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

Overview

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.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Culturing cell lines

1. 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 °C in a 5% CO₂ humidified incubator.
   NOTE: HK cells are normal human follicular dendritic cells and can be grown and expanded for ≤ 15 passages in vitro.

2.  Tagging and enrichment of luciferase labeled tumors

1. Implant tumors directly into the left and right flank of 6-8 week old female NOD/SCID mice.
2. Measure tumor growth bi-weekly using a digital caliper.
3. At 1 cm in diameter, transduce the tumor. Directly inject into the tumor a single dose of Luc/red fluorescent protein (RFP)-lentivirus (50 µL/tumor, 1:30 dilution from concentrated high titer lentivirus stock) using a 1 cc syringe with a 27 G needle.
   NOTE: The patient's tumor typically reaches 1 cm in diameter in 1-2 months. However, the growth rate is extremely variable and based on a number of factors, including tumor grade and type.

3. CRC mouse model

1. Anesthetize a 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.
2. Place the anesthetized mouse in supine position under a dissecting microscope, making sure to secure their snout to an isoflurane nose cone and 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.
3. Dilate the anal canal with curved lubricated blunt-tipped forceps to expose the distal anal and rectal mucosa. Remove feces.
4. Use a sterile 30 G removable needle on a 50 μL glass syringe to inject 10 μL of luciferase tagged tumor cells and HK cells (resuspend 10 µL of tumor cells per mouse for CRC model in complete RPMI media) 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.
5. Remove the mouse from the isoflurane nose cone. Observe it for 1 h following the procedure. Look for signs of distress, i.e., hunched back, labored breathing, etc.

4. Bioluminescent imaging

1. Monitor the primary tumor, liver, and lung metastatic burden weekly using a bioluminescent imaging system for luciferase activity.
   1. Obtain a mouse CRC experiment and weigh. Inject 150 mg/kg luciferin intraperitoneally and wait 5 min for the substrate to circulate in the mouse's body.
   2. Anesthetize the mouse with 2.5% isoflurane in 100% oxygen, 1 L/min in the induction chamber.
   3. Place the mouse in a BLI Imaging machine with the nose fixed in the nose cone. When exposing for image, make sure that the area of interest is facing the camera. For CRC injection, the ventral side should face the camera for each image. Image the mouse in supine position.

Materials

Name	Company	Catalog Number	Comments
RPMI-1640 Medium	American Type Culture Collection	110636
Isoflurane	Henry Schein Animal Health	108333
D-Luciferin (150 mg/kg)	Perkin Elmer	122796
glutamine (2 nM)	Fisher Scientific	35050061
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).
penicillin/streptomycin 100 mL (100 U/mL)	Fisher Scientific	15140-122
Gas
100% Oxygen	Airgas Inc	OX USP200
100% CO2	Airgas Inc	CD USPE
Mice
6-8 week old NOD/SCID Mice (female)	Jackson Lab	1303
Tools
15 mL Conical Tube	Sarstedt	11799
150 mm Tissue Culture Dish	USA Scientific Inc	CC7682-3614
Forceps	Symmetry Surgical Inc	06-0011
Surgical scissors	Symmetry Surgical Inc	02--2011
Equipment
5% CO2 humidified incubator	Thermo Scientific	3110
Bioluminescent (BLI) Imaging Machine	Perkin Elmer	CLS136334
BLI Imaging Machine Software	Perkin Elmer	CLS136335
Digital caliper	Fowler Tools and Instruments	54-115-330
Dissecting microscope	Precision Instruments LLC	(504) 228-0076
Isoflurane Induction Chamber	Perkin Elmer	119038