Abstract 52: A genomic editing approach for purification of viable quiescent cancer cells

Abstract

Abstract Recurrent and metastatic cancer lesions often undergo a period of dormancy, which is closely associated with cancer cell quiescence, a state whereby cancer cells exit the cell cycle and are reversibly arrested in G0 phase. Quiescent cancer cells are inherently resistant to cell death and refractory to therapeutic drugs. However, the mechanisms responsible for the resistance remain largely undefined. This is closely associated with the lack of understanding of their biological properties as a consequence of technical hurdles in the isolation and analysis of viable quiescent cells. Nevertheless, it is known that quiescent cells are characteristically negative for the proliferation marker Ki67 and express high levels of the cyclin-dependent kinase (CDK) inhibitor p27. In this study, we developed a CRISPR/Cas9-based system to fuse a green fluorescent protein (EGFP) gene with endogenous CDKN1B, the gene encoding p27, and a red fluorescent protein (mCherry) gene with endogenous MKI67, the gene encoding Ki67 in the genome of human melanoma cells. By using this system, we have successfully isolated viable p27high/Ki67- melanoma cells using fluorescence-activated cell sorting (FACS). The quiescent state of these cells was confirmed by dual nucleic acid staining (DNA with Hoechst-33342, and RNA with Pyronin Y). This approach provides a unique research tool for further characterization of the biological properties of quiescent cells. Comparative RNA-seq and proteomics analyses are being currently carried out to reveal molecular networks involved in regulating cell quiescence, and to identify molecular targets for overcoming resistance of quiescent melanoma cells to treatment. Citation Format: Ting La, Margaret Farrelly, Xu Guang Yan, Hamed Yari, Yuanyuan Zhang, Yuchen Feng, Seyed Hessamedin Tabatabaeehatambakhsh, Tao Liu, Lei Jin, Xu Dong Zhang. A genomic editing approach for purification of viable quiescent cancer 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 52.