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)^2,3,4. However, no clinically approved biomarkers are available for predicting the responses to chemotherapy or immunotherapy. There is a critical need to generate informative preclinical models that can improve the understanding of the mechanisms driving bladder cancer progression and develop predictive biomarkers for different treatment modalities.

A major obstacle in bladder translational research is the lack of preclinical models that recapitulate human bladder cancer pathogenesis and treatment responses^5,6. Multiple preclinical models have been developed, including in vitro 2D models (cell lines or conditionally reprogrammed cells), in vitro 3D models (organoids, 3D printing), and in vivo models (xenograft, carcinogen-induced, genetically engineered models, and patient-derived xenograft)^2,6. Genetically engineered mouse models (GEMMs) are useful for many applications in bladder cancer biology, including analyses of tumor phenotypes, mechanistic investigations of candidate genes and/or signaling pathways, and the preclinical evaluation of therapeutic responses^6,7. GEMMs can utilize site-specific recombinases (Cre-loxP) to control genetic deletions in one or more tumor suppressor genes. The process of generating desired GEMMs with multiple gene deletions is time-consuming, laborious, and expensive⁵. The overall goal of this method is to develop a rapid and efficient method of ex vivo Cre delivery for establishing bladder triple knockout (TKO) models from normal mouse urothelial cells carrying triple floxed alleles (Trp53, Pten, and Rb1)⁸. The major advantage of the ex vivo method is the fast workflow (1-2 weeks instead of years of mouse breeding). This article describes the protocol for harvesting normal urothelial cells with floxed alleles, ex vivo adenovirus transduction, organoid cultures, and in vitro and in vivo characterization in immunocompetent C57 BL/6J mice. This method can be further used to generate clinically relevant bladder cancer organoids in immunocompetent mice harboring any combination of floxed alleles.

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 all surfaces of the dissection area. Generate male triple floxed mice Trp53^f/f: Pten^f/f: Rb1^f/f (4 mice, 10 weeks old), as previously described in Dr. Goodrich's lab ⁸.
2. Euthanasia and incision
   1. Euthanize the mice by CO₂ asphyxiation using a 2.0 L/min flow rate, followed by cervical dislocation. Clean the dissection area with 70% ethanol and cover with a clean paper towel.
   2. Place the mouse on gauze in the dissection area. Spray 70% ethanol on the mouse abdomen and use sterile dissection scissors to make a lower midline abdominal incision exposing the bladder.
3. Dissecting the bladder
   1. Use forceps to grasp the bladder and gently pull up so that bilateral ureterovesical junctions are identified. Use scissors to remove the bladder fundus following the boundary line between the two ureter openings.
   2. Transfer the bladder into a sterile dish with 2 mL of DPBS. Remove fatty and connective tissue and put the bladder into a new dish with sterile 2 mL of DPBS. At this time, take the adenovirus out from -80 °C storage and keep it on ice.

2. Dissociation of urothelial cells

1. Preparation for cell dissociation
   1. Prepare all sterile instruments, including forceps, surgical scalpels (a size 10 blade with handle), sterile DPBS in 35 mm culture dishes, complete culture medium without charcoal-stripped FBS defined as CCM(-), collagenase/hyaluronidase mixture, recombinant enzyme for trypsin substitute, 10% charcoal-stripped FBS in DPBS with 10 µM fresh Y-27632, a 100 µm sterile cell strainer, and a 15 mL centrifuge tube.
      NOTE: The complete culture medium (CCM) comprises mammary epithelial cell growth medium supplemented with the provided growth factors and 5% charcoal-stripped FBS, 1% L-glutamine substitute, 100 µg/mL primocin, and 10 µM fresh Y-27632.
2. Mincing and digesting the bladder
   1. Transfer and wash the bladder again with 2 mL of sterile DPBS in a 35 mm dish in a biosafety cabinet.
   2. Collect bladder tissues from four mice into a dish with 1 mL of CCM(-) and mince each bladder into four equal pieces using a surgical blade. Use forceps to unfold the bladder to expose the urothelium surface to the medium.
   3. Transfer the bladder pieces and medium into a 15 mL centrifuge tube. Wash the dish with 2 mL of CCM(-) 2x and collect it in a centrifuge tube to a total volume of 5 mL.
   4. Add collagenase/hyaluronidase mixture to a final concentration of 300 U/mL collagenase and 100 U/mL hyaluronidase and place the tube horizontally in a 37 °C orbital incubating shaker for 30 min at 200 rpm.
   5. After tissue digestion, add an equal volume (5 mL) of 10% charcoal-stripped FBS in DPBS into the tube and centrifuge the tube at 200 x g for 5 min.
   6. Remove and discard the supernatant. Add 2 mL of trypsin substitute into the cell pellet and mix well. Put the tube in a cell incubator at 37 °C and 5% CO₂ for 4 min.
   7. After incubation, pipette up and down 10x with a standard P1000 tip. Add 5 mL of 10% charcoal-stripped FBS in DPBS.
   8. Strain the disassociated cells through a 100 µm sterile cell strainer. Collect the cell suspension and centrifuge at 200 x g for 5 min.
3. Counting and resuspending cells
   1. Remove and discard the supernatant. Resuspend the cell pellet with 1 mL of CCM.
   2. Count the number of cells using a hemocytometer and only plate 0.5 x 10⁶ cells into a well of a 24-well plate with 0.5 mL of fresh CCM. At this time, take out the matrix extracts of Engelbreth-Holm-Swarm mouse sarcoma cells (see Table of Materials) from -20 °C storage and thaw on ice. Pre-warm a 6-well plate in a 37 °C cell incubator.
      NOTE: The remaining cells can be centrifuged and frozen as cell pellets for non-virus transduction control.

