Tumor Cell Invasion Assay: A 3D In Vitro Native Matrix-based Method to Assess Epithelial-mesenchymal Cell Interactions

Abstract

Source: Ng, Y. Z. et al. Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion. J. Vis. Exp. (2014)

This video describes the technique of utilizing a native matrix to study tumor cell invasion. The technique used in this assay provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix.

Protocol

1. Preparation of Media and Reagents

1. Preparation of 200x of L-ascorbic acid 2-phosphate stock
   1. Dissolve 29 mg of L-ascorbic acid 2-phosphate per 5 mL of Dulbecco's Modified Eagle's Medium (DMEM) solution and filter through a 0.22 μm membrane filter. Store as 0.25 mL sterile aliquots at -20 °C.
   2. Add 0.25 mL aliquot of 200x L-ascorbic acid 2-phosphate stock to every 50 mL of fibroblast media (DMEM with 1% L-glutamine and 10% fetal bovine serum) on the day required, for a final concentration of 0.1 mM of L-ascorbic acid 2-phosphate.
2. Preparation of keratinocyte growth media
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Results

Figure 1. Workflow of generation of fibroblast-derived native matrix and the tumor invasion assay.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-Ascorbic acid 2-phosphate 	Sigma	A8960
DMEM with L-glutamine, 4,500 mg/L D-glucose, 110 mg/L sodium pyruvate  	Life Technologies	11995-073
100x Penicillin-Streptomycin  	Life Technologies	15070
Vaseline 	VWR	PROL28908.290
Clonal cylinders 	Sigma	Z370789
Nylon Net Filter Disc Hydrophilic 100 μm 25 μm diameter 100/pk 	Millipore	NY1H02500
Bent stainless steel wire mesh support  	Made in house		Dimensions were made so that the mesh would fit into 6-well plates