This research was supported by grants from the National Research Foundation of Korea to K.S: NRF-2017R1A2B4006043, NRF-2017M3C7A1047875, NRF-2017R1A5A1015366, Creative Economy Leading Technology Development Programme (SF317001A), POSCO (2018Y060) and the BK21 Plus Research Fellowship.

All procedures were approved and conducted under the guidelines of the Institutional Animal Care and Use Committee at POSTECH (IACUC number: POSTECH-2019-0055).
1. In Vitro Culture of Bladder Tumor Organoids
<ol>
	<li>Establish bladder tumor organoids from the murine bladder tumor (Figure 1A). 
	NOTE: The procedure for generating BBN-induced mouse bladder tumors is outlined in Shin et al.17.
	<ol>
		<li>Provide 0.1% BBN-containing water in a dark bottle to mouse ad libitum for 6 months. Change BBN-containing water 2x a week. 
		NOTE: A C57BL/6 male mouse with a body weight of approximately 25 g at 8&#8211;10 weeks of age was used. BBN-containing water can be administered to up to five mice in a single cage.</li>
		<li>After 6 months, euthanize the mouse using carbon dioxide inhalation and isolate the entire bladder tumor. Transfer it to a 90 mm Petri dish.</li>
		<li>Remove non-cancerous parts and necrotic regions using sterile surgical scissors and wash the bladder tumor fragments 2&#8211;3 times with cold 1x Dulbecco&#39;s phosphate-buffered saline (DPBS). Collect the fragments and transfer them to a new 90 mm Petri dish.</li>
		<li>Add 1 mL of Dulbecco&#39;s modified minimum essential medium (DMEM) with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).</li>
		<li>Mince the tumor tissue into pieces as small as possible (0.5&#8211;1 mm3) using a sterilized razor blade.</li>
		<li>Add 9 mL of DMEM with 10 mM HEPES, 250 &#181;g/mL collagenase type I, 250 &#181;g/mL collagenase type II, and 250 U/mL thermolysin. Incubate the minced tumor tissue for 1.5&#8211;2 h on an orbital shaker in an incubator (37 &#176;C, 5% CO2) to dissociate the fragments into the cell suspension. Transfer the cell suspension into a 50 mL tube. 
		NOTE: If the size of tumor harvested from the mouse is greater than 1 cm3, treat it with 2x the amount of thermolysin or increase the incubation time.</li>
		<li>Centrifuge the tube at 400 x g for 5 min at 4 &#176;C and aspirate the supernatant.</li>
		<li>Resuspend the pellet using 5 mL of ammonium-chloride-potassium (ACK) lysing buffer to lyse any red blood cells. Incubate the tube for 3&#173;&#8211;5 min at room temperature (RT) until the complete lysis of red blood cells. 
		NOTE: If the red blood cells are not observed, omit the lysing process.</li>
		<li>Add 20 mL of DMEM into the tube. Centrifuge the tube at 400 x g for 5 min at 4 &#176;C and aspirate the supernatant.</li>
		<li>Resuspend the pellet with 1 mL of 0.25% Trypsin-EDTA and 10 &#181;M Y-27632 dihydrochloride (Y-27632) to dissociate the pellet into single cells. Incubate the tube for 5 min in a 37 &#176;C water bath. 
		NOTE: Observe the tumor under a microscope to confirm complete dissociation into single cells. If chunks of cells persist, pipette the suspension further.</li>
		<li>Neutralize trypsin using 10 mL of DMEM with 10% fetal bovine serum (FBS). Filter the cell suspension through a 100 &#181;m cell strainer on a new 50 mL tube to remove the undigested debris.</li>
		<li>Centrifuge the tube at 400 x g for 5 min at 4 &#176;C and aspirate the supernatant.</li>
		<li>Coat a well in a 24 well plate using 150 &#181;L of ice-cold growth factor reduced basement membrane matrix (Table of Materials) and place the 24 well plate in an incubator (37 &#176;C, 5% CO2) for 30 min to solidify the basement membrane matrix. 
		NOTE: Thaw and maintain the basement membrane matrix at 4 &#176;C to prevent solidification before use.</li>
		<li>Resuspend the pellet using 1 mL of DMEM and count the cells using a hemocytometer. Transfer 3&#8211;4 x 104 tumor cells into a 1.5 mL microtube on ice.</li>
		<li>Centrifuge the microtube at 400 x g for 3 min at 4 &#176;C and carefully discard the supernatant.</li>
		<li>Resuspend the cells with 500 &#181;L of prewarmed organoid medium (Table 1) and 10 &#181;M Y-27632 and transfer them into the coated well. Place the 24 well plate in an incubator (37 &#176;C, 5% CO2).</li>
		<li>Extra bladder tumor cells can be stocked with 1 mL of DMEM containing 10% FBS, 1% penicillin/streptomycin, and 10% dimethyl sulfoxide (DMSO) in 1.5 mL cryovials. Place them in a cryovial freezing container and transfer the container to a -80 &#176;C freezer. After storage in the freezer overnight, transfer the cryovials into liquid nitrogen for long-term storage.</li>
