Tracking Normal and Chronic Myeloid Leukemia Progenitor Cell Cycle Kinetics in a Defined Niche Using Live Time Lapse Confocal Imaging with Fucci2BL a Novel Lentiviral Bicistronic Reporter
Authors: Pineda Gabriel , Lennon Kathleen M , Delos-Santos Nathaniel P , Lambert-Fliszar Florence , Riso Gennarina L , Marra Marco A , Morris Sheldon , Sakaue-Sawano Asako , Miyawaki Atsushi , Jamieson Catriona HM 
Published: October 2019

Abstract
Malignant reprogramming of progenitors into self-renewing cancer stem cells (CSCs) that have a predisposition for dormancy in protective niches has been implicated in therapeutic resistance of chronic myeloid leukemia (CML) and other CSC-driven malignancies. An unmet medical need for developing therapies that target niche dependent dormant human CSCs provides a compelling rationale for identifying key differences in gene expression at different cell cycle phases between normal and malignant progenitors in a CSC-supportive stromal co-culture system. Currently, few methods exist for quantifying cell cycle kinetics in live human leukemia stem cells (LSC). To date, efficient cell cycle transit time analysis in single live human leukemic progenitors derived from primary patient samples has been hampered by 1) decreased cell viability following transfection or transduction, 2) limited sample size, 3) dormancy of primitive progenitor populations thereby necessitating lentiviral rather than retroviral transduction and 4) increased apoptosis in the absence of a supportive microenvironment. To alleviate these challenges and improve transduction efficiency, we generated Fucci2BL, a lentiviral bicistronic reporter vector. Fucci2BL expresses mVenus-hGem(1/110) fused to mCherry-hCdt1(30/120) by the T2A peptide using an EF1 promoter that generates optimal levels of gene expression in progenitors. Initially, the reporter fidelity was characterized in 293A cells using flow cytometry and time-lapse confocal fluorescence microscopy. Time-lapse confocal fluorescence microscopy revealed normal cell morphology and distinct nuclear staining of either green or red fluorescence depending on the cell cycle stage. Once the fidelity of the Fucci2BL reporter was characterized, differences in gene expression levels between normal and malignant progenitors were analyzed. Whole transcriptome RNA-seq analysis revealed both cell cycle and DNA replication pathways were enriched in chronic phase CP (CML) compared to normal progenitors. Cell cycle kinetics between normal and chronic phase (CML) progenitors co-cultured in a niche were also analyzed using the Fucci2BL reporter. Normal progenitor cells on average transited the cell cycle within 26 hours while CP progenitor cells demonstrated a prolongation of transit through G1. In summary, the Fucci2BL system enables single transduction and single cell cycle tracking as well as gene expression changes in live primary progenitors in response to a niche. This robust lentiviral reporter can reproducibly distinguish cell cycle phases thereby providing an opportunity to quantitatively study the contribution of cell cycle kinetics to single cancer stem cell therapeutic resistance and to relapse.

Disclosures
Jamieson: J&J: Research Funding; GSK: Research Funding.