Summary
This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.
Abstract
Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.
Protocol
1. Sample Preparation Grow cells to ~70% confluent in their specific media with growth factors or FBS (e.g., DMEM with 10% FBS for MC-1 cells). Aspirate media from plate and gently wash several times with 1 x PBS (8 g/L of NaCl, 0.2 g/L of KCl, 1.15 g/L of Na2HPO4.7H2O, 0.2 g/L of KH2PO4, pH 7.3). Aspirate PBS and add 2 mL of 0.05% Trypsin in versene (0.014 g/L of phenol red and 0.2 g/L of EDTA-Na in 1 x PBS, pH 7.2). Gently rock plate to…
Discussion
Metastasis is the leading cause of death in cancer patients. It involves four major steps: detachment of cancer cells from their primary loci, their entry into circulation (intravasation), their exit from circulation (extravasation), and survival and growth in a distant organ. Metastasis in human is considered a slow process and often manifested after years of latency. To study its progression in a timely manner, the above relatively quick experimental assay was established in immunodeficient mice.4 Since its…
Acknowledgements
This protocol was tested and optimized initially in the laboratory of Dr. Richard Hynes (MIT). Funding is provided by the NYSTEM IDEA AWARD (to L.X.) and Ruth L. Kirschstein National Research Service Award (to L.X.).
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DMEM | Invitrogen | 10313-021 | ||
| FBS | Hyclone | SH30088.03 | ||
| HBSS | Invitrogen | 14175-095 | ||
| NaCl | Sigma-Aldrich | S9888 | ||
| KCl | Mallinckrodt Chemicals | 6838-04 | ||
| Na2 HPO4.7H2O | Mallinckrodt Chemicals | 7896-04 | ||
| KH2PO4 | Mallinckrodt Chemicals | 7100-12 | ||
| Phenol red | Sigma-Aldrich | P3532 | ||
| EDTA-Na | EMD Biosciences | 4010 | ||
| Trypan Blue | Invitrogen | 15250-061 | ||
| Trypsin 2.5% | Invitrogen | 15090-046 | ||
| Ethanol | Ultrapure | 200-CSGP | ||
| 50 ml Falcon Tube | VWR | 89039-656 | ||
| Microcentrifuge Tubes | Axygen Scientific | MCT-175-C | ||
| Falcon 70µm Cell Strainer | BD Biosciences | 352350 | ||
| 5ml Falcon Culture Tube | VWR | 60818-576 | ||
| Hemacytometer | Hausser Scientific | 15170-208 | ||
| 1 ml syringe | BD Biosciences | 329650 | ||
| 30 ½ gauge needle | BD Biosciences | 305106 | ||
| Mouse restrainer | Plas Labs Inc. | 551-BSRR | ||
| Surgical scissors | Fisher Scientific | 08-940 | ||
| Forceps | Fisher Scientific | 08-902 | ||
| Dissecting Microscope | Nikon | SMZ1500 | ||
| Benchtop Centrifuge | Thermoscientific | 75003491 |
References
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Cite This Article
Mohanty, S., Xu, L. Experimental Metastasis Assay. J. Vis. Exp. (42), e1942, doi:10.3791/1942 (2010).