HS-5 CM is enriched in inflammatory cytokines that promote the proliferation and survival of primary leukemic AML cells. (A) Freshly isolated AML cells were cultured in MCM or 25% HS-5 CM for 3 days, and cell viability assessed with the CTG assay. AML cells had a mean viability of 113% after 3 days in CM compared with 80% in MCM (113.0 ± 10.4 in CM and 80.2 ± 9.4 in MCM; n = 13; P < .05). Graphs show mean ± SEM. (B) Cytokine levels measured from HS-5 CM, primary MSCs CM from a patient with AML and BM supernatant fluid from a patient with AML and a healthy donor. (C) Phosphoflow analysis of phospho-ERK, phospho-AKT, phospho-STAT3, and phospho-STAT5 in AML patient cells treated with RPMI, 25% HS-5 CM, MCM, or RPMI supplemented with 10 ng/mL of IL-6, IL-8, MIP-3α, GM-CSF, G-CSF, or a combination of all the cytokines for 20 minutes, showing stimulation of STAT5 phosphorylation. (D) Detection of STAT5 phosphorylation in AML cells from 3 patients after 20 minutes of stimulation with different media conditions or individual cytokines (10 ng/mL). (E) Detection of phospho-STAT5 in AML patient cells treated for 1 hour with or without ruxolitinib (300 nM) after 20 minutes of stimulation with 25% CM or RPMI supplemented with 10 ng/mL GM-CSF, G-CSF, or a combination of the 2 cytokines. Error bars represent standard deviation of duplicates.