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Stephen Bustin Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford, UK |
Abstract
Studies aimed at elucidating the complex mechanisms driving colon cancer initiation, progression and therapeutics are hampered by the limitations of current models. Animal paradigms may not be wholly biologically relevant to human disease and cell cultures invariably have lost a number of specialised biochemical and behavioural properties of the parent tissues. We report the development of a long-term colorectal organotypic model based on multicellular cultures that mimic in vivo morphological features of neoplastic colonic mucosa. Colorectal cancer cell lines are cultured in a discrete environment consisting of a collagen matrix enclosing primary colonic fibroblasts, vascular smooth muscle and endothelial cells that is placed on a custom-designed support and suspended over growth medium. This approach approximates a three dimensional in vitro representation of the tumour microenvironment and results in the formation of large cellular aggregates that histologically resemble high-grade epithelial dysplasia or adenocarcinoma. Colon-specific differentiated structures develop in the constructs over a period of 21 days, with localised de-differentiation and evidence suggestive of epithelial-to-mesenchymal transition becoming apparent between days 28 and 56. Dissection of tissue sections allows the extraction and analysis of DNA, mRNA and miRNA from both the epithelial and stromal components of this model. When compared with two-dimensional adherent cell culture techniques, this protocol represents a more relevant system for the in vitro assessment of agents that regulate growth and progression in colorectal cancer.
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