Different gDNA content in the subpopulations of prostate cancer extracellular vesicles: apoptotic bodies, microvesicles, and exosomes

Abstract

Background: Extracellular vesicles (EVs) are cell-derived membrane vesicles. EVs contain several RNAs such as mRNA, microRNAs, and ncRNAs, but less is known of their genomic DNA (gDNA) content. It is also unknown whether the DNA cargo is randomly sorted or if it is systematically packed into specific EV subpopulations. The aim of this study was to analyze whether different prostate cancer (PCa) cell-derived EV subpopulations (apoptotic bodies, microvesicles, and exosomes) carry different gDNA fragments.

Methods: EV subpopulations were isolated from three PCa cell lines (LNCaP, PC-3, and RC92a/hTERT) and the plasma of PCa patients and healthy donors, and characterized by transmission electron microscopy, nanoparticle tracking analysis and total protein content. gDNA fragments of different genes were detected by real time quantitative PCR and confirmed by DNA sequencing.

Results: We report that the concentration of EVs was higher in the cancer patients than in the healthy controls. EV subpopulations differed from each other in terms of total protein and DNA content. Analysis of gDNA fragments of MLH1, PTEN, and TP53 genes from the PCa cell-derived EV subpopulations showed that different EVs carried different gDNA content, which could even harbor specific mutations. Altogether, these results suggest that both nucleic acids and proteins are selectively and cell-dependently packed into the EV subtypes.

Conclusions: EVs derived from PCa cell lines and human plasma samples contain double-stranded gDNA fragments which could be used to detect specific mutations, making EVs potential biomarkers for cancer diagnostics and prognostics.

Fig 1

Characterization of cell-derived extracellular vesicle subpopulations. A: Representative transmission electron microscopy images of different EV populations: apoptotic bodies (a1–c1), microvesicles (a2–c2) and exosomes (a3–c3) released from LNCaP, PC-3, RC92a/hTERT cell lines. The scale bar is included in each picture. B: Size distribution graphics of microvesicles (MVs) and exosomes (EXOs) derived from LNCaP, PC-3, and RC92a/hTERT cells were determined by NTA using Nanosight software. The mean particle size for each population is represented.

Fig 2

Particle concentration of microvesicles and exosomes from prostate cell lines and plasma samples. A: Barplots representing the nanoparticle tracking analysis results depicting the particle concentration of microvesicles (MVs) and exosomes (EXOs) per million cells per cell line. LNCaP MVs (n = 8); PC-3 MVs (n = 4); RC92a/hTERT MVs (n = 4); LNCaP EXOs (n = 6); PC-3 EXOs (n = 4); RC92a/hTERT EXOs (n = 5); error bars represent SE; P values were determined by unpaired Wilcoxon signed ranked test (upper bar plots) and paired Wilcoxon signed ranked test (lower bar plots). B: Nanoparticle tracking analysis of MVs and EXOs derived from cell lines and plasma samples. The concentration (particles/ml) is normalized per million cells for EVs derived from prostate cell lines. C: Dot plot showing the total EV particle concentration per milliliter of plasma for patients (n = 8) and healthy donors (n = 8); error bars represent SE; *P < 0.05, was determined by unpaired Student's t-test.

Fig 3

Total protein contents of EV subpopulations. A: Bar plots representing the total protein contents per million cells of microvesicles (MVs) (n = 11) and exosomes (EXOs) (n = 12) isolated from LNCaP, PC-3, and RC92a/hTERT prostate cancer cells. Error bars represent SE. P values determined by paired Wilcoxon signed ranked test. Comparison of the total protein content of EXOs and MVs between cell lines. LNCaP MVs (n = 5); PC-3 MVs (n = 3); RC92a/hTERT MVs (n = 3); LNCaP EXOs (n = 3); PC-3 EXOs (n = 4); RC92a/hTERT EXOs (n = 5); error bars represent SE; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student's t-test. B: Comparison of the total protein content between MVs and EXOs for LNCaP, PC-3, and RC92a/hTERT cells. Error bars represent SE; P values were determined by paired Wilcoxon signed ranked test. C: Dot plot showing the total EV protein content from patients (n = 8) and healthy donors (n = 8). Error bars represent SE; P values determined by unpaired Student's t-test. D: Linear regressions of the correlation between the EV concentration and total protein content per million cells for microvesicles (MVs) and exosomes (EXOs) isolated from prostate cancer cell lines. LNCaP MVs versus EXOs (R² = 0.905); PC-3 MVs versus EXOs (R² = 0.113); and, RC92a/hTERT MVs versus EXOs (R² = 0.623).

Fig 4

Presence of mutated DNA fragments in EV subpopulations. A: Relative ratio of MLH1, PTEN, and TP53 genomic DNA (gDNA) fragments assessed by qPCR from subpopulations of EVs derived from LNCaP, PC-3, and RC92a/hTERT cells, using GAPDH as a reference gene. The GAPDH gene was used as reference due to its constant presence in all the vesicle types (Supplementary Fig. S2). Asterisk (*) represents that the gene of interest was not detectable by qPCR or not present in the samples analyzed. Columns are the average of three experiments, each one measured in triplicates. Error bars represent SE. Apoptotic bodies: dark gray; microvesicles: gray; exosomes: light gray. B: Agarose gel electrophoresis of MLH1, PTEN, and TP53 gDNA fragments extracted of EVs derived from LNCaP, PC-3, and RC92a/hTERT cells (n = 3). Ladder (L), apoptotic bodies (ABs), microvesicles (MVs), exosomes (EXOs), supernatant from the last ultracentrifugation (SP), positive cell control (C+) and negative control (C−). C: Electropherograms displaying PTEN and TP53 mutations in ABs and EXOs. Part of genomic DNA sequences of MLH1, PTEN, and TP53 genes from LNCaP-derived vesicles, with no MLH1 mutation (left), PTEN mutation in codon 6 (Frame-shift mutations, delAA) (center) and TP53 mutation in codon 215 (CCC to CGC) (right). The arrows show the position of the mutations.

Fig 5

Bioanalyzer analysis of gDNA in plasma-derived EVs from prostate cancer patients. Pre-amplified EV-derived DNA isolated from 2 mL of plasma together with DNA markers were analyzed using a 2100 Bioanalyzer. Vertical axis (FU) represents the fluorescent units and horizontal axis shows the number of base pairs. The two picks at 50 and 10,380 bp represent the DNA markers. Apoptotic bodies (ABs); microvesicles (MVs); exosomes (EXOs). Representative images of prostate cancer patients are showed.