Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

Summary

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Abstract

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Introduction

The use of biomaterial in 3D tissue culture has enabled researchers to study cell behavior in the laboratory under physiological conditions more akin to an in vivo environment than that of one recapitulated with 2D adhesion and a plastic substrate. In particular, great strides forward have been made in modeling stratified epithelia with the adoption of 3D culture methods at the air-liquid interface^1-4. Such techniques faithfully mimic keratinocyte differentiation and tumor cell invasion enabling greater flexibility and fidelity for researchers studying these processes. The choice of biomaterial substrate to mimic the stromal environment has primari....

Protocol

This study was conducted according to the Declaration of Helsinki Principles and was approved by the appropriate Ethics Committees.

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 0.22 μm membrane filter. Store as 0.25 ml sterile aliquots in -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 requi....

Results

This technique opens up the possibility of examining and comparing the invasive behavior of tumor cells (in this case cSCC) under different 3D stromal environments.  Using this technique, not only C7-deficient native matrices that recapitulated the RDEB dermal environment can be generated, but also additional matrices that have been genetically engineered to over-express C7¹⁸. As seen in Figure 2, invasion of RDEB cSCC keratinocytes was significantly retarded in C7-overexpressing native matric.......

Discussion

Due to the nature of this experiment, the total time required for completion can be up to two months. Throughout this time, utmost care and sterile tissue culture practices must be employed to prevent microbial contamination.

Aside from its role as a cofactor in the synthesis of hydroxyproline and hydroxylysine of collagens, ascorbic acid stimulates collagen specific mRNA expression in fibroblasts²². The ascorbic acid of choice here is the more stable L-.......

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