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 5

EPHB2 on SEVs drives an ephrin-B–STAT3 angiogenic signaling cascade.

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EPHB2 on SEVs drives an ephrin-B–STAT3 angiogenic signaling cascade. (A)...
(A) Representative Western blots of phosphorylated (p–ephrin-B, p-STAT3, p-VEGFR2) and total (ephrin-B2, STAT3, VEGFR2, β-actin) proteins in cell lysates from HUVECs treated with PBS or SEVs, as indicated. Graphs show normalized quantitative analyses of the ratio of phosphorylated to total proteins from n = 3 Western blots. Box-and-whisker plots show median and 25th–75th percentile. Dunnett’s method was used for statistical analysis. *P < 0.05, ***P < 0.001. (B) Tube-formation assay of HUVECs treated with SEVs in the presence or absence of the Stat3 inhibitor Stattic. Representative images and analysis. Scale bar: 500 μm. Box-and-whisker plots show 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 < 0.001. (C) Model of SEV-induced angiogenesis pathway. EPHB2 on cancer cell–derived SEVs binds to ephrin-B2 on endothelial cells and activates ephrin-B2 reverse signaling with downstream phosphorylation and activation of STAT3. While phosphorylation of VEGFR2 downstream of ephrin-B2 reverse signaling has been reported (55), we did not detect changes due to SEV treatment, suggesting selective activation of ephrin reverse signaling pathways by SEVs.