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Open AccessArticle

Scanning Electron Microscopy of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles

1
Department of Medical Cell BioPhysics, University of Twente, 7522 NH Enschede, The Netherlands
2
Division of Clinical Studies, The Institute of Cancer Research, London SM2 5NG, UK
3
Prostate Cancer Targeted Therapy Group, The Royal Marsden NHS Foundation Trust, London SM2 5PT, UK
*
Authors to whom correspondence should be addressed.
Cancers 2018, 10(11), 416; https://doi.org/10.3390/cancers10110416
Received: 13 August 2018 / Revised: 24 October 2018 / Accepted: 30 October 2018 / Published: 31 October 2018
(This article belongs to the Special Issue Circulating Tumor Cells (CTCs))
To explore morphological features of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs), we developed a protocol for scanning electron microscopy (SEM) of CTCs and tdEVs. CTCs and tdEVs were isolated by immunomagnetic enrichment based on their Epithelial Cell Adhesion Molecule (EpCAM) expression or by physical separation through 5 μm microsieves from 7.5 mL of blood from Castration-Resistant Prostate Cancer (CRPC) patients. Protocols were optimized using blood samples of healthy donors spiked with PC3 and LNCaP cell lines. CTCs and tdEVs were identified among the enriched cells by fluorescence microscopy. The positions of DNA+, CK+, CD45− CTCs and DNA−, CK+, CD45− tdEVs on the CellSearch cartridges and microsieves were recorded. After gradual dehydration and chemical drying, the regions of interest were imaged by SEM. CellSearch CTCs retained their morphology revealing various shapes, some of which were clearly associated with CTCs undergoing apoptosis. The ferrofluid was clearly distinguishable, shielding major portions of all isolated objects. CTCs and leukocytes on microsieves were clearly visible, but revealed physical damage attributed to the physical forces that cells exhibit while entering one or multiple pores. tdEVs could not be identified on the microsieves as they passed through the pores. Insights on the underlying mechanism of each isolation technique could be obtained. Complete detailed morphological characteristics of CTCs are, however, masked by both techniques. View Full-Text
Keywords: scanning electron microscopy (SEM); circulating tumor cell (CTC); tumor-derived extracellular vesicle (tdEV); ferrofluid; CellSearch; microsieves scanning electron microscopy (SEM); circulating tumor cell (CTC); tumor-derived extracellular vesicle (tdEV); ferrofluid; CellSearch; microsieves
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Nanou, A.; Crespo, M.; Flohr, P.; De Bono, J.S.; Terstappen, L.W.M.M. Scanning Electron Microscopy of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles. Cancers 2018, 10, 416.

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