Proteomic analysis of plasma-derived exosomes during CLL evolution. (A) Molecular networks associated with proteins identified in plasma-derived exosomes during CLL evolution. The PPI networks were elucidated using the interaction model of FunRich software with the Vesiclepedia database, which focuses on statistically significant enriched genes and particular biological pathways in each subgroup. Different and specific networks according to the number of genes associated with the functions in the Prog-ddp subgroup are marked by red arrows: (1) Leukemic cell infiltration of secondary lymphoid organs, highlighted by genes related to the receptor sphingosine 1-phosphate (S1P1) pathway (P = 2.24e−4); (2) tumor proliferation network, involving genes of phosphatidylinositol 3-kinase (PI3K)/AKT kinase pathway (P = 2.24e−4); (3) survival and apoptosis network, involving genes implicated in the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) pathway; and (4) inflammation and oxidative stress networks, with genes involved in the AP-1 transcription factor pathway (P = 1.43e−4). (B) Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of CLL exosome proteins. LC-MS/MS data analysis was performed in accordance with the recently published PatternLab for proteomics 4.0 software (http://www.patternlabforproteomics.org) protocol for data analysis.16 The PatternLab approximately area-proportional Venn diagram module was used for pinpointing proteins exclusively identified in 1 biological condition. For the exosome protein-enriched sample, the analysis only considered proteins present in at least 3 of 5 patients for each biological condition. Junction plakoglobin (JUP) and S100-A9 (proteins in red font) were confirmed by specific monoclonal antibodies in immunoblot analysis. Plasma-derived exosomes were obtained from 10 patients (20 samples) corresponding to the 4 different subgroups (5 patients each) and subjected to 12% polyacrylamide gel (SDS-PAGE) electrophoresis. Representative immunoblots of both proteins at the different disease times are depicted. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL, high-density lipoprotein.
