Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

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 (Trp53^f/f: Pten^f/f: Rb1^f/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.......

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.......

Materials

Name	Company	Catalog Number	Comments
100 μm sterile cell strainer	Corning	431752
1 mL syringe	BD	309659
25G 1.5 inches needle	EXELINT International	26406
Adenovirus (Ad5CMVCre High Titer, 1E11 pfu/ml)	UI Viral Vector Core	VVC-U of Iowa-5-HT
C57 BL/6J	Jackson Lab	000664
Charcoal-stripped FBS	Gibco	A3382101
Collagenase/hyaluronidase	Stemcell Technologies	07912
Dispase	Stemcell Technologies	07913
DPBS, 1x	Corning	21-031-CV
L-glutamine substitute (GlutaMAX)	Gibco	35-050-061
Mammary Epithelial Cell Growth medium	Lonza	CC-3150
Matrix extracts from Engelbreth–Holm–Swarm mouse sarcomas (Matrigel)	Corning	CB-40234
Monoclonal mouse anti-CK20	DAKO	M7019	IF 1:100
Monoclonal mouse anti-CK7	Santa Cruz Biotechnology	SC-23876	IHC 1:50
Monoclonal mouse anti-p63	Abcam	ab735	IHC 1:100, IF 1:50
Monoclonal mouse anti-Upk3	Fitzgerald	10R-U103A	IHC 1:50
Monoclonal mouse anti-Vimentin	Santa Cruz Biotechnology	SC-6260	IF 1:100
Monoclonal rat anti-CK8	Developmental Studies Hybridoma Bank	TROMA-I-s	IF 1:100
HERAcell vios 160i CO2 incubator	Thermo Fisher	51033557
Polyclonal chicken anti-CK5	Biolegend	905901	IF 1:500
Primocin	InvivoGen	ant-pm-1
Recombinant enzyme of trypsin substitute (TrypLE Express Enzyme)	Thermo Fisher	12605036
Signature benchtop shaking incubator Model 1575	VWR	35962-091
Specimen Processing Gel (HistoGel)	Thermo Fisher	HG-4000-012
Surgical blade size 10	Integra Miltex	4-110
Sorvall T1 centrifuge	Thermo Fisher	75002383
Y-27632	Selleckchem	S1049