		<li>Change the medium every 2 days using 500 &#181;L of prewarmed organoid medium (Figure 1B).</li>
	</ol>
	</li>
	<li>Subculture bladder tumor organoids. 
	NOTE: Passage of bladder tumor organoids when they reach 100&#8211;150 &#181;m in diameter is recommended.
	<ol>
		<li>Add 500 &#181;L of collagenase/dispase to the organoid medium in the 24 well plate with tumor organoids. Pipette up and down the basement membrane matrix and the medium. Incubate for 20 min at 37 &#176;C and harvest the cells into a 15 mL tube. 
		NOTE: Examine the organoids isolated from the basement membrane matrix under a microscope. If the organoids are not detached from the basement membrane matrix, increase the incubation time or pipette more times.</li>
		<li>Add 5 mL of prewarmed DMEM, centrifuge the tube at 400 x g for 3 min at 4 &#176;C, and aspirate the supernatant.</li>
		<li>Resuspend the pellet using 1 mL of prewarmed 0.25% trypsin-EDTA and 10 &#181;M Y-27632. Incubate for 5 min in a 37 &#176;C water bath. Vigorously pipette the cells up and down and neutralize the trypsin using 5 mL of DMEM with 10% FBS.</li>
		<li>Centrifuge the tube at 400 x g for 3 min at 4 &#176;C and aspirate the supernatant.</li>
		<li>Resuspend the pellet using 1 mL of prewarmed organoid medium and count the number of single tumor cells.</li>
		<li>Repeat steps 1.1.14&#8722;1.1.18.</li>
	</ol>
	</li>
</ol>
2. Genetic Manipulation of Bladder Tumor Organoids Using Lentivirus-mediated Transduction (Figure 2A)
<ol>
	<li>Produce the GFP-expressing lentiviral particles.
	<ol>
		<li>On day 0, plate HEK 293T cells at a density of 5&#8211;6 x 106 cells per 10 cm cell culture plate in cell line culture medium (i.e., DMEM with 10% FBS and 1% penicillin/streptomycin).</li>
		<li>On day 1, prepare the DNA transfection solution including transfer plasmid containing GFP (8 &#181;g), lentiviral packaging plasmid (10 &#181;g of pCMVR 8.74 and 3 &#181;g of pMD2.G), and 1 mL of reduced serum medium (Table of Materials).</li>
		<li>Add 3 &#181;L of transfection reagent (Table of Materials) per 1 &#181;g of total plasmid according to the manufacturer&#39;s instructions and mix gently by pipetting. Incubate for 20 min at RT and add 9 mL of DMEM with 10% FBS.</li>
		<li>Aspirate the culture medium in the cell culture plate with HEK 293T cells. Carefully transfer 10 mL of the DNA transfection solution onto the HEK 293T cells and incubate in a cell culture incubator at 37 &#176;C.</li>
		<li>On day 3, observe the cells under a fluorescence microscope (excitation at 488 nm and emission at 512 nm) to determine the transfection efficiency. Almost 90%&#8211;100% of the cells in the entire cell population should express GFP.</li>
		<li>Collect the supernatant (containing the virus) and filtrate the supernatant with a 0.45 &#181;m polyethersulfone (PES) filter. 
		NOTE: Use a low protein-binding filter such as a PES filter.</li>
		<li>To concentrate the virus, centrifuge the virus supernatant at 98,768 x g in an ultracentrifuge for 2 h at 4 &#176;C in a swinging bucket rotor (Table of Materials) and carefully discard the supernatant.</li>
		<li>Resuspend the pellet in 2.5 mL of cold organoid medium.</li>
		<li>For long-term storage, aliquot 250 &#181;L of lentiviral medium into cryogenic vials and snap-freeze them using liquid nitrogen. Store the frozen viral stocks in a -80 &#176;C freezer.</li>
	</ol>
	</li>
	<li>Perform lentivirus-mediated transduction of the bladder tumor organoids.
	<ol>
		<li>On day 2, split the tumor organoids as described above (step 1.2) 12 h before the lentivirus-mediated transduction.</li>
		<li>On day 3, quickly thaw an aliquot (step 2.1.9) containing virus in a 37 &#176;C water bath and add the 250 &#181;L of organoid medium with 10 &#181;M Y-27632 and 8 &#181;g/mL hexadimethrine bromide.</li>
		<li>Replace the organoid medium in the 24 well plate with tumor organoids by 500 &#181;L of virus-containing medium and incubate for 12&#8211;16 h in an incubator (37 &#176;C, 5% CO2).</li>
		<li>On day 4, change the medium with 500 &#181;L of fresh organoid medium. 
		NOTE: After 12&#8211;16 h of incubation, the medium should be changed, because the medium containing lentivirus and hexadimethrine bromide is cytotoxic.</li>
		<li>On day 6, monitor the GFP signal from the tumor organoids 3 days after transduction under a fluorescence microscope (Figure 2B).</li>
