The authors thank Brian Reuter, Danielle Bertoni, Peter Miller, and Shannon McChesney who helped to initiate these studies for their excellent technical support. The authors also thank Heather Green Matrana, Margaret Variano, Sunil Talwar, and Maria Latsis for assistance in consenting patients and providing tumor specimens.

All methods described in these animal studies were conducted under the approved guidelines of the Institutional Animal Care and Use Committee of Ochsner Health System and in accordance with animal research guidelines. All patient tumors for this study were collected from consented patients undergoing cancer resection surgeries in accordance with the Ochsner Health System Investigative Review Board and the ethical standards of the Institutional Committee on Human Experimentation. Board-certified pathologists at Ochsner Health System determined the pathological diagnoses of patient specimens based on the microscopic features of tumor cells, their histological type, and grade level.
NOTE: The following protocol describes the steps for two separate xenograft models, a UCC model via the electrocauterization of the bladder wall to instill UCC cells and an intrarectal injection of CRC cells for study in a CRC model. All steps preparing for and monitoring the experiments are identical for both models, while sections 7 and 8 specifically describe the procedure for UCC instillation and CRC injection, respectively.
1. Culturing cell lines
<ol>
	<li>Grow HK cells in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 nM glutamine, 100 U/mL penicillin G, and 100 mg/mL streptomycin at 37 &#176;C in a 5% CO2 humidified incubator. 
	NOTE: HK cells are normal human follicular dendritic cells and can be grown and expanded for &#8804; 15 passages in vitro16.</li>
	<li>To prepare for an experiment, trypsinize the cells.
	<ol>
		<li>Remove media and add 2 mL of 1% trypsin in Hank&#8217;s balanced salt solution (HBSS) to cells. Place cells back in the 5% CO2 humidified incubator at 37 &#176;C for 4 min.</li>
		<li>Collect cells from dish into a 15 mL tube using a handheld pipet aid with a 10 mL serological pipet attached. Add 8 mL of complete RPMI-1640 medium.</li>
		<li>Combine 40 &#181;L of cells and 40 &#181;L of trypan blue in a single well of a 96 well plate. Add 10 &#181;L of mixture to a hemocytometer and count live cells. Add 1 million cells in 25 mL complete RPMI-1640 medium to a 150 mm sterile tissue culture-treated dish to continue growing the cells. 
		NOTE: HK cell suspension prepared in this step must be used within an hour to mix with tumor cells for injection.</li>
	</ol>
	</li>
</ol>
2. Patient specimen collection
<ol>
	<li>Collect the UCC tumors from consented patient 15 (BlCaPt15, pT3b N1 M0) and 37 (BlCaPt37, pT3b pN0 M0) at resection surgery.</li>
	<li>Collect CRC tumors from consented patient 155 (CoCaPt155, T1 N0 M0) and 302 (CoCaPt302, T1 N0 M0) at resection surgery.</li>
</ol>
3. Expansion of patient tumor
<ol>
	<li>Collect tumors at surgery in cold sterile McCoy&#8217;s medium containing penicillin G (500 U/mL) and streptomycin (500 mg/mL).</li>
	<li>Implant tumors directly into the left and right flank of 6&#8722;8 week old female NOD/SCID mice.
	<ol>
		<li>Mechanically mince tissues into small pieces (~1 mm3) using small surgical scissors.</li>
		<li>Implant tissue subcutaneously to the left and right flank using 13 G bone marrow aspiration biopsy needles. 
		NOTE: Implant a total volume of 8 mm3 divided evenly to both sides of flank.</li>
	</ol>
	</li>
</ol>
4. Tagging and enrichment of luciferase labeled tumors
<ol>
	<li>Measure tumor growth bi-weekly using a digital caliper.</li>
	<li>At 1 cm in diameter, transduce tumor. Directly inject into tumor with single dose of Luc/red fluorescent protein (RFP)-lentivirus (50 &#181;L/tumor, 1:30 dilution from concentrated high titer lentivirus stock) using a 1 cc syringe with a 27 G needle. 
	NOTE: The patient tumor typically reaches 1 cm in diameter in 1&#8722;2 months. However, the growth rate is extremely variable and based on a number of factors including tumor grade and type.</li>
	<li>Monitor tumor weekly by bioluminescent imaging (BLI) in live animals.
	<ol>
		<li>Weigh the mice. Inject conscious mouse with 150 mg/kg luciferin intraperitoneally and wait 5 min for the substrate to circulate in the mouse&#8217;s body.</li>
		<li>Anesthetize the mouse with 2.5% isoflurane in 100% oxygen, 1 L/min in an induction chamber.</li>
