Colon Organoids Research Articles

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

SummaryA major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1.

Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions and yield leads for developing ALDH1B1-targeting therapies.

Abstract 1416: Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models

Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide. The disease-initiating event in most CRC cases is the loss of function (LOF) of the adenomatous polyposis coli (APC) gene, accounting for 70-80% of CRC. Strategies to target cells that harbor the truncation mutation in APC, which leads to its LOF, may provide the opportunity to intercept tumorigenesis in CRC patients. High-risk CRC populations include patients with familial adenomatous polyposis (FAP), the inherited disorder caused by germline APC mutations. Aurora Kinase (AK), a family of serine/threonine kinases, is essential for proper cell division, and overexpression of AK has been associated with multiple heme and solid tumor malignancies, including CRC. Aurora kinases have been well-studied, both preclinically and clinically, as a target for therapeutic intervention across multiple cancers; however, the role of AK in CRC disease progression has not been fully explored. Here we report a synthetic lethal dependency between APC and AK which was identified in a human isogenic colon organoid model containing a genetically engineered APC LOF (APCLOF). Aurora A-specific, Aurora B-specific, and Pan-AK inhibitors were evaluated in isogenic APCLOF or APCWT colon organoids derived from multiple donors. All AK inhibitors demonstrated selective growth inhibition of the APCLOF organoid. The strongest synthetic lethal effect was observed with a pan-AK inhibitor showing greater than 1000-fold selectivity in the APCLOF colon organoids compared to APCWT. Interestingly, this activity diminished as oncogenic driver mutations in KRAS and/or p53 genes were introduced into the APCLOF organoids. These findings suggest that therapeutic benefit of AK inhibitors would be greater in early APCLOF driven adenomas as opposed to advanced CRC. Despite these encouraging results indicating therapeutic opportunity in early adenoma disease, on-target genotoxicity was observed in vitro through increases in micronuclei at pharmacologic doses. Therefore, although there is a high unmet need in FAP for therapeutic options, potential genotoxicity associated with AK inhibitor treatments may limit utility as an intervention strategy. Citation Format: Dana S. Gaffney, Eileen Vesely, David Pocalyko, Kurtis Bachman, Karla Wiehagen, Glenn Cowley, Victoria Wong, Prashanth Gokare, Peggy Guzzie-Peck, Sandy Weiner, Ann De Smedt, Jacqueline Kinyamu-Akunda, Tammy Bush, Zhuming Zhang, Robert Schulingkamp, Sylvia F. Boj, Rene Overmeer, Carla Verissimo, Roshni Nair. Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1416.

Abstract 152: Discovery and characterization of GSK3 allosteric activators

Abstract The intestinal epithelium undergoes constant regeneration and is tightly regulated. Disruption of its homeostasis can result in the development of colorectal cancer (CRC). Glycogen synthase kinase 3 (GSK3) is a kinase that plays a central role in multiple signaling pathways, including cellular proliferation, where it inhibits the activity of its substrates. GSK3 dynamics can be described as a multiple input multiple output system. Numerous upstream growth factors can regulate and thus inhibit GSK3 from acting on its many substrates. Mutations in these growth signaling pathways (e.g., Wnt and KRAS) have been well established to have a role in CRC progression. Therefore, aberrantly activated signaling due to these proteins are presumed to result in the constitutive inhibition of GSK3 activity in CRC, thus making it an ideal therapeutic target. The aim of this study is to discover an allosteric GSK3 small molecule activator that can restore GSK3 kinase activity within CRC. Method: We screened 50,000 small molecule compounds using a luciferase-based in-vitro kinase assay. We are further confirming our top hits using both chemical and cell-based assays. For the cell-based assays, we test the compounds in human colonic epithelial cells (HCEC) and its three isogenic cell lines, which mimic CRC progression, and monitor changes in GSK3 kinase activity indirectly, via changes in its substrate protein levels, and directly via a live-cell GSK3 specific reporter. In addition, the top hits are also tested in both normal colon and CRC organoids. Results: Prior to running our large screen, we performed various optimization steps to determine the ideal condition for our screening assay. Our screening assay scored a Z-Score of 0.5 and higher, which indicated that it was a high-quality screening assay. Additionally, we have developed two cell-based orthogonal HTS assays. One using immunofluorescence and single-cell quantification of 24 GSK3 substrates from various pathways in our cell lines. Second, we engineered a GSK3 live-cell reporter and expressed it in the HCEC cells and three isogenic cell lines and organoid models. Impact: There is a need for advancements in preventative or therapeutic targets against CRC. Research indicates that suppression of GSK3 enzymatic activity decreases sensitivity to certain chemotherapeutic and targeted therapies. Therefore, restoring GSK3 enzymatic activity in CRC may re-sensitize tumors to other targeted therapies and establish GSK3 as a potential therapeutic target. Citation Format: Reeba P. Varghese, Curtis A. Thorne. Discovery and characterization of GSK3 allosteric activators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 152.

