as the negative control. Comparative analysis was then performed on the inflammation and barrier function of the colons. In vitro analysis of mouse colonic organoid systems revealed the influence of RUS on LPS-induced apoptosis, cytokine, and chemokine expressions in the intestinal epithelium. It was found that RUS ameliorates murine colitis through activation of the Nrf2/NQO1 pathway which was presented as a decrease in inflammation score and downregulated levels of cytokine and chemokine synthesis, as well as increased intestinal permeability. Further, it was noted that RUS alleviated LPS-induced apoptosis in the intestinal epithelium cells through upregulation of the Nrf2/NQO1 signaling pathway in the mouse colonic organoids. In addition, ruscogenin (RUS) attenuated the levels of Bax and C-caspase-3 through activation of the Nrf2/HO1 signaling pathway both in vivo and in vitro. Therefore, it was evident that RUS can be applied as a potential alternative therapeutic agent in CD based on its protective effects on the barrier function and anti-inflammatory activity.

A12 STEPWISE COORDINATION OF COLONIC NEUTROPHILS AND INNATE LYMPHOID CELLS IN THE ONSET AND RESOLUTION OF CLOSTRIDIOIDES DIFFICILE TOXIN-INDUCED INJURY

BackgroundWhile our understanding and use of treatments for Clostridioides difficile infection (CDI) has improved, initial CDI still carries significant morbidity and mortality owing to heterogeneity in host immune responses. Further, host immunity is a critical modulator of fecal microbiota transplantation (FMT) success in CDI. Thus, understanding the host immune response during CDI is essential.AimsTo assess the cellular immune responses that trigger the onset and resolution of injury and inflammation in CDI.MethodsColonic injury and inflammation triggered by CDI was modelled in mice using intrarectal installation of C. difficile toxins A and B (TcdA/B). Colonic tissue was collected at various timepoints following TcdA/B exposure to assess gene expression (qPCR), cytokine production (ELISA) and immune cell responses (flow cytometry). Knockout mice and neutralizing antibodies were used to deplete cytokines or cells.ResultsExaminion of colonic gene expression at different times following TcdA/B exposure found a dominant transcriptional signature related to neutrophil adhesion and diapedesis. In addition to the typical neutrophil chemokines Cxcl1 and Cxcl2, TcdA/B exposure also increased expression of neutrophil effector genes including Elane (neutrophil elastase). Neutrophil influx in response to TcdA/B was a critical driver of intestinal injury as antibody-mediated depletion of neutrophils lead to significantly less damage in the colon following TcdA/B exposure. Along with neutrophil influx, there were high levels of antimicrobial gene expression in the colon after TcdA/B exposure including RegIIIγ, S100a8, and Socs3, all genes regulated by IL-22. Upon further investigation, IL-22 was a significant mediator in the host response to TcdA/B exposure as it was upregulated >150-fold in the colon and originated from type 3 innate lymphoid cells (ILC3). Further, TcdA/B exposure in IL-22-/- mice lead to significantly more colonic damage compared to wildtype (WT) mice. Subsequent screening of previously published RNAseq data from IL-22-treated mouse colonic organoids identified various upregulated proteins involved in immune regulation, including the gene Slpi that encodes a protein (secretory leukocyte peptidase inhibitor) that inhibits leukocyte proteases, including neutrophil elastase. While TcdA/B challenge robustly induced the expression of Slpi in the colon of WT mice, IL-22-/- mice failed to express increased levels of Slpi and had greater levels of neutrophil elastase activity in the colon.ConclusionsTogether these data suggest a stepwise immune response to TcdA/B where ILC3 produce IL-22 to induce epithelial release of SLPI that attenuates the damaging effects of early neutrophil responses. Strategies to upregulate IL-22 may help control damage triggered by CDI and promote resolution of injury.Funding AgenciesLloyd Sutherland Chair in GI Research, Canadian Research Chair

Limosilactobacillus reuteri ATCC 6475 metabolites upregulate the serotonin transporter in the intestinal epithelium.