3. Adenoviral transduction and plating organoids

CAUTION: Please be cautious when processing adenovirus. Laboratory personnel should follow biosafety level 2 practices and disinfect adenovirus with 10% bleach.

1. Add 2 µL of adenovirus (Ad5CMVCre; 1 x 10⁷ PFU/µL) into the 24-well plate. Mix well with the disassociated urothelial cells.
2. To enhance transduction efficacy, perform spinoculation by centrifuging the 24-well plate at 300 x g for 30 min at room temperature. Place the 24-well plate in a cell incubator at 37 °C and 5% CO₂ for 1 h.
3. Transfer cells and medium from the well into a 15 mL tube and centrifuge at 200 x g for 5 min. Remove and discard the supernatant, add 50 µL of CCM, and mix well with the cells at the bottom.
4. Add 140 µL of matrix extract and gently mix well to avoid air bubbles. Add the solution quickly into the pre-warmed 6-well plate at 50 µL/dome to create domes for organoids.
5. Transfer the plate into a 37 °C incubator with 5% CO₂ gently and allow the domes to solidify for 5 min. Flip the 6-well plate upside-down and continue solidification for an additional 25 min.
6. After incubation, add 2.5 mL of CCM to each well and place the plate in an incubator for organoid culture.

4. Organoid culturing and passaging

1. Change the medium every 3-4 days. Perform organoid culturing, passaging, and freezing as reported in Lee et al.⁹. Perform embedding of organoids using specimen processing gel as described in Fujii et al.¹⁰.
2. Remove the medium and add 1 mL of dispase to each well. Gently break the dome and mix well with a P1000 pipette tip.
3. Place the plate in a 37 °C incubator with 5% CO₂ for 30 min. Then, collect all the cells in a 15 mL centrifuge tube and wash the well with 4 mL of 10% charcoal-stripped FBS in DPBS. If the organoid cells appear as large clusters, perform Step 2.2.6. and Step 2.2.7. to get disassociated cells.
4. Centrifuge the tube at 200 x g for 5 min. Remove the supernatant and wash the cells with 1 mL of CCM.
5. Resuspend the cells in 70% matrix extract and follow Steps 3.4.-3.6. for culturing. Alternatively, cryopreserve the cells by resuspending in 90% charcoal-stripped FBS and 10% DMSO supplemented with 10 µM fresh Y-27632.

5. Organoid cells implantation into C57BL/6J mouse subcutaneously

1. Preparation for implantation
   1. Prepare 25G 1.5 in needles, a 1 mL syringe, 1 mg/mL dispase, matrix extract, 10% charcoal-stripped FBS in DPBS with 10 µM fresh Y-27632, and a 15 mL centrifuge tube.
2. Organoid cell collection
   1. After achieving a stable culture for at least 5 passages, collect the expanded organoid cells for in vivo injection. Follow Steps 4.2.-4.4. and resuspend cells in 1 mL of CCM.
3. Counting cells and subcutaneous injection
   1. Count the number of cells using a hemocytometer. After centrifugation at 200 x g for 5 min, resuspend 2 x 10⁶ cells in 100 µL of 50% matrix extract in DPBS. Place the suspension on ice and take it into a biosafety cabinet in an animal procedure room.
   2. Anesthetize two male C57BL/6J mice (10 weeks old) with 4% isoflurane and 1 L/min O₂ flow for approximately 4 min in a chamber. Once the mice have lost their righting reflex and their breathing pattern has become deeper and slower, transfer the mice to a non-rebreathing circuit with 2% isoflurane and 1 L/min O₂ flow. Apply vet ointment to protect the animals' eyes from traumatic injury.
   3. Shave one area around the injection site at the mouse's right flank and use 70% ethanol to cleanse, and then use a 25 G needle to directly inject the 100 µL cell suspension into the right flank under anesthesia.
   4. After injection, remove the inhalational anesthesia and place the mice in a cage under a heat lamp until recovered and mobilizing fully. Return the mice for housing and monitor tumor formation.

