The transition from proliferation to quiescence in glioblastoma stem-like cells requires Ca2+ signaling and mitochondria remodeling. Homo sapiens

Quiescence is a reversible cell-cycle arrest used by Cancer Stem Cells (CSCs) to evade killing following conventional therapies. Quiescent CSCs are therefore one of the main cause of cancer recurrence. In glioblastoma, the most common and aggressive primary brain tumors, the quiescent glioblastoma stem-like cells (GSCs) are localized in hypoxic and acidic microenvironments and microenvironmental changes can control cell cycle re-entering of the GSCs. Here, we show that proliferating GSCs isolated from patients can be induced and maintained in a quiescent state by lowering the extracellular pH. Through RNA-seq analysis we characterized the RNA signatures of quiescent versus proliferating GSCs. We identified genes involved in the control of Ca2+ signaling and differentially expressed between the two states. Using the bioluminescent Ca2+ reporter EGFP-aequorin targeted to the mitochondria or the cytosol we explored the changes in Ca2+ homeostasis occurring during the switch from proliferation to quiescence. This remodeling is controlled through store-operated channels (SOCs). SOCs play a causal role since the inhibition of the Ca2+ influx through SOC drives proliferating GSCs to quiescence. We showed that the switch to quiescence is characterized by both an increased capacity of GSCs’ mitochondria to capture Ca2+ and a dramatic and reversible change of mitochondrial morphology from a tubular to a donut shape. Our data suggest that the remodeling of the Ca2+ homeostasis and the reshaping of mitochondria during the transition from proliferation to quiescence constitute a protective mechanism that favors cancer stem-like cells’ survival and their aggressiveness in glioblastoma. Overall design: To establish RNA signatures of proliferative and quiescent GSLCs we have adopted the following strategy. Several experiments were performed in order to take into account different type of variabilities (Table S1); (1) Variability due to laboratories environments; experiments have been done in two laboratories ( Strasbourg and Toulouse) following identical protocols. The cells were obtained from the same master cell bank and the composition of the growing media was identical. (2) Cellular variability: two cell lines TG1 and OB1 were used. (3) Variability in inducing quiescence; the switch to quiescent state was obtained by either the non replacement of the medium during 9 days, acidification of the medium to pH 6.5 or 6.2 for 5 days or treatment of the cell by 10 µM SKF-96365 in the growing medium (pH 7.4) at day 1 and 3 and analysis at day 5. In normal medium at pH 7.4, the cells are in a proliferative state. Total RNA was extracted as described above and RNA quality was controlled with AATI Fragment Analyser (Advanced Analytical Technologies, Inc). RNA-seq was obtained from the IGBMC platform and the short reads were aligned using the reference genome hg38 (http://genomeast.igbmc.fr). The Qlucore software (http://www.qlucore.org) was used for the analysis.