CLL plasma-derived exosomes show increased levels of S100-A9 protein and are released by CLL cells. (A-B) S100-A9 quantification from plasma-derived exosomes between the different subgroups; immunoblot analysis was performed after exosome protein separation. A total of 50 μg of exosome protein from the different samples was transferred onto nitrocellulose and developed with specific anti–S100-A9 monoclonal antibody. HSP-70 protein was visualized as internal charge control. Values of S100-A9 were normalized to those of HSP-70 accordingly. Results showed that S100-A9 protein was significantly higher in the plasma exosomes at the time of disease progression, whereas no or low presence of this protein was found in the exosomes of the Prog-dt subgroup, the indolent group, or the matched HDs. *P < .05. (C) Release of S100-A9⁺ exosomes by CLL cells; in vitro analysis was performed with CD19⁺ cells (100 exp¹⁰  lymphocytes incubated O.N. with anti-IgM 15 μg/ml). AIM V medium 10 mL was collected and exosomes purified as previously described in Material and Methods. Results showed that CLL cells release exosomes carrying S100-A9 protein to the culture medium after incubation with anti-IgM. *S100-A9 homodimer. Immunoblot analysis was performed as described. ns, not significant.