Experimental Metastasis Assay

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

1. 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).
2. 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 facilitate cell detachment from plate. Observe the cells under a microscope. It usually takes 2-5 minutes for the cells to detach.
3. Add a....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	10313-021
FBS	Hyclone	SH30088.03
HBSS	Invitrogen	14175-095
NaCl	Sigma-Aldrich	S9888
KCl	Mallinckrodt Baker Inc.	6838-04
Na2 HPO4.7H2O	Mallinckrodt Baker Inc.	7896-04
KH2PO4	Mallinckrodt Baker Inc.	7100-12
Phenol red	Sigma-Aldrich	P3532
EDTA-Na	EMD Millipore	4010
Trypan Blue	Invitrogen	15250-061
Trypsin 2.5%	Invitrogen	15090-046
Ethanol	Ultrapure	200-CSGP
50 ml Falcon Tube	VWR international	89039-656
Microcentrifuge Tubes	Axygen Scientific	MCT-175-C
Falcon 70μm Cell Strainer	BD Biosciences	352350
5ml Falcon Culture Tube	VWR international	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 Instruments	SMZ1500
Benchtop Centrifuge	Thermo Fisher Scientific, Inc.	75003491