		<li>Place mouse on stomach in a BLI imaging machine with isoflurane flowing and image. Take sequential images to confirm the presence of Luc/RFP positive tumor regions (false-color bio-luminescent image). Return mouse to cage after imaging is complete.</li>
	</ol>
	</li>
</ol>
5. Select appropriate portion of tumor for enzymatic digestion
<ol>
	<li>On the day of UCC or CRC procedure, image mouse with luciferase tagged tumor as in steps 4.3.1&#8722;4.3.3. 
	NOTE: The length of time for the subcutaneous tumor to grow depends on the speed of tumor growth and the planned number of animals to be injected in the experiment.</li>
	<li>Harvest tumor from mouse flank and image.
	<ol>
		<li>Euthanize mouse by CO2 inhalation after imaging. Place mouse in the CO2 chamber, turn on gas at 1.4 L/min until respiratory arrest and leave on for 3 min. Follow this with cervical dislocation.</li>
		<li>Clean skin with 70% ethanol. Tent skin directly above tumor. With surgical scissors make a small incision in skin. Separate skin from tumor with scissors.</li>
		<li>Remove tumor and place in a sterile petri-dish. Image entire dish in an imaging machine.</li>
	</ol>
	</li>
	<li>Use sterile scissors or scalpel to separate luciferase-negative sections from luciferase positive sections in the tumor and re-image.</li>
	<li>Repeat until only most highly positive tumor pieces remain.</li>
</ol>
6. Enzymatic digestion of tumor
<ol>
	<li>Under laminar flow hood, mince luciferase positive tumor pieces (step 5.4) into the smallest possible pieces using sterile surgical scissors and put them into a sterile 50 mL conical tube. 
	NOTE: Mincing the tumor into the smallest possible pieces will yield more individual cells.</li>
	<li>Prepare digest solution by adding 10 mL of collagenase IV (1.5 mg/mL), 80 &#181;L of hyaluronidase (20 mg/mL), and 160 &#181;L of deoxyribonuclease I (0.1 mg/mL) to 40 mL of HBSS. Mix solution by inverting.</li>
	<li>Add 35&#8722;40 mL of the digest solution to minced tumor. Incubate at 37 &#176;C with continuous rotation for 2 h. 
	NOTE: Vigorously shake tube periodically throughout incubation to prevent tumor tissue from clumping.</li>
	<li>Filter entire digestion through sterile 100 &#181;m cell strainer followed by a 40 &#181;m cell strainer to remove debris. Save the flow through and discard debris.</li>
	<li>Wash free cells by adding 20 mL of HBSS and centrifuge at 329 x g for 5 min. Aspirate the supernatant and resuspend pellet in 30 mL of HBSS.</li>
	<li>Combine 10 &#181;L of cell solution and 90 &#181;L of trypan blue in a single well of a 96 well plate. Count live cells using a hemacytometer.</li>
	<li>Transfer 1 x 104 to 1 x 106 tumor cells per mouse to a sterile 15 mL conical tube. Add 3 x 105 HK cells from step 1.2.3 per mouse, to the same tube with tumor cells. 
	NOTE: Use sterile 50 mL conical tube if the total volume exceeds 15 mL. Always calculate more doses for additional animals per study group to account for loss of fluid during syringe use. For example, if a group contains 5 mice, make enough cells for 6 or 7 mice.</li>
	<li>Centrifuge at 329 x g for 5 min. Discard supernatant either by aspirating or pipetting.</li>
	<li>Resuspend cells in 50 &#181;L per mouse for UCC model or 10 &#181;L per mouse for CRC model in complete RPMI media. Keep cell suspension on ice until ready for use.</li>
</ol>
7. UCC mouse model
<ol>
	<li>Preparation of mice for procedure
	<ol>
		<li>Obtain six- to eight-week old female NOD/SCID mouse. Shave the lower backs of the mouse using hair removal cream. Anesthetize the mouse in an induction chamber with isoflurane (2.5% in 100% oxygen, 1 L/min).</li>
		<li>Once sedated, place mouse in supine position with its snout in an isoflurane nose cone and bare back firmly grounded on a dispersive electrode. 
		NOTE: Mouse is completely sedated if unresponsive to toe pinch.</li>
	</ol>
	</li>
	<li>Instill UCC cells prepared in step 6.9 to bladder using an angiocatheter (Figure 1Aa,Ab).
	<ol>
		<li>Set up a monopolar electrocautery machine and set to a power of 4 W. Lubricate a 24 G sterile angiocatheter with lubricating jelly and insert through urethra of the female mouse. 
		NOTE: Slight resistance may be felt. Gently push forward or remove angiocatheter and repeat. Do not force.&#160;If catheter bends on entry, insert a sterile guide wire (see 7.2.2) halfway into catheter to provide stability.</li>
		<li>Fully insert the&#160;0.025&#8221; fixed core straight guide wire 1 mm past the end of the angiocatheter. 
