An Optimized 3D Coculture Assay for Preclinical Testing of Pro- and Antiangiogenic Drugs
Daniela Unterleuthner1, Nina Kramer1, Karoline Pudelko1
11 Center for Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria.
Abstract:
Angiogenesis is a promising target for anticancer therapies, but also for treating other diseases with pathologic vessel development. Targeting the vascular endothelial growth factor (VEGF) pathway did not proof as effective as expected due to emerging intrinsic resistance mechanisms, as well as stromal contributions leading to drug insensitivity. Therefore, alternative strategies affecting the interaction of endothelial cells (ECs) with other stromal cells seem to be more promising. Human preclinical in vitro angiogenesis models successfully recapitulating these interactions are rare, and two-dimensional (2D) cell cultures cannot mimic tissue architecture in vivo. Consequently, models combining three-dimensionality with heterotypic cell interaction seem to be better suited. Here, we report on an improved human fibroblast-EC coculture assay mimicking sprouting angiogenesis from EC-covered microbeads resembling existing endothelial structures. Culture conditions were optimized to assess pro- and antiangiogenic compounds. Important characteristics of angiogenesis, that is, the number of sprouts and branch points, sprout length protrusion, and overall vessel structure areas, were quantified. Notably, the endothelial sprouts display lumen formation and basal membrane establishment. In this model, angiogenesis can be inhibited by genetic interference of pro-angiogenic factors expressed in the fibroblasts. Moreover, bona fide antiangiogenic drugs decreased, whereas pro-angiogenic factors increased vessel formation in 24-well and 96-well settings, demonstrating the applicability for screening approaches.
Insights
Developing new human preclinical models for angiogenesis is crucial for cancer therapies. This study presents an improved 3D co-culture assay for studying vessel formation and drug responses.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Vascular Biology
Background:
- Angiogenesis is a key target for cancer therapies, but resistance to VEGF pathway inhibitors necessitates alternative strategies.
- Existing 2D models fail to replicate complex tissue architecture and cell interactions crucial for angiogenesis.
- There is a need for advanced in vitro models that mimic in vivo angiogenesis, including heterotypic cell interactions.
Purpose of the Study:
- To develop and optimize an improved human preclinical in vitro angiogenesis model.
- To create a three-dimensional (3D) co-culture assay that recapitulates sprouting angiogenesis.
- To validate the model's utility for assessing pro- and antiangiogenic compounds and genetic interference.
Main Methods:
- Developed a human fibroblast-endothelial cell (EC) co-culture assay using EC-covered microbeads to mimic endothelial structures.
- Optimized culture conditions for assessing angiogenesis.
- Quantified key angiogenesis parameters including sprout number, branch points, sprout length, and vessel area.
Main Results:
- The model successfully mimics sprouting angiogenesis with lumen formation and basal membrane establishment.
- Genetic interference of fibroblast-derived pro-angiogenic factors inhibited angiogenesis.
- Antiangiogenic drugs reduced, while pro-angiogenic factors enhanced, vessel formation in the model.
Conclusions:
- The developed 3D co-culture assay provides a more physiologically relevant platform for studying angiogenesis.
- This model is suitable for screening antiangiogenic and pro-angiogenic compounds.
- It offers a valuable tool for advancing anticancer therapies and understanding other diseases involving pathologic vessel development.
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