A correlative microscopy approach with live cell imaging, immunocytochemistry, 3-D holographic imaging and FESEM for characterization of hypoxia-driven EMT in T84 colon adenocarcinoma cells.

Abstract number
European Microscopy Congress 2020
Corresponding Email
[email protected]
LSA.6 - Applications of correlative microscopy of biological systems
Dr Tzipi Cohen Hyams (1, 2, 4), Dr Srinivasa Pothula (2, 1), Associate Professor Murray C. Killingsworth (1, 3, 2, 4)
1. Ingham Institute for Applied Medical Research
2. South Western Sydney Clinical School, niversity of New South Wales
3. Anatomical Pathology, New South Wales Health Pathology
4. School of Medicine, Western Sydney University

correlative microscopy, Hypoxia, FESEM, immunocytochemistry, 3-D holographic,  adenocarcinoma

Abstract text

Background: Hypoxia is a prominent feature of solid tumour growth often resulting in angiogenesis from the surrounding vasculature to return the growing mass to tissue homeostasis. Hypoxia is also known to promote epithelial to mesenchymal transition (EMT), which has been shown to enable metastatic spread of tumour cells. To investigate these phenomena and search for relevant biomarkers we have developed an in-vitro colon adenocarcinoma model which employs detailed characterization by live cell imaging, immunocytochemistry, electron microscopy, CLEM and 3-D holographic imaging. 

Methods: The initial characterization of T84 cells is done using live cell time-lapse video imaging (Nikon Biostation, Japan) to assess cell viability and/or reaction to hypoxia. Hypoxia was applied for 2-6 hours to determine the time point when irreversible cell death occurred. We then subjected cells to a shortened period of hypoxia followed by normoxia to investigate cell recovery after the hypoxic event. The cells were then fixed with PFA at various timepoints. This was followed by permeabilization and immunolabelling for EpCam, vimentin and actin with visualisation by antibody fragment conjugated quantum dot nanoparticles. Intact and labelled cell colonies are then imaged by LSM 800 with Airyscan (Carl Zeiss, Germany) and holographic microscopy (NanoLive Fluo, Switzerland) to assess colony features in 3-D. The cells are then further fixed with 2.5% glutaraldehyde, scraped from the culture dish and then prepared as for routine transmission electron microscopy. Correlative electron microscopy was carried out with a GeminiSEM 300 FESEM system (Carl Zeiss, Germany).

Results: T84 cells in their normal resting state were observed to divide at regular intervals and displayed characteristic EpCam, actin and vimentin staining. They were shown to be able to withstand 6 hours of hypoxia with 100% CO2 and then recover. During this process, EMT was observed. Hypoxia caused a relocation of these markers which corresponded with the generation of EMT cells. Electron microscopy and CLEM confirmed the immunostaining results.

Conclusion: Correlation of complementary histological and cytological imaging modalities provides an unprecedented holistic view of tumour cell growth and development in-vitro. Moreover, the adenocarcinoma cell culture model system as developed can be manipulated to replicate hypoxic conditions encountered during tumour growth. The present study demonstrates features of hypoxia-driven EMT and baseline normoxic immunostained data with correlated ultrastructure and 3-D holographic imaging.