Laminin 2-enriched extracellular vesicles of oral squamous cell carcinoma cells enhance in vitro lymphangiogenesis via integrin 3-dependent uptake by lymphatic endothelial cells

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 3, 3 and 2, 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 2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin 2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/2-mediated EV uptake was dependent on integrin 3 but not 1, 4 or 6. Collectively, the uptake of laminin 2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC. What's new? Oral squamous cell carcinoma (OSCC) cell sublines derived via in vivo selection in previous work exhibit high capacities for lymphangiogenesis. It remains unclear, however, how these sublines interact with lymphatic endothelial cells (LECs) to promote lymphangiogenesis. Here, laminin-332 was found to be highly expressed in extracellular vesicles (EVs) form metastatic OSCC cells. Clinically, laminin-332-bearing EV can serve as a diagnostic biomarker for lymphatic metastasis. The uptake of laminin-332-rich EVs by LECs is dependent on integrin 3, suggesting further investigation of inhibited EV uptake as a therapeutic strategy against tumor lymphangiogenesis.