Abstract 171: ERK dynamics are essential for colon tissue patterning

Abstract Maintaining the correct ratio of cellular niches in space and time allows tissues to perform their complex functions and create patterns. This maintenance of tissue patterning is often dysregulated during cancer development. In order to study tissue patterning, temporal and spatial resolution of communication between single cells is required. To explore how this complex process is controlled, we developed a patient-derived organoid screening platform to study both tissue patterning and kinase pathway dynamics in single cells across hundreds of conditions. Unlike normal and polyp-derived organoids, tumor organoids displayed a lack of patterning reminiscent of in vivo dysplasia. Using this system, we discovered that waves of Erk signaling induced by apoptotic cells are critical for maintaining tissue patterning in human colonic organoids. Using a small molecule-based screen, we discovered novel kinases which limit Erk activation across tissues and help maintain patterning. Perturbation of Erk restricting kinases results in dysfunctional colonic wound repair and re-establishment of tissue patterns in vivo. Citation Format: Kelvin W. Pond, Jayati Chakrabarti, Curtis A. Thorne, Andrew L. Paek. ERK dynamics are essential for colon tissue patterning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 171.

Regulation of the Intestinal Extra-Adrenal Steroidogenic Pathway Component LRH-1 by Glucocorticoids in Ulcerative Colitis.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) and can be treated with glucocorticoids (GC), although some patients are unresponsive to this therapy. The transcription factor LRH-1/NR5A2 is critical to intestinal cortisol production (intestinal steroidogenesis), being reduced in UC patients. However, the relationship between LRH-1 expression and distribution with altered corticosteroid responses is unknown. To address this, we categorized UC patients by their steroid response. Here, we found that steroid-dependent and refractory patients presented reduced glucocorticoid receptor (GR)-mediated intestinal steroidogenesis compared to healthy individuals and responder patients, possibly related to increased colonic mucosa GR isoform beta (GRβ) content and cytoplasmic LRH-1 levels in epithelial and lamina propria cells. Interestingly, an intestinal epithelium-specific GR-induced knockout (GRiKO) dextran sodium sulfate (DSS)-colitis mice model presented decreased epithelial LRH-1 expression, whilst it increased in the lamina propria compared to DSS-treated control mice. Mechanistically, GR directly induced NR5A2 gene expression in CCD841CoN cells and human colonic organoids. Furthermore, GR bound to two glucocorticoid-response elements within the NR5A2 promoter in dexamethasone-stimulated CCD841CoN cells. We conclude that GR contributes to intestinal steroidogenesis by inducing LRH-1 in epithelial cells, suggesting LRH-1 as a potential marker for glucocorticoid-impaired response in UC. However, further studies with a larger patient cohort will be necessary to confirm role of LRH-1 as a therapeutic biomarker.

Altered gut microbiota and metabolites profile are associated with reduced bone metabolism in ethanol-induced osteoporosis.

