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

Summary

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.

Abstract

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.

Introduction

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 bl....

Protocol

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 ....

Results

In the UCC PDOX model, UCC patients' 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%) mice.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Avidin-biotin-peroxidase	Vector Labs Inc	PK-6100
Biotinylated secondary antibody	Vector Labs Inc	BA-1000
Collagenase IV (1.5 mg/mL)	Worthington Biochemical Corporation	LS004189
Deoxyribonuclease I (0.1 mg/mL)	Sigma	D4263
D-Luciferin (150 mg/kg)	Perkin Elmer	122796
Formalin (10% neutral buffered)	Leica	46129
glutamine (2 nM)	Fisher Scientific	35050061
Hair Removal Cream	Church & Dwight Co., Inc	1 (800) 248-8820
Hanks Balanced Salt Solution (HBSS)	Fisher Scientific	SH30016.02
Hyaluronidase (20 mg/mL)	Sigma	H3884
Isoflurane	Henry Schein Animal Health	108333
Luc/RFP-lentivirus	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).
McCoy’s medium	Life Technologies	110862
penicillin/streptomycin 100 mL (100 U/mL)	Fisher Scientific	15140-122
RPMI-1640 Medium	American Type Culture Collection	110636
Trypan Blue	Sigma	T6146
Trypsin/EDTA	Life Technologies	15400-054
Name	Company	Catalog Number	Comments
Gas
100% Oxygen	Airgas Inc	OX USP200
100% CO2	Airgas Inc	CD USPE
Name	Company	Catalog Number	Comments
Mice
6-8 week old NOD/SCID Mice (male)	Jackson Lab	001303
6-8 week old NOD/SCID Mice (female)	Jackson Lab	001303
Name	Company	Catalog Number	Comments
Immunohistochemistry
Hematoxylin	Sigma	GHS232
Ki-67 Rabbit Monoclonal Antibody	Thermo Scientific	RM-9106-S
Name	Company	Catalog Number	Comments
Tools
40 µm cell strainer	Fisher Scientific	08-771-1
100 µm cell strainer	Fisher Scientific	08-771-19
15 mL Conical Tube	Sarstedt	11799
50 mL Conical tube	Sarstedt	15762
150 mm Tissue Culture Dish	USA Scientific Inc	CC7682-3614
96 Well plate	USA Scientific Inc	CC7682-7596
Forceps	Symmetry Surgical Inc	06-0011
Surgical scissors	Symmetry Surgical Inc	02-2011
Name	Company	Catalog Number	Comments
Equipment
5% CO2 humidified incubator	Thermo Scientific	3110
Bioluminescent (BLI) Imaging Machine	Perkin Elmer	CLS136334
BLI Imaging Machine Software	Perkin Elmer	CLS136334
Centrifuge	Beckman	366830
Deconvoluting Microscope	Intelligent Imaging Innovations	Marianas
Deconvoluting Microscope Imaging Software	Intelligent Imaging Innovations	+1 (303) 607-9429 x1
Digital caliper	Fowler Tools and Instruments	54-115-330
Dissecting microscope	Precision Instruments LLC	(504) 228-0076
Electrosurgical generator	ValleyLab	FORCE1C20
Isoflurane Induction Chamber	Perkin Elmer	119038
Microtome	American Optical Corporation	829
Pipet Aid	Fisher Healthcare	13-681-15E
Serological pipet (10 mL)	Sarstedt	86.1254.001