cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Preparations
2.2. Cell Culture
2.3. Plate Reader Binding Assay
2.4. Ex vivo Incubation of Tissue Specimens with GST-cCPE
2.5. Generation and Characterization of Patient-Derived Gastric Cancer Organoids
2.5.1. Gastric Tissue Processing
2.5.2. Gastric Organoid Culture
2.6. Live-Cell Imaging
2.6.1. Live-Cell Imaging of HT-29/B6 with AF647-GST-cCPE and YFP-cCPE
2.6.2. Live-Cell Imaging of Colonic Ulcers
2.6.3. Live-Cell Imaging of Gastric Organoids
2.7. Histology and Immunochemistry
2.8. Quantification of cCPE Binding in Whole Mount Samples
2.9. Flow Cytometry
2.9.1. Flow Cytometry for Assessing YFP-cCPE Binding
2.9.2. Flow Cytometry of Gastric Organoids
2.10. Targeted DNA Sequencing
3. Results
3.1. YFP-cCPE-wt and YFP-cCPE-SSS Bind Specifically to CLDN4 and Other Claudins on the Cell Surface
3.2. cCPE Fusion Proteins Visualize Non-Junctional Claudins and TJ Diffusion Barriers in Colonocyte Islands
3.3. Use of cCPE Variants for Detection of Delocalized Non-Junctional Claudins in Ex Vivo Models of Rat and Human Colon Tissue
3.4. Use of YFP-cCPE to Probe the Expression, Polar Localization and Dysregulation of Claudins in Human Gastric Cancer Organoids
3.4.1. Establishment of Patient-Derived Gastric Organoids as a 3D Testing Platform for Cancer-Related Dedifferentiation
3.4.2. Characterization of Epithelial Differentiation in Gastric Tissue
3.4.3. Claudin Expression, as Well as Differential Cell Polarity and Junctional Integrity, is Largely Conserved between Parental Tissues and Organoids
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Patient | Histology | Subtype | Morphology | T | N | M | G | R | L | V | Neoadjuvant Therapy | Tumor Regression |
---|---|---|---|---|---|---|---|---|---|---|---|---|
GC_1 | AC | Intestinal (Lauren) | Glandular, regressive changes | 3 | 1 | 1 | - | 0 | 0 | 0 | Yes | 1b |
GC_2 | AC + high grade dysplasia | Mucinous (WHO 2019) | Mucinous | 1b | 0 | 0 | 3 | 0 | 0 | 0 | No | - |
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Waldow, A.; Beier, L.-S.; Arndt, J.; Schallenberg, S.; Vollbrecht, C.; Bischoff, P.; Farrera-Sal, M.; Loch, F.N.; Bojarski, C.; Schumann, M.; et al. cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids. Pharmaceutics 2023, 15, 1980. https://doi.org/10.3390/pharmaceutics15071980
Waldow A, Beier L-S, Arndt J, Schallenberg S, Vollbrecht C, Bischoff P, Farrera-Sal M, Loch FN, Bojarski C, Schumann M, et al. cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids. Pharmaceutics. 2023; 15(7):1980. https://doi.org/10.3390/pharmaceutics15071980
Chicago/Turabian StyleWaldow, Ayk, Laura-Sophie Beier, Janine Arndt, Simon Schallenberg, Claudia Vollbrecht, Philip Bischoff, Martí Farrera-Sal, Florian N. Loch, Christian Bojarski, Michael Schumann, and et al. 2023. "cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids" Pharmaceutics 15, no. 7: 1980. https://doi.org/10.3390/pharmaceutics15071980