The serotonin transporter (SERT) readily takes up serotonin (5-HT), thereby regulating the availability of 5-HT within the intestine. In the absence of SERT, 5-HT remains in the interstitial space and has the potential to aberrantly activate the many 5-HT receptors distributed on the epithelium, immune cells and enteric neurons. Perturbation of SERT is common in many gastrointestinal disorders as well as mouse models of colitis. Select commensal microbes regulate intestinal SERT levels, but the mechanism of this regulation is poorly understood. Additionally, ethanol upregulates SERT in the brain and dendritic cells, but its effects in the intestine have never been examined. We report that the intestinal commensal microbe Limosilactobacillus (previously classified as Lactobacillus) reuteri ATCC PTA 6475 secretes 83.4 mM ethanol. Consistent with the activity of L. reuteri alcohol dehydrogenases, we found that L. reuteri tolerated various levels of ethanol. Application of L. reuteri conditioned media or exogenous ethanol to human colonic T84 cells was found to upregulate SERT at the level of mRNA. A 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+) uptake assay confirmed the functional activity of SERT. These findings were mirrored in mouse colonic organoids, where L. reuteri metabolites and ethanol were found to upregulate SERT at the apical membrane. Finally, in a trinitrobenzene sulphonic acid model of acute colitis, we observed that mice treated with L. reuteri maintained SERT at the colon membrane compared with mice receiving phosphate buffered saline vehicle control. These data suggest that L. reuteri metabolites, including ethanol, can upregulate SERT and may be beneficial for maintaining intestinal homeostasis with respect to serotonin signalling.

P015 Differential expression in colitis-associated cancer compared to Crohn′s disease and ulcerative colitis

Abstract Background Crohn’s disease (CD) and ulcerative colitis (UC) are autoimmune-mediated conditions of chronic inflammation affecting the intestinal mucosa, that pose a lifelong risk to patients to develop a colitis-associated carcinoma (CAC). Much of current knowledge on mechanisms in CAC development result from murine CAC models, while studies on human CAC carcinogenesis are scarce. Thus, in our present study, we aimed to contribute to the understanding of colitis-associated carcinogenesis by comparative analysis of gene expression in human samples and further validation of these findings in colon organoids. Methods RNA isolation was done from microdissected surgical colon specimen from 60 patients that suffered from either UC, CD, UC-CAC, CD-CAC or inflammation-free healthy controls (10 patients per group). Nanostring nCounterTM technology with a gene panel comprising >630 genes was performed to examine genes focusing on mucosal immunology, epithelial barrier/polarity. nSolverTM data analysis software was used for primary data analysis and statistics. Patients were grouped using expression patterns (by heat maps), differential expression and pathway analysis. For organoid cultures, crypts were isolated from mouse colon and cultured using the R-Spondin method. Organoids were subjected to osteopontin treatment. Consecutively, RNA was isolated. This was followed by gene expression analysis of core EMT (epithelial to mesenchymal transition) transcription factors (TFs) focusing on Twist1, Snai1, Snai2 by RT-PCR. Results Heat maps generated from the expression data revealed close-to-optimal grouping recapitulating the clinical subgroups. Differential expression comparing CD with CD-CAC and UC with UC-CAC identified 203 and 271 differentially expressed genes, respectively. Genes most significantly upregulated included SPP1/Osteopontin (OPN), GRHL2 and EMT signature gene as fibronectin1 and ZEB1, generating the hypothesis of OPN driving EMT and thereby inducing inflammation-associated carcinogenesis. Furthermore, mouse colon organoids treated with OPN (50ng/µl) revealed an increased expression by 3fold of core the EMT TFs Twist1, Snai1, Snai2. Conclusion We provide a comprehensive quantitative gene expression analysis for CAC with comparison of gene expression in CAC to the respective underlying IBD, CD and UC. Identified upregulated or downregulated genes allow to allocate signal transduction pathways important for CAC carcinogenesis. OPN as the most upregulated gene in CAC in our gene panel might be crucial for regulating EMT in CAC carcinogenesis. Thus, our findings uncover the role of OPN in CAC carcinogenesis.

