BM stromal cells induce S100A8 and S100A9 in AML cells. (A) FACS-sorted primary CD33⁺CD34⁺ AML blasts (n = 6) were cultured for 24 hours in the absence (w/o) or presence (w) of a confluent layer of the human BM-derived stroma cell line HS-5 and subsequently analyzed by microarray analysis (left). The heat map (right) shows the top 30 highest differentially expressed genes in the group of AML blasts cultured in the presence of HS-5 cells. (B) Publicly available data from TCGA (LAML data set) was divided into S100A8^hi/S100A9^hi and S100A8^lo/S100A9^lo groups based on the mean expression, and depicted as survival curves using the Mantel-Cox test for calculation of significance. (C) AML cell lines OCI-AML3 and MOLM-13 were cultured for 48 hours in the presence or absence of HS-5 cells (contact; OCI-AML, n = 11; MOLM-13, n = 10) or HS-5 CM (CM; OCI-AML, n = 5; MOLM-13, n = 5). Gene expression of S100A8 and S100A9 was analyzed by qPCR and is depicted as the fold change of treated/untreated cells (untreated set as 1). (D) Frequency of S100A8/S100A9^hi cells as shown in the representative pseudocolored flow cytometry plot was determined in AML cell lines (OCI-AML, n = 27; MOLM-13, n = 27) cultured for 48 hours in the absence or presence of HS-5 CM. (E) AML cell lines (OCI-AML, n = 13; MOLM-13, n = 11) were cultured for 48 hours in the absence or presence of CM from MSCs of 7 patients with AML (AML-MSC CM, patient ID 11-17; supplemental Table 1) and analyzed for the frequency of the S100A8/A9^hi population by flow cytometry. (F) Log₂-transformed normalized counts of the S100A8 and S100A9 gene expression (from the TCGA LAML data set) were correlated by the Spearman test. (G) The S100A8/A9^hi population among matched-pair PB- and BM-derived AML blasts (n = 10; patient ID 1-10; supplemental Table 1) was analyzed by flow cytometry. Data are expressed as the standard error of mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. FACS, fluorescence-activated cell sorting; qPCR, quantitative real-time polymerase chain reaction.