Protective effects of metformin in the pro-inflammatory cytokine induced intestinal organoids injury model.

Pathogenesis of inflammatory bowel disease (IBD) accompanies disrupted intestinal tight junctions. However, many approaches of therapeutics for IBD are focused only on anti-inflammatory effects and most cellular experiments are based on two-dimensional cell lines which have insufficient circumstances of intestine. Thus, here, we used three-dimensional structure intestinal organoids to investigate effects of metformin in the in vitro IBD condition. In this study, we focused on both tight junctions and the levels of inflammatory cytokines. Metformin enhances the intestinal barrier in injured intestine via upregulation of AMP-activated protein kinase, dysfunction of which contributes to the pathogenesis of intestinal diseases. We aim to investigate the effects of metformin on cytokine-induced injured intestinal organoids. Tumor necrosis factor-alpha (TNF-α) was used to induce intestinal injury in an organoid model, and the effects of metformin were assessed. Cell viability and levels of inflammatory cytokines were quantified in addition to tight junction markers. Furthermore, 4 kDa FITC-dextran was used to assess intestinal permeability. The upregulation of inflammatory cytokine levels was alleviated by metformin, which also restored the intestinal epithelium permeability in TNF-α-treated injury organoids. We confirmed that claudin-2 and claudin-7, representative tight junction markers, were also protected by metformin treatment. This study confirms the protective effects of metformin, which could be used as a therapeutic strategy for inflammatory intestinal diseases.

Magnetic resonance imaging of pancreatic islets using tissue-adhesive particles containing iron oxide nanoparticles.

Post-transplantation tracking of pancreatic islets is a prerequisite for advancing cell therapy to treat type 1 diabetes. Magnetic resonance imaging (MRI) has emerged as a safe and non-invasive technique for visualizing cells in clinical applications. In this study, we proposed a novel MRI contrast agent formulation by encapsulating iron oxide nanoparticles (IONPs) in poly(lactic-co-glycolic acid) (PLGA) particles functionalized with a tissue adhesive polydopamine (PD) layer (IONP-PLGA-PD MS). Intriguingly, our particles facilitated efficient and robust labeling through a one-step process, allowing for the incorporation of a substantial amount of IONPs without detrimental impacts on the viability and functionality of pancreatic islets. The MRI signals emanating from islets labeled using our particles were found to be stable over 30 days in vitro and 60 days when transplanted under kidney capsules of diabetic mice. These results suggest that our approach provides a potential platform for monitoring the fate of pancreatic islets after transplantation.

Anti-cancer effects of metformin in a 3D co-culture model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) presents with condensed stroma that contributes to its high invasive capability. Although metformin adjuvant treatment has been suggested to improve the survival times of patients with PDAC, the mechanism responsible for that benefit has been investigated only in two-dimensional cell lines. We assessed the anti-cancer effect of metformin in a three-dimensional (3D) co-culture model to quantify the migration behavior of patient-derived PDAC organoids and primary pancreatic stellate cells (PSCs). At a concentration of 10 µM, metformin reduced the migratory ability of the PSCs by downregulating the expression of matrix metalloproteinase-2 (MMP2). In the 3D direct co-cultivation of PDAC organoids and PSCs, metformin attenuated the transcription of cancer stemness-related genes. The reduced stromal migratory ability of PSCs was associated with the downregulation of MMP2, and MMP2 knockdown in PSCs reproduced their attenuated migratory ability. The anti-migration effect of a clinically relevant concentration of metformin was demonstrable in a 3D indirect co-culture model of PDAC consisting of patient-derived PDAC organoids and primary human PSCs. The metformin suppressed PSC migration via MMP2 downregulation and attenuated cancer stemness factors. Furthermore, oral administration of metformin (30 mg/kg) strikingly suppressed the growth of PDAC organoids xenograft in immunosuppressed mice. These results indicate metformin could offer the potential approach as an effective therapeutic drug for PDAC.

