Generation of a stable fluorescent cell cycle reporter myeloma cell line for real-time evaluation of microenvironment-responsive and drug-induced cell cycle alterations

Purpose of the study Multiple myeloma (MM) is the second most common blood cancer in U.S. Unfortunately, a significant percentage of patients develop refractory disease1, despite intensive treatment (i.e. lenalidomide and bortezomib). These poor clinical outcomes are thought to be driven by the emergence of a population of syndecan-1 negative (CD138-) quiescent myeloma-initiating cells with enhanced self-renewal capacity2,3. De-regulation of the cell cycle may play an important role in the generation and maintenance of these malignant cells during MM progression and relapse. However, most cell cycle analytical techniques involve fixation of cells or tissues, thereby precluding isolation of viable cells from different stages of the cell cycle for molecular analyses. The FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator)4 system facilitates direct evaluation of the cell cycle in various functional contexts. The aim of this study was to generate a MM cell line stably expressing fluorescent cell cycle indicators delivered using lentiviral vectors, thus allowing quantification and visualization of cell cycle dynamics in MM cells in bone marrow stromal co-cultures and in response to drug treatment. Project description The fluorescence ubiquitin cell cycle indicator (FUCCI) system is comprised of two vectors expressing fluorescent markers that are detectable during the G0-G1/S or S/G2/M phases of the cell cycle. The MM cell line NCI-H929, which harbors a CD138- subpopulation, underwent sequential rounds of lentiviral transduction with these two vectors. Using fluorescence activated cell sorting (FACS), we purified cells that were stably co-transduced with both FUCCI vectors (H929-FUCCI). H929-FUCCI cells were co-cultured with human or mouse bone marrow stromal cell lines (HS or OP9), along with primary MM stromal cells. H929-FUCCI plated on stromal monolayers for 24h were treated with lenalidomide (1µM-10µM, 24h), and then collected for cell cycle quantification by FACS. Results and conclusion Culture of H929-FUCCI cells on human stromal cell lines or primary MM stroma significantly increased the percentage of putative myeloma-initiating cells (CD138-). Additionally, an increased proportion of these CD138- cells were in the S/G2/M phase, an effect that was not observed in co-culture with mouse stromal cells. Treatment of H929-FUCCI cells with the immunomodulatory drug lenalidomide significantly increased the proportion of surviving H929-FUCCI cells in G0-G1 phases, with a corresponding decrease in the proportion of cells in S/G2/M phase, indicative of G1 cell cycle arrest. Co-culture with human bone marrow stromal cells diminished this effect, suggesting that cell cycle de-regulation of MM cells in the bone marrow niche may contribute to drug resistance. The generation of a novel cell cycle reporter myeloma cell line provides a valuable tool for precise evaluation of the effects of specific microenvironments as well as MM targeted therapies on cell cycle alterations in in vitro and in vivo niche models. This lays the foundation for developing new treatment strategies targeting cell cycle regulation of quiescent MM-initiating cells residing in the protective bone marrow niche. References 1. Kumar, S. K. et al. Improved survival in multiple myeloma and the impact of novel therapies. Blood 111, 2516–20 (2008). 2. Matsui, W. et al. Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Res. 68, 190–197 (2008). 3. Kawano, Y. et al. Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells. Int. J. Oncol. 43, 1809–16 (2013). 4. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–98 (2008).