Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment

• Published in: Cells (Basel, Switzerland) Switzerland), 2019-08, Vol.8 (8), p.937
• Main Authors: Nanou, Afroditi, Zeune, Leonie L, Terstappen, Leon W M M
• Format: Article
• Language: English
• Subjects: ACCEPT; Adenocarcinoma - pathology; Adenomatous polyposis coli; Antibodies; Automation; Biomarkers; Blood; Breast; Breast Neoplasms - pathology; Cancer; Carcinoma, Non-Small-Cell Lung - pathology; Carcinoma, Non-Small-Cell Lung - ultrastructure; CD45 antigen; Cell adhesion molecules; Cell Count; CellSearch; Cloning; Colorectal Neoplasms - pathology; Datasets; EasyCount slides; EpCAM enrichment; Epithelial Cell Adhesion Molecule - metabolism; Epithelial cells; Erythrocytes; Extracellular vesicles; Extracellular Vesicles - metabolism; Extracellular Vesicles - pathology; Female; Fluorescence microscopy; Humans; immunofluorescence imaging; leukocyte-derived extracellular vesicles; Leukocytes; Leukocytes - metabolism; Lung Neoplasms - pathology; Lung Neoplasms - ultrastructure; Metastases; Metastasis; Monoclonal antibodies; Neoplastic Cells, Circulating - pathology; Non-small cell lung carcinoma; Open source software; Prostate; Small cell lung carcinoma; Tumor cells
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells8080937

Large tumor-derived Extracellular Vesicles (tdEVs) detected in blood of metastatic prostate, breast, colorectal, and non-small cell lung cancer patients after enrichment for Epithelial Cell Adhesion Molecule (EpCAM) expression and labeling with 4',6-diamidino-2-phenylindole (DAPI), phycoerythrin-conjugated antibodies against Cytokeratins (CK-PE), and allophycocyanin-conjugated antibody against the cluster of differentiation 45 (CD45-APC), are negatively associated with the overall survival of patients. Here, we investigated whether, similarly to tdEVs, leukocyte-derived EVs (ldEVs) could also be detected in EpCAM-enriched blood. Presence of ldEVs and leukocytes in image data sets of EpCAM-enriched samples of 25 healthy individuals and 75 metastatic cancer patients was evaluated using the ACCEPT software. Large ldEVs could indeed be detected, but in contrast to the 20-fold higher frequency of tdEVs as compared to Circulating Tumor Cells (CTCs), ldEVs were present in a 5-fold lower frequency as compared to leukocytes. To evaluate whether these ldEVs pre-exist in the blood or are formed during the CellSearch procedure, the blood of healthy individuals without EpCAM enrichment was labelled with the nuclear dye Hoechst and fluorescently tagged monoclonal antibodies recognizing the leukocyte-specific CD45, platelet-specific CD61, and red blood cell-specific CD235a. Fluorescence microscopy imaging using a similar setup as the CellSearch was performed and demonstrated the presence of a similar population of ldEVs present at a 3-fold lower frequency as compared to leukocytes.