		<li>On day 10, passage and stock the organoids 7 days after transduction as described in step 1.2, to maintain the genetically modified tumor organoid lines.</li>
	</ol>
	</li>
</ol>
3. Orthotopic Transplantation of Bladder Organoid (Figure 3A)
<ol>
	<li>Prepare the bladder tumor organoids for orthotopic transplantation.
	<ol>
		<li>Before transplantation, culture the bladder tumor organoids for 5&#8211;7 days, as described above (step 1.2).</li>
		<li>Add 500 &#181;L of collagenase/dispase to organoid medium in a 24 well plate with the tumor organoids. Pipette up and down the basement membrane matrix and medium. Incubate for 20 min at 37 &#176;C and collect the cells into a 15 mL tube.</li>
		<li>Add 5 mL of prewarmed DMEM, centrifuge the tube at 400 x g for 3 min at 4 &#176;C, and aspirate the supernatant.</li>
		<li>Resuspend the pellet with 1 mL of DMEM and transfer the solution into a 90 mm Petri dish.</li>
		<li>Under a microscope, pick up the 10&#8211;100 tumor organoids by using a p200 micropipette and collect them into a microtube on ice.</li>
		<li>Centrifuge the tube at 400 x g for 3 min at 4 &#176;C and carefully discard the supernatant.</li>
		<li>Maintain the cell pellet on ice until the mice are ready for surgery.</li>
	</ol>
	</li>
	<li>Submucosal bladder wall transplantation 
	NOTE: This procedure is modified from the protocol published by Fu et al18.
	<ol>
		<li>Prepare an 8 to 10 week-old male nude mouse (CAnN.Cg-Foxn1nu/Crl) at least 1 week before the experiment to allow it to acclimate to a new environment. Inject enrofloxacin (5 mg/kg) subcutaneously 24 h before surgery.</li>
		<li>Clean the bench surface by soap and water. Autoclave the surgical instruments prior to the surgical procedure and perform surgery using sterile instruments.</li>
		<li>Keep the 29 G insulin syringe, pipette tips, and basement membrane matrix on ice. Administer ketoprofen (5 mg/kg) subcutaneously before administration of anesthesia.</li>
		<li>Anesthetize the mouse with 4% isoflurane in an induction chamber. Once general anesthesia achieved, lay the mouse in a supine position and maintain anesthesia by mask inhalation of 2% vaporized isoflurane. 
		NOTE: If the anesthetization time is over 30 min, apply eye ointment to both eyes using a cotton swab to avoid corneal drying.</li>
		<li>Apply povidone-iodine with a sterile gauze and wipe it down with 70% ethanol. Repeat 3x with a new gauze or a cotton swab each time.</li>
		<li>Cover the anus and the surgical field using disposable, sterile surgical drapes.</li>
		<li>Using a dissecting microscope for magnification, make a small transverse incision (smaller than 1.5 cm) in the skin and muscular wall of the lower midline abdomen with sterile surgical scissors. Expose the bladder from the abdominal cavity and support it with saline-soaked cotton swabs. 
		NOTE: If the bladder is full of urine, gently press the bladder to decompress it slightly.</li>
		<li>Resuspend the organoid pellets (step 3.1.7) in 80 &#181;L of organoid medium containing 50% high-concentration basement membrane matrix (Table of Materials).</li>
		<li>Inject the organoid suspension into the anterior aspect of the bladder dome using the 29 G insulin syringe under a dissecting microscope.</li>
		<li>Close the inner layer of the abdominal wall with antibacterial absorbable suture and then close the outer layer with 4-0 nylon suture. Disinfect the surgical site with povidone-iodine and 70% ethanol.</li>
		<li>Allow the mouse to recover under an infrared irradiator 10&#8211;15 min. Monitor the mouse until it regains consciousness and motility.</li>
		<li>One day after surgery, check the general condition of the mouse and anastomotic leakage. Administer ketoprofen (5 mg/kg) once daily for 3 days post-operatively and treat enrofloxacin (5 mg/kg) once daily for 10 days post-operatively.</li>
		<li>When incision site has healed (10-14 days after surgery), remove the sutures. Monitor the growth of the mouse bladder tumor for 2&#8211;3 weeks after the tumor organoid injection.</li>
		<li>If bladder tumor growth is observed, euthanize the mouse using carbon dioxide inhalation, and harvest the entire bladder tumor. Wash it using cold DPBS (Figure 3B)16.</li>
		<li>To analyze the bladder tumor histology, stain the paraffin-embedded section of the tissue using hematoxylin and eosin (H and E) staining (Figure 3B)16.</li>
	</ol>
	</li>
</ol>

