Abstract
Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500–3000 nm) reminiscent of apoptosomes, (ii) EVs (100–500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80–300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20–200 nm) containing unidentified vesicles/particles, and (v) EVs (10–70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producing a GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.
Highlights
Extracellular vesicles (EVs) are small particles surrounded by lipid membrane that are released by cells of diverse organisms, including eukaryotes and prokaryotic cells
We have developed stable cell lines that express palmitoylation signal (Palm) fused with green fluorescent proteins and with tandem dimer Tomato, which visualize the membrane of the cells and their EVs [20]
Since Fr. 1–6, Fr. 7–9, and Fr. 10–20 prepared using the size exclusion chromatography and concentration filters (SEC-CF) method showed a tendency to reduce the viability of HSC-3, we further examined whether Fr. 1–3, Fr. 4–6, Fr. 7–9, Fr. 10–12, Fr. 13–15, Fr. 16–18, or Fr. 19–21 of macrophageEVs could reduce the viability of HSC-3 cells
Summary
Extracellular vesicles (EVs) are small particles surrounded by lipid membrane that are released by cells of diverse organisms, including eukaryotes and prokaryotic cells. EVs exist in different body fluids such as blood, urine, saliva, bile, ascites, amniotic fluid, breast milk, pleural ascites, synovial fluid, and cerebral spinal fluid [1]. EVs contain various molecular cargo types, including proteins, nucleic acids, lipids, minerals, and metabolites [1,2,3]. Earlier studies classified EVs into the following three types: (1) exosomes (50–200 nm) originated from the endosome; (2) ectosomes, called microvesicles (MVs) or microparticles (MPs) (100–1000 nm), generated by the budding and shedding of the plasma membrane of cells; and (3) apoptotic bodies (1–5 μm), known as apoptosome, originated via blebbing of the plasma membrane [4,5,6]. Non-membranous nanoparticles termed exomeres (~35 nm) involve metabolic enzymes and microtubule, hypoxia, coagulation proteins, glycosylation, and mTOR signaling [15]
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