		NOTE: The wire is marked with tape prior to the procedure to indicate the 1 mm stopping point and insure consistency.</li>
		<li>Hold the monopolar pin to the guide wire for 1 s allowing for electrical irritation of the bladder mucosa.</li>
		<li>Attach a fresh sterile angiocatheter to 1 cc luer-lok syringe and draw up 200 &#181;L of collected cells from step 6.9. 
		NOTE: At least 100 &#181;L is lost from the angiocatheter to the syringe. Compensate for loss volume when calculating volume of cell suspension needed.</li>
		<li>Remove guide wire and angiocatheter from mouse urethra. Insert angiocatheter with syringe of cells attached into urethra. 
		NOTE: Advancement should be easier than before.</li>
		<li>Instill 50 &#181;L of cells to mouse bladder. Wait a few seconds before removing the angiocatheter to allow for cells to adhere to the bladder wall. 
		NOTE: Cells remain in the bladder and develop into a primary tumor.</li>
	</ol>
	</li>
	<li>Remove mouse from isoflurane nose cone and grounding pad. Observe mouse for 1 h following procedure. Look for signs of distress, i.e., hunched back, labored breathing, etc.</li>
</ol>
8. CRC mouse model
<ol>
	<li>Anesthetize six-to-eight-week old male NOD/SCID mouse with isoflurane (2.5% in 100% oxygen, 1 L/min) in the induction chamber. Confirm sedation with a toe pinch.</li>
	<li>Place anesthetized mouse in supine position under a dissecting microscope, making sure to secure their snout to an isoflurane nosecone and to secure their front limbs with tape for stability. 
	NOTE: Loupes may be used instead of a dissecting microscope. A small object can be used to improve visibility and angle when placed under the base of the tail, elevating the anus. Typically, small sections of gauze are rolled into a cylinder shape of 1-inch diameter.</li>
	<li>Dilate the anal canal with curved lubricated blunt-tipped forceps to expose the distal anal and rectal mucosa. Remove feces.</li>
	<li>Use a sterile 30 G removable needle on a 50 &#956;L glass syringe to inject 10 &#956;L of tumor and HK cells (from step 6.9) into the distal posterior rectal submucosa 1 to 2 mm above the anal canal. The bevel of the needle should be covered by mucosa. Be careful not to pass into the pelvic cavity.</li>
	<li>Remove mouse from isoflurane nose cone. Observe mouse for 1 h following procedure. Look for signs of distress, i.e., hunched back, labored breathing, etc.</li>
</ol>
9. Bioluminescent imaging
<ol>
	<li>Monitor the primary tumor, liver, and lung metastatic burden weekly using a bioluminescent imaging system for luciferase activity.
	<ol>
		<li>Obtain a mouse from UCC or CRC experiment and weigh. Inject 150 mg/kg luciferin intraperitoneally and wait 5 min for the substrate to circulate in the mouse&#8217;s body.</li>
		<li>Anesthetize mouse with 2.5% isoflurane in 100% oxygen, 1 L/min in the induction chamber.</li>
		<li>Place mouse in BLI Imaging machine with nose fixed in nosecone. When exposing for the image, make sure that the area of interest is facing the camera. For UCC and CRC injection, the ventral side should face the camera for each image. Image mouse in supine position.</li>
	</ol>
	</li>
</ol>
10. Harvesting organs and tumor
<ol>
	<li>When the primary tumor luminescence radiance reaches 1 x 1011 photons or if mice exhibit signs of distress (i.e., weight loss, hunched back, harsh/labored breathing, etc.), euthanize mice by CO2 inhalation (as in step 5.2.1) after luciferin injection and whole-body imaging.</li>
	<li>Remove liver and lung, place in a petri dish and image to identify any metastases. Remove tumor, weigh and image. Fix organs and tumor in 10% neutral buffered formalin for 48 h at ambient temperature. 
	NOTE: Clean/wipe scissors and forceps between each organ to avoid transfer of tissue.</li>
</ol>
11. Histological evaluation
<ol>
	<li>Embed formalin fixed tissues in paraffin and slice the tissues at 5 &#181;m thickness on a microtome for hematoxylin and eosin (H&#38;E) and immunohistochemical (IHC) staining. 
	NOTE: All H&#38;E staining of paraffin slides was done in the pathology laboratory of Ochsner Health System, and all IHC staining in this paper was performed in a research laboratory of Ochsner Health System after high-temperature antigen retrieval using Ki67 and cytokeratin 20 antibodies, followed by biotinylated secondary antibody, and avidin-biotin-peroxidase complexes according to manufacturer instructions13,17.</li>
</ol>