6. Tumor collection and in vivo propagation

1. Follow the preparation Step 2.1.1. Euthanize mice by CO₂ asphyxiation using a 2.0 L/min flow rate followed by cervical dislocation after a tumor of ~2.0 cm diameter (nearly 2-3 weeks) is formed. Transfer the mice to a clean dissection area, spray 70% ethanol on the mouse flank tumor area, and use sterile dissection scissors and forceps to make an incision to separate the tumor.
2. Wash the tumor in a 60 mm dish with 5 mL of DPBS and use scissors to remove the connective tissue. Cut one piece of the fresh tumor and sink it with 4% paraformaldehyde in DPBS for 48 h and send for paraffin embedding and sectioning for histology analysis.
3. Process the other half of the fresh tumor for cell disassociation. Briefly, mince the fresh tumor into 1 mm³ pieces for dissociation in a 60 mm dish with 5 mL of CCM(-). Then, after following Steps 2.2.4.-2.2.8., inject the disassociated cells into new C57BL/6J mice for in vivo passaging following Step 5.3. or keep as in vitro organoid culture following Steps 3.4.-3.6., or freeze directly in liquid nitrogen using 90% charcoal-stripped FBS and 10% DMSO with 10 µM fresh Y-27632.
4. If needed, recover the frozen cells by thawing them rapidly in a 37 °C water bath and washing with CCM(-). After this, resuspend them in 100 µL of 50% matrix extract in DPBS for direct injection into mice for in vivo tumor formation. Use at least 2 x 10⁶ thawed cells for one in vivo injection.

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 urothelial cells from triple floxed mice with membrane-targeted tdTomato/membrane-localized enhanced green fluorescent protein (mT/mG) were transduced with adenovirus Cre. Green fluorescent protein (GFP) was detected in nearly 100% of cells, indicating high efficiency of adenovirus transduction (Figure 1B).

The stable TKO organoids were established following standard organoid protocols (in vitro with more than 5 passages; Figure 2A). H&E and immunofluorescence (IF) of in vitro organoid sections (Figure 2A) demonstrated a morphology of high-grade urothelial carcinoma with positive expression of urothelial lineage markers CK5 and p63¹¹. PCR analysis revealed recombined alleles in the TKO organoids, whereas unrecombined floxed alleles were only detected in urothelial cells untreated with adenovirus (Figure 2B). A total of 2 x 10⁶ primary ex vivo organoids were injected into C57BL/6J for initial subcutaneous (SQ) tumor formation in 8 weeks. Then, SQ tumor (passage 1) was collected and passaged in mice. In general, 2-3 weeks were needed to form a 2 cm SQ tumor (passages 2-5) (Figure 2C). The immunohistochemistry (IHC) of TKO in vivo xenograft displayed positive expression of CK7 (patchy), CK5, and p63 and negative expression of CK8 and Uroplakin 3 (Figure 2D). To detect contamination of mesenchyme cells, the TKO tumors were stained for vimentin. H&E stain showed the tumor on the left and the capsule on the right demarcated by the yellow line. Positive vimentin was detected only in the tumor capsule or stroma (Figure 2E).

Figure 1: Adenovirus-Cre mediated gene deletions in mouse urothelial cells with loxP sites. (A) The workflow of TKO organoid generation via ex vivo adenovirus Ad5CMVCre. A short-time 30 min enzyme digestion was sufficient to disassociate urothelial cells from the underlying submucosa and muscularis propria. The disassociated urothelial cells were transduced with Ad5CMVCre for TKO organoids and subsequently injected into C57BL/6J mice. (B) Triple floxed urothelial cells with mT/mG (constitutive transgene expression of red fluorescent protein that converts to green fluorescent protein following Cre recombination) were transduced with Ad5CMVCre. Nearly 100% of cells were GFP positive, indicating highly efficient transduction and Cre recombination. Please click here to view a larger version of this figure.