ObjectiveChronic heavy drinking causes ethanol‐induced osteoporosis (EIO). The present study aimed to explore the role of GM in EIO.Material and MethodsA rat EIO model was established by chronic ethanol intake. Taking the antibiotic application as the matched group of dysbacteriosis, an integrated 16S rRNA sequencing and liquid chromatography–tandem mass spectrometry‐based metabolomics in serum and faeces were applied to explore the association of differential metabolic phenotypes and screen out the candidate metabolites detrimental to ossification. The colon organoids were used to track the source of 5‐HT and the effect of 5‐HT on bone formation was examined in vitro . ResultsCompared with antibiotics application, ethanol‐gavaged decreased the BMD in rats. We found that both ethanol and antibiotic intake affected the composition of GM, but ethanol intake increased the ratio of Firmicutes to Bacteroidetes. Elevated serotonin was proved to be positively correlated with the changes of the composition of GM and faecal metabolites and inhibited the proliferation and mineralization of osteogenesis‐related cells. However, the direct secretory promotion of serotonin was absent in the colon organoids exposed to ethanol.ConclusionThis study demonstrated that ethanol consumption led to osteoporosis and intestinal‐specific dysbacteriosis. Conjoint analysis of the genetic profiles of GM and metabolic phenotypes in serum and faeces allowed us to understand the endogenous metabolite, 5‐HT, as detrimental regulators in the gut‐bone axis to impair bone formation.

Transcriptomic Response to Calcium in Normal Colon Organoids is Impacted by Colon Location and Sex.

A chemopreventive role for calcium in colorectal cancer is still unclear. Here, we identify mechanisms through which calcium supplementation may reduce risk. Calcium supplementation increased differentiation and altered expression of colorectal cancer-related genes in a large study of patient-derived colon organoids. These findings were influenced by colon location and sex.

P-112 Oleic acid promotes the malignant transformation of Kras-mutant colonic organoids via the expansion of tumorigenic stem cells and abnormal Paneth cells through upregulation of NFATc family

Although the five-year survival rate of colorectal cancer (CRC) is significantly high with localized stage (90%), only 38% of the patients are diagnosed at this stage. Therefore, finding high risk factors of CRC could prevent the disease at the early stage. Obesity and high-fat diet consumption are risk factors of CRC. In detail, oleic acids (OA) were found to accumulate in the adipose tissues of obese patients as well as being the most common long chain fatty acid in dietary lipid. The role of OA in colorectal cancer development is still controversary. On the other hand, organoids, which can be derived from adult tissue stem cells, are the miniatures of organs. Recent works suggest organoids are useful tools to dissect mechanisms involved in cancer development. The current work aims to utilize crypt stem cell-derived organoids to dissect how obesity can contribute to CRC development. Organoids were derived from Lgr5 + -EGFP mice (normal colon control), Lgr5 + -EGFP-creER; LSL-KrasG12D mice (colonic epithelium harbored KrasG12D mutation) and AOM/DSS-induced CRC mice (cancerous colon control). For in-vitro, the impact of OA on the malignant transformation of Kras-mutant colonic organoids was assessed by MTT proliferation assay, 2D transwell migration and invasion assay, irinotecan-resistant test and colony formation assay. The tumor formation in-vivo was induced by subcutaneous engrafting of OA-treated organoids on NOD/SCID mice. RNAseq was done to screen some differential expressed genes when treating OA. The existence of stem cell and abnormal Paneth cell population was demonstrated by immunostaining, flow cytometry and ELISA. OA enhanced the malignant of Kras-mutant colonic organoids through increasing proliferation, migration, invasion, drug resistance and colony formation. Besides, mice which were engrafted with OA-trained Lgr5-Kras organoids possessed larger tumors. The colonic stem cell population which were represented by positive Lgr5-GFP + cells were upregulated in OA-treated group. Interestingly, there is an existence of abnormal Lyz + /CD24 + Paneth cells in OA-treated Kras-mutant organoids. This suggested that OA may promote abnormal differentiation of colonic progenitor cells. Indeed, treatment of OA upregulated ATOH1 and GFI1, which contributed to select between goblet/Paneth and enteroendocrine cell fates. Besides, NFAT family, among which NFATc3 expressed the most, significantly increased in OA-treated organoids. The upregulation of NFATc was induced by inhibition of proteasomal degradation resulting from low expression of the complex RNF123, USP18 and TRIM30A. In addition, inhibition of the nuclear translocalization of NFATc family by VIVIT demonstrated the decline of stemness markers as well as Paneth cell markers. Finally, we showed the role of NFATc, especially NFATc3, in promoting the malignancy of OA-treated Kras-mutant organoids. Our findings suggested the impact of oleic acid on promoting the malignant transformation of Kras-mutant colonic organoids through NFATc-relating pathway. The expansion of tumorigenic stem cells via induction of abnormal Paneth cell, may contribute to the potential of cancer-toward metaplasia in Kras-mutant organoids when exposing to high level of oleic acid. Oleic acid is an unfavorable factor of colorectal cancer. The frequent usage of OA, which may benefit to cardiovascular diseases, may lead to the increased risk of CRC in latent sensitive Kras-mutant individuals.