DAMP-modulating nanoparticle for successful pancreatic islet and stem cell transplantation.

Cell therapy is targeted at many organs, but locally or systemically delivered cells are shortly able to survive resulting from the immune/inflammation reactions and irregular cell targeting. Here we explore the multimodal nanoparticle having anti-inflammation and magnetic guidance for successful cell transplantation. We design magnetic resonance (MR)-active glycyrrhizin-chitosan coated superparamagnetic iron oxide nanoparticle (SPIO@Chitosan-GL) to inhibit release of inflammatory damage-associated molecular pattern (DAMP) protein and to offer noninvasive monitoring after intrahepatic transplantation of pancreatic islets and mesenchymal stem cell (MSC) spheroids. Intracellular delivered SPIO@Chitosan-GL is not cytotoxic to pancreatic islets and MSC spheroids and attenuate DAMP release from them. Also, therapeutic cells labeled with SPIO@Chitosan-GL are magnetically localized to the intended lobe of liver during transplantation procedure. If necessary, partial hepatectomy can be performed to remove the localized therapeutic cells for protection of the remaining liver lobes from systemic inflammation. Therapeutically, the cells selectively localized in the liver can treat blood glucose in diabetic mice to normal levels with DAMP modulation, and are visualized using in vivo MR imaging for over 4 weeks. Collectively, DAMP-modulating SPIO@Chitosan-GL can be used in multimodal nanomedince for attenuating the inflammation reaction by transplanted cells and for noninvasively long-term monitoring of transplanted cells.

Alternative splicing variant of NRP/B promotes tumorigenesis of gastric cancer.

Gastrointestinal cancer is associated with a high mortality rate. Here, we report that the splice variant of NRP/B contributes to tumorigenic activity in highly malignant gastric cancer through dissociation from the tumor repressor, HDAC5. NRP/B mRNA expression is significantly higher in the human gastric cancer tissues than in the normal tissues. Further, high levels of both the NRP/B splice variant and Lgr5, but not the full-length protein, are found in highly tumorigenic gastric tumor cells, but not in non-tumorigenic cells. The loss of NRP/B markedly inhibits cell migration and invasion, which reduces tumor formation in vivo. Importantly, the inhibition of alternative splicing increases the levels of NRP/B-1 mRNA and protein in AGS cells. The ectopic expression of full-length NRP/B exhibits tumor-suppressive activity, whereas NRP/B-2 induces the noninvasive human gastric cancer cells tumorigenesis. The splice variant NRP/B-2 which loses the capacity to interact with tumor repressors promoted oncogenic activity, suggesting that the BTB/POZ domain in the N-terminus has a crucial role in the suppression of gastric cancer. Therefore, the regulation of alternative splicing of the NRP/B gene is a potential novel target for the treatment of gastrointestinal cancer. [BMB Reports 2022; 55(7): 348-353].

Development of organoid-based drug metabolism model.

Cytochrome P450 (CYP) gene superfamily catalyzes oxidative metabolism of a wide variety of drugs, carcinogens, and endogenous biomolecules in the liver and intestinal organs. In vitro assay platforms such as primary hepatocyte and immortalized liver-derived cell lines have been developed to evaluate drug effects. However, several limitations have been suggested regarding discrepancies between in vitro and in vivo assays. In this study, we aimed to investigate drug metabolism and toxicity based on mouse small intestinal and liver organoids derived from resident stem cells. At first, expressions and activities of CYP subfamilies (CYPs) in intestinal and liver organoids were investigated. Organoids treated with three CYPs-inducers dexamethasone (Dex), ß-naphthoflavone (BNF), and 1,4-bis-2-(3, 5-dichloropyridyloxy)-benzene (TCPOBOP) were evaluated for CYPs activities. The CYPs-induced intestinal and liver organoids were confirmed to digest more docetaxel, as colon cancer cell-line survived more in CYPs-induced organoid's medium than in non-induced organoid's medium. Then, the activity of docetaxel in a co-culture platform of mouse liver organoids and human pancreatic tumoroids was measured. We obtained significant statistical values on CYPs-induced metabolic activities: cell survival rates of pancreatic tumoroids co-cultured with docetaxel-treated undifferentiated, differentiated, and CYPs-induced differentiated organoids were 66.05 ±â€¯2.14%, 89.20 ±â€¯2.67%, and 101.90 ±â€¯0.94%, respectively. To sum up, gene expression modification and drug metabolism evaluation were able to be done with organoids as done with tissues. In vivo-like in vitro investigation on drug toxicity may potentially be done with organoids as a stepping bridge to the clinical trial.

