TY - JOUR
T1 - Exosome-based molecular transfer activity of macrophage-like cells involves viability of oral carcinoma cells
T2 - Size exclusion chromatography and concentration filter method
AU - Lu, Yanyin
AU - Eguchi, Takanori
AU - Sogawa, Chiharu
AU - Taha, Eman A.
AU - Tran, Manh Tien
AU - Nara, Toshiki
AU - Wei, Penggong
AU - Fukuoka, Shiro
AU - Miyawaki, Takuya
AU - Okamoto, Kuniaki
N1 - Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021
Y1 - 2021
N2 - Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and im-munology. 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.
AB - Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and im-munology. 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.
KW - Exosomes
KW - Extracellular vesicles
KW - Heat shock proteins
KW - Macrophage
KW - Molecular transfer
KW - Oral carcinoma
KW - Size exclusion chro-matography and concentration filter (SEC-CF) method
UR - http://www.scopus.com/inward/record.url?scp=85107397449&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85107397449&partnerID=8YFLogxK
U2 - 10.3390/cells10061328
DO - 10.3390/cells10061328
M3 - Article
C2 - 34071980
AN - SCOPUS:85107397449
VL - 10
JO - Cells
JF - Cells
SN - 2073-4409
IS - 6
M1 - 1328
ER -