Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method

Cells

Takuya Miyawaki¹  Takanori Eguchi²  Shiro Fukuoka²  Kuniaki Okamoto²  Chiharu Sogawa²  ManhTien Tran²  Penggong Wei²  Yanyin Lu²  EmanA. Taha²  Toshiki Nara³ 

[1] Department of Dental Anesthesiology and Special Care Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan;Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan;Research Program for Undergraduate Students, Okayama University Dental School, Okayama 700-8525, Japan;

关键词: macrophage;    exosomes;    extracellular vesicles;    molecular transfer;    size exclusion chromatography and concentration filter (SEC-CF) method;    heat shock proteins;   

DOI  :  10.3390/cells10061328

来源: DOAJ

【 摘 要 】

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 producinga 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.

【 授权许可】

Unknown