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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Abstract

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Introduction

Bladder cancer is the fourth most common cancer in men and affects more than 80,000 people annually in the United States1. Platinum-based chemotherapy has been the standard of care for patients with advanced bladder cancer for more than three decades. The landscape of bladder cancer treatment has been revolutionized by the recent Food and Drug Administration (FDA) approval of immunotherapy (anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors), erdafitinib (a fibroblast growth factor receptor inhibitor) and enfortumab vedotin (an antibody-drug conjugate)2,3,

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Roswell Park Comprehensive Cancer Center, Buffalo, NY (1395M, Biosafety 180501 and 180502).
NOTE: Perform Steps 1-3 on the same day.

1. Dissection of mouse bladder

  1. Preparation for dissection
    1. Prepare all the sterile instruments including scissors, forceps, sterile DPBS in 35 mm culture dishes, 70% ethanol, sterile gauze, and clean paper towels. Clean a.......

Representative Results

The workflow of adenovirus-Cre mediated gene deletions in mouse urothelial cells is shown in Figure 1A. The accompanying video demonstrates how the urothelial cells are disassociated from the fundus of the bladder and how the triple floxed cells are transduced ex vivo with Ad5CMVCre. Figure 1A showed that minimal submucosa and muscle cells were disassociated after enzyme digestion. To confirm the efficiency of adenovirus-Cre delivery, disassociated urot.......

Discussion

GEMMs have been the gold standards for cancer modeling initiated from normal cells, allowing the consequences of potential oncogenic perturbations (oncogene activation and/or loss of tumor suppressors) to be tested rigorously. Here, a rapid and efficient protocol is provided to generate bladder cancer organoids via ex vivo gene editing of normal mouse urothelial cells carrying floxed alleles in genes of interest. H&E and IHC staining demonstrate that TKO organoids exhibit histology consistent with high-grade.......

Acknowledgements

This research work was supported in part by NIH Grants, K08CA252161(Q.L.), R01CA234162, and R01 CA207757 (D.W.G.), P30CA016056 (NCI Cancer Center Core Support Grant), the Roswell Park Alliance Foundation, and the Friends of Urology Foundation. We thank Marisa Blask and Mila Pakhomova for proofreading the manuscript.

....

Materials

NameCompanyCatalog NumberComments
100 μm sterile cell strainerCorning431752
1 mL syringeBD309659
25G 1.5 inches needleEXELINT International26406
Adenovirus (Ad5CMVCre High Titer, 1E11 pfu/ml)UI Viral Vector CoreVVC-U of Iowa-5-HT
C57 BL/6JJackson Lab000664
Charcoal-stripped FBSGibcoA3382101
Collagenase/hyaluronidaseStemcell Technologies07912
DispaseStemcell Technologies07913
DPBS, 1xCorning21-031-CV
L-glutamine substitute (GlutaMAX)Gibco35-050-061
Mammary Epithelial Cell Growth mediumLonzaCC-3150
Matrix extracts from Engelbreth–Holm–Swarm mouse sarcomas (Matrigel)CorningCB-40234
Monoclonal mouse anti-CK20DAKOM7019IF 1:100
Monoclonal mouse anti-CK7Santa Cruz BiotechnologySC-23876IHC 1:50
Monoclonal mouse anti-p63Abcamab735IHC 1:100, IF 1:50
Monoclonal mouse anti-Upk3Fitzgerald10R-U103AIHC 1:50
Monoclonal mouse anti-VimentinSanta Cruz BiotechnologySC-6260IF 1:100
Monoclonal rat anti-CK8Developmental Studies Hybridoma BankTROMA-I-sIF 1:100
HERAcell vios 160i CO2 incubatorThermo Fisher51033557
Polyclonal chicken anti-CK5Biolegend905901IF 1:500
PrimocinInvivoGenant-pm-1
Recombinant enzyme of trypsin substitute (TrypLE Express Enzyme)Thermo Fisher12605036
Signature benchtop shaking incubator Model 1575VWR35962-091
Specimen Processing Gel (HistoGel)Thermo FisherHG-4000-012
Surgical blade size 10Integra Miltex4-110
Sorvall T1 centrifugeThermo Fisher75002383
Y-27632SelleckchemS1049

References

  1. Siegel, R. L., Miller, K. D., Fuchs, H. E., Jemal, A. Cancer statistics, 2021. CA: A Cancer Journal for Clinicians. 71 (1), 7-33 (2021).
  2. Zhu, S., et al. Preclinical models for bladder cancer research. Hematology/Oncology Clinics of North America

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