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Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method
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 aime...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228134/ https://www.ncbi.nlm.nih.gov/pubmed/34071980 http://dx.doi.org/10.3390/cells10061328 |
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author | Lu, Yanyin Eguchi, Takanori Sogawa, Chiharu Taha, Eman A. Tran, Manh Tien Nara, Toshiki Wei, Penggong Fukuoka, Shiro Miyawaki, Takuya Okamoto, Kuniaki |
author_facet | Lu, Yanyin Eguchi, Takanori Sogawa, Chiharu Taha, Eman A. Tran, Manh Tien Nara, Toshiki Wei, Penggong Fukuoka, Shiro Miyawaki, Takuya Okamoto, Kuniaki |
author_sort | Lu, Yanyin |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8228134 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-82281342021-06-26 Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method Lu, Yanyin Eguchi, Takanori Sogawa, Chiharu Taha, Eman A. Tran, Manh Tien Nara, Toshiki Wei, Penggong Fukuoka, Shiro Miyawaki, Takuya Okamoto, Kuniaki Cells Article 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. MDPI 2021-05-27 /pmc/articles/PMC8228134/ /pubmed/34071980 http://dx.doi.org/10.3390/cells10061328 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Lu, Yanyin Eguchi, Takanori Sogawa, Chiharu Taha, Eman A. Tran, Manh Tien Nara, Toshiki Wei, Penggong Fukuoka, Shiro Miyawaki, Takuya Okamoto, Kuniaki Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method |
title | Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method |
title_full | Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method |
title_fullStr | Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method |
title_full_unstemmed | Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method |
title_short | Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method |
title_sort | exosome-based molecular transfer activity of macrophage-like cells involves viability of oral carcinoma cells: size exclusion chromatography and concentration filter method |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228134/ https://www.ncbi.nlm.nih.gov/pubmed/34071980 http://dx.doi.org/10.3390/cells10061328 |
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