Adenoma-Derived Organoids for Precision Therapy

Human colonic organoids derived from adult tissue biopsies are based on the ability of isolated somatic epithelial stem cells to reconstitute the structure and function of the colon, offering new opportunities for studying the biology of the large intestine in both health and disease. These colonoids may also function as efficient platforms for drug screening and discovery. Here, we describe the establishment of human colonic organoids derived from healthy, and adenomatous polyp tissues. We then demonstrate that organoids grown from adenomas of familial adenomatous polyposis (FAP) patients harboring nonsense mutations in the tumor suppressor gene adenomatous polyposis coli (APC), can be used to establish a personalized therapeutic strategy which relies on nonsense mutation readthrough therapy.

Colon Tumors in Enterotoxigenic Bacteroides fragilis (ETBF)-Colonized Mice Do Not Display a Unique Mutational Signature but Instead Possess Host-Dependent Alterations in the APC Gene.

ABSTRACTEnterotoxigenic Bacteroides fragilis (ETBF) is consistently found at higher frequency in individuals with sporadic and hereditary colorectal cancer (CRC) and induces tumorigenesis in several mouse models of CRC. However, whether specific mutations induced by ETBF lead to colon tumor formation has not been investigated. To determine if ETBF-induced mutations impact the Apc gene, and other tumor suppressors or proto-oncogenes, we performed whole-exome sequencing and whole-genome sequencing on tumors isolated after ETBF and sham colonization of Apcmin/+ and Apcmin/+Msh2fl/flVC mice, as well as whole-genome sequencing of organoids cocultured with ETBF. Our results indicate that ETBF-induced tumor formation results from loss of heterozygosity (LOH) of Apc, unless the mismatch repair system is disrupted, in which case, tumor formation results from new acquisition of protein-truncating mutations in Apc. In contrast to polyketide synthase-positive Escherichia coli (pks+ E. coli), ETBF does not produce a unique mutational signature; instead, ETBF-induced tumors arise from errors in DNA mismatch repair and homologous recombination DNA damage repair, established pathways of tumor formation in the colon, and the same genetic mechanism accounting for sham tumors in these mouse models. Our analysis informs how this procarcinogenic bacterium may promote tumor formation in individuals with inherited predispositions to CRC, such as Lynch syndrome or familial adenomatous polyposis (FAP).IMPORTANCE Many studies have shown that microbiome composition in both the mucosa and the stool differs in individuals with sporadic and hereditary colorectal cancer (CRC). Both human and mouse models have established a strong association between particular microbes and colon tumor induction. However, the genetic mechanisms underlying putative microbe-induced colon tumor formation are not well established. In this paper, we applied whole-exome sequencing and whole-genome sequencing to investigate the impact of ETBF-induced genetic changes on tumor formation. Additionally, we performed whole-genome sequencing of human colon organoids exposed to ETBF to validate the mutational patterns seen in our mouse models and begin to understand their relevance in human colon epithelial cells. The results of this study highlight the importance of ETBF colonization in the development of sporadic CRC and in individuals with hereditary tumor conditions, such as Lynch syndrome and familial adenomatous polyposis (FAP).

Tofacitinib Downregulates TNF and Poly(I:C)-Dependent MHC-II Expression in the Colonic Epithelium.

