Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

Abstract

Source: Okazawa, Y., et al. High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor. J. Vis. Exp. (2018).

This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.

Protocol

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

1. Dissection of CRC PDXs from Mice

Experimental procedures for dissection of CRC PDXs are outlined in Figure 1B (step 2).

1. Allow the tumor to grow subcutaneously to ~1 cm³ in size, which usually takes approximately 1-3 months.
2. Anesthetize the mouse with isoflurane inhalation using the small animal inhalation anesthesia device and disinfect the skin with 70% ethanol.
3. Place the mouse in a prone po....

Results

Figure 1: Schematic representation of the generation of metastases by the PDX-derived CRC organoids labeled with GFP lentivirus in NOG mice. (A) Implantation of small pieces of the CRC tissue subcutaneously into NOG mice (step 1).......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NOD/Shi-scid IL2Rγ null (NOG) mice	 The Central Institute for Experimental Animals,Kanagawa, Japan 		Breed 6-week-old male mice under germ-free and specific pathogenfree conditions
Wound clips 2×10mm 	Natsume manufacturing, Japan 	#C-21-S 	Autoclave before use
Zeiss Axioplan 2 stereofluorescence microscope	 Zeiss
SW55Ti swinging bucket rotor	 Beckman Coulter
pCMV-dR8.2 dvpr 	Gift from Dr. Robert A. Weinberg
PRRL-GFP vector	Gift from Dr. Robert A. Weinberg
pCMV-VSV-G	Gift from Dr. Robert A. Weinberg
5 ml polypropylene centrifuge tubes 	Beckman Coulter 	326819
Minisart 0.45 µm filter 	Sartorius stedim 	17598-K
FuGENE 6 transfection regent 	Roche	 11814 443001
CRC organoid culture medium with 1% or 5% FCS			DMEM/F-12 with GlutaMAX™ supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 ng/ml hEGF and 10 µM Y27632, a ROCK inhibitor. Store at 4°C. Use within 1 month.
Culture medium 	Gibco		DMEM/F-12 with GlutaMAX™ supplement supplemented with 5% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Store at 4°C. Use within 1 month
Hamilton syringe needle size:22 gauge 	Tokyo Science, Japan 		Disinfect with 70% alcohol and sterile PBS.
6-well plate 	BMBio 	#92006
12-well plate 	BMBio 	#92412
15ml conical tube 	Sumitono Bakelite 	MS-57150
Microtube 	Eppendorf 	#0030120086 	Autoclave before use
50ml conical tube 	Sumitono Bakelite 	MS-57500
Hemocytometer 	Erma 	#03-202-1
40μm cell strainer 	Corning 	#352340
Matrigel basement membrane matrix	Corning 	#354234 	Store aliqupts at -20°C. Place on ice until use
Collagenase type 1 	Sigma 	#C1030 	150 mg/ml collagenase type1 in 1×PBS. Store aliqupts at -20°C for up to 1 year
Accutase 	Innovate Cell Technologies 	#5V2623A	 Store at 4°C.
DMEM/F-12 with GlutaMAX™	 Gibco 	#10565018 	Store at 4°C. Warm at 37°C before use
Cell banker 1plus 	ZENOAQ 	#628 	Store at 4°C. Use within 1 month
Penicillin 	Gibco 	#15140122	Store at 4°C. Use within 1 month
Streptomycin	Gibco 	#15140122	Store at 4°C. Use within 1 month
hEGF 	PEPROTECH	#AF-100-15 	Store at -20°C. Add to medium on same day as use
Y27632, a ROCK inhibitor	 Wako 	#253-00591	Store at -20°C. Add to medium on same day as use