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

Published: April 30, 2023

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). Allow the tumor to grow subcutaneously to ~1 cm3 in size, which usually takes approximately 1-3 months. Anesthetize the mouse…

Representative 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). The CRC tissue surgically dissected from the patient was cut into pieces and implanted subcutaneously into NOG mice. s.c.: subcut…

Divulgaciones

The authors have nothing to disclose.

Materials

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
a Zeiss Axioplan 2 stereofluorescence microscope  Zeiss
the 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
the 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

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Citar este artículo
Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells. J. Vis. Exp. (Pending Publication), e20335, doi: (2023).

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