Abstract
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.
Highlights
During the last decades, Extracellular Vesicles (EVs) have emerged as promising disease biomarkers bearing similar membrane and cargo composition as their originating cells [1,2,3]
We identified in the digitally stored CellSearch images some CD45+, DAPI, CK- objects of similar size to tumor-derived Extracellular Vesicles (tdEVs) that we baptized leukocyte-derived Extracellular Vesicles
After carefulexamination examinationof of immunofluorescence images the CellSearch cartridges, After careful thethe immunofluorescence images of theofCellSearch cartridges, CD45+, CD45+, DAPI, CKobjects, that resemble in size our previously reported
Summary
Extracellular Vesicles (EVs) have emerged as promising disease biomarkers bearing similar membrane and cargo composition as their originating cells [1,2,3]. For nucleic acid analysis, the membrane encapsulated nucleic acid cargo is protected from enzymatic degradation, and it can circulate for a longer time compared to cell-free DNA (cfDNA) [4,5]. It has been demonstrated that EVs in biofluids of cancer patients are significantly elevated when compared to the respective numbers of healthy individuals [10,11]. That finding implies that more cell types secrete EVs in response to the
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