The elusive complexity of membranous extracellular vesicle (EV) and membrane-less extracellular particle (EP) populations released from various cellular sources contains clues as to their biological functions and diagnostic utility. In this study, we employed optimized multicolor nano-flow cytometry, structured illumination (SIM), and atomic force microscopy (AFM) to bridge sensitive detection at the single EV/EP level and high-throughput analysis of cancer cell secretomes. We applied these approaches to particles released from intact cells driven by several different transforming mechanisms or to cells under therapeutic stress imposed by pharmacological inhibition of their oncogenic drivers, such as epidermal growth factor receptor (EGFR). We demonstrate a highly heterogeneous distribution of biologically relevant elements of the EV/EP cargo, including oncoproteins (EGFR), clotting factors (tissue factor), prometastatic integrins (ITGA6, ITGA4), tetraspanins (CD63), and genomic DNA across the entire particulate secretome of cancer cells. We observed that targeting EGFR activity with irreversible kinase inhibitors (dacomitinib) triggers emission of DNA containing EP/EV subpopulations, including particles (chromatimeres) harboring both EGFR and DNaseresistant chromatin. While nano-flow cytometry enables quantification of these changes across the entire particular secretome, SIM reveals individual molecular topography of EV/EP subsets and AFM exposes some of their physical properties, including the presence of nanofilaments and other substructures. We describe differential uptake rates of distinct EV subsets, resulting in preferential internalization of exosome-like small EVs by cancer cells to the exclusion of larger EVs. Thus, our study illustrates the potential of nano-flow cytometry coupled with high-resolution microscopy to explore the cancer-related EV/EP landscape.
BackgroundMitral annular calcification (MAC) is associated with risk of stroke. This study aimed to define the morphological and functional characteristics of MAC that are related to stroke.
MethodsA total of 460 subjects with MAC from transthoracic echocardiography in a single center from 2012 to 2016 was retrospectively reviewed. Subjects were classified into two groups according to history of stroke [Group 1 (n = 366): without stroke; Group 2 (n = 94): with stroke]. Morphological and functional features of MAC on echocardiogram were scored from 0 to 3 according to MAC mobility, presence of echodense mass with central echolucencies in the periannular region suggesting caseous necrosis, and functional stenosis.
ResultsSignificantly more patients in group 2 were men and had history of diabetes mellitus, dyslipidemia, atrial fibrillation, or infective endocarditis. Although MAC thickness and extent did not differ between the two groups, group 2 showed a considerably higher MAC score than group 1 (0.50 ± 0.77 vs. 0.23 ±0.52 p<0.001) as a result of the higher prevalence of each component in group 2 [mobility (22 vs. 11%, p = 0.003), echodense mass with central areas of echolucencies suggesting caseous necrosis (23 vs. 7%, p<0.001), and functional mitral stenosis (12 vs. 7%, p = 0.042)]. On logistic regression analysis, MAC score was independently associated with stroke and showed significant incremental value to demographic factors and comorbidities in association with stroke in a consecutive manner.
ConclusionsIn conclusion, morphological and functional characteristics of MAC had incremental value in association with stroke over traditional risk factors. MAC score consisting of MAC mobility, typical echodense mass with central echolucencies suggesting caseous necrosis, and PLOS ONE | https://doi.
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