Interleukin-37 regulates innate immune signaling in human and mouse colonic organoids

Intestinal epithelial cells (IEC) reside in close proximity to the gut microbiota and are hypo-responsive to bacterial products, likely to prevent maladaptive inflammatory responses. This is in part due to their strong expression of Single Ig IL-1 related receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and toll-like receptor signaling. IL-37 is an anti-inflammatory cytokine that inhibits innate signaling in diverse cells by signaling through SIGIRR. Despite the strong expression of SIGIRR by IEC, few studies have examined whether IL-37 can suppress their innate immune signaling. We characterized innate immune responses of human and murine colonoids to bacteria (FliC, LPS) and host (IL-1β) products and the role of IL-37/SIGIRR in regulating these responses. We demonstrated that human colonoids responded only to FliC, but not to LPS or IL-1β. While colonoids derived from different donors displayed significant inter-individual variability in the magnitude of their innate responses to FliC stimulation, all colonoids released a variety of chemokines. Interestingly, IL-37 attenuated these responses through inhibition of p38 and NFκB signaling pathways. We determined that this suppression by IL-37 was SIGIRR dependent, in murine organoids. Along with species-specific differences in IEC innate responses, we show that IL-37 can promote IEC hypo-responsiveness by suppressing inflammatory signaling.

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids

Background & AimsDiligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids.MethodsMouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors.ResultsIn contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures.ConclusionOur study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

Immunomodulation of dendritic cells by Lactobacillus reuteri surface components and metabolites.

BackgroundLactic acid bacteria are commensal members of the gut microbiota and are postulated to promote host health. Secreted factors and cell surface components from Lactobacillus species have been shown to modulate the host immune system. However, the precise role of L. reuteri secreted factors and surface proteins in influencing dendritic cells (DCs) remains uncharacterized.HypothesisWe hypothesize that L. reuteri secreted factors will promote DC maturation, skewing cells toward an anti‐inflammatory phenotype. In acute colitis, we speculate that L. reuteri promotes IL‐10 and dampens pro‐inflammatory cytokine production, thereby improving colitis.Methods & ResultsMouse bone marrow‐derived DCs were differentiated into immature dendritic cells (iDCs) via IL‐4 and GM‐CSF stimulation. iDCs exposed to L. reuteri secreted factors or UV‐irradiated bacteria exhibited greater expression of DC maturation markers CD83 and CD86 by flow cytometry. Additionally, L. reuteri stimulated DCs exhibited phenotypic maturation as denoted by cytokine production, including anti‐inflammatory IL‐10. Using mouse colonic organoids, we found that the microinjection of L. reuteri secreted metabolites and UV‐irradiated bacteria was able to promote IL‐10 production by DCs, indicating potential epithelial‐immune cross‐talk. In a TNBS‐model of acute colitis, L. reuteri administration significantly improved histological scoring, colonic cytokine mRNA, serum cytokines, and bolstered IL‐10 production.ConclusionsOverall these data demonstrate that both L. reuteri secreted factors and its bacterial components are able to promote DC maturation. This work points to the specific role of L. reuteri in modulating intestinal DCs.New & Noteworthy Lactobacillus reuteri colonizes the mammalian gastrointestinal tract and exerts beneficial effects on host health. However, the mechanisms behind these effects have not been fully explored. In this article, we identified that L. reuteri ATTC PTA 6475 metabolites and surface components promote dendritic cell maturation and IL‐10 production. In acute colitis, we also demonstrate that L. reuteri can promote IL‐10 and suppress inflammation. These findings may represent a crucial mechanism for maintaining intestinal immune homeostasis.

AdipoRon Attenuates Wnt Signaling by Reducing Cholesterol-Dependent Plasma Membrane Rigidity

