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 He...

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	<li>Sundlisaeter, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphangiogenesis+in+colorectal+cancer--prognostic+and+therapeutic+aspects.">Lymphangiogenesis in colorectal cancer--prognostic and therapeutic aspects.</a> International Journal of Cancer. Journal international du cancer. 121, 1401-1409 (2007).</li><li>Gout, S., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cancer+microenvironment+in+metastasis:+focus+on+colon+cancer.">Role of cancer microenvironment in metastasis: focus on colon cancer.</a> Cancer Microenvironment. 1, 69-83 (2008).</li><li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2018.">Cancer statistics, 2018.</a> CA: a Cancer Journal for Clinicians. 68, 7-30 (2018).</li><li>Hautmann, R. E., de Petriconi, R. C., Pfeiffer, C., Volkmer, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radical+cystectomy+for+urothelial+carcinoma+of+the+bladder+without+neoadjuvant+or+adjuvant+therapy:+long-term+results+in+1100+patients.">Radical cystectomy for urothelial carcinoma of the bladder without neoadjuvant or adjuvant therapy: long-term results in 1100 patients.</a> European Urology. 61, 1039-1047 (2012).</li><li>Stein, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radical+cystectomy+in+the+treatment+of+invasive+bladder+cancer:+long-term+results+in+1,054+patients.">Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients.</a> Journal of Clinical Oncology. 19, 666-675 (2001).</li><li>Lerner, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rationale+for+en+bloc+pelvic+lymph+node+dissection+for+bladder+cancer+patients+with+nodal+metastases:+long-term+results.">The rationale for en bloc pelvic lymph node dissection for bladder cancer patients with nodal metastases: long-term results.</a> The Journal of Urology. 149, 758-764 (1993).</li><li>Poulsen, A. L., Horn, T., Steven, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radical+cystectomy:+extending+the+limits+of+pelvic+lymph+node+dissection+improves+survival+for+patients+with+bladder+cancer+confined+to+the+bladder+wall.">Radical cystectomy: extending the limits of pelvic lymph node dissection improves survival for patients with bladder cancer confined to the bladder wall.</a> The Journal of Urology. 160, 2015-2019 (2020).</li><li>Margolin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+stromal+cells+enhance+drug-resistant+colon+cancer+cell+tumor+formation+through+SDF-1alpha/CXCR4+paracrine+signaling.">Lymph node stromal cells enhance drug-resistant colon cancer cell tumor formation through SDF-1alpha/CXCR4 paracrine signaling.</a> Neoplasia. 13, 874-886 (2011).</li><li>Vermeulen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+activity+defines+colon+cancer+stem+cells+and+is+regulated+by+the+microenvironment.">Wnt activity defines colon cancer stem cells and is regulated by the microenvironment.</a> Nature Cell Biology. 12, 468-476 (2010).</li><li>Margolin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+roles+of+tumor-initiating+cells+and+the+lymph+node+stromal+microenvironment+in+human+colorectal+cancer+extranodal+metastasis+using+a+unique+humanized+orthotopic+mouse+model.">The critical roles of tumor-initiating cells and the lymph node stromal microenvironment in human colorectal cancer extranodal metastasis using a unique humanized orthotopic mouse model.</a> FASEB Journal. 29, 3571-3581 (2015).</li><li>Gills, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+patient-derived+orthotopic+xenograft+model+enabling+human+high-grade+urothelial+cell+carcinoma+of+the+bladder+tumor+implantation,+growth,+angiogenesis,+and+metastasis.">A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis.</a> Oncotarget. 9, 32718-32729 (2018).</li><li>Hidalgo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discovery. 4, 998-1013 (2014).</li><li>Hiroshima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+mouse+models+of+cancer+need+to+be+orthotopic+in+order+to+evaluate+targeted+anti-metastatic+therapy.">Patient-derived mouse models of cancer need to be orthotopic in order to evaluate targeted anti-metastatic therapy.</a> Oncotarget. 7, 71696-71702 (2016).</li><li>Kim, H. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+establishment+features+of+orthotopic+murine+bladder+cancer+models.">Tumor establishment features of orthotopic murine bladder cancer models.</a> Korean Journal of Urology. 53, 396-400 (2012).</li><li>Hadaschik, B. 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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%) ...

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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 ...

