Fig. 6. Lipidomic analyses reveal that ABCA12 reduces intracellular ceramide abundance to maintain cancer stemness.

(A) Lipid species significantly down-regulated in SOX9^high cells (fold change > 2) in C3/TAg;Sox9-GFP tumor (n = 5). (B) Level of ceramide lipids in Sox9^neg, Sox9^low, and Sox9^high C3/TAg tumor cells. (C) Lipid pathway enrichment analyses of the lipids identified in (A). AGE, advanced glycation end products; RAGE, receptor for AGE. (D) Flow cytometry measuring the abundance of ceramide in the SOX9^neg, Sox9^low, and Sox9^high C3/TAg tumor cells (n = 7). (E) Abundance of ceramide in the SOX9^neg, Sox9^low, and Sox9^high PyMT tumor cells (n = 5). (F) Abundance of ceramide species in sgNT and sgAbca12 C3/TAg organoids (n = 3). (G) Ceramide levels in C3/TAg organoids treated with dimethyl sulfoxide (DMSO) or D-NMAPPD for 5 days as measured by flow cytometry (n = 5). (H) Effect of C6-ceramide or D-NMPAAD on C3/TAg organoid formation (n = 6). (I) Ceramide levels in PyMT organoids treated with DMSO or D-NMAPPD (10 μM) for 5 days (n = 5). (J) Effect of C6-ceramide or D-NMPAAD on PyMT organoid formation (n = 3). (K and L) Organoid-forming efficiency of C3/TAg [(K), n = 5] or PyMT [(L), n = 3] tumor cells pretreated with D-NMAPPD (20 μM) for 3 days. (M) Ceramide levels in C3/TAg organoids treated with DMSO or Fumonisin B1 (FB1; 10 μM) for 5 days (n = 4). (N and O) Effect of FB1 on organoid formation by SOX9^neg/low C3/TAg [(N), n = 2] or SOX9^neg/low PyMT [(O), n = 3] tumor cells. All data are represented as means ± SEM. P values were determined by one-way ANOVA [(D) and (E)], or with Tukey’s test [(H), (J), (N), and (O)], or paired two-tailed t test [(G), (I), and (K) to (M)]. ***P < 0.001, **P < 0.01, and *P < 0.05.