Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Summary

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells.  

Abstract

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.

Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.

By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.   

Introduction

Organotypic co-cultures are tissue-engineered models which aid in reconstructing the in vivo tumor microenvironment in 3-dimensions by juxtaposing malignant epithelial cells and stromal cells in a collagen gel containing essential extracellular matrix components^1-3. Principally conceived as a method of measuring tumor invasion, organotypics reduce reliance on in vivo animal models, and avoid the shortcomings of other in vitro techniques such as Transwell assays, in which invading cells are forced artificially into a mono-dispersed state^2-4. The inclusion of stromal cells in these models, such as fibroblasts, reflects th....

Protocol

1. Establishment of Primary Fibroblast Cultures from Colonic Explants

1. Obtain samples of normal human colon mucosa tissue directly from the surgical operating theatre, and suspend in 7-10 ml of PBS supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml Fungizone.
2. In the laboratory, place the specimen at the center of a 10-cm tissue culture dish and wash 3 times with PBS/Pen-strep/Fungizone. Do not aspirate after the final wash.
3. With sterile forceps and scalpel, cut the tissue into small pieces (approximately 2mm) and place each piece at the center of a cross drawn with the scalpel blade in a 12-well p....

Results

We have applied the above method to multiple combinations of CRC cell lines and stromal cells. One example presented here is SW480 epithelial CRC cell lines with primary human ex vivo colonic fibroblasts. 3-dimensional co-cultures are constructed (Figure 1), subjected to microscopy and laser capture microdissection (Figure 2), and analyzed by microRNA (miRNA) profiling (Figure 3) for comparison of differentially expressed miRNAs between cells at the invasive tum.......

Discussion

Here we describe a method to specifically isolate and characterize tumor cells which have acquired the capacity to invade the cancer associated stroma during the earliest stages of metastatic progression in a 3-dimensional co-culture construct of epithelial and stromal cells.

Co-culture models comprised of CRC epithelial cells juxtaposed with a synthetic stroma containing ex vivo human colonic fibroblasts were used to study CRC invasion in 3-dimensions. This physiologically relev.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Colorectal cancer cell lines - example shown SW480	ATCC	ATCC CCL-228
Collagen	BD Biosciences	354265
Matrigel	BD Biosciences	354234
Nylon membrane	Merck Millipore	VVLP01300
Metal grid	The Mesh Company	WSS20-A4	themeshcompany.com
Laser microdissection platform	Leica Microsystems	Leica AS LMD
Membrane mounted slides	Molecular devices
Cresyl Violet	Merck Millipore	1052350025