Figure 1. Acetate secretion by stromal cells increases in AML-stroma co-cultures of several AML cell lines and primary AML cells in direct contact.

(A) Section of ¹H-NMR spectra, corresponding to the methyl group of acetate, from extracellular medium samples of SKM-1 cells cultured alone (blue), MS-5 cells cultured alone (red) and SKM-1 and MS-5 cells in co-culture (green) after 24 hours. (B) Extracellular acetate levels in AML cell lines (SKM-1, Kasumi-1 and HL-60) cultured alone (blue), MS-5 cells cultured alone (red) and AML and MS-5 cells in co-culture in direct contact (green) at 0, 20 and 24 hours of incubation. Each point represents the mean of n=3 independent experiments and error bars represent standard deviations. (C) Extracellular acetate levels in AML cell lines cultured alone (blue), MS-5 cells cultured alone (red) and AML and MS-5 cells in co-culture separated by a 0.4 µm permeable membrane (green) at 0, 20, and 24 hr of incubation. Each point represents the mean of n=3 independent experiments and error bars represent standard deviations. (D) Extracellular acetate levels in AML cell lines and MS-5 cells in co-culture (black) for 24 hr and after being separated and cultured alone in the same medium until 48 hr (blue for AML and red for MS-5). Each point represents the mean of n=3 independent experiments and error bars represent standard deviations. (E) Extracellular acetate levels in MS-5 cells cultured alone and primary patient-derived AML cells co-cultured with MS-5 cells at 48 hr. Each set of points represents an independent experiment (n=4). (F) Extracellular acetate levels in MS-5 cells cultured alone and healthy donor-derived peripheral blood mononuclear CD34⁺ (PBMC) cells co-cultured with MS-5 cells at 48 hr. Each set of points represents an independent experiment (n=3). For E and F, symbols (circles or triangles) indicate same cell culture medium composition was used. (G), Acetate levels in bone marrow extracellular fluid of C57BL6/J mice 6 months after transplantation with bone marrow nucleated cells isolated from control or MLL-AF9 transgenic mice. For B, C, and D unpaired Student’s t-tests were applied for each condition (black brackets) for G a Mann-Whitney test was applied (black brackets). p-values are represented by n.s. for not significant * for p-value <0.05, ** for p-value <0.01 and *** for p-value <0.001.

Figure 1—figure supplement 1. Other extracellular metabolite levels in co-cultures with SKM-1 and MS-5 cells.

Sections of ¹H-NMR spectra from extracellular medium samples of SKM-1 cells cultured alone (blue), MS-5 cells cultured alone (red) and SKM-1 and MS-5 cells in co-culture (green) after 24 hr of culture, corresponding to glucose, lactate, glutamate, and glutamine.

Figure 1—figure supplement 2. Proliferation in co-culture, co-culturing AML cells with an unrelated cell line (HeLa).

(A) CFSE cell proliferation analysis in AML cell lines alone or in co-culture with MS-5 cells. The population of living cells was gated, and 1500 cells for each condition were randomly selected and plotted. The geometric mean for each population and time point was compared between cells alone or in co-culture by performing an unpaired unpaired Student’s t-test and p-values were represented as n.s. for not significant. Each histogram is representative of n=3 independent experiments. (B), Extracellular acetate levels in SKM-1 cells cultured alone (blue), HeLa cells cultured alone (red) and AML and HeLa cells in co-culture (green) at 0, 20, and 24 hr. Each point represents the mean of n=3 independent experiments and error bars represent standard deviation. An unpaired Student’s t-test was applied for each condition (black brackets) and p-values are represented by n.s. for not significant.