Fig. 3: Follower cells exhibit elevated glucose uptake, glycolysis, and are less reliant on OXPHOS.

a OCR, an indication of OXPHOS, and ECAR, an indication of glycolysis, were measured using Seahorse Bioscience XFe96 Analyzer. Basal respiration rate is calculated by subtracting OCR after the addition of antimycin and rotenone from the OCR before the addition of any inhibitors. Error bars represent the mean ± SEM (n = 5 biologically independent samples). A two-tailed unpaired Student’s t-test was used to analyze statistical significance between leader and follower cell groups; p < 0.0001. b Basal OCR/ECAR ratio was calculated and graphed. Error bars represent the mean ± SEM (n = 5 biologically independent samples). A two-tailed unpaired Student’s t-test was used to analyze statistical significance between leader and follower cell groups; p < 0.0001. c The lactate concentration in leader or follower cell culture media were evaluated. Error bars represent the mean ± SEM (n = 3 biologically independent samples). A two-tailed unpaired Student’s t-test was used to analyze statistical significance between leader and follower cell groups; p < 0.0001. d [³H]2-deoxyglucose uptake of leader cells and follower cells was measured at 37 °C for 6 min as described in the methods. Error bars represent the mean ± SEM (n = 3 biologically independent samples). A two-tailed unpaired Student’s t-test was used to analyze statistical significance between leader and follower cell groups; p = 0.0070. e Metabolic profiling of leader cells and follower cells was performed and the concentration of metabolites (pmol/10⁶ cells) in glycolysis pathway were graphed. Error bars represent the mean ± SEM (n = 3 biologically independent samples). f Cellular lysates were prepared and evaluated for GLUT1 protein expression with GAPDH as a loading control. Relative densitometry is normalized to H1299. Repeated three times independently with similar results. g Representative confocal images of invading chains from H1299 spheroids embedded in Matrigel and allowed to invade for 24 h, then fixed and stained with phalloidin and GLUT1. White arrowheads designate leader cells. Scale bars = 50 µm. h Results shown in (g) are quantified. Error bars represent the mean ± SEM (n = 9 cells examined over three independent experiments). A two-tailed unpaired Student’s t-test was used to analyze statistical significance between leader and follower cell groups; p = 0.0021. i) H1299 spheroids embedded in Martigel were allowed to invade for 24 h and then stained with CellTracker Red and 2-NBDG and fixed. Representative confocal images are shown. White arrowheads designate leader cells. Scale bar = 100 µm. j 2-NBDG and CellTracker Red fluorescence intensity were quantified and are shown here as a ratio of 2-NBDG normalized to CellTracker. Error bars represent the mean ± SEM (n = 19 cells examined over three independent experiments). An ordinary one-way ANOVA with a Tukey’s multiple comparisons test was used to determine significance; Leader and second cell p = 0.0424, Leader and follower p < 0.0001, second cell and follower p < 0.0001.