Fig 2: CD151 regulates the secretion of many proteins via exosomes. Western blot analysis of WCLs (A) and exosomes (B) showing CD151 was only present in the WT WCLs and exosomes compared with CD151KO WCLs and exosomes. β-actin and flotillin-1 were used as loading controls for (A) and (B), respectively. Heat map of all quantified protein in WCLs (C) and exosomes (D) performed based on Euclidean distance by the package “pheatmap” in R, revealing that the profiles of total cellular protein were alike, but the profiles of total exosomal proteins were obviously different. The heat map input was the relative intensities of whole quantified proteins. The intensity of each protein in each sample was divided by the average intensity of the same protein in all six samples, and log2 transformation was done to scale the heat map input. The red color indicates increased protein abundance, and the blue color indicates decreased protein abundance. WCLs, whole-cell lysates.

Fig 3: Volcano plot of quantified proteins of WCLs and exosomes after knocking out CD151.A, volcano plot of quantified proteins of exosomes. B, volcano plot of quantified proteins of WCLs. The threshold for determining DEPs is indicated by the dashed lines (adjusted p value < 0.01, fold change > 1.5). Red dots indicate significantly upregulated proteins, and green dots indicate significantly downregulated proteins. DEPs, differentially expressed proteins; WCLs, whole-cell lysates.

Fig 4: Workflow for characterization of TNBC-related changes in serum and cell line–derived exosome proteome.A, five hundred microliter of serum was collected from ten patients with TNBC and 17 healthy donors, and exosomes were purified by ultracentrifugation. Exosomes were lysed, and 100 μg of exosomal protein was digested. The peptides in each individual sample and the pooled samples were randomly distributed and labeled across three 10-plex TMT experiments. After labeling, samples were mixed within each TMT experiment and separated into seven fractions. Tandem MS data were obtained using an Orbitrap Fusion Lumos instrument, and proteins were identified using Proteome Discoverer 2.2. B, the serum exosome proteome data were normalized and analyzed. DEPs were determined by t test and further characterized through gene enrichment and network analysis. Representative protein CD151 was validated by Western blotting. C, CD151 was knocked out in MDA-MB-231 cells by using CRISPR–Cas9. Quantitative proteomics approach was performed to reveal the proteomes of CD151-deleted exosomes and cells. DEP, differentially expressed protein; TMT, tandem mass tag; TNBC, triple-negative breast cancer.

Fig 5: Exosomal CD151 promotes TNBC cell migration and invasion.A–D, 231-CD151KO-Exo decreases migration of MDA-MB-468 (A and C) and MDA-MB-231 (B and D) cells compared with 231-Control-Exo group. E–H, 231-CD151KO-Exo decreases invasion of MDA-MB-468 (E and G) and MDA-MB-231 (F and H) cells compared with 231-Control-Exo group. Migrated cell number and invaded cell number was determined by ImageJ software. Error bars represent mean ± SEM. Significance was determined using one-way ANOVA (∗p < 0.05). ns, not significant; TNBC, triple-negative breast cancer.