Angiogenesis is a multi-step physiological process which 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. Tubules formed in co-culture assays composed of both endothelial and stroma-producing cells were significantly more heterogeneous and more closely resembled capillaries than mono cell culture models utilizing 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 an aortic endothelial cell line TeloHAEC-GFP and an hTERT immortalized adipose-derived mesenchymal stem cell line in a specially formulated medium containing VEGF supplement. 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 which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining, indicating that the system has physiological relevance. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots with the anti-cancer drug, ramucirumab. More importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as sunitinib and bevacizumab. We also tested four HIF-1 inhibitors identified in previous high-throughput screens and found that those compounds inhibited tubule formation in the co-culture system. These results suggest that the co-culture system can mimic the hypoxic environment in solid tumors. Previously the authors optimized the system for 384-well performance and here we report further optimization of the system into a 1536-well high-throughput format and a shortening of the assay time frame to 3 days. Using this format, we evaluated 2816 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 were also identified from this study. Among the inhibitors, some compounds were proven to be involved in the hypoxia-inducible factor-1α and the nuclear factor-kappa B 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.

Citation Format: Chaozhong Zou, Shuaizhang LI, Chia-Wen Hsu, Menghang Xia, Metewo S. Enuameh. Development of a high-throughput screening co-culture angiogenesis assay system using hTERT immortalized primary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3868.