Correlative light and electron microscopy (CLEM) analysis of the tumour microenvironment in non-melanoma skin cancer

Session

Poster Session Two

Authors

Dr Emma McDermott (1, 2), Dr Kerry Thompson (1, 2), Dr Alanna Stanley (2)

Affiliations

1. Anatomy Imaging and Microscopy (AIM) Facility
2. University of Galway

Abstract text

Skin cancer is the most diagnosed caner in Ireland. Cancer initiation is a complex multistep process of cellular and neoplastic transformation, its progression is reliant on local tissue invasion and distant metastasis. The acquisition of these latter capabilities requires the activation of epithelial mesenchymal transition (EMT) where the polarized epithelial cell phenotype is lost and a highly motile mesenchymal phenotype, with increased resistance to apoptosis is assumed. Degradation of the basement membrane enables cells to move from the epithelial layer to the underlying connective tissue layer and is the final step of EMT [1]. EMT, and ultimately tumour progression, is reliant on cell-to-cell and cell-ECM interactions, in particular at the invasive front and within what is known as the tumour microenvironment (TME) [2]. Extracellular vesicles (EVs), membrane encased nano-sized structures released from cells, mediate intracellular communication through targeted delivery of their bioactive components [3]. Tumour cell derived EVs, mediated by hypoxia inducible factors (HIFs), have emerged as key regulators for cancer progression including the formation of a favourable microenvironment, recruitment of non-cancerous cells, cancer associated fibroblast (CAF) interactions and regulation of EMT [4, 5]. An essential component in all these processes is the remodelling of the cytoskeletal. 

Reactive oxygen species (ROS), can act as signalling molecules and maintain cellular homeostasis, but have also been extensively linked to the development and progression of many cancers [6, 7]. It is known that cytoskeletal reorganisation occurs as a result of an increase in ROS but also as a stage in EMT and EV release [8, 9]. Rho GTPases are primarily known as key regulators of the actin cytoskeleton, but they are also involved with the regulation of ROS [7,10]. Interestingly, Rho GTPases, the actin cytoskeleton, HIFs and EVs release are all sensitive to redox regulation [10-12].

The vast expanse of in-vitro methods, animal and molecular studies have largely elucidated the mechanisms and key players involved in the regulation of the above processes. However, to truly understand how the localisation and spatial mapping of these molecules relates to subcellular and extracellular structures, a correlative light and electron microscopy (CLEM) protocol can be applied to patient samples. CLEM combines the versatility of fluorescence microscopy with the resolution of EM, enabling analysis of larger volumes of tissues followed by the in-depth ultrastructural analysis of more precise areas of interest. Basal and squamous cell carcinomas represent a good model for investigating these processes as they can be non-aggressive or aggressive i.e invading the surrounding tissue, but still not become metastatic. In approximately 5% of cases, particularly in patients with high risk features including recurrence of carcinoma or immunosuppression, these carcinomas become metastatic. 

This study investigates changes in the basement membrane, EMT markers (E-cadherin, vimentin), actin cytoskeleton (phalloidin), EVs (CD40), NOX complexes and CAFs (alpha-smooth muscle actin) in patient samples diagnosed with squamous and basal cell carcinoma. Labelling is via fluoronanogold secondary antibodies to allow for both immunofluorescence and transmission electron microscopy analysis. These targets are investigated in the context of ultrastructural environment, and additional qualitative and quantitative analysis on cell-cell junctions, basement membrane integrity, changes in ECM will be carried out.

 

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