Dataset Information

A Microwell-Based Intestinal Organoid-Macrophage Co-Culture System to Study Intestinal Inflammation.

ABSTRACT: The mammalian intestinal epithelium contains more immune cells than any other tissue, and this is largely because of its constant exposure to pathogens. Macrophages are crucial for maintaining intestinal homeostasis, but they also play a central role in chronic pathologies of the digestive system. We developed a versatile microwell-based intestinal organoid-macrophage co-culture system that enables us to recapitulate features of intestinal inflammation. This microwell-based platform facilitates the controlled positioning of cells in different configurations, continuous in situ monitoring of cell interactions, and high-throughput downstream applications. Using this novel system, we compared the inflammatory response when intestinal organoids were co-cultured with macrophages versus when intestinal organoids were treated with the pro-inflammatory cytokine TNF-α. Furthermore, we demonstrated that the tissue-specific response differs according to the physical distance between the organoids and the macrophages and that the intestinal organoids show an immunomodulatory competence. Our novel microwell-based intestinal organoid model incorporating acellular and cellular components of the immune system can pave the way to unravel unknown mechanisms related to intestinal homeostasis and disorders.

SUBMITTER: Kakni P

PROVIDER: S-EPMC9736416 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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A Microwell-Based Intestinal Organoid-Macrophage Co-Culture System to Study Intestinal Inflammation.

Kakni Panagiota P Truckenmüller Roman R Habibović Pamela P van Griensven Martijn M Giselbrecht Stefan S

International journal of molecular sciences 20221206 23

The mammalian intestinal epithelium contains more immune cells than any other tissue, and this is largely because of its constant exposure to pathogens. Macrophages are crucial for maintaining intestinal homeostasis, but they also play a central role in chronic pathologies of the digestive system. We developed a versatile microwell-based intestinal organoid-macrophage co-culture system that enables us to recapitulate features of intestinal inflammation. This microwell-based platform facilitates ...[more]

PMID: 36499691

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Project description:BackgroundGemcitabine resistance (GR) is a significant clinical challenge in pancreatic adenocarcinoma (PAAD) treatment. Macrophages in the tumor immune-microenvironment are closely related to GR. Uncovering the macrophage-induced GR mechanism could help devise a novel strategy to improve gemcitabine treatment outcomes in PAAD. Therefore, preclinical models accurately replicating patient tumor properties are essential for cancer research and drug development. Patient-derived organoids (PDOs) represent a promising in vitro model for investigating tumor targets, accelerating drug development, and enabling personalized treatment strategies to improve patient outcomes.MethodsTo investigate the effects of macrophage stimulation on GR, co-cultures were set up using PDOs from three PAAD patients with macrophages. To identify signaling factors between macrophages and pancreatic cancer cells (PCCs), a 97-target cytokine array and the TCGA-GTEx database were utilized. The analysis revealed CCL5 and AREG as potential candidates. The role of CCL5 in inducing GR was further investigated using clinical data and tumor sections obtained from 48 PAAD patients over three years, inhibitors, and short hairpin RNA (shRNA). Furthermore, single-cell sequencing data from the GEO database were analyzed to explore the crosstalk between PCCs and macrophages. To overcome GR, inhibitors targeting the macrophage-CCL5-Sp1-AREG feedback loop were evaluated in cell lines, PDOs, and orthotopic mouse models of pancreatic carcinoma.ResultsThe macrophage-CCL5-Sp1-AREG feedback loop between macrophages and PCCs is responsible for GR. Macrophage-derived CCL5 activates the CCR5/AKT/Sp1/CD44 axis to confer stemness and chemoresistance to PCCs. PCC-derived AREG promotes CCL5 secretion in macrophages through the Hippo-YAP pathway. By targeting the feedback loop, mithramycin improves the outcome of gemcitabine treatment in PAAD. The results from the PDO model were corroborated with cell lines, mouse models, and clinical data.ConclusionsOur study highlights that the PDO model is a superior choice for preclinical research and precision medicine. The macrophage-CCL5-Sp1-AREG feedback loop confers stemness to PCCs to facilitate gemcitabine resistance by activating the CCR5/AKT/SP1/CD44 pathway. The combination of gemcitabine and mithramycin shows potential as a therapeutic strategy for treating PAAD in cell lines, PDOs, and mouse models.

Project description:Acute graft-versus-host disease (GvHD) remains the biggest clinical challenge and prognosis-determining complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T cells are acceptedly key mediators of alloreactivity against host tissues and here especially the gut. In support of previous studies, we found that the intestinal intra-epithelial lymphocyte (IEL) compartment was dynamically regulated in the course of MHC class I full mismatch allo-HSCT. However, while intestinal epithelial cell (IEC) damage endangers the integrity of the intestinal barrier and is a core signature of intestinal GvHD, the question whether and to what degree IELs are contributing to IEC dysregulation is poorly understood. To study lymphoepithelial interaction, we employed a novel ex vivo T cell/organoid co-culture model system. Here, allogeneic intra-epithelial T cells were superior in inducing IEC death compared to syngeneic IEL and allogeneic non-IEL T cells. The ability to induce IEC death was predominately confined to TCRβ+ T cells and was executed in a largely IFNγ-dependent manner. Alloreactivity required a diverse T cell receptor (TCR) repertoire since IELs genetically modified to express a TCR restricted to a single, non-endogenous antigen failed to mediate IEC pathology. Interestingly, minor histocompatibility antigen (miHA) mismatch was sufficient to elicit IEL-driven IEC damage. Finally, advanced live cell imaging analyses uncovered that alloreactive IELs patrolled smaller areas within intestinal organoids compared to syngeneic controls, indicating their unique migratory properties within allogeneic IECs. Together, we provide here experimental evidence for the utility of a co-culture system to model the cellular and molecular characteristics of the crosstalk between IELs and IEC in an allogeneic setting ex vivo. In the light of the emerging concept of dysregulated immune-epithelial homeostasis as a core aspect of intestinal GvHD, this approach represents a novel experimental system to e.g. screen therapeutic strategies for their potential to normalize T cell/IEC- interaction. Hence, analyses in pre-clinical in vivo allo-HSCT model systems may be restricted to hereby positively selected, promising approaches.

Dataset Information

A Microwell-Based Intestinal Organoid-Macrophage Co-Culture System to Study Intestinal Inflammation.

Publications

A Microwell-Based Intestinal Organoid-Macrophage Co-Culture System to Study Intestinal Inflammation.

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