Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization—A New Standardized Approach in Bladder Cancer Research
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Generation of Standardized BCa Organoids
2.3. Wnt Ligand Treatment
2.4. Histology and DAB Immunostaining
2.5. Immunofluorescence
2.6. Drug and Radiation Response
2.7. Statistical Analysis
3. Results
3.1. Morphological Characterization of BCa Orgs
3.1.1. Bladder-like Self-Organization
3.1.2. Urothelial Differentiation
3.1.3. Proliferation Status
3.2. Ligand-Dependent Wnt Pathway Activation
3.3. Response to Anti-Tumor Therapies
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Cell Line | Acc.-No. | Origin (Gender, Age, Stage) | Invasiveness | Grade | Subtype |
---|---|---|---|---|---|
RT-4 | ACC-412 | male, 63 y, T2 | NMIBC | 1 | luminal |
RT-112 | ACC-418 | female, n.n., n.n. | NMIBC | 2 | luminal |
T-24 | ACC-376 | female, 81 y, n.n. | MIBC | 3 | mixed |
CAL-29 | ACC-515 | female, 80 y, T2 | MIBC | 4 | luminal |
Antigen | Specificity | Host | Source | Cat.-No. | Dilution | |
---|---|---|---|---|---|---|
Cytokeratin Pan | panCK | Epithelial cells | Ms | Sigma-Aldrich, Munich, Germany | C2931 | 1:400 |
Vimentin | VIM | VIM-positive cells, e.g., fibroblasts | Ms | Sigma-Aldrich, Munich, Germany | V6389 | 1:400 |
Alpha Smooth muscle cell actin | αSMCA | αSMCA-positive cells, e.g., detrusor myocytes | Ms | Sigma-Aldrich, Munich, Germany | A2547 | 1:1000 |
Cytokeratin 7 | CK7 | Poorly differentiated epithelial cells | Ms | Abcam, Cambridge, UK | ab9021 | 1:400 |
Cytokeratin 13 | CK13 | Moderately differentiated epithelial cells | Ms | Abcam, Cambridge, UK | ab101001 | 1:50 |
Cytokeratin 20 | CK20 | Well-differentiated epithelial cells | Rb | Abcam, Cambridge, UK | Ab53120 | 1:100 |
Uroplakin-II | UPL-II | Terminally differentiated urothelial cells | Rb | Novus Biologicals, Abingdon, UK | NBP2-33389 | 1:50 |
Uroplakin-III | UPL-III | Terminally differentiated urothelial cells | Ms | Abcam, Cambridge, UK | ab78196 | 1:30 |
KI67 | Nuclei of proliferating cells | Ms | Dako, Glostrup, Denmark | M0722 | 1:50 | |
Claudin 4 | CL4 | Tight junction protein CL4 | Rb | Abcam, Cambridge, UK | ab15104 | 1:400 |
Zonula occludens-1 | ZO-1 | Tight junction protein ZO-1 | Ms | BD Biosciences, Franklin Lakes, NJ, USA | 610966 | 1:200 |
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Berndt-Paetz, M.; Han, S.; Weimann, A.; Reinhold, A.; Nürnberger, S.; Neuhaus, J. Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization—A New Standardized Approach in Bladder Cancer Research. Biomedicines 2023, 11, 2958. https://doi.org/10.3390/biomedicines11112958
Berndt-Paetz M, Han S, Weimann A, Reinhold A, Nürnberger S, Neuhaus J. Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization—A New Standardized Approach in Bladder Cancer Research. Biomedicines. 2023; 11(11):2958. https://doi.org/10.3390/biomedicines11112958
Chicago/Turabian StyleBerndt-Paetz, Mandy, Shanfu Han, Annett Weimann, Annabell Reinhold, Sandra Nürnberger, and Jochen Neuhaus. 2023. "Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization—A New Standardized Approach in Bladder Cancer Research" Biomedicines 11, no. 11: 2958. https://doi.org/10.3390/biomedicines11112958
APA StyleBerndt-Paetz, M., Han, S., Weimann, A., Reinhold, A., Nürnberger, S., & Neuhaus, J. (2023). Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization—A New Standardized Approach in Bladder Cancer Research. Biomedicines, 11(11), 2958. https://doi.org/10.3390/biomedicines11112958