Background:
Bladder cancer (BCa) is the most frequent malignancy in the urinary tract. Despite great progress in our understanding of BCa in the past decades, we still lack significant improvement in the development of new BCa chemotherapeutics, which is largely attributed to the fact that 2D tumor models are used as the predominant platform for cell-based assays.
The conventional 2D tumor models, although simple and convenient, fail to recreate an in vivo-like tumor microenvironment (TME). 3D tumor models more faithfully recapitulate the complexity of TME than 2D-based models. 3D-organoid models reproduce many biological characteristics of the real solid tumor, including biochemical gradients, different proliferating states, complex cell-cell interaction, ECM deposition, and chemo-resistance, thus providing a promising tool in tumor biology research.
There have been many studies about the patient-derived BCa organoids. However, we still lack a research about the development and characterization of multiple bladder organoid models that mimicked both normal bladder tissue and bladder tumors representing the entire range of malignant grades.
Aims:
The study aimed to investigate the formation and characteristics of hetero-typed bladder organoids derived from the three major cell types in the human bladder, including either non-cancerous urothelial cells or different bladder cancer cell lines.
Hypotheses:
1. Bladder-like organoids can formed by self-organization from mixed bladder cell suspension
2. The degree of histological organization depends on the urothelial cells used
3. The different cell types can be identified in the bladder organoids by immunohistochemistry
Materials and Methods:
We used RT4, RT112, T24, and CAL29 cells (transitional cancer cell lines, histological grade: G1-G4), non-malignant HBLAK (bladder epithelium progenitors), primary human bladder fibroblasts (hBF), and human bladder smooth muscle cells (hBSMC) in this study. The following figure displays the construction process of hetero-typed bladder organoids by ultra-low attachment (ULA) 96-well microplate method.
At day 4 post seeding, bladder organoids were harvested, fixed, processed, and sectioned. Then, we characterized the bladder organoids by histology and immunohistochemistry (IHC) staining. Bladder organoids were stained for panCK, Vimentin, α-SMCA, CK7, CK13, CK20, Ki67, Claudin4, ZO-1, and fibronectin and the immunoreactivity (IR) of specific antigen was analyzed using ImageJ plus IHC profiler plugin. We also did H&E and Crossmon staining to investigate the histology and ECM deposition of the bladder organoids.
Results:
Mixed cell suspensions self-organized into compact organoids in the ULA plate within 24 hours after seeding. At day 4 post seeding, the organoids were grown to 650-1,000 μm in diameter, with BCa organoids significantly bigger than HBLAK organoids.
The morphology of bladder organoids greatly varied depending on the urothelial cells used. Besides, urothelial cells were mainly located in the periphery and supportive cells (hBF and hBSMC) were in the core of the organoids. High-grade RT112, T24 and CAL29 organoids showed significantly thicker urothelial cell layers and more urothelial cells in the periphery than low-grade RT4 organoids and non-malignant HBLAK organoids.
The CK7, CK13 and CK20-IR greatly varied between the organoid-cultured urothelial cells. HBLAK cells showed significant lower panCK, CK7, CK13 and CK20-IR than BCa cells. CK-IR was higher in RT4 and RT112 cells than in T24 cells. CAL29 organoids showed a stratified layering: CK7-IR in superficial cells, CK13-IR in intermediate cells, and CK20 in all cells.
The urothelial cells in organoid culture consisted of not only proliferating cells but also a large portion of quiescent cells. High-grade RT112, T24 and CAL29 cells (30-50%) showed a significantly higher proliferation index than the low-grade RT4 (2%) and non-malignant HBLAK (6.6%) cells.
All urothelial cells in organoid culture expressed Claudin4(CLDN4) positively. In addition, high-grade T24 and CAL29 cells showed higher Claudin4-IR than low-grade RT4 and RT112 cells. In contrast, all bladder organoids showed very low ZO-1-IR.
Fibronectin and Crossmon staining indicated fibronectin, collagen, and reticular fibers deposition in the bladder organoids. Besides, high-grade T24 and CAL29 cells showed remarkably higher fibronectin-IR than low-grade RT4 and RT112 cells.
Discussion:
All urothelial cells were able to form compact and reproducibly sized organoids with hBSMC and hBF by spontaneous cell aggregation in ULA 96-well plate.
