Abstract A46: Real time niche tracking of cancer stem cell cycle kinetics using a novel bi-cistronic lentiviral reporter.

Human leukemia stem cells (LSC) are quiescent self-renewing cells that drive leukemic transformation of myeloproliferative neoplasms and myelodysplastic syndromes. Currently, there are few methods to quantitatively visualize cell cycle progression in (LSC) and to interrogate stem cell cycle gene regulatory networks in live cells. Human LSC are characterized as quiescent self-renewing cells and current methods require sequential transduction and clonal selection. These methods and conditions are not feasible for studying primary patient samples and LSC, which are generally limited in cell numbers. To alleviate this challenge, we generated a lentiviral biscistronic vector encoding FUCCI2 (fluorescent ubiquitination-based cell-cycle indicator) probes. We characterized the fidelity of the cell cycle by confirming normal cell division and distinct nuclear staining of either green or red fluorescence in both transiently and stably transduced 293 cells. Further, analysis by flow cytometry of 293 cell confirmed that S/G2/M and G1/G0 phases of cell cycle correlate with expression of fluorescent reporters expressed from the bicistronic lentiviral vector and recapitulate FACS based analysis using traditional Ki67 and 7AAD staining. Recently, using this lentiviral biscistronic reporter we succeeded in tracking cell cycle progression in CD34+ cells from CML primary patient samples co-cultured with SL/M2 cells. Time-lapse confocal imaging of these samples reveals distinct differences within the population of CML cells. In summary using this new Dual FUCCI2 lentiviral reporter makes it possible to track real time cell cycle kinetics in cancer stem cells. Future studies will include measuring LSC, cell cycle gene regulatory networks in live cells at different stages of the cell cycle.

Citation Format: Gabriel Pineda, Kathleen M. Lennon, Florence Lambert-Fliszar, Gennarina L. Riso, Catriona Jamieson. Real time niche tracking of cancer stem cell cycle kinetics using a novel bi-cistronic lentiviral reporter. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A46.