Major Histocompatibility Complex (MHC)-I and -II genes are upregulated in intestinal epithelial cells (IECs) during active inflammatory bowel diseases (IBD), but little is known about how IBD-relevant pro-inflammatory signals and IBD drugs can regulate their expression. We have previously shown that the synthetic analog of double-stranded RNA (dsRNA) Polyinosinic:polycytidylic acid (Poly(I:C)), induces interferon stimulated genes (ISGs) in colon organoids (colonoids). These ISGs may be involved in the induction of antigen presentation. In the present study, we applied colonoids derived from non-IBD controls and ulcerative colitis patients to identify induction and effects of IBD-drugs on antigen presentation in IECs in the context of Tumor Necrosis Factor (TNF)-driven inflammation. By RNA sequencing, we show that a combination of TNF and Poly(I:C) strongly induced antigen-presentation gene signatures in colonoids, including expression of MHC-II genes. MHC-I and -II protein expression was confirmed by immunoblotting and immunofluorescence. TNF+Poly(I:C)-dependent upregulation of MHC-II expression was associated with increased expression of Janus Kinases JAK1/2 as well as increased activation of transcription factor Signal transducer and activator of transcription 1 (STAT1). Accordingly, pre-treatment of colonoids with IBD-approved pan-Janus Kinase (JAK) inhibitor Tofacitinib led to the downregulation of TNF+Poly(I:C)-dependent MHC-II expression associated with the abrogation of STAT1 activation. Pre-treatment with corticosteroid Budesonide, commonly used in IBD, did not alter MHC-II expression. Collectively, our results identify a regulatory role for IBD-relevant pro-inflammatory signals on MHC-II expression that is influenced by Tofacitinib.

Mapping the epithelial\u2013immune cell interactome upon infection in the gut and the upper airways

Increasing evidence points towards the key role of the epithelium in the systemic and over-activated immune response to viral infection, including SARS-CoV-2 infection. Yet, how viral infection alters epithelial–immune cell interactions regulating inflammatory responses, is not well known. Available experimental approaches are insufficient to properly analyse this complex system, and computational predictions and targeted data integration are needed as an alternative approach. In this work, we propose an integrated computational biology framework that models how infection alters intracellular signalling of epithelial cells and how this change impacts the systemic immune response through modified interactions between epithelial cells and local immune cell populations. As a proof-of-concept, we focused on the role of intestinal and upper-airway epithelial infection. To characterise the modified epithelial–immune interactome, we integrated intra- and intercellular networks with single-cell RNA-seq data from SARS-CoV-2 infected human ileal and colonic organoids as well as from infected airway ciliated epithelial cells. This integrated methodology has proven useful to point out specific epithelial–immune interactions driving inflammation during disease response, and propose relevant molecular targets to guide focused experimental analysis.

Klebsiella pneumoniae Cross‐feeds Clostridioides difficile and Enhances Colonic Pro‐inflammatory Responses

BackgroundRecent in‐depth analysis of the human gut microbiota demonstrates that many intestinal disorders are polymicrobial in origin and manifestation. Select microbes can interact synergistically with pathobionts to induce virulence traits, modify environmental niches, and modulate host responses. In the setting of Clostridioides difficile infection, polymicrobial interactions are incompletely understood. C. difficile is an intestinal pathogen responsible for 500,000 infections and 30,000 deaths annually in the US. Using stool‐seeded bioreactors with mucus‐coated inserts, we identified Klebsiella pneumoniae in the mucus‐associated microbiota along with C. difficile.HypothesisWe hypothesized that K. pneumoniaemay enhance C. difficile traits required for colonization.Methods & ResultsTo test this hypothesis, we cultured 11 K. pneumoniae strains with four C. difficile strains (representing 4 different ribotypes) in a fully defined bacteria media ZMB1. We observed that cell‐free supernatant from all K. pneumoniae strains enhanced the growth of all C. difficile strains, indicating that metabolites produced by K. pneumoniae enhanced C. difficile growth. Importantly, C. difficile grew well with K. pneumoniae bacteria and both bacteria were identified in co‐cultures by gram staining. Non‐targeted metabolomics revealed that K. pneumoniae shifted the metabolism of C. difficile away from amino acid utilization. K. pneumoniae metabolites also suppressed toxin production in an in vitro model of nutrient starvation as assessed by live‐imaging of LifeAct expressing Vero cell rounding. These findings further confirm the ability of K. pneumoniae to cross‐feed C. difficile. Although K. pneumoniae is well known for robust biofilm production, K. pneumoniae did not significantly enhance C. difficile biofilm formation, indicating that biofilm synthesis is not a significant component of C. difficile‐Klebsiella interactions. RNA sequencing of mouse colonic inside‐out organoids incubated with live C. difficile and/or K. pneumoniae revealed increased pro‐inflammatory responses when the two microbes were added together compared to either microbe alone or media controls.ConclusionsThese results suggest that K. pneumoniae can cross‐feed and shift the metabolism of C. difficile, as well as enhance the pro‐inflammatory capacity of C. difficile. We speculate that interspecies interactions could be among the earliest events that occur as C. difficile colonizes the host. As a result, we believe that an improved understanding of how mucus associated microbes like K. pneumoniae interact with C. difficile will continue to clarify the mechanisms of C. difficile infection.

