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Updated: Apr 14, 2026
In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing
Published on: May 11, 2021
Silencing of Eps8 inhibits in vitro angiogenesis
Elisa Cappellini1, Claudia Vanetti1, Lucia M Vicentini1
1Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Vanvitelli 32, 20129 Milano, Italy.
Aims:
Eps8 is an actin-binding protein which has been proposed as a regulator of cancer cell motility and invasion. However, nothing much is known about its contribution to the invasive properties of endothelial cells (ECs), and more generally to angiogenesis.
Main Methods:
Expression and silencing of Eps8 were evaluated by western blot analysis. The effect of Eps8 silencing on cell number and VEGF-induced signaling was tested with standard methods. Migration was evaluated by scratch wound assay and morphogenesis with 2-dimensional (2-D) tube formation and 3-dimensional (3-D) sprouting assays. Actin cytoskeleton was visualized by immunofluorescence.
Key Findings:
We found that silencing of Eps8 profoundly affected the ability of human ECs to migrate and to undergo tube formation and sprouting in 2-D and 3-D in vitro assays, respectively. Notably, capillary-like outgrowth was strictly depending on Eps8 expression also in human tumor-derived ECs.
Significance:
Our data demonstrate for the first time the involvement of Eps8 in the morphological processes required for in vitro angiogenesis, and suggest that this protein might represent a common target for the design of new anticancer drugs, acting at the same time on both tumor and endothelial cells.
Insights
Eps8 protein is crucial for endothelial cell (EC) migration, tube formation, and sprouting, processes vital for angiogenesis. Targeting Eps8 could offer a dual approach for anticancer drug development, affecting both tumor and ECs.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Eps8 is an actin-binding protein implicated in cancer cell motility and invasion.
- Its role in endothelial cell (EC) invasiveness and angiogenesis remains largely unexplored.
Purpose of the Study:
- To investigate the contribution of Eps8 to endothelial cell migration, invasion, and angiogenesis.
- To explore Eps8 as a potential therapeutic target in cancer treatment.
Main Methods:
- Western blot analysis to assess Eps8 expression and silencing.
- Standard assays to evaluate cell number and VEGF-induced signaling.
- Scratch wound, 2-D tube formation, and 3-D sprouting assays for migration and morphogenesis.
- Immunofluorescence to visualize the actin cytoskeleton.
Main Results:
- Eps8 silencing significantly impaired human EC migration, 2-D tube formation, and 3-D sprouting.
- Capillary-like outgrowth in human tumor-derived ECs was dependent on Eps8 expression.
- Eps8 affects actin cytoskeleton organization crucial for angiogenesis.
Conclusions:
- Eps8 plays a critical role in the morphological processes essential for in vitro angiogenesis.
- Eps8 represents a potential common target for novel anticancer drugs targeting both tumor and endothelial cells.
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