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This study was partially supported by the Ochsner Translational Medicine Research Initiative Grant 2014. The authors declare no conflict of interest.

Metastatic disease is responsible for most cancer patient mortalities. In pre-clinical therapeutic tests, it is crucial to establish mouse models that most closely emulate human tumor growth with spontaneous distant organ metastases. Using murine models with implanted patient tumor derived cancer cells (xenografts) allows for a better understanding of tumor biology and predictive biomarkers as well as testing and prediction of antineoplastic effects of novel therapies18. Many models have been used...

It has been shown that lymph node (LN) metastasis is a poor prognostic indicator in many solid organ malignancies, including high-grade urothelial cell carcinoma (UCC) of the bladder and colorectal cancer (CRC)1,2. Over half of the patients with muscle-invasive UCC (MIUCC), despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years. Metastatic CRC is a leading cause of cancer-related death in the US.
An estimated 81,190 new patients and 17,240 cancer specific deaths are expected to occur in 2018 in the United States due to UCC of the bladder3,4. While patients will predominantly (70%) present with non-muscle invasive disease, 30% will have MIUCC5. Despite the curative therapy (radical cystectomy [RC] with or without systemic chemotherapy) for clinically localized disease, half of the patients with MIUCC of the bladder will still develop metastases and die within 5 years3. Lymph node involvement is found in approximately 20%&#8722;25% of patients having undergone RC6,7,8. Five-year survival rate in LN positive patients is less than 35% even after RC, suggesting LN involvement as a crucial negative predictor for the prognosis in UCC patients.
Colorectal cancer is the third most common cancer diagnosed in both men and women in the United States. The patient outcomes largely depend on tumor characteristics and tumor microenvironment, such as depth of invasion, LN involvement, and distant organ metastases. Although the mortality rate in CRC decreased in the last decade due to screening and effective surgeries, it is estimated that almost 50% of CRC patients will develop metastases or recurrent disease9.
Small animal models provide an expeditious, reproducible, and modifiable platform to study tumor progression and different metastatic patterns. There are currently no described xenograft models that consistently mimic CRC and UCC metastasis seen in patients. The primary route of cancer distant metastasis is via lymphatic spread. New research suggests that the LNs&#160;provide tumors with a unique microenvironment, and are not only simply stationary targets where cancer cells transiently pass, but also play an integral role by interacting with cancer cells in the metastatic process. Indeed, our studies discovered that, in addition to educating and promoting tumor progression and metastases, the LN stromal microenvironment is also responsible for drug resistance in CRC10,11. Our lab recently confirmed the tumorigenic effects of LN stromal cells (LNSCs) on CRC and UCCs using patient-derived orthotopic xenograft (PDOX) mouse models12,13.