The increasing prevalence of adult and adolescent obesity and its associated risk of colorectal cancer reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in obese individuals. Adiponectin is an adipose tissue-derived adipokine, whose levels are reduced during obesity. Both epidemiological and preclinical data indicate that adiponectin suppresses colon tumorigenesis. We have previously demonstrated that both adiponectin and AdipoRon, a small-molecule adiponectin receptor agonist, suppress colon cancer risk in part by reducing the number of Lgr5+ stem cells in mouse colonic organoids. However, the mechanism by which the adiponectin signaling pathway attenuates colon cancer risk remains to be addressed. Here, we have hypothesized that adiponectin signaling supports colonic stem cell maintenance through modulation of the biophysical properties of the plasma membrane (PM). Specifically, we investigated the effects of adiponectin receptor activation by AdipoRon on the biophysical perturbations linked to the attenuation of Wnt-driven signaling and cell proliferation as determined by LEF luciferase reporter assay and colonic organoid proliferation, respectively. Using physicochemical sensitive dyes, Di-4-ANEPPDHQ and C-laurdan, we demonstrated that AdipoRon decreased the rigidity of the colonic cell PM. The decrease in membrane rigidity was associated with a reduction in PM free cholesterol levels and the intracellular accumulation of free cholesterol in lysosomes. These results suggest that adiponectin signaling plays a role in modulating cellular cholesterol homeostasis, PM biophysical properties, and Wnt-driven signaling. These findings are noteworthy because they may in part explain how obesity drives colon cancer progression.

Abstract 2601: HMGA1 is induced by procarcinogenic bacteria within the microbiome where it drives expansion in the colon stem cell pool and tumorigenesis

Abstract Cancer cells undergo chromatin remodeling and epigenetic reprogramming to hijack stem cell transcriptional networks and drive tumorigenesis, although the molecular underpinnings remain unclear. High Mobility Group A (HMGA1) chromatin remodeling proteins are architectural transcription factors that bind AT-rich sequences where they “open” chromatin and recruit transcriptional complexes to modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis, but silenced postnatally in differentiated tissues. HMGA1 is re-expressed in aggressive cancers where high levels portend adverse clinical outcomes. In colorectal cancer, HMGA1 is among the genes most overexpressed compared to normal colon epithelium. We previously found that HMGA1 induces genes involved in EMT and drives tumor progression in colon cancer models. Hmga1 transgenic mice develop aberrant proliferation and polyposis in the small intestine and colon. In intestinal stem cells, Hmga1 amplifies Wnt signals to enhance self-renewal. Surprisingly, Hmga1 also induces Sox-9 to drive Paneth cell differentiation and “build” a stem cell niche. To determine how HMGA1 functions in the colon, we examined mouse models with varied levels of Hmga1. Hmga1 overexpression in colon stem cells leads to expansion of the colon stem cell pool. Both goblet cells and colonic mucous are increased. In DSS models of inflammatory bowel disease, Hmga1 enhances repair and tissue regeneration, while Hmga1 overexpression promotes aberrant proliferation and polyposis with advancing age. To determine how HMGA1 is induced during carcinogenesis, we examined mice harboring a heterozygous Apc mutation (Min+/-) following colonization with the human symbiotic bacteria, enterotoxigenic Bacterioides fragilis (ETBF). ETBF triggers increased Wnt signaling by inducing E-cadherin cleavage with release of membrane bound β-catenin, leading to enhanced distal colon tumorigenesis in mice. Further, ETBF colonization in humans is linked to colon cancer. ETBF colonization in mice induces Hmga1 and increases stem cell self-renewal in preliminary studies. Inflammatory cytokine genes are also induced. Bacteroides fragilis toxin (BFT) induces Hmga1 in wildtype and Min+/- mouse colon organoids. HMGA1 also increases modestly in human HT29/C1 cells after exposure to BFT. Studies are underway to identify transcriptional networks and epigenetic alterations governed by HMGA1 in this setting. This work not only provides new insights into the role of HMGA1 in colon epithelial homeostasis by maintaining both the stem cell pool and promoting regeneration following injury, but also suggests that HMGA1 can be aberrantly induced by signals from the microbiome to promote tumorigenesis. Our results also highlight the interactions between the microbiome and HMGA1 as a potential therapeutic opportunity in colon carcinogenesis. Citation Format: Linda M S Resar, Lingling Xian, Jawara Allen, Shaoguang Wu, Lionel Chia, Cynthia Sears. HMGA1 is induced by procarcinogenic bacteria within the microbiome where it drives expansion in the colon stem cell pool and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2601.

