cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids

Author:

Waldow Ayk1,Beier Laura-Sophie12,Arndt Janine34,Schallenberg Simon5ORCID,Vollbrecht Claudia5,Bischoff Philip567ORCID,Farrera-Sal Martí3ORCID,Loch Florian N.8ORCID,Bojarski Christian9,Schumann Michael9,Winkler Lars10ORCID,Kamphues Carsten11,Ehlen Lukas34,Piontek Jörg1ORCID

Affiliation:

1. Clinical Physiology/Nutritional Medicine, Medical Department, Division of Gastroenterology, Infectiology, Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany

2. Laboratory of Mucosal Barrier Pathobiology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA

3. Berlin Institute of Health (BIH), Charité—Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), 13353 Berlin, Germany

4. Department of Anesthesiology and Intensive Care Medicine, Charité—Universitätsmedizin Berlin, 10117 Berlin, Germany

5. Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Berlin Institute of Health, Institute of Pathology, 10117 Berlin, Germany

6. Berlin Institute of Health, Charité—Universitätsmedizin Berlin, 10178 Berlin, Germany

7. German Cancer Consortium (DKTK), Partner Site Berlin, and German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

8. Department of General and Visceral Surgery, Campus Benjamin Franklin, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany

9. Medical Department, Division of Gastroenterology, Infectiology, Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany

10. Experimental Pharmacology & Oncology Berlin-Buch GmbH, 13125 Berlin, Germany

11. Park-Klinik Weißensee, Department of General-Visceral and Minimally-Invasive Surgery, 13086 Berlin, Germany

Abstract

Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.

Funder

Deutsche Forschungsgemeinschaft

Stiftung Oskar-Helene-Heim

Publisher

MDPI AG

Subject

Pharmaceutical Science

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