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 1

Small extracellular vesicles (SEVs) derived from HNSCC cells drive tumor angiogenesis.

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Small extracellular vesicles (SEVs) derived from HNSCC cells drive tumor...
(A) Hypothesis for study. EVs mediate angiogenesis and lymphangiogenesis to respectively drive distant and locoregional metastasis. (B) Representative images of tongue tumors derived from SCC61, OSC19, Detroit 562, MOC1, and MOC2 cells stained for CD31 (black, identifies endothelial cells). SCC61, n = 4; OSC19, Detroit 562, MOC1, and MOC2, n = 5. Ten images for each tumor. Scale bar: 100 μm. (C) Plot of CD31+ vessel area per total tumor area in tongue tumors. SCC61, n = 4; OSC19, Detroit 562, MOC1, and MOC2, n = 5. Total tumor area and CD31-stained area were calculated using ImageJ. (D) SEV secretion rate of cell lines, calculated from nanoparticle tracking analysis of purified vesicles obtained from a known final number of cells over 48 hours. SCC61, n = 4; OSC19, n = 7; Detroit 562, n = 5; MOC1, n = 11; and MOC2, n = 8. (E) Linear regression models were performed to analyze relationship between SEV secretion rates and blood vessel density in tumors for various cell lines. Adjusted R2 of the linear regression model = 0.7822. (F) HUVECs were incubated for 12 hours in serum-free media plus PBS (control), conditioned media of cancer cells (CM), CM ultracentrifuged to remove SEVs (CM-SEV), or serum free media plus purified cancer cell SEVs (5 × 107 and 1 × 108). (Left) Representative images of HUVEC cells cultured with PBS (control) or OSC19/Detroit 562 SEVs (5 × 107). ImageJ outlining of vessels for automated analysis is shown. Scale bar: 500 μm. (Right) Quantification of relative total tube length and junction numbers for the indicated groups. Plots show the average of ≥ 3 technical replicates per condition for each n value from ≥ 3 independent experiments. For C, D, and F, box-and-whisker plots show median and 25th–75th percentile. Tukey-Kramer method was used in C and D, and Dunnett’s method was used in F for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001.