TY - JOUR
T1 - Laminin γ2-enriched extracellular vesicles of oral squamous cell carcinoma cells enhance in vitro lymphangiogenesis via integrin α3-dependent uptake by lymphatic endothelial cells
AU - Wang, Ssu Han
AU - Liou, Gunn Guang
AU - Liu, Szu Heng
AU - Chang, Jeffrey S.
AU - Hsiao, Jenn Ren
AU - Yen, Yi Chen
AU - Chen, Yu Lin
AU - Wu, Wan Ling
AU - Chang, Jang Yang
AU - Chen, Ya Wen
N1 - Funding Information:
Key words: oral squamous cell carcinoma, lymphatic endothelial cells, extracellular vesicles, laminin γ2, integrin α3 Abbreviations: DLS: dynamic light scattering; ELISA: enzyme-linked immunosorbent assay; EV: extracellular vesicle; FACS: fluorescence-activated cell sorting; IVIS: in vivo imaging system; LC–MS/MS: liquid chromatography-tandem mass spectrometry; LECs: lymphatic endothelial cells; OSCC: oral squamous cell carcinoma; shRNA: short hairpin ribonucleic acid; SILAC: stable isotope labeling by amino acids in cell culture; TEM: transmission electron microscopy Additional Supporting Information may be found in the online version of this article. *S.-H.W., G.-G.L. and S.-H.L. contributed equally to this work Conflict of interest: The authors declare no potential conflicts of interest. Grant sponsor: Ministry of Science and Technology, Taiwan; Grant numbers: MOST 106-2314-B-400-026-MY3; Grant sponsor: National Health Research Institutes, Taiwan; Grant numbers: NHRI CA-107-PP-04, NHRI CA-105-PP-04 DOI: 10.1002/ijc.32027 History: Received 1 Nov 2018; Accepted 19 Nov 2018; Online 28 Nov 2018 Correspondence to: Ya-Wen Chen, Ph.D., National Institute of Cancer Research, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 35053, Taiwan, Tel.: 886-37-246-166 ext. 35103, Fax: 886-37-586-463, E-mail: [email protected]
Funding Information:
The authors thank Dr. Wen-Chun Hung (National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan) for providing LECs, Ms. Pei-Ying Lin (National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan) for technical support and the protein core in Institute of Biological Chemistry and Institute of Molecular Biology, Academia Sinica (Taipei, Taiwan) for protein identification. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica. The authors thank Taiwan Bioinformatics Institute Core Facility for assistance on using Oncomine (National Core Facility Program for Biotechnology, MOST 105-2319-B-400-002). We are also grateful to the Tissue Bank, Research Center of Clinical Medicine, National Cheng Kung University Hospital (Tainan, Taiwan) for providing clinical samples.
Publisher Copyright:
© 2018 UICC
PY - 2019/6/1
Y1 - 2019/6/1
N2 - Oral squamous cell carcinoma (OSCC) LN1-1 cells previously showed greater capacities for lymphangiogenesis and lymph node metastasis compared to their parental OEC-M1 cells, in addition to an ability to enhance the migration and tube formation of lymphatic endothelial cells (LECs). Purified by a series of differential centrifugations and characterized using electron microscopy, dynamic light scattering and western blot, LN1-1 cell-derived extracellular vesicles (LN1-1 EVs) were shown to promote LEC migration, tube formation and uptake by LECs more effectively than did OEC-M1 cell-derived EVs (OEC-M1 EVs). Using stable isotope labeling with amino acids in cell culture/liquid chromatography–tandem mass spectrometry-based proteomic platform, the laminin-332 proteins, including laminin α3, β3 and γ2, were validated as highly expressed proteins in LN1-1 EVs. Clinically, a higher level of laminin-332 was detected in plasma EVs from OSCC patients with lymph node metastasis than in both healthy controls and OSCC patients without lymphatic metastasis, suggesting EV-borne laminin-332 as a novel and noninvasive biomarker for the detection of lymph node metastasis in OSCC. The knockdown of laminin γ2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin γ2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/γ2-mediated EV uptake was dependent on integrin α3 but not β1, β4 or α6. Collectively, the uptake of laminin γ2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC.
AB - Oral squamous cell carcinoma (OSCC) LN1-1 cells previously showed greater capacities for lymphangiogenesis and lymph node metastasis compared to their parental OEC-M1 cells, in addition to an ability to enhance the migration and tube formation of lymphatic endothelial cells (LECs). Purified by a series of differential centrifugations and characterized using electron microscopy, dynamic light scattering and western blot, LN1-1 cell-derived extracellular vesicles (LN1-1 EVs) were shown to promote LEC migration, tube formation and uptake by LECs more effectively than did OEC-M1 cell-derived EVs (OEC-M1 EVs). Using stable isotope labeling with amino acids in cell culture/liquid chromatography–tandem mass spectrometry-based proteomic platform, the laminin-332 proteins, including laminin α3, β3 and γ2, were validated as highly expressed proteins in LN1-1 EVs. Clinically, a higher level of laminin-332 was detected in plasma EVs from OSCC patients with lymph node metastasis than in both healthy controls and OSCC patients without lymphatic metastasis, suggesting EV-borne laminin-332 as a novel and noninvasive biomarker for the detection of lymph node metastasis in OSCC. The knockdown of laminin γ2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin γ2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/γ2-mediated EV uptake was dependent on integrin α3 but not β1, β4 or α6. Collectively, the uptake of laminin γ2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC.
KW - extracellular vesicles
KW - integrin α3
KW - laminin γ2
KW - lymphatic endothelial cells
KW - oral squamous cell carcinoma
UR - http://www.scopus.com/inward/record.url?scp=85059918171&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85059918171&partnerID=8YFLogxK
U2 - 10.1002/ijc.32027
DO - 10.1002/ijc.32027
M3 - Article
C2 - 30485433
AN - SCOPUS:85059918171
SN - 0020-7136
VL - 144
SP - 2795
EP - 2810
JO - International Journal of Cancer
JF - International Journal of Cancer
IS - 11
ER -
