Name: ex vivo organoid model of adenovirus-cre mediated gene deletions in mouse urothelial cells
Uploaded: 2022-05-05T15:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells at JoVE.com

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...

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><tbody><tr><td>100 &amp;mu;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&amp;ndash;Holm&amp;ndash;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 Legend RT</td><td>Thermo Fisher</td><td>75004377</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 urot...

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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 ...

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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

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Medicine

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

Patient-derived organoids (PDOs) are a powerful tool in translational cancer research, reflecting both the genetic and phenotypic heterogeneity of the disease and response to personalized anti-cancer therapies. Here, a consolidated protocol to generate human primary bladder cancer PDOs in preparation for the evaluation of phenotypic analyses and drug responses is detailed.

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Biology

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense and transmit information about its environment to the underlying tissues, promoting voiding function and behavior. Disruption of the urothelial barrier, or its sensory/transducer function, can lead to disease. Studying these complex events is hampered by lack of simple strategies to alter gene and protein expression in the urothelium. Methods are described here that allow investigators to generate large amounts of high-titer adenovirus, which can then be used to transduce rodent urothelium with high efficiency, and in a relatively straightforward manner. Both cDNAs and small interfering RNAs can be expressed using adenoviral transduction, and the impact of transgene expression on urothelial function can be assessed 12 h to several days later. These methods have broad applicability to studies of normal and abnormal urothelial biology using mouse or rat animal models.

Methods are described for the generation of large amounts of recombinant adenoviruses, which can then be used to transduce the native rodent urothelium allowing for expression of transgenes or downregulation of endogenous gene products.

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense ...

Patient-derived Tumor Xenografting: A Technique to Generate Experimental Mouse Model for Evaluating Urothelial Cell Carcinoma

Source: Moret, R. et al. <a href="https://www.jove.com/video/59223" target="_blank">Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.</a> J. Vis. Exp. (2019)
This video describes the procedure to generate an experimental xenograft murine model, which can be used for studying urothelial cell carcinoma progression.

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

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Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

Ksenografacja guza pochodząca od pacjenta: technika generowania eksperymentalnego modelu myszy do oceny raka urotelialnego