Phenotypic Selection Screening Reveals Cancer Stem Cell Therapeutic Targets

Cancer stem cells (CSCs) represent an important subpopulation of tumor cells that are untouched and perhaps even expanded after chemotherapy and radiation. This population has been shown in murine models of cancer to be solely responsible for the regrowth of tumors following chemotherapy, underscoring the importance of therapeutically targeting this population. Therefore, we sought to find novel targets against CSCs using a phenotypic selection screen. Using a designed ankyrin repeat (DARpin) library, we panned cells disaggregated from tumor spheres from three patient-derived xenograft models of late stage pancreatic cancer to enrich for DARPins that bind CSCs. Once released from the cells, the DARPins were further analyzed for binding to both normal, pancreatic cancer total tumor cells and pancreatic CSCs in order to choose targets that showed either pancreatic cancer-specific or pancreatic CSC-specific binding. These DARPins were then tested in a sphere formation assay, an EZH2-based high content imaging screen, and a standard proliferation assay. Those DARPins showing good cancer-specific binding profiles were also tested for internalization and killing using a saporin-conjugated secondary antibody. Using this approach, we identified 30 DARPins of interest for either direct inhibitory antibody approaches or for potential antibody drug conjugate or nanoparticle delivery approaches. Identification of the proteins bound by each of the DARPins was done using either immunopreciptation followed by liquid chromatography tandem-mass spectrometry or through Retrogenix9s cell expression microarray technology. Identified targets included known CSC antigens such as integrin alpha6 (CD49f) as well as other potentially novel targets. These results highlight the possibilities of finding therapeutic targets for CSCs and opens up an exciting possibility for novel therapies. Citation Format: Elaine M. Hurt, Matt Flynn, Suneetha Thomas, Lilian van Vlerken-Ysla, Alan Sandercock, Steven Rust, Minter Ralph, Robert Hollingsworth. Phenotypic selection screening reveals cancer stem cell therapeutic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4218. doi:10.1158/1538-7445.AM2015-4218