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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#181;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 &#181;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&#39;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&#39;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) 
			Growth Factor 
			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 &#181;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 &#181;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/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/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/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

Research

JoVE Journal

Cancer Research

9.6K Views.  Pohang University of Science and Technology. 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.

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

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

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 ...

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 ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

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 ...

Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in esophageal adenocarcinoma (EAC). Patient-derived organoids (PDOs) have recently emerged as a remarkable preclinical model in a variety of cancers. However, there are still limited protocols available for developing EAC PDOs. Once the PDOs are established, the propagation and cryopreservation are essential for further downstream analyses. Here, two different methods have been standardized for EAC PDOs subculture and cryopreservation, i.e., with and without single cell digestion. Both methods can reliably obtain appropriate cell viability and are applicable for a diverse experimental setup. The current study demonstrated that subculturing EAC PDOs with single cell digestion is suitable for most experiments requiring cell number control, uniform density, and a hollow structure that facilitates size tracking. However, the single cell-based method shows slower growth in culture as well as after re-cultivation from frozen stocks. Besides, subculturing with single cell digestion is characterized by forming hollow structures with a hollow core. In contrast, processing EAC PDOs without single cell digestion is favorable for cryopreservation, expansion, and histological characterization. In this protocol, the advantages and disadvantages of subculturing and cryopreservation of EAC PDOs with and without single cell digestion are described to enable researchers to choose an appropriate method to process and investigate their organoids.

This protocol describes the methods of subculture and cryopreservation of esophageal adenocarcinoma organoids with and without single cell digestion to enable researchers to choose appropriate strategies based on their experimental design.

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in ...