Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

Summary

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

Abstract

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an "in-incubator" video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

Introduction

Cell invasion and migration are involved in the dissemination of cancer cells, which is the main cause of resistance to treatment¹ and can lead to locoregional or metastatic recurrence after cancer treatment². The epithelial-mesenchymal transition (EMT) is the initial process of cell invasion-migration in which cancer cells switch from an epithelial to a mesenchymal phenotype. E-Cadherin is an extracellular marker of the epithelial phenotype³, and increased expression of N-cadherin and vimentin is characteristic of the mesenchymal phenotype⁴. Migration also depends on the i....

Protocol

NOTE: The Boyden chamber and scratch assays without inclusion of the ECM are referred to as the migration assay, and the same assays with the ECM is referred to as the invasion assay.

1. Boyden Chamber Assay

NOTE: This protocol is adapted for the SQ20B cell line, which is derived from a recurrent Head and Neck Squamous Cell Carcinoma (HNSCC) laryngeal cancer and was obtained from John Little (Boston, MA, USA).

Day 1

1. Cell Seeding
   1. Seed 2 10⁶ SQ20B cells in 12 mL of culture medium (CM) in a 175 cm² flask 72 h ....

Results

We report here two different methods to analyze cell invasion and migration. Figure 1 shows the Boyden chamber experiment. The inserts are placed into a companion plate with chemoattractant medium, and the cells are seeded in CM. The membrane can be uncoated (migration assay) or coated (invasion assay). Cells are seeded into the upper chamber in s-CM. The lower chamber is filled with CM as a chemoattractant. The cells are fixed before the doubling time.

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Discussion

We report here two different modalities to study the cell invasion and migration process. The analysis of this process is important to understanding the factors involved in metastatic recurrence, which might be explained by increased motility of a subpopulation of cancer cells called cancer stem cells^10,11.

The Boyden chamber experiment is one the most frequently used techniques to explore cell invasion and migration. One advantage of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal Calf Serum Gold	GE Healthcare	A15-151
Hydrocortisone water soluble	Sigma-Aldrich	H0396-100MG
Penicillin/Streptomycin 100 X	Dominique Dutscher	L0022-100
DMEM	Gibco	61965-026
F12 Nut Mix (1X) + GlutaMAX-I	Gibco	31765-027
EGF	Promega	G5021	The solution must be prepared just before use because it is very unstable
Z1 coulter particle	Beckman Coulter	6605698
Optical microscope	Olympus	CKX31
SQ20B cell line	Gift from the John Little’s Laboratory	-
Wound Maker	Essen Bioscience	4494	Store in safe and dry place
96-well ImageLock Plate	Essen Bioscience	4379
CoolBox 96F System with CoolSink 96F	Essen Bioscience	1500-0078-A00
CoolBox with M30 System	Essen Bioscience	1500-0078-A00
Boyden Insert	Dominique Dutcher	353097
Boyden Coated Insert	Dominique Dutcher	354483	Store at -20 °C
Companion 24-well Plate	Dominique Dutcher	353504
BD Matrigel standard	BD BioScience	BD 354234	Store at -20°C.
RAL 555 Staining Kit	RAL Diagnostics	361550	Store in safe and dry place
Microcentrifuge tubes	Eppendorf	33511