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An endothelial cell competition assay for determinants of the response to targeted anti-angiogenics
Michael M Halford1, Michael Y He1,2, Nancy Amin3
1Tumour Angiogenesis Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
Abstract:
Anti-angiogenics, inhibitors of pathological blood vessel growth, are an important class of targeted agent for the treatment of common cancers and ocular conditions. However, efficacy is compromised by the absence of biomarkers to guide patient selection or inform the management of resistance. We describe an assay for modified endothelial cell (EC) responses to the VEGF-A-neutralizing monoclonal antibody bevacizumab as part of a biomarker discovery program. ECs are transduced by lentivector expressing an experimental or non-silencing shRNA, each co-expressed with a different fluorescent protein. A 1:1 mixed cell population is then cultured with bevacizumab or control antibody under VEGF-A-dependent conditions. A normalized ratio of surviving cells, obtained by flow cytometry analysis, reflects EC resistance or sensitization to bevacizumab mediated by the experimental shRNA. With reagents prepared, the protocol takes 10 days and rigorously quantifies the impact of gene perturbation on the EC response to bevacizumab or other targeted anti-angiogenics.
Insights
Researchers developed a novel assay to identify biomarkers for anti-angiogenic therapies like bevacizumab. This assay measures endothelial cell (EC) responses to predict treatment resistance or sensitivity, aiding patient selection.
Area of Science:
- Oncology
- Vascular Biology
- Biotechnology
Background:
- Anti-angiogenic agents are crucial for treating cancers and ocular diseases by inhibiting pathological blood vessel growth.
- Current limitations include a lack of biomarkers for patient selection and managing treatment resistance.
- Endothelial cell (EC) response assays are needed to guide targeted therapy efficacy.
Purpose of the Study:
- To develop and validate a novel assay for discovering biomarkers that predict endothelial cell (EC) response to anti-angiogenic drugs.
- To quantify the impact of gene perturbations on EC sensitivity or resistance to bevacizumab.
- To facilitate patient selection and resistance management for targeted anti-angiogenic therapies.
Main Methods:
- Utilized lentiviral vectors for shRNA-mediated gene knockdown in ECs, co-expressing fluorescent proteins for identification.
- Created a 1:1 mixed EC population with experimental or non-silencing shRNA.
- Cultured mixed ECs with bevacizumab or control antibody under VEGF-A-dependent conditions and analyzed survival ratios via flow cytometry.
Main Results:
- The assay successfully quantified modified EC responses to bevacizumab.
- A normalized ratio of surviving cells indicated EC resistance or sensitization mediated by specific shRNAs.
- The assay provides a rigorous method to assess gene perturbation effects on anti-angiogenic drug response.
Conclusions:
- This assay serves as a valuable tool for biomarker discovery in targeted anti-angiogenic therapy.
- It enables the assessment of EC sensitivity and resistance mechanisms.
- The 10-day protocol offers a standardized method for evaluating drug response in oncology and ophthalmology.
