Figure 2. Modelling the induction of CD44^HiCD24^Hi cells.

(a) Schematic shows experimental design used to derive mathematical parameters of population dynamics. Treatment of MDA-MB-231 breast cancer cells with 25 nM docetaxel (DTX) for 24 h induces phenotype plasticity rather than providing a selection pressure. In parallel, starting cells with different permutations and combinations of CD24 and CD44 expression levels were used, and the expression of CD44CD24 was monitored over defined time points. (b) Population dynamics modelling derived from experimental data indicates temporal kinetics of breast cancer cells in distinct compartments over 5 days (CD44^Lo described as non-CSC). Left panel shows dynamics of distinct phenotypes under basal conditions, right panel demonstrates population dynamics under chemotherapy pressure. (c) Schematic shows subpopulation transition dynamics and predictive contribution of each population under chemotherapy pressure or basal state, saturated to equilibrium. Arrow weights denote prevalence of conversion. Loops indicate propensity to replicate or transition. (d) Treatment-naive 231-parental cells were sorted into CD44^HiCD24^Hi, CD44^HiCD24^Lo, CD44^LoCD24^Hi and CD44^LoCD24^Lo subpopulations, which were subsequently exposed to high-dose docetaxel (100 nM) for 48 h and re-analyzed by FACS for CD44^HiCD24^Hi subset expressed as % of total population. ‘Basal’ denotes the change in % of CD44^HiCD24^Hi subset in parental cells treated with vehicle. Data are mean±s.e.m. (ANOVA analysis, N=7, #P<0.05, *P<0.05 **P<0.01 versus basal group). (e) Depletion of intrinsic CSC population with salinomycin (5 μM, 48 h) was confirmed by reduction of a CSC (CD44^Hi/CD24^Lo) and enrichment of a non-CSC phenotype (CD44^LoCD24^Hi) expressed as fold change from vehicle-treated cells (error bars indicate s.e.m., N=5, *P<0.05 **P<0.01). (f) Chemo-tolerant cells generated from parent (DTC) and salinomycin-selected (Sal-DTC) MDA-MB-231 cells were analyzed by FACS for CD44^Hi/CD24^Hi, and results are expressed as % of total population (Data shown are mean±s.e.m., n=8, ANOVA analysis ***P<0.001, NS, not significant). (g) Graph shows mean fluorescent intensity (MFI) from FACS analysis of CD44 and CD24 expression in MDA-MB-231-parent, -DTC, -Sal-DTC or in a population of -expanded (E)-DTC and -Sal-DTC, demonstrating a reversal to parental phenotype when the chemotolerant cells are expanded over time (Data shown are mean±s.e.m. n=5, *P<0.05 **P<0.01). (h) Graph shows cell viability of each indicated population to docetaxel or doxorubicin, quantified by MTS cytotoxicity assay as % of viability in vehicle-treated control. All data shown are mean±s.e.m. from independent replicates (ANOVA analysis, n=8, ***P<0.001 versus parent cells).