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The authors declare no competing financial interests.

This protocol describes the experimental procedures to culture and maintain bladder tumor organoids derived from carcinogen-induced murine bladder tumors.
In this protocol, there are several experimental steps in which the procedures might need some troubleshooting. First, the number of tumor cells that are initially seeded is a critical factor because low numbers of tumor cells in culture (&#60;2 x 104 cells) mostly lead to cell death due to lack of interactions among tumor cells. In contrast, beginning with too many cells (&#62;5 x 104 cells) at seeding leads to overcrowded organoids, resulting in difficulty when handling cultures with poor growth of each organoid. It is strongly suggested that multiple plates with different numbers of cells be established at the beginning to optimize the experimental conditions. Identifying the right number of initial tumor cells is crucial to achieve the highest cell viability and to establish successful bladder tumor organoids. Also, in long-term culture of over 2 weeks without passaging, most tumor organoids stop growing, potentially due to inadequate supply of nutrients at the center of the organoids and the depletion of growth factor in the basement membrane matrix. Therefore, subculturing organoids in a timely manner is a critical step to maintain tumor organoid culture.
Second, the production of high-titer lentiviral particles is critical for the efficient genetic manipulation of tumor organoids. To troubleshoot virus titer-related issues, it is strongly suggested that the virus titers be determined before viral transduction every time because lentiviral constructs tend to produce viral particles with varying efficiency. If tumor organoids exhibit low viability following viral infection, it is likely that the viral titers are potentially too high. It is strongly suggested to use lower amount of virus in this case. Third, during orthotopic transplantation of BBN-induced bladder tumor organoids, it is critical to maintain the integrity of the bladder wall. In case that the injection reaches the lumen of the bladder by penetrating the bladder wall layer, the experiment should be terminated and discarded. If possible, the monitoring of bladder tumor growth using an ultrasound imaging system is recommended.
One limitation of the current techniques is the absence of the tumor microenvironment or stroma in these organoids. To overcome this issue, it is strongly suggested that the orthotopic transplantation of tumor organoids use an in vivo system to mimic the native tumor microenvironment. In the future, it will be necessary to develop 3D in vitro organoid systems that are composed of tumor organoids with other components of tumor stroma.
One of the major implications of our technique is that, in orthotopic transplantation of tumor organoids, only 10 bladder tumor organoids can induce tumor growth in the bladder. Compared to the conventional tumor transplantation experiments that require 5 x 105&#8211;1 x 106 single bladder tumor cells, our methods are much more efficient and robust. Another significant difference is that the organoids can be diversely manipulated using various lentiviral vectors, such as lentiviral constructs containing short-hairpin RNA, the CRISPR&#8211;Cas9 system, or genes of interest. These would be powerful tools to add to current organoid technology. Overall, the experimental approaches presented here can facilitate the establishment of in vitro tumor models that can improve our understanding of the pathogenesis of bladder cancer rather than using 2D bladder cancer cell lines.
This method was able to establish bladder tumor organoids derived from a carcinogen-induced murine bladder tumor. The article provides a description of the lentivirus-mediated experimental procedures through which the genetic modifications are introduced and stably maintained in bladder tumor organoids. In addition, a procedure for orthotopic transplantation of tumor organoids is included. In combination with current in vivo cancer models, this technique will be a useful tool to study the molecular basis of bladder tumorigenesis.

