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…
開示
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 |
