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.

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
<ol>
	<li>Preparation for dissection
	<ol>
		<li>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 Trp53f/f: Ptenf/f: Rb1f/f (4 mice, 10 weeks old), as previously described in Dr. Goodrich&#39;s lab 8.</li>
	</ol>
	</li>
	<li>Euthanasia and incision
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dissecting the bladder
	<ol>
		<li>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.</li>
		<li>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 &#176;C storage and keep it on ice.</li>
	</ol>
	</li>
</ol>
2. Dissociation of urothelial cells
<ol>
	<li>Preparation for cell dissociation
	<ol>
		<li>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 &#181;M fresh Y-27632, a 100 &#181;m 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 &#181;g/mL primocin, and 10 &#181;M fresh Y-27632.</li>
	</ol>
	</li>
	<li>Mincing and digesting the bladder
	<ol>
		<li>Transfer and wash the bladder again with 2 mL of sterile DPBS in a 35 mm dish in a biosafety cabinet.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#176;C orbital incubating shaker for 30 min at 200 rpm.</li>
		<li>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.</li>
		<li>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 &#176;C and 5% CO2 for 4 min.</li>
		<li>After incubation, pipette up and down 10x with a standard P1000 tip. Add 5 mL of 10% charcoal-stripped FBS in DPBS.</li>
		<li>Strain the disassociated cells through a 100 &#181;m sterile cell strainer. Collect the cell suspension and centrifuge at 200 x g for 5 min.</li>
	</ol>
	</li>
	<li>Counting and resuspending cells
	<ol>
		<li>Remove and discard the supernatant. Resuspend the cell pellet with 1 mL of CCM.</li>
		<li>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 &#176;C storage and thaw on ice. Pre-warm a 6-well plate in a 37 &#176;C cell incubator. 
		NOTE: The remaining cells can be centrifuged and frozen as cell pellets for non-virus transduction control.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Add 2 &#181;L of adenovirus (Ad5CMVCre; 1 x 107 PFU/&#181;L) into the 24-well plate. Mix well with the disassociated urothelial cells.</li>
	<li>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 &#176;C and 5% CO2 for 1 h.</li>
	<li>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 &#181;L of CCM, and mix well with the cells at the bottom.</li>
	<li>Add 140 &#181;L of matrix extract and gently mix well to avoid air bubbles. Add the solution quickly into the pre-warmed 6-well plate at 50 &#181;L/dome to create domes for organoids.</li>
	<li>Transfer the plate into a 37 &#176;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.</li>
	<li>After incubation, add 2.5 mL of CCM to each well and place the plate in an incubator for organoid culture.</li>
</ol>
4. Organoid culturing and passaging
<ol>
	<li>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.</li>
	<li>Remove the medium and add 1 mL of dispase to each well. Gently break the dome and mix well with a P1000 pipette tip.</li>
	<li>Place the plate in a 37 &#176;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.</li>
	<li>Centrifuge the tube at 200 x g for 5 min. Remove the supernatant and wash the cells with 1 mL of CCM.</li>
	<li>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 &#181;M fresh Y-27632.</li>
</ol>
5. Organoid cells implantation into C57BL/6J mouse subcutaneously
<ol>
	<li>Preparation for implantation
	<ol>
		<li>Prepare 25G 1.5 in needles, a 1 mL syringe, 1 mg/mL dispase, matrix extract, 10% charcoal-stripped FBS in DPBS with 10 &#181;M fresh Y-27632, and a 15 mL centrifuge tube.</li>
	</ol>
	</li>
	<li>Organoid cell collection
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Counting cells and subcutaneous injection
	<ol>
		<li>Count the number of cells using a hemocytometer. After centrifugation at 200 x g for 5 min, resuspend 2 x 106 cells in 100 &#181;L of 50% matrix extract in DPBS. Place the suspension on ice and take it into a biosafety cabinet in an animal procedure room.</li>
		<li>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&#39; eyes from traumatic injury.</li>
		<li>Shave one area around the injection site at the mouse&#39;s right flank and use 70% ethanol to cleanse, and then use a 25 G needle to directly inject the 100 &#181;L cell suspension into the right flank under anesthesia.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Tumor collection and in vivo propagation
<ol>
	<li>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.</li>
	<li>Wash the tumor&#160;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.</li>
	<li>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 &#181;M fresh Y-27632.</li>
	<li>If needed, recover the frozen cells by thawing them rapidly in a 37 &#176;C water bath and washing with CCM(-). After this, resuspend them in 100 &#181;L 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.</li>
</ol>

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The authors have nothing to disclose.

