Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer

Figures

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  Figure 1. Quality assessment of the EVs isolation process A. Transmission electron microscopy images of EVs isolated from post-DRE urine; B. Linealregression analysis showing a good correlation between the number of EVs counted by NTA and the total amount of protein recovered from the same sample; C. Western blot analysis of TSG101, CD81 and Rab5, described in the literature as EVs markers, were performed in EVs isolated from post-DRE urine. Benign and PCa samples were pooled to obtain representative and sufficient material.

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  Figure 2. Abundance changes and diagnostic/prognostic evaluation of targeted proteins in urinary EVs Volcano plots represent differentially expressed target proteins in urinary EVs from A. PCa vs. benign patients and B. from high- vs. low-grade patients; C. Western blot of TGM4 and ADSV in a selected set of benign and PCa samples; D. Western blot of CD63, SPHM, TSG101 and PSA in a selected set of low and high-grade PCa samples. CD81 was used as EVs related marker.

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  Figure 3. Protein-based panels for PCa diagnosis and prognosis A-B. Scatter plots representing the protein abundance levels of selected proteins that are part of diagnostic and prognostic panels, respectively; C-D. ROC curves of protein-based panels for PCa diagnosis (ADSV + TGM4) and prognosis (CD63 + GLPK5 + SPHM + PSA + PPAP).

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  Figure 4. Assessment of PCa diagnostic biomarkers in tissue microarrays A. Tissue microarrays images with immunohistochemistry results for ADSV and TGM4; B. Scatter plots representing immunohistochemistry scores (IHC) of ADSV and TGM4 in benign and PCa FFPE tissue from PCa patients; C. Diagnostic performance represented by a ROC curve of ADSV and TGM4 individually. Prognostic value of TGM4 represented as IHC score (D) and ROC curve (E) between low vs. high-grade patients. Relation between TGM4 and PCa progression is showed as IHC score (F) and ROC curve (G) between patients with and without biochemical recurrence (BCR). Values that are significantly different by the Mann-Whitney test from the control group are indicated by p-value < 0.001 (*** vs. benign; +++ vs. No BCR).

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  Figure 5. Assessment of aggressive PCa biomarkers in tissue microarrays A-B. Scatter plots representing immunohistochemistry scores (IHC) of CD63, GLPK5 and SPHM in PCa FFPE tissue from low and high-grade PCa patients; C. ROC curve of CD63 biomarker performance; D-E. Scatter plots representing IHC scores of CD63, GLPK5 and SPHM in PCa FFPE tissue from patients with and without biochemical recurrence (BCR); F. ROC curve of GLPK5 biomarker performance. Significantly different values were assessed by the Mann-Whitney test and are indicated by *p-value < 0.05.