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. 2020 Nov 3;32(5):829–843.e9. doi: 10.1016/j.cmet.2020.09.001

Figure 3.

Figure 3

BMSCs Support Leukemic Blast Survival, Chemoresistance, and Bioenergetics

(A) Scheme showing the different culture conditions and cells tested.

(B) Frequency of alive (AnnexinV−DAPI−) cells 24 h after AraC treatment (n = 19).

(C) Frequency of alive human AML cells monocultured (M) or cocultured or cocultured (C) with human mesenspheres or adherent BMSCs for 24 h under AraC treatment (n = 3–5). ∗p < 0.05; One-way ANOVA and Bonferroni comparisons.

(D and E) Frequency of alive CD45+ (D, n = 8) cells or CD34+ HSPCs (E, n = 3) from human AML BM 24 h after AraC in monoculture (black) or coculture (red) with BMSCs expanded as mesenspheres from AML patients (n = 8). ∗p < 0.05; ∗∗p < 0.01; paired two-tailed t test.

(F) OCR indicating mitochondrial respiration after oligomycin, FCCP and antimycin A/rotenone treatment in leukemic blasts cultured alone or previously cocultured with mesenspheres for 24 h in presence of AraC.

(G–J) Seahorse measurement of basal respiration (G), maximal respiration (H), spare respiratory capacity (I), and ATP content (J) in murine leukemic blasts monocultured (black columns) or cocultured with BMSCs (red columns); n = 6 independent experiments. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; unpaired two-tailed t test.

(K) Levels of the TCA cycle metabolites fumarate, ɑ-KG, and malate measured by LC-MS in murine leukemic blasts monocultured (black columns) or cocultured with BMSCs (red columns); n = 5 independent experiments with 4 biological replicates each.

(L) Frequency of AML blasts uptaking MitoTracker Red+ mitochondria previously stained in BMSCs only, before the coculture with AML cells under AraC treatment; n = 4 independent experiments; ∗p < 0.05; paired two-tailed t test.

(M) Representative flow cytometry diagrams showing CD45 (green) and MitoTracker Red (red) fluorescence in murine AML blasts monocultured or cocultured with BMSCs, which were previously stained with MitoTracker Red to label their mitochondria. The frequencies of gated cell populations are indicated. The red box highlights the increased frequency of BMSC-derived mitochondria in AML cells after AraC treatment.