AbstractDuring development and disease progression, cells and tissue undergo mechanical changes and alter their response to external physical cues. This is also true during the onset of colorectal cancer, the second most common cause of cancer deaths in the Western world. Before the changes that occur during cancer onset can be understood, a better appreciation is needed of the mechanical forces and properties involved. Therefore, there is a drive to develop novel tools to accurately measure biomechanical properties at different spatial scales.
3D cell models are a useful method to understand the mechanical properties at cell and tissue scale, as they provide a more physiologically relevant environment for cells than 2D cell culture. Additional insight may be achieved by complementing experimental approaches with computational models to reveal how the properties of individual cells work as a system to generate the mechanical properties of 3D tissue structures.
In this work, Madine-Darby Canine Kidney (MDCK) cells and cysts were used as a model for epithelial tissue. These cells can be genetically modified to mimic the onset of colorectal cancer, by the expression of a truncated Adenomatous polyposis coli (APC) protein: N-APC. N-APC is present in most cases of colorectal cancer and provides a useful tool to study the early changes in colorectal cancer. MDCK cells form spherical cysts up to 100 μm in diameter with a hollow lumen when cultured in an extracellular matrix (ECM) gel.
Atomic Force Microscopy (AFM) experiments have revealed that MDCK cells expressing N-APC grown at continued confluency were significantly stiffer than cells expressing only WT-APC. Cells expressing N-APC had an average stiffness of 4.59 ± 1.30 kPa in the nuclear region, while the average stiffness of WT cells was 2.90 ± 0.59 kPa. This is the first time a mechanical effect of N-APC expression has been observed.
Adapting AFM for use on 3D tissue models is challenging, as these structures are grown embedded in an ECM gel and cannot be probed directly. This work describes a novel protocol that allowed the AFM cantilever to come in contact with a MDCK cyst surface. These experiments have been used to validate a Finite Ele- ment (FE) model of the mechanical behaviour of an MDCK cyst, that predicted the deformation behaviour of a cyst being compressed by an AFM cantilever.
Alternatively, microultrasound (μUS) imaging was evaluated as an approach to provide additional useful information about 3D tissue models, with the benefit that direct contact with the sample is not required. Quantitative analysis of the μUS signal allowed the calculation of the apparent speed of sound through an MDCK cysts, which was on average 1112.96 ± 383.87 m/s. This was significantly lower than the apparent speed of sound through a spheroid (a 3D structure that does not contain a lumen) such as HCT-116 spheroids, which had an average apparent speed of sound of 1494.68 ± 951.82 m/s. This suggests that the speed of sound depends on the geometrical features of a spherical structure. Indeed, FE modelling has confirmed that stronger interference effects occur for a sound wave interacting with a cyst compared to a spheroid due to surface and Lamb waves.
In addition, cellular and molecular markers for mechanical stress were identified to determine the effect of static compression applied with a weighted coverslip: compressed cells had nuclei with smoother nuclear envelopes (decrease in amount of folded nuclear envelopes of 32 % ± 41 % for WT-MDCK cells) and an increased vinculin signal (increase of signal intensity of 22 % ± 10 % for WT-MDCK cells) at the junctional membrane.
Finally, the use of acoustic radiation force (ARF) was explored as a means of applying compression to cells. While preliminary experiments that exposed the apical surface of samples to continuous acoustic radiation did not reveal an ob- servable effect on the cells, a FE model has been developed to aid further design of ARF experiments.
This project explored a range of different tools for studying the mechanical properties of cells and tissue, specifically in the context of 3D models. Experimental approaches have been complemented with FE models to increase understanding of how mechanical properties relate over different length scales, from cells to tissue structures, and how acoustic waves interact with cells and tissue structures. In the future, these approaches could permit further investigation of how the mechanical properties of cells and tissues change during the onset of colorectal cancer.
|Date of Award||2019|
|Supervisor||Inke Nathke (Supervisor) & Sandy Cochran (Supervisor)|