The development of a novel 3D migration assay to study the effects of cell signalling and microenvironment on the migratory behaviour of colorectal cancer

Abstract

Colorectal cancer is one of the mostly commonly diagnosed cancers for both men and women in the UK, with a poor survival rate compared to other Western countries and other commonly diagnosed cancer types. Part of the reason for poor prognosis for patients is the diagnosis of the disease at an advanced stage of progression, which has been shown to have a severe impact on patient survival rates. Due to this, the signalling events surrounding the adoption of an invasive phenotype may provide the opportunity to develop therapies to limit the spread of tumours from their original site and improve patient prognosis.

It has previously been highlighted that the culture of cells in standard two-dimensional (2D) induces alterations to gene expression via the imposition of a microenvironment which does not reflect the microenvironment experienced by cancer cells in vivo. This limits the accuracy of data obtained using 2D migration assays and can help to account for the failure of some anti-migratory compounds to be effective in in vivo or clinical screening, after showing promise in conventional cell culture tests.

This project has aimed to develop a novel three-dimensional (3D) migration assay based on Alvetex® technology, which provides a more biologically relevant microenvironment in vitro, to investigate the role of cell signalling and microenvironment in determining the migratory behaviour of colorectal cancer cells. Through extensive optimisation, the 3D culture of two colorectal cancer cell lines, SW480 and SW620, was established to allow for the assessment of cell migration via histological processing, in addition to the assessment of other behavioural traits via the use of commercial biochemical assays.

Modulation of both the Wnt and Insulin-like Growth Factor (IGF)-I signalling pathways via small molecule inhibitors and exogenous protein application has highlighted compounds which alter the migratory behaviour of the colorectal cancer cell lines, results which were not reflected in counterpart 2D scratch wound assays. This underlines the need to use culturing techniques which better reflect the biological system in question, as the anti-migratory applications of these compounds have the potential to be missed if subjected to 2D screening only.

The biological relevance of the model developed here was also increased by altering the culture microenvironment by the addition of extracellular matrix (ECM) coatings or co-culture, demonstrating that this model can be adapted to recreate a variety of microenvironments depending on the aims of the research undertaken.

Together, the data presented in this thesis demonstrates the suitability of this novel culture system to assess the migratory behaviour of colorectal cancer cells in vitro, with the possibility of the adaption of this system to assess the behaviour of other cancer types.