The compactness of bladder organoids might reflect the adhesion between the cells and the expression of underlying adhesion molecules. The size of organoids and number of urothelial cells in the periphery could be influenced by multiply factors. The organoids formed a bladder-like structure with outside urothelial cells and a core of supportive cells, indicating they could be useful tools in the testing of anti-cancer drugs.
The cytokeratin expression profiles reflect the differentiation status of tumors. All urothelial cells retained the CK7/CK13/CK20 expression patterns of their original tissues in organoid culture, supporting that the bladder organoids mimicked both the normal bladder urothelium and bladder tumors of different grades. In addition, RT112 and CAL29 organoids formed a stratified urothelium.
Urothelial cells in organoid culture were at different proliferation stages, better mimicking the in vivo bladder tumors in terms of proliferation and cell heterogeneity than 2D-based BCa models. Besides, the organoid-cultured urothelial cells retained the proliferation characteristics of their original tissues.
Bladder organoids showed abundant fibronectin deposition, which could affect their response to drug treatments. Besides, the fibronectin expression level in BCa cells was correlated with their primary origins, supporting the view that the BCa cells in organoid culture retained the invasive and metastatic feature of their parental bladder tumors. The fibronectin, collagen, and reticular fibers deposition indicated bladder organoids mimicked the in-situ situation of bladder tumors in respect to ECM deposition.
The expression of CLDN4 in urothelial cells indicated the formation of para-cellular barriers in the bladder organoids, which could limit the penetration and diffusion of anticancer drugs into bladder organoids. Especially, the RT112 and CAL29 organoids showed high expression of CLDN4 on the apical membrane, indicating that they could be helpful in the investigation of drug penetration into tumor tissues.
The ULA microplate method is easy, fast, and suitable for massive production of reproducibly sized organoids. The imageJ plus IHC profiler plugin method was able to perform fast and automatic quantitative analysis for DAB-stained IHC images and comparisons of the expression of specific antigens in formalin-fixed tissues
Conclusion:
The bladder organoids represented a bladder-like architecture by self-organization, with a peripheral urothelium surrounding a supportive core of hBF and SMCs. These organoids exhibit characteristics of the in situ normal urothelium and bladder tumors of different grades in cell composition, proliferation, stratified urothelium, epithelial diferentiation, and ECM deposition. Thus, they can be useful tools in cancer biology research and anti-cancer drug development.:1. Title page
2. Table of contents
3. List of Abbreviations
4. Introduction
4.1 Bladder cancer
4.2 Currently available bladder cancer models
4.3 Advantages of 3D cell culture over 2D cell culture
4.4 3D tumor spheroid/organoid models
4.5 Spheroids/organoids construction methods
4.6 Application of 3D organoid culture in bladder cancer research
4.7 Limitations of previous research of bladder cancer organoids
5. Aims
5.1 Hypothesis
5.2 Tasks
6. Materials and Methods
6.1 Cell lines used in the study
6.2 Monolayer culture of the cell lines
6.3 Bladder organoids construction by ULA microplate method
6.4 Fixation, processing, sectioning, and size measurement
6.5 Histology and immunohistochemistry
6.6 Image acquisition
6.7 Image analysis with ImageJ plus IHC profiler plugin
6.8 Statistical analysis
7. Results
7.1 Spontaneous formation of packed bladder organoids in the ULA plate
7.2 Bladder-like self-organization of the organoids
7.3 Immunohistochemical characterization of the organoids
7.4 Expression of urothelial cell differentiation makers
7.5 Proliferation of urothelial cells in 3D organoid culture
7.6 Tight junction protein expression in the organoids
7.7 ECM deposition in bladder organoids
8. Discussion
8.1 Organoid models in cancer research
8.2 Morphology and Size
8.3 Bladder-like internal structure
8.4 Formation of a stratified urothelium in bladder organoids
8.5 Proliferation characteristics of the urothelial cells in organoid culture
8.6 ECM deposition in bladder organoids
8.7 TJ protein expression in bladder organoids
8.8 Possible application of bladder organoids
8.9 Review of methods used in this project
8.10 Limitations of the research
9. Summary of the work
10. References
11. Appendix
12. Declaration of independence
13. Curriculum vitae
14. Acknowledgment
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:82260 |
| Date | 17 November 2022 |
| Creators | Han, Shanfu |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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