Abstract C227: High-throughput anchorage independence assay for screening inhibitors of cancer metastasis.

Abstract

Abstract Tumor cells are capable of expanding without the requirement for cell adherence which is an indicator for their ability to form metastatic tumors in vivo. To do so, cells need to overcome cell cycle inhibitory signaling and/or apoptosis. This so-called anchorage-independent growth is an indicator of oncogenic transformation and deregulated cell growth and can be mimicked in vitro by culturing cells in soft agar. Our aim was to set up a high-throughput assay to identify inhibitors of anchorage-independent growth. Cancer cell lines were mixed with agar and seeded into 96-well plates. After polymerization, medium was added to the agar and subsequently refreshed twice a week. Depending on the cell line used the colonies in the agar were fixed after 8-14 days of culture and stained with phalloidin. The BD Pathway 435 was used to generate two collapsed stacks of 20 images per well. The inhibition of colony formation was analyzed using the Attovision software with an in-house developed algorithm. This approach was optimized using adenoviral knock-down constructs targeting known targets involved in anchorage independence as well as tool compounds which inhibited colony formation. This assay will be used for screening an adenoviral knock-down library to identify novel targets involved in anchorage independence as well as testing of compounds directed against the identified targets. We successfully developed an automated high-throughput anchorage independence assay in multiple cancer cell lines. This assay can be used for discovery and validation of novel targets and compounds for their potential to inhibit uncontrolled growth of cancer cells. Discovery of novel targets in metastasis may lead to the development of small molecule compounds or antibody therapeutics in cancer therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C227. Citation Format: Remko de Pril, Carla Oerlemans-Bergs, Edo Elstak, Ilhem Maghrani, Richard Janssen. High-throughput anchorage independence assay for screening inhibitors of cancer metastasis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C227.