Laminin γ2‐enriched extracellular vesicles of oral squamous cell carcinoma cells enhance in vitro lymphangiogenesis via integrin α3‐dependent uptake by lymphatic endothelial cells

Abstract

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.

Extracellular Vesicles in Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders: A Systematic Review

“…Laminin-332 in cancer cells has been shown to promote cell growth, invasion and metastasis [55]. In accord with this, Wang et al (2019) [44] found that Laminin-332 proteins (laminin α3, β3 and γ2) were upregulated in LN1-1 cell line EVs versus the levels found in the EV of the OEC-M1 cell line, from which LN1-1 cells were derived (>1.5 fold) and the number of laminin γ2-positive gold particles per EV was greater in LN1-1 EVs than in OEC-M1 EVs (p < 0.01) [44]. Comparing 10 healthy controls and 20 OSCC, ELISA detected a significantly higher level of plasma EV-borne laminin-332 in lymph node positive OSCC patients than in lymph node negative OSCC patients, which were both higher than healthy controls (p < 0.01).…”

“…Comparing 10 healthy controls and 20 OSCC, ELISA detected a significantly higher level of plasma EV-borne laminin-332 in lymph node positive OSCC patients than in lymph node negative OSCC patients, which were both higher than healthy controls (p < 0.01). The protein load ex vivo fluorescent signals from the cervical lymph nodes of mice orthotopically implanted with PKH-26-labeled laminin γ2-deficient EVs (from LN1-1 LAMC2-knockdown cells) demonstrated a significant decrease in uptake relative to the corresponding controls (p < 0.01), suggesting a reduced ability to drain into lymph nodes in comparison with the control EVs [44].…”

“…Plasma analysis showed that relative to healthy controls, patients with HNSCC with active disease have elevated levels of exosomes that interact with HUVECs and stimulate their proliferation [43]. Wang et al (2019) [44] found that incubation of human dermal lymphatic endothelial cells (LEC) with metastatic subline LN1-1-EVs did not affect growth but significantly increased migration and tube formation of LEC when compared to LEC incubation with non-metastatic parent cell line OEC-M1-derived EVs [44].…”

Emerging functions and clinical applications of exosomes in human oral diseases

“…Similarly, the level of PD-L1 carried by plasma exosomes instead of soluble PD-L1 level was correlated with disease severity, the UICC stage and the lymph node status [64]. Moreover, Wang et al have identified a higher expression of laminin-332 in plasma exosomes from OSCC patients with lymph node metastasis [65]. Li et al found that serum exosomal miR-21 level was closely associated with HIF-1a/HIF-2a expression, T stage, and lymph node metastasis [20].…”

Peer Review #1 of "Exosomal cargoes in OSCC: current findings and potential functions (v0.1)"

“…In Kozaki and colleagues' study, they found that the increase of HSP90-rich exosomes is positively correlated to the increased invasive capacity of OSCC; moreover the knockdown of HSP90α and HSP90β decreased the metastatic capacity and survivability of OSCC cells (Ono et al 2018). Observing that OSCC LN1-1 cells were more aggressive in lymphatic node metastasis than OEC-M1 cells, Wang et al used stable isotope amino acid labeling to reveal that higher laminin-332 protein levels in tumor cell-derived exosomes was a major cause for the superior lymphangiogenesis ability of OSCC LN1-1 cells, as laminin-332 promotes lymphatic endothelial cell migration and tube formation (Wang et al 2019a). Li et al reported that PF4V1, CXCL7, F13A1, and ApoA1 could affect OSCC lymph node metastasis, and the mechanism remains unknown (Li et al 2019a).…”