The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

Summary

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

Abstract

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

Introduction

The in vitro invasion assay can be a useful tool when measuring a cell's ability to migrate through a protein-coated membrane, analogous to the first steps in metastasis. A key feature of malignant cancer cells is their ability to migrate through and invade nearby tissues. Cancer that has spread or metastasized poses more treatment challenges and has lower rates of long-term survival, while localized tumors are easier to treat and have higher rates of long-term survival. In order to metastasize, cancer cells must leave the primary tumor and migrate into the circulatory or lymphatic system, a process which requires passing through the extracellular matrix and basem....

Protocol

All experiments and methods were performed as authorized by Villanova University Institutional Animal Care and Use Committee (IACUC).

1. Gene Expression in Cultured Mouse Mammary Tumor Cells

1. First, prepare the cell lines to be tested.
2. Use a breeding colony of BALB/cV mice. These mice carry the BALB/cV strain of mouse mammary tumor virus, transmitted to pups in milk^6,7. Fifty percent of breeding females develop mammary tumors by 10 months of age.
   1. To establish a tumor cell line, sacrifice a tumor-bearing dam using an IACUC-approved pr....

Results

This method of in vitro invasion through a protein matrix was used to assess the aggressive phenotypes and oncogenic cell behaviors of mouse mammary tumor cells with altered expression of the zinc finger protein ZC3H8⁸. In conjunction with other approaches that also examine cell migration and growth in 3D environments, it was found that higher levels of expression of Zc3h8 in tumor cell lines, or by promoter-mediated expression from a plasmid, resulted in rapid rat.......

Discussion

The in vitro invasion assay is an inexpensive, rapid, quantitative, and straightforward method for studying the factors promoting cancer cell invasion. Breast cancer is the most commonly diagnosed cancer among women. Of the three major subtypes of breast cancer, triple negative, (or ER-, PR-, HER2/neu-), is the most aggressive, most likely to metastasize, and most deadly⁹. Therefore, understanding the genes and expression that result in metastasis can help find new therapeutic targets and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	Corning	353504
Antibiotic-Antimycotic (100x)	ThermoFisher	15240062
BALB/c mice
Cell Culture Incubator
Cell Culture Treated Flasks
Clinical cenrifuge
Cotton swab	Puritan	25-806
Crystal Violet	Sigma Aldrich	C0775
Distilled water
DMEM	ThermoFisher	10566-016	high glucose, GlutaMAX
Ethanol
FBS	Sigma Aldrich	F2442-500ML
Forcepts
Glass Slide	VWR	16004-422
HBSS	ThermoFisher	14025076	no calcium, no magnesium
Hemocytometer
Imersion oil
Invasion Chambers (24-well)	Corning	354480	Cat. #354481 for 6-well
Light Microscope
Lipofectamine Transfection Reagent
Paraformaldehyde	Sigma Aldrich	P6148
PBS
Scalpel, disposable			#11
shRNA
Sterile Transfer pipet
Trypsin-EDTA	ThermoFisher	25200056