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 &#38; 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&#8217;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Gas </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% CO2</td>
			<td>Airgas Inc</td>
			<td>CD USPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mice</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immunohistochemistry</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2 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 ...

patient-derived-orthotopic-xenograft-models-for-human-urothelial-cell

Research

JoVE Journal

Cancer Research

10.0K Views.  Ochsner Clinic Foundation. 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.

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

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

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 ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

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 ...