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Screening assay for blood vessel maturation inhibitors
Chenglai Fu1, Anita van der Zwan, Stephanie Gerber
1Department of Oncology, Johns Hopkins University, Baltimore, MD, USA.
Abstract:
In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.
Insights
Developing new anti-cancer therapies requires effective screening. This study presents a novel in vitro assay to identify compounds that reduce pericyte coverage, potentially overcoming resistance to anti-angiogenic treatments.
Area of Science:
- Oncology
- Vascular Biology
- Drug Discovery
Background:
- Resistance to anti-angiogenic agents targeting the VEGF pathway is a significant challenge in cancer therapy.
- Increased pericyte coverage of tumor vasculature is implicated in this resistance.
- Existing in vitro assays for studying vessel maturation and pericyte association are not high-throughput.
Purpose of the Study:
- To develop a novel, scalable in vitro assay for blood vessel maturation.
- To facilitate the screening of compound libraries for inhibitors of pericyte recruitment.
- To aid in the development of strategies to overcome resistance to anti-angiogenic therapies.
Main Methods:
- Developed a novel in vitro assay using human umbilical vein endothelial cells (HUVECs) and mesenchymal progenitor cells (10T1/2).
- Mesenchymal progenitor cells differentiate into pericyte-like cells, visualized via GFP reporter under alpha smooth muscle actin promoter (SMP-8).
- Vascular structures and associated mural cells form in a single optical plane, enabling automated image analysis.
Main Results:
- The assay successfully forms lumenized vascular structures with associated pericyte-like cells in one optical plane.
- Automated data capture and image analysis are feasible due to the assay's design.
- The assay was validated for its ability to screen for inhibitors of pericyte recruitment.
Conclusions:
- This novel in vitro assay provides a valuable tool for high-throughput screening.
- It can identify new agents to inhibit pericyte recruitment, potentially breaking resistance to anti-angiogenic therapies.
- The assay supports the development of more effective cancer treatments.