Developing PDOX models provides an important platform for translational cancer research14,15. By maintaining the principal histologic&#160;and genetic characteristics of their donor tumor, PDOX models&#160;remain stable across passages and make good platforms for translational cancer research12,15. PDOX models are being used for preclinical drug evaluation, biomarker identification, and preclinical evaluation of personalized medicine strategies allowing for prediction of clinical outcomes. Currently, there are no described xenograft models that consider the importance of LN involvement and are capable of consistently reproducing primary tumor and distant organ metastasis in CRC and UCC. In this study, we describe the development of PDOX models in NOD/SCID mice with reproduction of metastatic CRC and UCC diseases with LNSC involvement.&#160;

<table><tbody><tr><td>Avidin-biotin-peroxidase</td><td>Vector Labs Inc</td><td>PK-6100</td><td></td></tr><tr><td>Biotinylated secondary antibody</td><td>Vector Labs Inc</td><td>BA-1000</td><td></td></tr><tr><td>Collagenase IV (1.5 mg/mL)</td><td>Worthington Biochemical Corporation</td><td>LS004189</td><td></td></tr><tr><td>Deoxyribonuclease I (0.1 mg/mL)</td><td>Sigma</td><td>D4263</td><td></td></tr><tr><td>D-Luciferin (150 mg/kg)</td><td>Perkin Elmer</td><td>122796</td><td></td></tr><tr><td>Formalin (10% neutral buffered)</td><td>Leica</td><td>46129</td><td></td></tr><tr><td>glutamine (2 nM)</td><td>Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Hair Removal Cream</td><td>Church &amp;amp; Dwight Co., Inc</td><td>1 (800) 248-8820</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (HBSS)</td><td>Fisher Scientific</td><td>SH30016.02</td><td></td></tr><tr><td>Hyaluronidase (20 mg/mL)</td><td>Sigma</td><td>H3884</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>108333</td><td></td></tr><tr><td>Luc/RFP-lentivirus</td><td>From our collaborators. See reference 13: Gills, J. et al. A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis. Oncotarget. 9, 32718-32729, doi:10.18632/oncotarget.26024 (2018).</td><td></td><td></td></tr><tr><td>McCoy&amp;rsquo;s medium</td><td>Life Technologies</td><td>110862</td><td></td></tr><tr><td>penicillin/streptomycin 100 mL (100 U/mL)</td><td>Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>RPMI-1640 Medium</td><td>American Type Culture Collection</td><td>110636</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T6146</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Gas &lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100% Oxygen</td><td>Airgas Inc</td><td>OX USP200</td><td></td></tr><tr><td>100% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas Inc</td><td>CD USPE</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Mice&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6-8 week old NOD/SCID Mice (male)</td><td>Jackson Lab</td><td>001303</td><td></td></tr><tr><td>6-8 week old NOD/SCID Mice (female)</td><td>Jackson Lab</td><td>001303</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Immunohistochemistry&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma</td><td>GHS232</td><td></td></tr><tr><td>Ki-67 Rabbit Monoclonal Antibody</td><td>Thermo Scientific</td><td>RM-9106-S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Tools&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>100 &amp;micro;m cell strainer</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>15 mL Conical Tube</td><td>Sarstedt</td><td>11799</td><td></td></tr><tr><td>50 mL Conical tube</td><td>Sarstedt</td><td>15762</td><td></td></tr><tr><td>150 mm Tissue Culture Dish</td><td>USA Scientific Inc</td><td>CC7682-3614</td><td></td></tr><tr><td>96 Well plate</td><td>USA Scientific Inc</td><td>CC7682-7596</td><td></td></tr><tr><td>Forceps</td><td>Symmetry Surgical Inc</td><td>06-0011</td><td></td></tr><tr><td>Surgical scissors</td><td>Symmetry Surgical Inc</td><td>02-2011</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Equipment&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5% CO&lt;sub&gt;2&lt;/sub&gt; humidified incubator</td><td>Thermo Scientific</td><td>3110</td><td></td></tr><tr><td>Bioluminescent (BLI) Imaging Machine</td><td>Perkin Elmer</td><td>CLS136334</td><td></td></tr><tr><td>BLI Imaging Machine Software</td><td>Perkin Elmer</td><td>CLS136334</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>366830</td><td></td></tr><tr><td>Deconvoluting Microscope</td><td>Intelligent Imaging Innovations</td><td>Marianas</td><td></td></tr><tr><td>Deconvoluting Microscope Imaging Software</td><td>Intelligent Imaging Innovations</td><td>+1 (303) 607-9429 x1</td><td></td></tr><tr><td>Digital caliper</td><td>Fowler Tools and Instruments</td><td>54-115-330</td><td></td></tr><tr><td>Dissecting microscope</td><td>Precision Instruments LLC</td><td>(504) 228-0076</td><td></td></tr><tr><td>Electrosurgical generator</td><td>ValleyLab</td><td>FORCE1C20</td><td></td></tr><tr><td>Isoflurane Induction Chamber</td><td>Perkin Elmer</td><td>119038</td><td></td></tr><tr><td>Microtome</td><td>American Optical Corporation</td><td>829</td><td></td></tr><tr><td>Pipet Aid</td><td>Fisher Healthcare</td><td>13-681-15E</td><td></td></tr><tr><td>Serological pipet (10 mL)</td><td>Sarstedt</td><td>86.1254.001</td><td></td></tr></tbody></table>

