Figure 3.
S100A8/A9hiAML cells display enhanced fatty acid metabolism. (A) Gene expression of the fatty acid translocase CD36 was determined in FACS-sorted S100A8/A9hi and S100A8/A9lo OCI-AML (n = 3) and MOLM-13 (n = 3) cells cultured for 48 hours in the presence of HS-5 CM by qPCR and is shown as the fold change (S100A8/A9lo cells were set as 1). (B) Surface levels of CD36 were determined by flow cytometry on S100A8/A9hi and S100A8/A9lo cells among the AML cell lines cultured for 48 hours in the presence of CM from either HS-5 cells (left; OCI-AML, n = 4; MOLM-13, n = 4) or AML-MSCs isolated from 6 patients (right; OCI-AML, n = 12; MOLM-13, n = 12, patient ID 11-16; supplemental Table 1). (C) Surface levels of CD36 were analyzed by flow cytometry in matched-pair (n = 10; patient ID 1-10; supplemental Table 1) PB- and BM-derived S100A8/A9hi and S100A8/A9lo AML blasts, as representatively shown in histograms (left) and summarized in a bar graph (right). (D) Localization of S100A8 and S100A9 was visualized by fluorescence microscopy (left, scale bars = 20 μm). MOLM-13 cells (n = 5) were cultured in the absence (top) or presence (bottom) of HS-5 CM and stained for the cell membrane (WGA, green), S100A8 (left, red) or S100A9 (right, red), and the nucleus (blue, DAPI). Co-localization of S100A8/S100A9 and WGA were calculated with ZEN software (bar graphs; Zeiss). (E) A proximity ligation was performed with a Duolink Flow kit (Sigma-Aldrich) with antibodies against S100A8, S100A9, and CD36. The representative images show the bright-field (left, BF) and the fluorescence signal (right) in MOLM-13 cells. Bars represent 20 μm for 20× magnification. (F) Long-chain fatty acid uptake by AML cell lines (OCI-AML, n = 5; MOLM-13, n = 5) cultured for 48 hours in absence or presence of HS-5 CM was measured by flow cytometry using the fluorescent probe Bodipy FLC16 based on the median fluorescence intensity (MdFI). (G) Long-chain fatty acid uptake was analyzed in AML cell lines cultured for 48 hours in the absence or presence of HS-5 CM, with or without ruxolitinib (OCI-AML, n = 2; MOLM-13, n = 3). (H) Oxygen consumption rate (OCR; as a surrogate for oxidative phosphorylation) was determined for MOLM-13 cells (n = 3) cultured for 48 hours in the absence (absence) or presence of HS-5 CM (CM) at baseline and after consecutive injection of SSO and etomoxir, which enabled calculation of the dependence on exogenous (exo) and endogenous (endo) fatty acids for fueling mitochondrial respiration. (I) S100A8/A9hi frequency was analyzed in AML cell lines (OCI-AML, n = 5; MOLM-13, n = 4) cultured for 48 hours in the presence of HS-5 CM, with or without the CD36 inhibitor SSO. Data are expressed as the standard error of the mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ns, not significant. DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; qPCR, quantitative real-time polymerase chain reaction.

S100A8/A9hiAML cells display enhanced fatty acid metabolism. (A) Gene expression of the fatty acid translocase CD36 was determined in FACS-sorted S100A8/A9hi and S100A8/A9lo OCI-AML (n = 3) and MOLM-13 (n = 3) cells cultured for 48 hours in the presence of HS-5 CM by qPCR and is shown as the fold change (S100A8/A9lo cells were set as 1). (B) Surface levels of CD36 were determined by flow cytometry on S100A8/A9hi and S100A8/A9lo cells among the AML cell lines cultured for 48 hours in the presence of CM from either HS-5 cells (left; OCI-AML, n = 4; MOLM-13, n = 4) or AML-MSCs isolated from 6 patients (right; OCI-AML, n = 12; MOLM-13, n = 12, patient ID 11-16; supplemental Table 1). (C) Surface levels of CD36 were analyzed by flow cytometry in matched-pair (n = 10; patient ID 1-10; supplemental Table 1) PB- and BM-derived S100A8/A9hi and S100A8/A9lo AML blasts, as representatively shown in histograms (left) and summarized in a bar graph (right). (D) Localization of S100A8 and S100A9 was visualized by fluorescence microscopy (left, scale bars = 20 μm). MOLM-13 cells (n = 5) were cultured in the absence (top) or presence (bottom) of HS-5 CM and stained for the cell membrane (WGA, green), S100A8 (left, red) or S100A9 (right, red), and the nucleus (blue, DAPI). Co-localization of S100A8/S100A9 and WGA were calculated with ZEN software (bar graphs; Zeiss). (E) A proximity ligation was performed with a Duolink Flow kit (Sigma-Aldrich) with antibodies against S100A8, S100A9, and CD36. The representative images show the bright-field (left, BF) and the fluorescence signal (right) in MOLM-13 cells. Bars represent 20 μm for 20× magnification. (F) Long-chain fatty acid uptake by AML cell lines (OCI-AML, n = 5; MOLM-13, n = 5) cultured for 48 hours in absence or presence of HS-5 CM was measured by flow cytometry using the fluorescent probe Bodipy FLC16 based on the median fluorescence intensity (MdFI). (G) Long-chain fatty acid uptake was analyzed in AML cell lines cultured for 48 hours in the absence or presence of HS-5 CM, with or without ruxolitinib (OCI-AML, n = 2; MOLM-13, n = 3). (H) Oxygen consumption rate (OCR; as a surrogate for oxidative phosphorylation) was determined for MOLM-13 cells (n = 3) cultured for 48 hours in the absence (absence) or presence of HS-5 CM (CM) at baseline and after consecutive injection of SSO and etomoxir, which enabled calculation of the dependence on exogenous (exo) and endogenous (endo) fatty acids for fueling mitochondrial respiration. (I) S100A8/A9hi frequency was analyzed in AML cell lines (OCI-AML, n = 5; MOLM-13, n = 4) cultured for 48 hours in the presence of HS-5 CM, with or without the CD36 inhibitor SSO. Data are expressed as the standard error of the mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ns, not significant. DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; qPCR, quantitative real-time polymerase chain reaction.

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