Abstract 5292: Cell-autonomous suppression of colon carcinogenesis by type-2 cGMP-dependent protein kinase

Abstract A growing body of evidence demonstrates that cGMP signaling has anti-tumor effects in the colon, and could harbor novel targets for colon cancer prevention. The tumor suppressive components that function downstream of cGMP remain largely understudied. In the present work we have characterized the expression of cGMP-dependent protein kinases (PKG1, PKG2) in normal and cancerous tissue from humans. PKG1 was highly expressed in both normal and tumor tissue, where it localized to supportive cells in the lamina propria and stroma (respectively). In contrast, PKG2 expression was notably decreased in tumors compared to matched normal tissue, where it was identified in the epithelium by IHC. Neither PKG isoform was detected at the RNA or protein level in colon cancer cell lines. To interrogate a potential tumor-suppressor role of PKG2 in the colon epithelium we bred PKG2 knockout (KO) mice into a C57BL/6J background. These animals exhibited increased intestinal turnover as reflected by crypt hyperplasia (Ki67) and elevated luminal apoptosis (cleaved caspase 3). When subjected to AOM/DSS treatment, the PKG2 KO mice developed almost twice the number of polyps per mouse as wild type siblings (1.75-fold, p=0.0037). Mouse colon organoids were used to confirm that PKG2 was the only isoform expressed in the epithelium, where it was observed in both proliferating and differentiating epithelial compartments. Colon organoids derived from PKG2 KO mice exhibited more rapid proliferation, and a reduced ability to differentiate compared to wild type controls. Taken together our results show that PKG2 is the central target of cGMP in the colon epithelium, where it suppresses carcinogenesis by controlling proliferation in a cell autonomous manner. Citation Format: Bianca N. Islam, Yali Hou, Sarah K. Sharman, Darren D. Browning. Cell-autonomous suppression of colon carcinogenesis by type-2 cGMP-dependent protein kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5292.

Development of Collagen-Based 3D Matrix for Gastrointestinal Tract-Derived Organoid Culture.

Organoid is a cell organization grown in a three-dimensional (3D) culture system which represents all characteristics of its origin. However, this organ-like structure requires supporting matrix to maintain its characteristics and functions. Matrigel, derived from mouse sarcoma, has often been used as the supporting matrix for organoids, but the result may not be desirable for clinical applications because of the unidentified components from the mouse sarcoma. On the other hand, natural characteristics of collagen emphasize toxic-free friendly niche to both organoid and normal tissue. Hence, this study attempts to develop a new, collagen-based matrix that may substitute Matrigel in organoid culture. Collagen-based matrix was made, using type 1 collagen, Ham's F12 nutrient mixture, and bicarbonate. Then, characteristics of mouse colon organoids were analyzed by morphology and quantitative messenger RNA (mRNA) expression, revealing that the mouse colon organoids grown in the collagen-based matrix and in Matrigel had quite similar morphology, specific markers, and proliferative rates. Mouse small intestine–derived organoids, stomach-derived organoids, and human colon–derived organoids were also cultured, all of which were successfully grown in the collagen-based matrix and had similar properties compared to those cultured in Matrigel. Furthermore, possibility of organoid transplantation was observed. When mouse colon organoids were transplanted with collagen matrix into the EDTA-colitis mouse model, colon organoids were successfully engrafted in damaged tissue. For that reason, the use of collagen-based matrix in organoid culture will render organoid cultivation less expensive and clinically applicable.

Abstract B38: In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis

Abstract In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis that rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR/Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis. Citation Format: Jatin Roper, Tuomas Tammela, Naniye Malli Cetinbas, Adam Akkad, Ali Roghanian, Steffen Rickelt, Mohammad Almeqdadi, Katherine Wu, Matthias Oberli, Francisco Sánchez-Rivera, Yoona Park, Xu Liang, George Eng, Martin S. Taylor, Roxana Azimi, Dmitriy Kedrin, Rachit Neupane, Semir Beyaz, Ewa T. Sicinska, Yvelisse Suarez, James Yoo, Lillian Chen, Lawrence Zukerberg, Pekka Katajisto, Vikram Deshpande, Adam Bass, Philip N. Tsichlis, Jacqueline Lees, Robert Langer, Richard O. Hynes, Jianzhu Chen, Arjun J. Bhutkar, Tyler Jacks, Ömer H. Yilmaz. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B38.

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis that rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer predisposing mutations. Autochthonous tumor formation is induced by CRISPR–Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/ Δ;KrasG12D/+;Trp53Δ/ Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.