Bladder cancer is the most prevalent urinary tract cancer, with approximately 165,000 patients dying annually1. Among the various types of bladder cancer, muscle-invasive urothelial carcinoma exhibits an aggressive phenotype, and its 5 year survival rate is lower than 50%2. Novel therapeutic options for invasive urothelial tumors have not been expanded over the last few decades1.
Cancer cell lines have been extensively used for drug screening3. Although favorable results have been observed in numerous drug candidates in cancer cell lines, poor results are reported in clinical trials4. Following increased adaptation to in vitro two-dimensional (2D) culture environments, it has become increasingly difficult to recapitulate native tumors in cell lines. Animal cancer models or patient-derived tumor xenografts can be used to address the limitations observed in bladder cancer cell lines. However, animal cancer models are time and resource intensive. Therefore, improved disease models have been on demand for years and a novel model system, organoids, has been developed to overcome the shortcomings of existing models5.
An organoid is a multicellular 3D construct that can recapitulate in vitro the physiological characteristics of its corresponding in vivo organ. Normal and tumor organoids can be derived from either pluripotent or adult stem cells, and primary tumor cells, respectively5,6. Over the last several years, tumor organoids have been established from a large number of diverse tumor tissues7, including colon8,9, bladder10, pancreas11,12, prostate13, liver14, and breast15 tumor tissues. Such tumor organoids mimic their original tumors phenotypically and genetically. Due to their similarity to in vivo tumor tissues and their numerous practical applications, researchers have adopted them as novel disease models in the study of cancer pathogenesis.
Here, the procedures for the establishment of tumor organoids from a carcinogen-induced murine invasive urothelial tumor16 are laid out. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) is used as a carcinogen to induce invasive urothelial carcinoma in mice17 and the tumor organoids, which exhibit the pathological characteristics of mouse muscle-invasive bladder tumors, are established from the BBN-induced murine bladder cancer16. The method to genetically manipulate the tumor organoids is illustrated using lentivirus-mediated transduction to develop a model system for studying the molecular basis of the development of bladder cancer. In addition, a method for transplanting organoids orthotopically into a bladder to investigate the role of the native bladder environment in bladder cancer is described.