I3C-MEDIATED PROTECTION AGAINST COLITIS DEPENDENT ON AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS

Abstract Many current treatments for ulcerative colitis (UC) often causes side effects warranting safe therapeutic strategies to control the pathogenesis of colitis. Current study was aimed to ascertain the possible role of aryl hydrocarbon receptor (AhR) in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. We investigated how intestinal regulatory mediators were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis and had a higher disease score with increased inflammation in the colon compared to the controls. Also, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Transcriptome analysis of RNA isolated from enriched CECs of experimental mice showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice compared to controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids validated these results. In summary, AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.

Characterizing the Capacity of Acinetobacter calcoaceticusto Grow in Intestinal Conditions and Modulate the Gut Epithelium

BackgroundAcinetobacter species can occupy several ecological niches, including the mammalian intestine. Acinetobacter has been identified in the human fecal microbiota and it is speculated that the gut can serve as a reservoir for this pathobiont. The Acinetobacter calcoaceticus–Acinetobacter baumannii complex is a common cause of drug‐resistant pneumonia, urinary tract, skin and soft tissue infections. A. calcoaceticus has been considered to have relatively low virulence compared to its counterpart A. baumannii since colonization is more frequently noted more than infection clinically. However, few studies have dissected the interaction between A. calcoaceticus and the host, particularly in the gut. In this study, we sought to examine the ability of A. calcoaceticus strains to survive environmental stressors found in the gut. We also examined the reciprocal interactions of A. calcoaceticus strains on the host using colonic organoids.Methods & ResultsThree commercially available strains of A. calcoaceticus and four clinical isolates were grown aerobically in brain heart infusion (BHI) media, subcultured in a fully defined bacterial media termed ZMB1 and exposed to various concentrations of NaCl, ethanol, bile salts and pHs that were designed to mimic the gut. All A. calcoaceticusstrains grew robustly at NaCl concentrations that mirrored the small intestine, but differences were observed between strains at higher NaCl concentrations. Additionally, all strains grew well at pH 7 and pH 6, and exhibited growth reduction at pHs 5 and 4. All strains tolerated several concentrations of bile salts and ethanol; suggesting tolerance for intestinal stressors. Incubation of live A. calcoaceticus strains with inside‐out jejunum and colonic organoids significantly increased pro‐inflammatory cytokines (IL‐1α, KC/IL‐8, MCP‐1 and TNF) and decreased MUC2 and MUC13 transcripts, without altering tight junctions.ConclusionsCollectively, these data demonstrate that A. calcoaceticus is well adapted to the gastrointestinal tract and points to the potential for Acinetobacter to influence the gut epithelium. Of note, we observed differences between lab adapted strains and clinical isolates, highlighting strain variation and indicating that clinical isolate strains should be incorporated into future work.

Human iPSC colon organoid function is improved by exposure to fecal fermentates.

The host–microbe interaction is critical for intestinal homeostasis. By‐products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by‐product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human‐induced pluripotent (iPSC) colon organoid model. SCFA and BCFA's of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin A, SCFA transport monocarboxylate transporter‐1, (6.2‐fold, 2.1‐fold, and 1.8‐fold, respectively; p < 0.05). Mucin production (mucin‐2, mucin‐4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin‐3) was noted by immunofluorescence in 25% supernatant‐ treated cells. A dose–response increase in barrier function was observed over the 72‐h time course, with a twofold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (p < 0.05). To further investigate host effects, clarified supernatants from a continuous multistage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL‐1β, 6.15 pg/ml; IL‐6, 27.58 pg/ml; TNFα, 4.49 pg/ml; p < 0.05) in DC‐treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by‐products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

Cell-intrinsic Aryl Hydrocarbon Receptor signalling is required for the resolution of injury-induced colonic stem cells

