Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

Abstract

Longitudinal tracking is a powerful approach to understand the biology of single cells. In cancer therapy, outcome is determined at the molecular and cellular scale, yet relationships between cellular response and cell fate are often unknown. The selective inhibitor of nuclear export, selinexor, is in development for the treatment of various cancers. Selinexor covalently binds exportin-1, causing nuclear sequestration of cargo proteins, including key regulators of the cell cycle and apoptosis. The cell cycle effects of selinexor and the relationships between cell cycle effects and cell fates, has not been described for individual cells. Using fluorescent cell cycle indicators we report the majority of cell death after selinexor treatment occurs from a protracted G1-phase and early S-phase. G1- or early S-phase treated cells show the strongest response and either die or arrest, while those treated in late S- or G2-phase progress to mitosis and divide. Importantly, the progeny of cell divisions also die or arrest, mostly in the next G1-phase. Cells that survive selinexor are negative for multiple proliferation biomarkers, indicating a penetrant, arrested state. Selinexor acts quickly, shows strong cell cycle selectivity, and is highly effective at arresting cell growth and inducing death in cancer-derived cells.

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level

“…2B). Consistent with previous studies, this effect on single-cell spatiotemporal dynamics was rapid, as the apparent difference was noticeable as early as 1 h following treatment, reflecting an accumulation of S/G2-M cells (Vassilev et al, 2006;Marcus et al, 2015). Automated single-cell duration measurement showed that the increase in the S/G2-M population was correlated with the residence time in the green phase (representing this population), the latter also exhibiting a dose-dependent trend (Fig.…”

“…Although the ambiguity might have stemmed from interline variation, the understanding so far has been drawn from cell-population-based experiments, phase-contrast images or low-content microscopy, precluding the precise discrimination between cells that die in mitosis and cells that die following a mitotic attempt. More recently, several qualitative studies using FUCCI-expressing cells have reported the emergence of CDT1 expression following prolonged mitosis (Honda-Uezono et al, 2012;Kaida et al, 2011;Marcus et al, 2015). Through indepth single-cell analyses, we found this phenomenon to be far more common than previously implied.…”

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

“…These compounds are termed selective inhibitors of nuclear export (SINE) and their anti-cancer effects are currently under investigation 31,32 . SINE treatment results in strong cell cycle phenotypes and cell death 33,34 . This example shows a cell that progresses through G1-phase with normal kinetics (approximately 6 hours), but experiences delay in S-phase progression as indicated by the period with both red and green signal (approximately 3 hours in control but 10 in SINE treated).…”

Loss of p53 expression in cancer cells alters cell cycle response after inhibition of exportin-1 but does not prevent cell death

“…Additionally, there are small molecules that block the ubiquitination of p53 via inhibition of the E3-ubiquitin ligase MDM2 (HDM2), namely nutlin-3a [12]. Nuclear accumulation of p53, cell cycle effects, apoptosis, and senescentlike growth arrest are noted after treatment of cells with selinexor [13][14][15] or nutlin-3a [12,16], consistent with the involvement of p53. Some studies indicate cancer cells with mutated TP53 show response to treatment with SINE, and others report that the response shows some p53-dependence [14,15,17].…”

“…To follow cell loss directly over time we used longterm time-lapse microscopy and longitudinal tracking after treatment with 1 μM selinexor; 1 μM provides nearly maximal effects (Figure 1), has been used extensively in vitro [8,13,17], and is relevant in patients [24,37]. TP53-matched HT-1080 and MCF7 cell lines expressing FUCCI are used; HCT116 FUCCI cells lines could not be obtained due to poor degradation of the G1-phase indicator peptide, mKO2-hCdt1 (30/120).…”