patient-derived orthotopic xenograft models for human urothelial cell carcinoma and colorectal cancer tumor growth and spontaneous metastasis

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

In the UCC PDOX model, UCC patients&#39; BlCaPt15 or BlCaPt37 cells were intra-vesically (IB) instilled in the presence of HK cells into female NOD/SCID mouse bladder (Figure 1A). Twenty-five out of thirty (83.3%) animals generated primary tumors and displayed time dependent primary tumor growth based on weekly BLI (Figure 1B,C and Table 1). Similarly, in the CRC PDOX model, 31 out of 32 (96.9%) ...

Watch this Scientific Journal Video about Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis at JoVE.com

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

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

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

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Research

JoVE Journal

Cancer Research

10.1K Views.  Ochsner Clinic Foundation. Modellen zijn nodig om consequent nodig om kankermetastase na te bootsen door de interactie met lymfeklieren. Bij patiënten met zowel colorectale kanker als urotheliale celkanker van de blaas. Onze orthotopische xenografische modellen beschreven in dit document kunnen helpen bij het beantwoorden van deze belangrijke biologische vragen en het testen van nieuwe therapieën efficiënt en klinisch relevante wijze.Onze orthotopische xenograft tumoren geproduceerd in muizen lijken op de tum...

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

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

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

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

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

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

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

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

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

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

Medicine

Culture of Bladder Cancer Organoids as Precision Medicine Tools

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

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

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

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 ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

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

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

Source: Moret, R. et al. Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and ...

JoVE Encyclopedia of Experiments

Urinary Tract Cancer

In vivo studies

Generating PDOX Mouse Model of Colorectal Cancer: Studying Progression of Colorectal Cancer in a Murine Model

Source: &#160;Moret, R. et al. <a href="https://www.jove.com/v/59223"> Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.</a> J. Vis. Exp. (2019).
This video describes the protocol for generating a patient-derived orthotopic xenograft colorectal cancer mouse model to study the progression of colorectal cancer cells in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. This murine model helps in understanding primary tumor growth and metastases pattern.

Source: &#160;Moret, R. et al.  Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and ...

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

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