Plasma-derived exosomes with S100-A9 cargo promote NF-κB pathway activation in leukemic cells. (A-B) NF-κB pathway is activated after in vitro incubation with plasma-derived exosomes (Exo) carrying S100-A9. PBMCs of unmutated and mutated CLLs were incubated in RPMI 1640 and fetal bovine serum with plasma-derived exosomes (S100-A9⁺) extracted from Prog-ddp and plasma-derived exosomes without S100-A9 extracted from Prog-dt samples (90 minutes; 60 μg/ml of total protein). Negative and positive exosomes were categorized by immunoblot. NF-κB activation was determined as the relative percentage by densitometry of the corresponding signal from phosphorylated IκB-α/total IκB-α. “Without exosomes” indicates basal phosphorylation of IκB-α in the PBMCs of CLLs. Significant changes were observed in the rate of IκB-α phosphorylation after incubation with S100-A9⁺ plasma-derived exosomes compared with their counterparts (S100-A9⁻ plasma-derived exosomes; Wilcoxon signed rank test, P = .0078). In this experiment, PBMCs at disease onset from 3 different patients, 2 with unmutated and 1 with mutated CLL (CLLs 10 and 13 and CLL 25, respectively) were incubated with plasma-derived exosomes from 3 different CLLs (Exo1, CLL 02; Exo2, CLL 05; Exo3, CLL 07) extracted at disease onset (S100-A9⁻) and during disease progression (S100-A9⁺). NF-κB pathway activation after incubation with plasma-derived exosomes carrying S100-A9 was also evidenced by p65 nuclear translocation. After 2 hours of incubation with plasma-derived exosomes (with or without S100-A9), PBMCs of the 3 CLLs were collected and stained with anti-IgM, anti-CD5, and anti-p65 transcription factor. A representative figure of the 3 patients with CLL studied is provided in Figure 5B. Green color indicates p65, and yellow staining corresponds to IgM⁺ cells. DNA staining with methyl green was performed. More than 95% of IgM cells for CLL 10 and 13 and 72% for CLL 25 were positive for CD5 marker (estimated by flow cytometry analysis and confocal microscopy; data not shown). Leukemic cells after incubation with exosomes carrying S100-A9 with increased nuclear localization of p65 transcription factor are highlighted by white asterisks. Scale bar, 5μm (lower right). (C) Phosphorylation of IκB-α in PBMCs of CLL after 90-minute incubation with increasing concentrations of plasma-derived exosomes carrying S100-A9. Data are reported as relative changes of phosphorylated IκB-α/total IκB-α. (D) Monoclonal antibody against S100-A9 was used to block the interaction of S100-A9⁺ exosomes with leukemic cells. To confirm the specificity of NF-κB activation, 60 μg of CLL plasma-derived exosomes (S100-A9⁺) were extracted from patient 01 and preincubated for 60 minutes with polyclonal antibody anti–S100-A9 at 6°C or with isotype control. Next, PBMCs from 4 different patients were incubated for 90 minutes at 37°C with these cocktails (S100-A9⁺ exosomes plus anti–S100-A9 antibody, S100-A9⁺ exosomes, and S100-A9⁺ exosomes plus isotype control antibody). Low levels of IκB-α phosphorylation were found after PBMC incubation with anti–S100-A9 (data not shown). This unspecific activation was subtracted to obtain the values corresponding to the “exosomes S100-A9⁺ plus anti–S100-A9 group. Our results showed a significant reduction in the 4 samples in the ratio p-IκB-α / IκB-α after CLL PBMCs were incubated with S100-A9⁺ exosomes plus anti–S100-A9 antibody (mean, 22) compared with CLL PBMCs incubated only with S100-A9⁺ exosomes (mean, 44.95; 1-way analysis of variance P < .01). Significant differences were found between CLL PBMCs incubated only with S100-A9⁺ exosomes compared with untreated CLLs (mean, 10.40; without exosomes).