Fig. 5: Loss of ether lipids decreases metastasis and cancer cell stemness.

a Representative confocal images of extravasated tdTomato-labeled pB3 WT and AGPS KO cells from an in vitro microvascular network (see methods), over 24 h. Data representative of two independent replicates (scale bar = 100 µm). b Quantification of extravasated pB3 WT and AGPS KO cells from microvascular network. Data represent the mean percentage of extravasated cells per device +/− SEM (n = 5 technical replicates). Significance was calculated using unpaired, two-tailed t-test. Data representative of two independent experiments. c Quantification of extravasated tdTomato-labeled PyMT-1099 cell line derivatives from microvascular network. Data represent the mean percentage of extravasated cells per device +/− SEM (n = 6 technical replicates). Significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test; ns, not significant. Data representative of two independent experiments. d Representative in vivo imaging system (IVIS) images of overall metastatic burden in C57BL/6 female mice following intracardiac injection of pB3 WT (n = 5) and AGPS KO (n = 5) cells. e Quantification of metastatic burden in C57BL/6 female mice following intracardiac injection of pB3 WT and AGPS KO cells (n = 5). Graph shows the mean +/− SEM and statistical significance was calculated using two-tailed Mann-Whitney test. f Representative images of H&E-stained sections of harvested kidneys from C57BL/6 female mice following intracardiac injection of pB3 WT or pB3 AGPS KO cells (scale bar = 1,000 µm). g Gross images of primary tumors derived from pB3 WT control cells and pB3 AGPS KO cells. h Tumor growth kinetics of primary tumors derived from pB3 WT control cells and pB3 AGPS KO cells (n = 5 mice per group). Data shown as mean +/− SEM. i Bar graph showing the average weight of primary tumors derived from pB3 WT and AGPS KO cells (n = 5 mice). Graph shows the mean +/− SEM and statistical significance was calculated using two-tailed Mann-Whitney test; ns, not significant. j Estimated number of cancer stem cells (CSCs) per 10,000 cells as calculated by extreme limiting dilution analysis (ELDA) software. Tumor-initiating capacity was assessed following implantation of pB3 WT or pB3 AGPS KO cells into the mammary fat pad of C57BL/6 mice. P values, two-sided χ2 pairwise test. k Table showing the number of mice with palpable primary tumors at 121 d post orthotopic implantation of PyMT-1099 WT or AGPS KO cells +/− pretreatment with TGF-β into female NSG mice. l Quantification of lung metastases for aforementioned experiment. Data represents mean number of lung metastases +/− SEM (n = 8 for WT and AGPS KO + TGF-β, n = 7 for WT + TGF-β). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test; ns, not significant. m Representative images of H&E-stained lungs harvested from female NSG mice from aforementioned experiment. Arrows indicate metastases. Scale bar = 2000 µm.