Models are needed to consistently needed to mimic cancer metastasis through the interaction with lymph nodes. In patients with both colorectal cancer and urothelial cell cancer of the bladder. Our orthotopic xenographic models described in this paper can help answer these important biologic questions and test new therapies efficiently and in a clinically relevant manner.Our orthotopic xenograft tumors produced in mice resemble the patients'tumor. In the presence of lymph node stromal cells both models have a higher tumor implantation and distant organ metastasis rates and locations that mimic human metastatic patterns. Also, both animal models have zero procedural-related mortality.After removing patient derived tumors from euthanized mice, use steril surgical scissors to mince luciferase positive tumor pieces as small as possible in a petri dish under a laminar flow hood. Transfer the tumor pieces into a sterile 50 mL conical tube. To prepare digest solution, add 10 mL of collagenase type 4, 80 microliters of hyaluronidase and 160 microliters of deoxyribonuclease type 1 to 40 mL of HBSS and mix by inverting.Add 35-40 mL of the digest solution to minced tumor and incubate at 37 degrees Celsius with continuous rotation for 2 hours with periodic vigorous shaking. To remove debris after digestion, filter the digested tumor through sterile 100 micrometer cell strainer followed by filtration through a 40 micrometer cell strainer saving the flow through in a 50 mL tube each time and discarding the debris. To wash free cells, add 20 mL of HBSS to the flow through and centrifuge at 329 times gravity for 5 minutes.Aspirate the supernatant and resuspend the pellet in 30 mL of HBSS. Transfer 100, 000 tumor cells to a sterile 15 mL conical tube and add 300, 000 previously prepared fresh HK cells. Centrifuge the cells at 329 times gravity for 5 minutes and discard the supernatant by aspiration.Resuspend the cells in complete RPMI medium and keep on ice until ready for use. To prepare the animal for UCC cell injection use hair removal cream to shave the lower back of a 6-8 week old female NOD-SCID mouse. To administer UCC cells into a bladder, first set up a monopolar electrocautery machine to a power of 4 watts.After anesthetizing the mouse, place it in supine position with its snout in a isoflurane nose cone and bare back firmly grounded on a dispersed electrode. Lubricate a 24 gauge angiocatheter with lubricating jelly and then insert the angiocatheter gently but firmly through the mouse urethra. If catheter bends on entry, insert guide wire halfway into catheter to provide stability.Then, fully insert the 0.64 millimeter fixed core straight guide wire 1 millimeter past the end of the angiocatheter. Hold the monopolar pin to the guide wire for one second allowing for an electrical irritation of the bladder mucosa. Attach a fresh, sterile angiocatheter to 1 mL leur lock syringe and draw up to 200 microliters of UCC cells prepared in previous steps.Remove guide wire and angiocatheter from the urethra and then insert angiocatheter with syringe of cells attached to it. Inject 50 microliters of the cells to the mouse bladder and to allow the cells to adhere to the bladder wall, wait a few seconds before removing the angiocatheter. Finally, remove the mouse from isoflurane nose cone and grounding pad and observe it for 1 hour following procedure for signs of distress.To create CRC mouse model, place an anesthetized 6-8 week old male NOD-SCID mouse in supine position under a dissecting microscope securing the snout to and isoflurane nose cone and the front limbs with tape for stability. Place a small object such as a small gauze section under the base of the tail elevating the anus to improve visability and angle. Use curved lubricated blunt tipped forceps to dilate the anal canal and expose the distal anal and rectal mucosa and remove feces.Connect a sterile 30 gauge removable needle to a 50 microliter glass syringe. Draw 10 microliters of tumor and HK cells cell suspension prepared in previous steps into the syringe. Inject the 10 microliters into the distal posterior rectal submucosa 1-2 millimeters above the anal canal making sure not to pass into the pelvic cavity.Finally, remove the mouse from isoflurane nose cone and observe it for one hour following procedure for signs of distress. To weekly monitor the primary tumor, liver and lung metastatic burden in either mouse model, first, weigh the mouse. Inject 150 mg/kg luciferin intra-peritoneally and wait 5 minutes for the substrate to circulate in the body.Place the anesthetized mouse in a bioluminescent imaging system with nose fixed in nose cone making sure that the area of interest, which is the ventral side of the animal is facing the camera for each image. Image mouse in supine position weekly for luciferase activity. After slicing and staining paraffin-embedded isolated tissues, observe them using a high-powered microscope.In the UCC mouse model, 83.3 percent of animals generated primary tumors and displayed time-dependent primary tumor growth based on weekly bioluminescence measurements. In the CRC mouse model, 96.9%of mice grew primary tumors. Depending on the patient tumor, mouse tumor growth had a different latency period, which reflects the difference in the patients'clinical characteristics.In both intra-vesicle and intra-rectal models, tumor cell injection generated orthotopic primary tumors. Furthermore, 33.3%and 53.1%of mice instilled with UCC cells and CRC cells with HK cells, respectively, developed distant organ metastasis. Histopathology of xenografts in the UCC model was compared to primary patient bladder carcinoma.The staining results from xenografts were similar to those in the original surgical biopsies, confirming similar tissue morphology. Antibody to Ki67, a human cell proliferation marker, shows positive nuclear staining indicating highly proliferating, fast-growing human tumor cells. Similarly, in the CRC model, H&E staining indicates the similarity of architecture between xenografts and patient tumors.Antibody staining against cytokeratin 20 also showed similar tumor growth pattern in both CRC models. The key to these procedures is to treat the animals gently, but firmly. Practice with a steady hand is the best way to perform these techniques safely and achieve the best outcomes.Once these models are made, multiple different forms of cancer therapies can be tested, including combinations of current drugs as well as newly developed therapies prior to clinical trials in patients. It is important to remember that the handling of both cancer cells and the needles should be performed by experienced personnel for their safety as well as to achieve optimal results.

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