Profiling of urinary extracellular vesicle protein signatures from patients with cribriform and intraductal prostate carcinoma in a cross-sectional study

Abstract

Prognostic tests and treatment approaches for optimized clinical care of prostatic neoplasms are an unmet need. Prostate cancer (PCa) and derived extracellular vesicles (EVs) proteome changes occur during initiation and progression of the disease. PCa tissue proteome has been previously characterized, but screening of tissue samples constitutes an invasive procedure. Consequently, we focused this study on liquid biopsies, such as urine samples. More specifically, urinary small extracellular vesicle and particles proteome profiles of 100 subjects were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). We identified 171 proteins that were differentially expressed between intraductal prostate cancer/cribriform (IDC/Crib) and non-IDC/non-Crib after correction for multiple testing. However, the strong correlation between IDC/Crib and Gleason Grade complicates the disentanglement of the underlying factors driving this association. Nevertheless, even after accounting for multiple testing and adjusting for ISUP (International Society of Urological Pathology) grading, two proteins continued to exhibit significant differential expression between IDC/Crib and non-IDC/non-Crib. Functional enrichment analysis based on cancer hallmark proteins disclosed a clear pattern of androgen response down-regulation in urinary EVs from IDC/Crib compared to non-IDC/non-Crib. Interestingly, proteome differences between IDC and cribriform were more subtle, suggesting high proteome heterogeneity. Overall, the urinary EV proteome reflected partly the prostate pathology.

Keywords: Cribriform; Intraductal prostate carcinoma; Liquid chromatography mass spectrometry; Proteomics; Urinary extracellular vesicles and particles.

Fig. 1

Urinary EV characterization by western blot and NTA. (A) CD63 and Alix western blot of representative urinary EV samples from each major clinical groups. (B) Non-EV markers GRP75 (endoplasmic reticulum marker) and TOM20 (mitochondrial marker) as markers of transmembrane, lipid-bound and soluble proteins associated to other intracellular compartments than plasma membrane and endosomes. The process of transforming the original blot image into the final annotated image is outlined at the end of the supplementary documentation. NTA particle size distribution analysis of a representative urinary EV sample from (C) BPD and (D) IDC/Crib (insert represents a zoom in). Particle size mode distribution (E) and number of particles per protein amount (F) across major clinical subgroups. The central line inside each box represents the median value. Whiskers extend from the edges of the box to the minimum and maximum values within 1.5 times the interquartile range (IQR). Individual data points beyond the whiskers are considered outliers and are plotted individually. “c” indicates a mitochondrial enriched fraction from the large diffuse B cell lymphoma cell line HT.

Fig. 2

Urinary EV characterization by MS and TEM. (A) Small EV protein marker expression comparison with expression observed in published studies on small EVs isolated from cell lines (based on ultracentrifugation or polyethylene glycol (PEG)), bronchoalveolar lavage (BAL), plasma (P), diffuse large B cell lymphoma (DLBCL), healthy donor in DLBCL cohort (HD), and urinary samples (current study). General exosomes or small EV markers are color-coded in black, established exosome markers are in green, potential contaminant markers from other subcellular organelles are in red and markers from large EVs or microsomes are indicated in blue. (B) TEM 4000 × magnification, (C) TEM 20,000 × magnification.

Fig. 3

Overview of regulated proteins for three pairwise comparisons. (A) Table summary of up- and down-regulated proteins applying a 0.05 adjusted P value and no threshold on effect size. The numbers in parenthesis represent regulated proteins with similar thresholds but applying regular P values. Volcano plots for the pairwise comparisons (B) non-IDC/non-Crib versus BPD, (C) IDC/Crib versus BPD, (D) cancer versus BPD, (E) ISUP ≥ 2 versus ISUP = 1 and (C) ISUP ≥ 2 versus ISUP = 1 considering adjustment of other clinical variables. Red horizontal line indicates 0.05 P value threshold. Red vertical lines indicate two-fold differential regulation. The number of regulated proteins indicated in the titles is based on P values and adjusted P values with an effect size greater than twofold.

Fig. 4

Volcano plots for the pairwise comparisons. (A) Number of significantly regulated proteins for each of the pairwise comparisons given as N_{adjust-p-value} (N_{regular-p-value}). (B) IDC/Crib versus non-IDC/non-Crib, (C) crib versus non-IDC/non-Crib, (D) cribriform and IDC versus non-IDC/non-Crib, and (E) IDC versus non-IDC/ non-Crib. (F) Crib versus IDC. Red horizontal line indicates 0.05 P value thresholds. Red vertical lines indicate two-fold differential regulation. The number of regulated proteins indicated in the titles is based on P values and adjusted P value with an effect size greater than twofold.

Fig. 5

Box plot displaying the distribution of iBAQ values across increasing levels of Gleason score. The box plot showcases the median (represented by the horizontal line inside the box), interquartile range (IQR; the box’s height), and the minimum and maximum values (whiskers) within each level. Increasing trends are displayed for (A) HIST1H2BE, (B) JCHAIN, (C) HPX, and (D) SERPINA1. P value calculated by Jonckheere’s test.

Fig. 6

Functional enrichment analysis of cancer hallmark proteins based on significant regulated proteins for pairwise comparisons to control: Heatmap summarizing the functional enrichment of cancer hallmark proteins for each of the three pairwise comparisons to control based on (A) all regulated proteins, (B) up-regulated proteins and (C) down-regulated proteins. . Functional analysis based on significant regulated proteins for pairwise comparisons of different histological subgroups to non-IDC/non-Crib and cribriform: Functional enrichment against cancer hallmarks was performed for (D) all regulated proteins (p < 0.05), (E) all up-regulated proteins, and (F) all down-regulated proteins for each of the pairwise comparisons.

Fig. 7

Heatmap overview of the 20 most frequent reported differential regulated proteins from current study compared with previous studies.