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&#38;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 transduction12,13. 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 GEMMs14,15,16,17. 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 editing5,13,19,20.
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.

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 responses5,6. 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 responses6,7. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#956;m sterile cell strainer</td>
			<td>Corning</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>25G 1.5 inches needle</td>
			<td>EXELINT International</td>
			<td>26406</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenovirus (Ad5CMVCre High Titer, 1E11 pfu/ml)</td>
			<td>UI Viral Vector Core</td>
			<td>VVC-U of Iowa-5-HT</td>
			<td></td>
		</tr>
		<tr>
			<td>C57 BL/6J</td>
			<td>Jackson Lab</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Charcoal-stripped FBS</td>
			<td>Gibco</td>
			<td>A3382101</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase/hyaluronidase</td>
			<td>Stemcell Technologies</td>
			<td>07912</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>07913</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine substitute (GlutaMAX)</td>
			<td>Gibco</td>
			<td>35-050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Mammary Epithelial Cell Growth medium</td>
			<td>Lonza</td>
			<td>CC-3150</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix extracts from Engelbreth&#8211;Holm&#8211;Swarm mouse sarcomas (Matrigel)</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-CK20</td>
			<td>DAKO</td>
			<td>M7019</td>
			<td>IF 1:100</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-CK7</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-23876</td>
			<td>IHC 1:50</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-p63</td>
			<td>Abcam</td>
			<td>ab735</td>
			<td>IHC 1:100, IF 1:50</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-Upk3</td>
			<td>Fitzgerald</td>
			<td>10R-U103A</td>
			<td>IHC 1:50</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-Vimentin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-6260</td>
			<td>IF 1:100</td>
		</tr>
		<tr>
			<td>Monoclonal rat anti-CK8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I-s</td>
			<td>IF 1:100</td>
		</tr>
		<tr>
			<td>HERAcell vios 160i CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>51033557</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal chicken anti-CK5</td>
			<td>Biolegend</td>
			<td>905901</td>
			<td>IF 1:500</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant enzyme of trypsin substitute (TrypLE Express Enzyme)</td>
			<td>Thermo Fisher</td>
			<td>12605036</td>
			<td></td>
		</tr>
		<tr>
			<td>Signature benchtop shaking incubator Model 1575</td>
			<td>VWR</td>
			<td>35962-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Processing Gel (HistoGel)</td>
			<td>Thermo Fisher</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade size 10</td>
			<td>Integra Miltex</td>
			<td>4-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall T1 centrifuge</td>
			<td>Thermo Fisher</td>
			<td>75002383</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo organoid model of adenovirus-cre mediated gene deletions in mouse urothelial cells

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.

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&#38;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&#38;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63855/63855fig01.jpg" /> 
Figure 1: Adenovirus-Cre mediated gene deletions in mouse urothelial cells with loxP sites. (A)&#160;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. <a href="https://www.jove.com/files/ftp_upload/63855/63855fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63855/63855fig02.jpg" /> 
Figure 2: Characterization of TKO organoids via ex vivo adenovirus-Cre recombinase. (A) The disassociated Trp53f/f: Ptenf/f: Rb1f/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&#38;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&#38;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&#38;E and IF (vimentin). <a href="https://www.jove.com/files/ftp_upload/63855/63855fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells at JoVE.com

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

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.

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

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

ex-vivo-organoid-model-adenovirus-cre-mediated-gene-deletions-mouse

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.6K Views.  Roswell Park Comprehensive Cancer Center. 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.

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...