S100A8/A9^highAML cells display signs of myeloid differentiation. (A-C) AML cell lines were analyzed by flow cytometry for the myeloid maturation markers CD11b and CD14 in S100A8/A9^hi and S100A8/A9^lo cells after 48 hours of culture in the presence of HS-5 CM (A; OCI-AML, n = 5; MOLM-13, n = 5), AML-MSC CM from 6 patients (B; OCI-AML, n = 12; MOLM-13, n = 12, patient ID 11-16; supplemental Table 1), and recombinant human IL-6 (C; OCI-AML, n = 3; MOLM-13, n = 3), based on median fluorescence intensity (MdFI). (D) The myeloid maturation markers CD11b and CD14 were analyzed by flow cytometry in matched-pair (n = 10; patient ID 1-10; supplemental Table 1) PB- and BM-derived S100A8/A9^hi and S100A8/A9^lo AML blasts, as shown in representative histograms (left) and as summarized by fold change (right; S100A8/A9^lo set as 1). (E-F) Surface levels of CD11b and CD14 were analyzed in S100A8/A9^hi and S100A8/A9^lo (OCI-AML, n = 4; MOLM1,3 n = 4) populations after culture for 48 hours with HS-5 CM in the absence or presence of the pan-Jak inhibitor ruxolitinib, as indicated. (G) Surface levels of CD11b and CD14 were analyzed in S100A8/A9^hi cells among the AML cell lines (OCI-AML, n = 5; MOLM-13, n = 5) after 48 hours of culture with HS-5 CM in the absence or presence of the CD36 inhibitor SSO. Data are expressed as the standard error of mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.