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.

Gastrointestinal Epithelial Organoid Cultures from Postsurgical Tissues.

An organoid is a cellular structure three-dimensionally (3D) cultured from self-organizing stem cells in vitro, which has a cell population, architectures, and organ specific functions like the originating organs. Recent advances in the 3D culture of isolated intestinal crypts or gastric glands have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells. Thus far, gastrointestinal organoids have been extensively used for generating gastrointestinal disease models. This protocol describes the method of isolating a gland or crypt using stomach or colon tissue after surgery and establishing them into gastroids or colonoids.

LINCS L1000 dataset-based repositioning of CGP-60474 as a highly potent anti-endotoxemic agent.

Sepsis is one of the most common clinical syndromes that causes death and disability. Although many studies have developed drugs for sepsis treatment, none have decreased the mortality rate. The aim of this study was to identify a novel treatment option for sepsis using the library of integrated network-based cellular signatures (LINCS) L1000 perturbation dataset based on an in vitro and in vivo sepsis model. Sepsis-related microarray studies of early-stage inflammatory processes in patients and innate immune cells were collected from the Gene Expression Omnibus (GEO) data repository and used for candidate drug selection based on the LINCS L1000 perturbation dataset. The anti-inflammatory effects of the selected candidate drugs were analyzed using activated macrophage cell lines. CGP-60474, an inhibitor of cyclin-dependent kinase, was the most potent drug. It alleviated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in activated macrophages by downregulating the NF-κB activity, and it reduced the mortality rate in LPS induced endotoxemia mice. This study shows that CGP-60474 could be a potential therapeutic candidate to attenuate the endotoxemic process. Additionally, the virtual screening strategy using the LINCS L1000 perturbation dataset could be a cost and time effective tool in the early stages of drug development.

Organoid-based epithelial to mesenchymal transition (OEMT) model: from an intestinal fibrosis perspective.

The current in vitro or in vivo intestinal fibrosis models have many limitations. Recent advancements in the isolation and culturing of organoids has led to development of various three-dimensional (3D) intestinal disease models with in vivo physiology. In this study, we generated an organoid-based epithelial to mesenchymal transition (OEMT) model, which could be used as a novel intestinal fibrosis model. Intestinal epithelial organoids (IEOs) were isolated and cultured from the small intestines of normal mice. IEOs were treated with transforming growth factor- ß1 (TGF-ß1) or Tumor necrosis factor-α (TNF-α) to evaluate their phenotypic change. Raw 264.7 cells (macrophage) stimulated with lipopolysaccharide were co-cultured with IEOs in growth media with or without TGF-ß1. TGF-ß1 alone slightly induced epithelial to mesenchymal transition (EMT) in the IEOs but mainly disrupted them. Macrophage released cytokines synergistically induced mesenchymal phenotypic changes in TGF-ß1 stimulated intestinal organoids. TNF-α and TGF-ß1 synergistically induced proliferation of mesenchymal cells as well as EMT in the IEOs. We generated a novel OEMT model based on our finding that TNF-α and TGF-ß synergistically induce type 2 EMT in IEOs. This 3D EMT model with in vivo physiology could be used to study EMT associated intestinal fibrosis.