Cell morphology. Cell shape was primarily examined to check for particular morphological phenotypes that have been linked to cell invasion (Xxxxx and Xxxxxxxxx, 2015). Cancer cells are normally in contact with extracellular matrix which involves a crucial part of the tumour microenvironment influencing cancer progression and metastasis (Xxxxxxx and Xxxxx, 2011). Immunohistochemical analysis on primary breast tumour samples has in fact suggested that fibronectin expression correlates with poor prognosis and progression free survival (Xxx et al., 2013). In order to examine cell morphology while trying to retain some aspects of the tumour microenvironment, cells were seeded on fibronectin coated glass coverslips and stained with TRITC phalloidin that stains the actin filaments as a marker of cell shape. Cell morphology features such as cell area and circularity were thus measured. The relative cell elongation parameter was obtained by subtracting the circularity value from 1.
Cell morphology. (A) Cells were seeded on fibronectin coated coverslips. Cells were subsequently fixed and stained with TRITC Phalloidin (red) indicating the actin cytoskeleton and DAPI (blue) indicating the nuclei. Scale bar = 10μm. Cells were imaged and analysis was performed on ImageJ to obtain (B) the cell area and (C) elongation index. Analysis was performed on 30 cells per experiment. Bars represent the mean values and error bars represent the SEM (N=3). The first observation that was made is that unlike the normal MCF10A cells, all the breast cancer cell lines that were evaluated grew as single cells while no colony forming capacity was observed (figure 3.5A). The absence of colony forming cells indicates the loss of ability to form cell-cell interactions. This is one of the primary characteristics of epithelial to mesenchymal transition (EMT) which is accompanied by loss of e-cadherin expression that normally maintains cell-cell junctions and tissue integrity (Thiery, 2002). This feature is consistent with the aggressive behaviour of triple negative breast cancer. The cells were also evaluated in terms of other morphological features such as their area and elongation. In terms of their area, this feature varied a lot across the cell panel with the basal-like HCC38 cells appearing to have the largest cell area (figure 3.5A, 3.5B). The MB-231 and MB- 436 which also express high levels of IQGAP3 (figure 3.2B) were shown to be the most elongated cells consistent with their mesenchymal phenotype (figure 3.5C) but also have small cell area (figure 3.5B). Nonetheless, levels of IQGAP3 expression do not appear to strongly correlate with a particular morphological feature.
Cell morphology. The time course of changes in cellular morphology by light microscopy following exposure to the placebo or Propanc compounds will be examined. If Propanc induces a change in morphology this might be indicative of a change in the expression of integrins (integrins are ‘receptors’ found on cell membranes that bind the extracellular matrix and bring about changes in cell shape (cytoskeleton), cell mobility and cell cycle). The expression of markers of epithelial cell-cell contacts (E-cadherin, beta-catenin) can also be investigated. This approach might be crucial to understanding how the drugs cross the epithelial barrier in the rectum. To address this question in more detail, it is likely that a model for transport across a single epithelial cell layer will need to be developed in the long term.
