Exosome proteomic analyses identify inflammatory phenotype and novel biomarkers in African American prostate cancer patients

Abstract

African American men face a stark prostate cancer (PCa)-related health disparity, with the highest incidence and mortality rates compared to other races. Additional and innovative measures are warranted to reduce this health disparity. Here, we focused on the identification of a novel serum exosome-based "protein signature" for potential use in the early detection and better prognosis of PCa in African American men. Nanoparticle tracking analyses showed that compared to healthy individuals, exosome concentration (number/ml) was increased by ~3.2-fold (P ˂ 0.05) in the sera of African American men with PCa. Mass spectrometry-based proteomic analysis of serum exosomes identified seven unique and fifty-five overlapping proteins (up- or downregulated) in African Americans with PCa compared to healthy African Americans. Furthermore, ingenuity pathway analyses identified the inflammatory acute-phase response signaling as the top pathway associated with proteins loaded in exosomes from African American PCa patients. Interestingly, African American PCa E006AA-hT cells secreted exosomes strongly induced a proinflammatory M2-phenotype in macrophages and showed calcium response on sensory neurons, suggesting a neuroinflammatory response. Additionally, proteomic analyses showed that the protein Isoform 2 of Filamin A has higher loading (2.6-fold) in exosomes from African Americans with PCa, but a lesser loading (0.6-fold) was observed in exosomes from Caucasian men with PCa compared to race-matched healthy individuals. Interestingly, TCGA and Taylor's dataset as well as IHC analyses of PCa tissue showed a lower Filamin A expression in tissues of PCa patients compared with normal subjects. Overall, these results support the usefulness of serum exosomes to noninvasively detect inflammatory phenotype and to discover novel biomarkers associated with PCa in African American men.

Keywords: biomarker; exosomes; health disparity; inflammation; prostate cancer.

Figure 1

Characterization of exosomes size and concentration by NTA and TEM. Exosomes were isolated by ultracentrifugation method from the serum of African American and Caucasian men either healthy or with PCa. Exosomes size and concentration were characterized by NTA. A, Exosome concentration (number per ml of serum); B, exosome size (nm); C‐D, exosome size distribution and representative TEM images (98 000×) for African American and Caucasian men. Data for A and B represent mean ± SEM of 3‐6 samples

Figure 2

Mass spectrometry analysis of proteins loaded in exosomes. Exosomes isolated from the serum of African American and Caucasian men either healthy or with PCa were analyzed by mass spectrometry. Number of proteins in exosomes from each group is presented by Venn diagram

Figure 3

Exosomes from African American PCa E006AA‐hT cells promote proinflammatory phenotype in macrophages and sensory neurons. A, THP1 cells were cultured in chambered coverslip along with PMA (100 ng/mL) for 12 h followed by treatment with Exo^Normoxic or Exo^Hypoxic (10 µg) and processed for arginase‐1 expression (green) by confocal microscopy. DAPI (blue) was used to stain nuclei. Representative images are shown at 60×. B, A change in intracellular calcium concentration of cultured DRG neurons to exosomes was measured by Ca²⁺ imaging. Representative traces of DRG neurons stimulated with Exo^Normoxic and Exo^Hypoxic (10 µg/mL) are presented. Rapid and transient cytosolic Ca²⁺ elevations were recorded, which was evoked by successive application (vertical arrow) of vehicle, Exo^Normoxic and Exo^Hypoxic (10 μg/mL each) and 1 μmol/L capsaicin. N = 30 neurons

Figure 4

Filamin A expression in PCa determined through TCGA database and Taylor's cohort. Box‐whisker plots showing the expression of Filamin A in TCGA based on (A) normal versus primary tumor, (B) patient's race, (C) patient's Gleason score, and (D) patient's age. (E) Box‐whisker plots showing the expression of Filamin A in Taylor's cohort based on major cancer stages

Figure 5

Filamin A expression in PCa tissues. PCa tissue sections from African American and Caucasian men were stained for Filamin A expression by IHC as described in methods, and representative photomicrographs are shown. A,B, Filamin A staining in benign prostate glands; C‐F, images for Filamin A staining in tumor area