Abstract
In this study scanning near-field optical microscopy (SNOM) has been utilised in conjunction with quantum dot labelling to interrogate the biomolecular composition of cell membranes. The technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The technique has been applied to investigate cell-cell adhesion in human epithelial cells. This has been realised through immunofluorescence labelling of the cell-cell adhesion protein E-cadherin. Moreover, a dual labelling protocol has been optimised to facilitate a comparative study of the adhesion mechanisms and the effect of aberrant adhesion protein expression in both healthy and cancerous epithelial cells. This study reports clear differences in the morphology and phenotype of healthy and cancerous cells. In healthy prostate epithelial cells (PNT2), E-cadherin was predominantly located around the cell periphery and within filopodial extensions. The presence of E-cadherin appeared to be enhanced when cell-cell contact was established. In contrast, examination of metastatic prostate adenocarcinoma cells (PC-3) revealed no E-cadherin labelling around the periphery of the cells. This lack of functional E-cadherin in PC-3 cells coincided with a markedly different morphology and PC-3 cells were not found to form close cell-cell associations with their neighbours. We have demonstrated that with a fully optimised sample preparation methodology, multiplexed quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.
Highlights
Cellular adhesion plays an important role in maintaining the architecture of tissues and organs and is vital for their correct functioning
Following previous investigations of adhesion mechanisms in the healthy epithelial cell line PNT2 [22], a dual labelling methodology has been developed to facilitate the study of adhesion molecules in other cell lines
Gene expression analysis revealed a significant down-regulation in E-cadherin expression in the PC-3 cell line compared to the control cell line, PNT2
Summary
Cellular adhesion plays an important role in maintaining the architecture of tissues and organs and is vital for their correct functioning. A concerted effort has been made by many researchers to determine the relationship between cellular adhesion and the metastatic capacity of many cancers [1,2]. E-cadherin is one of the principle mediators of cell-cell adhesion in epithelial tissues and has been extensively examined to determine its role in cancer progression and metastasis. It has been demonstrated that the loss of E-cadherin expression or function is linked to increased invasive potential [3], metastatic potential [4] and poorer patient prognosis [5,6] This relationship is relevant to prostate cancers which have a propensity to metastasise and form secondary tumours (primarily skeletal), resulting in a poor patient prognosis [7,8]. A more detailed understanding of adhesion mechanisms could lead to the development of novel cancer treatments as indicated by the preliminary studies carried out by Zhou et al [11] and Mao et al [12]
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