<table><tbody><tr><td>0.45 &amp;micro;m syringe filter (PES membrane)</td><td>Millipore</td><td>SLHP033RS</td><td></td></tr><tr><td>10 cm culture plate</td><td>Eppendorf</td><td>0030-702-115</td><td></td></tr><tr><td>90 mm Petri dish</td><td>SPL</td><td>10090</td><td></td></tr><tr><td>100 &amp;micro;m cell strainer</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>15 mL conical tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>29 G 1/2 insulin syringe</td><td>SHINA</td><td>B299473538</td><td></td></tr><tr><td>3 mL syringe</td><td>Norm-ject</td><td>N7.A03</td><td></td></tr><tr><td>50 mL conical tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>A8301</td><td>Tocris</td><td>2939</td><td>stock concentration: 25 mM</td></tr><tr><td>Absolute ethanol</td><td>Daejung</td><td>4023-2304</td><td></td></tr><tr><td>Absorbable suture</td><td>Henry Schein</td><td>039010</td><td></td></tr><tr><td>Advanced DMEM/F-12</td><td>Thermo</td><td>12634028</td><td></td></tr><tr><td>Ammonium-chloride-potassium (ACK) lysing buffer</td><td>Thermo</td><td>A1049201</td><td></td></tr><tr><td>B-27</td><td>Gibco</td><td>17504-044</td><td>stock concentration: 50X</td></tr><tr><td>BBN(N-butyl-N-(4-hydroxybutyl) nitrosamine)</td><td>Tokyo Chemical Industry</td><td>B0938</td><td></td></tr><tr><td>Blue nylon 5/0-13mm</td><td>AILEE</td><td>NB521</td><td></td></tr><tr><td>C57BL Mouse</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>CAnN.Cg-Foxn1nu/Crl (nude mouse)</td><td>Charles River</td><td>194</td><td></td></tr><tr><td>Collagenase type I</td><td>Thermo</td><td>17100017</td><td>stock concentration: 20 mg/mL</td></tr><tr><td>Collagenase type II</td><td>Thermo</td><td>17100015</td><td>stock concentration: 20 mg/mL</td></tr><tr><td>Collagenase/dispase</td><td>Sigma</td><td>10269638001</td><td>stock concentration: 1 mg/mL</td></tr><tr><td>Cyrovial</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>DMEM(Dulbecco&#039;s modified minimum essential media)</td><td>Gibco</td><td>11965-118</td><td></td></tr><tr><td>DMSO(Dimethyl sulfoxide)</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>DPBS(Dulbecco&#039;s phosphate-buffered saline)</td><td>Welgene</td><td>LB 001-02</td><td></td></tr><tr><td>Enrofloxacin (Baytril)</td><td>Bayer Healthcare</td><td>DIN: 02169428</td><td></td></tr><tr><td>FBS(Fetal bovine serum)</td><td>Millipore</td><td>ES009B-KC</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050061</td><td>100X</td></tr><tr><td>HEK 293T</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)</td><td>Welgene</td><td>BB001-01</td><td></td></tr><tr><td>Isoflurane</td><td>Hana Pharm Co., Ltd.</td><td></td><td></td></tr><tr><td>Ketoprofen (Anafen)</td><td>Merial</td><td>DIN: 02150999</td><td></td></tr><tr><td>Matrigel growth factor reduced (GFR)&lt;br/&gt; Growth Factor&lt;br/&gt; Reduced (GFR)</td><td>Corning</td><td>354230</td><td>use for organoid culture in plate</td></tr><tr><td>Matrigel high concentration (HC)</td><td>Corning</td><td>354248</td><td>use for organoid transplantation</td></tr><tr><td>1.5 mL microtube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>LT1 transfection reagent</td><td>Mirus Bio</td><td>MIR 2300</td><td></td></tr><tr><td>murine EGF(epidermal growth factor)</td><td>Peprotech</td><td>315-09</td><td>stock concentration: 100 &amp;micro;g/mL</td></tr><tr><td>N-acetyl-L-cysteine</td><td>Sigma</td><td>A9165</td><td>stock concentration: 200 mM</td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td>stock concentration: 1M</td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>pCMV.R 8.74</td><td>Addgene</td><td>22036</td><td>Packaging plasmid</td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140122</td><td>100X</td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td>Envelope plasmid</td></tr><tr><td>Polybrene(hexadim ethrine bromide)</td><td>Sigma</td><td>H9286</td><td>stock concentration: 2 &amp;micro;g/mL</td></tr><tr><td>pSiCoR</td><td>Addgene</td><td>11579</td><td>Lentiviral plasmid</td></tr><tr><td>Razor blade</td><td></td><td></td><td></td></tr><tr><td>Saline buffer</td><td>JW Pharmaceutical</td><td></td><td></td></tr><tr><td>SW41Ti swinging bucket rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Thermolysin, Bacillus thermoproteolyticus</td><td>Millipore</td><td>58656-2500KUCN</td><td>stock concentration: 250 KU/mL</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200072</td><td></td></tr><tr><td>Ultracentrifugation tube</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Abmole</td><td>M1817</td><td>stock concentration: 10 mM</td></tr></tbody></table>

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.