The aryl hydrocarbon receptor (AHR) is an environmental sensor that integrates microbial and dietary cues to influence physiological processes within the intestinal microenvironment, protecting against colitis and colitis-associated colorectal cancer development. Rapid tissue regeneration upon injury is important for the reinstatement of barrier integrity and its dysregulation promotes malignant transformation. Here we show that AHR is important for the termination of the regenerative response and the reacquisition of mature epithelial cell identity post injury in vivo and in organoid cultures in vitro. Using an integrative multi-omics approach in colon organoids, we show that AHR is required for timely termination of the regenerative response through direct regulation of transcription factors involved in epithelial cell differentiation as well as restriction of chromatin accessibility to regeneration-associated Yap/Tead transcriptional targets. Safeguarding a regulated regenerative response places AHR at a pivotal position in the delicate balance between controlled regeneration and malignant transformation.

Synthesis and Characterization of Size- and Charge-Tunable Silver Nanoparticles for Selective Anticancer and Antibacterial Treatment

Advances in the research of nanoparticles (NPs) with controlled charge and size are driven by their potential application in the development of novel technologies and innovative therapeutics. This work reports the synthesis, characterization, and comprehensive biological evaluation of AgNPs functionalized by N,N,N-trimethyl-(11-mercaptoundecyl) ammonium chloride (TMA) and trisodium citrate (TSC). The prepared AgNPs were well characterized in terms of their morphological, spectroscopic and functional properties and biological activities. The implementation of several complementary techniques allowed not only the estimation of the average particle size (from 3 to 40 nm depending on the synthesis procedure used) but also the confirmation of the crystalline nature of the NPs and their round shape. To prove the usefulness of these materials in biological systems, cellular uptake and cytotoxicity in microbial and mammalian cells were determined. Positively charged 10 nm Ag@TMA2 revealed antimicrobial activity against Gram-negative bacteria with a minimum inhibitory concentration (MIC) value of 0.17 μg/mL and complete eradication of Escherichia coli (7 logs) for Ag@TMA2 at a concentration of 0.50 μg/mL, whereas negatively charged 10 nm Ag@TSC1 was effective against Gram-positive bacteria (MIC = 0.05 μg/mL), leading to inactivation of Staphylococcus aureus at relatively low concentrations. In addition, the largest 40 nm Ag@TSC2 was shown to exhibit pronounced anticancer activity against murine colon carcinoma (CT26) and murine mammary gland carcinoma (4T1) cells cultured as 2D and 3D tumor models and reduced toxicity against human HaCaT keratinocytes. Among the possible mechanisms of AgNPs are their ability to generate reactive oxygen species, which was further evaluated in vitro and correlates well with cellular accumulation and overall activity of AgNPs. Furthermore, we confirmed the anticancer efficacy of the most potent Ag@TSC2 in hiPSC-derived colonic organoids and demonstrated that the NPs are biocompatible and applicable in vivo. A pilot study in BALB/c mice evidenced that the treatment with Ag@TSC2 resulted in temporary (>60 days) remission of CT26 tumors.

Fourier light-field imaging of human organoids with a hybrid point-spread function

Volumetric interrogation of the cellular morphology and dynamic processes of organoid systems with a high spatiotemporal resolution provides critical insights for understanding organogenesis, tissue homeostasis, and organ function. Fluorescence microscopy has emerged as one of the most vital and informative driving forces for probing the cellular complexity in organoid research. However, the underlying scanning mechanism of conventional imaging methods inevitably compromises the time resolution of volumetric acquisition, leading to increased photodamage and inability to capture fast cellular and tissue dynamic processes. Here, we report Fourier light-field microscopy using a hybrid point-spread function (hPSF-FLFM) for fast, volumetric, and high-resolution imaging of entire organoids. hPSF-FLFM transforms conventional 3D microscopy and enables exploration of less accessible spatiotemporally-challenging regimes for organoid research. To validate hPSF-FLFM, we demonstrate 3D imaging of rapid responses to extracellular physical cues such as osmotic and mechanical stresses on human induced pluripotent stem cells-derived colon organoids (hCOs). The system offers cellular (2–3 μm and 5–6 μm in x-y and z, respectively) and millisecond-scale spatiotemporal characterization of whole-organoid dynamic changes that span large imaging volumes (>900 μm × 900 μm × 200 μm in x, y, z, respectively). The hPSF-FLFM method provides a promising avenue to explore spatiotemporal-challenging cellular responses in a wide variety of organoid research.