EPHB2 carried on small extracellular vesicles induces tumor angiogenesis via activation of ephrin reverse signaling
Shinya Sato, Suhas Vasaikar, Adel Eskaros, Young Kim, James S. Lewis, Bing Zhang, Andries Zijlstra, Alissa M. Weaver
Shinya Sato, Suhas Vasaikar, Adel Eskaros, Young Kim, James S. Lewis, Bing Zhang, Andries Zijlstra, Alissa M. Weaver
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Research Article Angiogenesis Oncology

EPHB2 carried on small extracellular vesicles induces tumor angiogenesis via activation of ephrin reverse signaling

Abstract

Angiogenesis is a key process that allows nutrient uptake and cellular trafficking and is coopted in cancer to enable tumor growth and metastasis. Recently, extracellular vesicles (EVs) have been shown to promote angiogenesis; however, it is unclear what unique features EVs contribute to the process. Here, we studied the role of EVs derived from head and neck squamous cell carcinoma (HNSCC) in driving tumor angiogenesis. Small EVs (SEVs), in the size range of exosomes (50–150 nm), induced angiogenesis both in vitro and in vivo. Proteomic analysis of HNSCC SEVs revealed the cell-to-cell signaling receptor ephrin type B receptor 2 (EPHB2) as a promising candidate cargo to promote angiogenesis. Analysis of patient data further identified EPHB2 overexpression in HNSCC tumors to be associated with poor patient prognosis and tumor angiogenesis, especially in the context of overexpression of the exosome secretion regulator cortactin. Functional experiments revealed that EPHB2 expression in SEVs regulated angiogenesis both in vitro and in vivo and that EPHB2 carried by SEVs stimulates ephrin-B reverse signaling, inducing STAT3 phosphorylation. A STAT3 inhibitor greatly reduced SEV-induced angiogenesis. These data suggest a model in which EVs uniquely promote angiogenesis by transporting Eph transmembrane receptors to nonadjacent endothelial cells to induce ephrin reverse signaling.

Authors

Shinya Sato, Suhas Vasaikar, Adel Eskaros, Young Kim, James S. Lewis, Bing Zhang, Andries Zijlstra, Alissa M. Weaver

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Figure 3

SEVs carry EPHB2 and induce ephrin reverse signaling.

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SEVs carry EPHB2 and induce ephrin reverse signaling. (A) Left: Represen...
(A) Left: Representative Western blots of EPHB2 in whole cell lysates (WCL) and SEVs of OSC19, Detroit 562, and SCC61. Right top: Western blot comparing EPHB2, CD63, and TSG101 in OSC19 whole cell lysates and SEVs (loading 5 μg in each lane). Right bottom: Western blot comparing EPHB2, HSP70, and TSG101 in OSC19 SEVs and LEVs (loading 5 μg in each lane). (B) Top: Representative Western blots of phospho-ephrin-B (p–ephrin-B), ephrin-B2, and β-actin in cell lysates from HUVECs treated with Fc-EPHB2, PBS (–), or 5 × 107 OSC19 SEVs (+), as indicated. Bottom: p–ephrin-B/ephrin-B2 ratio from 3 independent experiments (median and 25th–75th percentile). Dunnett’s method was used for statistical analysis. (C) Schematic of blocking experiments in which preincubation of Fc–ephrin-B2 with SEVs binds EPHB2 and blocks its binding to ephrin-B ligands on HUVECs. (D) Tube-formation assay. HUVECS were cultured with PBS (control), OSC19 SEVs with or without preincubation with recombinant ephrin-B2 (Fc–ephrin-B2), or Fc–ephrin-B2 alone. Top: Representative images. Scale bar: 500 μm. Bottom: Quantification of relative total tube length and junction number. Dot plots represent median and 25th–75th percentile. Tukey-Kramer method was used for statistical analysis using the average of ≥ 3 technical replicates per condition for each n value from ≥ 3 independent experiments. *P < 0.05; ***P < 0.001 when compared with no added SEVs condition; #P <0.05; ###P < 0.001 when compared with +SEVs condition.