In vitro culture of mouse bladder tumor organoids 
The number of tumor cells dissociated from an ~1 cm3 BBN-induced tumor is at least 4 x 105 cells. When the cells are initially seeded in the basement membrane matrix, non-cancerous cells and debris may be observed. Debris was gradually diluted out by continuing the subculture. Figure 1B shows images of the cultured organoids at different time points. If the tumor cells do not form tumor organoids, the cells are potentially dead during the dissociation step. In such a case, dissociation procedures including incubation time with the enzyme need to be adjusted to increase cell viability.
Expression of GFP in bladder tumor organoids using lentivirus-mediated genetic manipulation 
Bladder tumor organoids exhibited strong GFP signals with successful lentiviral infection (Figure 2B). After concentration, a total of 250 &#181;L of virus-containing media was enough to infect 3 x 104 single tumor cells on the basement membrane matrix, maintaining 90%&#8211;100% infection efficiency. GFP signals could be detected from the bladder tumor organoids 3 days after lentiviral transduction. If the fluorescence signals are low, the efficiency of viral infection is potentially low. This can be due to numerous factors, such as low viral titer, and the procedures need to be adjusted accordingly.
Orthotopic transplantation of bladder tumor organoids 
A bladder tumor allograft obtained from BBN-induced bladder tumor organoids is presented in Figure 3B16. Bladder tumor allografts were harvested 3 weeks after orthotopic transplantation. The histology of the transplanted bladder tumor was analyzed using H and E staining. Orthotopic transplants of tumor organoids can grow as bladder tumors for 2&#8211;3 weeks.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60469/60469fig01.jpg" /> 
Figure 1: In vitro culture of mouse bladder tumor organoids. (A) Schematic diagram for the establishment of mouse bladder tumor organoids. (B) Representative images for the culture of bladder tumor organoids at different time points. Mouse bladder tumor organoids were established and cultured over 9 days. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60469/60469fig01large.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/60469/60469fig02.jpg" /> 
Figure 2: Expression of GFP in bladder tumor organoids using lentivirus-mediated genetic manipulation. (A) Schematic diagram of lentiviral transfection and transduction of bladder tumor organoids. (B) Representative images of bladder tumor organoids expressing GFP. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60469/60469fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60469/60469fig03.jpg" /> 
Figure 3: Orthotopic transplantation of bladder tumor organoids. (A) Schematic diagram of orthotopic transplantation of bladder tumor organoids to a nude mouse. (B) Representative images of bladders and H and E stained sections from mice orthotopically transplanted with bladder tumor organoids. Magnified views of the boxed regions in the middle panels are shown in the left panels. Scale bar = 500 &#181;m. This figure was reproduced from Figure 1&#8211;Figure Supplement 1, Kim et al.16, published under the Creative Commons Attribution 4.0 International Public License (CC BY 4.0; https://creativecommons.org/licenses/by/4.0/). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60469/60469fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Mouse bladder tumor organoids medium</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12 (Basic medium)</td>
			<td>10 mM HEPES(pH 7.4)</td>
		</tr>
		<tr>
			<td>10 mM Nicotinamide</td>
			<td>0.5x Serum-free supplement</td>
		</tr>
		<tr>
			<td>2 mM L-alanyl-L-glutamine dipeptide</td>
			<td>1% Penicillin/Streptomycin</td>
		</tr>
		<tr>
			<td>1 mM N-acetyl-L-cysteine</td>
			<td>50 ng/mL Murine epidermal growth factor</td>
		</tr>
		<tr>
			<td>1 &#181;M A 83-01</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Composition of bladder tumor organoid medium.

Watch this Scientific Journal Video about Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids at JoVE.com

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

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 ...

culture-manipulation-orthotopic-transplantation-mouse-bladder-tumor

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.7K Views.  Pohang University of Science and Technology. Tumor organoïden zijn op grote schaal gebruikt als een in vitro kanker model om tumorbiologie te bestuderen. Ons protocol biedt een gedetailleerde procedure om te isoleren, cultuur, en genetisch manipuleren blaas tumor organoïden die dienen als een kritisch instrument om verschillende aspecten in de kanker biologie te bestuderen. Met behulp van ons protocol, kunnen we genetisch manipuleren de blaas tumor organoïden uit te drukken of knock-out van het gen van ons belang.Ook kunnen we...

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

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Medicine

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

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 ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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 ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

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.

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.

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

Bladder Tumor Organoid Model: A Method for Orthotopic Transplantation of Bladder Organoids into Recipient Murine Bladder

Source: Kim, Y. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60469" target="_blank">Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids.</a>&#160;J. Vis. Exp.&#160;(2020)
In this video, we describe the orthotopic transplantation of cultured bladder tumor organoids into the murine bladder to study the role of the organ environment on developing tumors.

Source: Kim, Y. et al. Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids.&#160;J. Vis. Exp.&#160;(2020)
In this ...

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

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