Figure 2: Characterization of TKO organoids via ex vivo adenovirus-Cre recombinase. (A) The disassociated Trp53^f/f: Pten^f/f: Rb1^f/f urothelial cells were transduced with adenovirus (Ad5CMVCre) to generate TKO organoids (bright field). The TKO organoids were embedded with speciment processing gel and stained for H&E and immunofluorescence. (B) Genomic DNA was extracted from triple floxed urothelial cells (lane 2) and TKO organoids (lane 4) and amplified by PCR to detect floxed alleles or Cre recombined alleles of the Trp53, Rb1, and Pten. PCR bands were stained with ethidium bromide. Lane 1 and lane 3 were negative and positive recombined controls. (C) A gross TKO SQ tumor (passage 4) was shown on day 17 after SQ injection. (D) Tumor tissue sections from the TKO SQ xenografts were stained with H&E, IF (CK5, CK8), or IHC (CK7, p63, Upk3). Abbreviations: CK5 = Cytokeratin 5; CK20 = Cytokeratin 20; CK8 = Cytokeratin 8; CK7 = Cytokeratin 7; Upk3 = Uroplakin III. (E) Tumor tissue sections from the TKO SQ xenografts were stained with H&E and IF (vimentin). Please click here to view a larger version of this figure.

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 urothelial urothelium, with squamous differentiation and a basal-like subtype both as in vitro organoids and as in vivo xenografts. The TKO organoids can be used in vitro for studying the effects of Trp53, Rb1, and Pten loss, drug screening, and the molecular assessment of signaling pathways. Rapid tumor formation in C57BL/6J mice will enable in vivo studies designed to evaluate treatment responses to systemic therapies like chemotherapy or immunotherapy.

The urothelium is composed of a layer of basal cells (positive for CK5 and p63), 1-2 layers of intermediate cells (Uroplakins and p63 positive), and umbrella cells (Uroplakins, CK8 and CK20 positive)⁶. A critical step in the method is the limited time (30 min) of tissue disassociation instead of a prolonged (4 hours) enzyme digestion, which allows minimal contamination of non-urothelial submucosa cells (Figure 1A). A longer disassociation time may increase the percentage of stromal cells, such as endothelial cells and fibroblast cells. The ex vivo transduction step of adenovirus is highly effective. After ex vivo adenovirus Cre transduction, GFP was visualized in nearly 100% of urothelial cells with mT/mG reporter alleles (Figure 1B).

There are limitations to the ex vivo method. First, the disassociated cells are not pre-selected before adenovirus transduction. For instance, cells are not differentiated for urothelial cells vs. non-urothelial cells, or luminal cells vs. basal cells. Cell sorting with EpCAM and/or CD49f can be used to sort pure epithelial cells and/or stem cells before ex vivo transduction^12,13. Second, the adenovirus driving Cre expression with CMV promoter used in this protocol targets a wide range of cell types after tissue disassociation (urothelial vs. non-urothelial, basal vs. luminal cells). This nonspecific targeting may lead to a selection bias causing overgrowth of cells with the most oncogenic potential. Cell type-specific adenovirus vectors have been used topically in both lung cancer and bladder cancer GEMMs^14,15,16,17. Future studies using ex vivo cell type-specific adenovirus (adenovirus with a CK8 promoter or a CK5 promoter) may address cell-of-origin questions of whether the TKO organoids are derived from basal or luminal cells¹⁸. CK20 or Upk2 promoter-specific adenovirus (not available to our knowledge) can also be designed to transduce umbrella cells in the urothelium. However, future studies are needed to examine the efficiency and specificity of these promoter-specific adenoviruses for ex vivo bladder infection. There may be a discrepancy in adenovirus-Cre efficiency between in vivo and ex vivo transduction due to the host environment¹⁸. Third, the ex vivo method is limited by the lack of tumor environment, which may impact the in vivo tumorigenesis and histomorphology. Despite these limitations, a major advantage of the ex vivo method is the fast workflow (1-2 weeks instead of years of mouse breeding). The second advantage of the ex vivo approach is that adenovirus-Cre (Ad5CMVCre) is commercially available, straightforward, and nearly 100% efficient for viral transduction and Cre recombination (Figure 1B). In contrast, alternative ex vivo methods using CRISPR editing or lentiviral transduction require further clone selection due to less efficient genomic editing^5,13,19,20.

In summary, the ex vivo adenovirus approach described is convenient, fast, and efficient in generating bladder cancer models utilizing any combination of genes of interest (floxed alleles) related to human bladder cancer. These models can be combined with cell type-specific adenovirus and cell sorting to address the impact of the cell of origin on bladder cancer phenotype and to evaluate treatment responses to immunotherapies in a setting of defined genetic mutations.

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 Legend RT	Thermo Fisher	75004377
Sorvall T1 centrifuge	Thermo Fisher	75002383
Y-27632	Selleckchem	S1049