Angiogenesis is a multi-step physiological process that is involved in a large number of normal and disease state processes; in vitro angiogenesis models provide useful tools to study these processes, one of which is the analysis of tubule formation. Several papers report that tubules formed in co-culture assays that are composed of both endothelial and stroma-producing cells were significantly more heterogeneous and more closely resembled capillaries than mono cell culture models that utilized only endothelial cells to generate tubules in an extracellular matrix. Current co-culture models using primary cells have donor variability and inconsistent results due to lot-to-lot variation.
In this study, we established an in vitro co-culture model system consisting of an assay-ready mixture of the aortic endothelial cell line TeloHAEC-GFP (GFP-tagged, human telomerase reverse transcriptase [hTERT]-immortalized human aortic endothelial cells) and an hTERT-immortalized, adipose-derived mesenchymal stem cell line (hTERT-MSCs) in a specially formulated medium containing VEGF supplement (Angio-Ready™ Angiogenesis Assay System; ATCC® ACS-2001-2™ and ATCC® ACS-2001-10™). Both cell lines were immortalized by hTERT alone and have been well-characterized, showing that the cells retain the most important characteristic of their primary counterparts. The new co-culture system forms functional tubular structures in less than 7 days; additionally, the hTERT-MSC cells that surround the tubular structures have undergone transformation as evidenced by elevated positive αSMA staining (a marker of smooth muscle cells), indicating that the system has physiological relevance.
Notably, our results showed that the co-culture system has minimal lot-to-lot variation as indicated by the treatment of three lots with the anti-cancer drug ramucirumab (Cyramza®), which targets the VEGF pathway. More importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as sunitinib (SUTENT®) and bevacizumab (Avastin®). We also tested four hypoxia inducible factor-1 (HIF-1) inhibitors identified in previous high-throughput screens and found that those compounds inhibited tubule formation in the Angio-Ready™ co-culture system.
These results suggest that the co-culture system can mimic the hypoxic environment in solid tumors. Previously, the authors optimized the Angio-Ready™ system for 384-well performance, and here we report further optimization of the system into a 1,536-well, high-throughput format and a shortening of the assay time frame to 3 days. Using this format, we evaluated 2,816 drugs from The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC), and 35 potent inhibitors (IC50 ≤1 μM) were identified. Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors (e.g., thimerosal and podofilox) were also identified from this study. Among the inhibitors, some compounds were proven to be involved in the hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-kappa B (NF-κB) pathways.
These results demonstrate that the co-culture model described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.