Large oncosomes in human prostate cancer tissues and in the circulation of mice with metastatic disease

Abstract

Oncosomes are tumor-derived microvesicles that transmit signaling complexes between cell and tissue compartments. Herein, we show that amoeboid tumor cells export large (1- to 10-μm diameter) vesicles, derived from bulky cellular protrusions, that contain metalloproteinases, RNA, caveolin-1, and the GTPase ADP-ribosylation factor 6, and are biologically active toward tumor cells, endothelial cells, and fibroblasts. We describe methods by which large oncosomes can be selectively sorted by flow cytometry and analyzed independently of vesicles <1 μm. Structures resembling large oncosomes were identified in the circulation of different mouse models of prostate cancer, and their abundance correlated with tumor progression. Similar large vesicles were also identified in human tumor tissues, but they were not detected in the benign compartment. They were more abundant in metastases. Our results suggest that tumor microvesicles substantially larger than exosome-sized particles can be visualized and quantified in tissues and in the circulation, and isolated and characterized using clinically adaptable methods. These findings also suggest a mechanism by which migrating tumor cells condition the tumor microenvironment and distant sites, thereby potentiating advanced disease.

Figure 1

Characterization and detection of shed tumor cell–derived vesicles. A: Prostate cancer (PC3 and DU145), bladder cancer (253J), and glioblastoma (U87) cell lines are stained with CTxB and imaged by confocal microscopy. Scale bar = 10 μm. Arrowhead denotes a large vesicle released in the extracellular space. B: CTxB-labeled DU145 cells showing large, bulbous membrane protrusions and several large vesicles released into the surrounding environment. Arrowhead points to a large membrane bleb resulting in a released vesicle. C: Nucleic acid extracted from vesicles shed from LNCaP/MyrAkt1 cells. Total RNA/DNA is treated with RNase A or DNase 1 and samples electrophoresed through 2% agarose (D). E: FACS analysis of vesicles, isolated from the medium of WPMY-1 and LNCaP/MyrAkt1 cells treated with epidermal growth factor (50 ng/mL), fixed, permeabilized, and stained with PI. The dot plots depict forward scatter signal (FSC) and FL2 (PI). The red gates surround PI-positive events, graphed on the right. P < 2.2e-16. F: Left panel, photomicrograph of FITC-labeled gelatin matrix loaded with a biochemical preparation of vesicles shed from LNCaP/MyrAkt1 cells, showing large zones of proteolytic clearance. Original magnification, ×40. Trypsin is used as a positive control; a general protease inhibitor and vehicle serve as negative controls. Right panel, shed vesicles (SVs) are analyzed via gelatin zymography (substrate gel electrophoresis), showing active MMP9 and MMP2. G: Top panel, LNCaP/MyrAkt1 cells are stained with FITC-CTxB. Two membrane vesicles are large. Bottom panel, FITC-labeled gelatin after exposure to SVs. The CTxB-labeled membrane blebs (n = 150; G) and gelatin degradation spots (n = 135; H) are measured by AxioVision version 4.5 (Zeiss) and plotted graphically (mode, 3.1 to 4 μm).

Figure 2

Large oncosomes are bioactive and can be identified by FACS. A: LNCaP/MyrAkt1-derived vesicles, stained with HA-FITC–labeled antibody to detect MyrAkt1, are analyzed by FACS. Unstained vesicles are used as a negative control (gray shaded). LNCaP/MyrAkt1 cells are stained with Alexa-594–CTxB and FITC-HA–labeled antibody to detect MyrAkt1. Original magnification, ×40. Arrowheads, a shed large oncosome. B: Shed vesicles (SVs) and/or cell lysates (Cs) from LNCaP/MyrAkt1 and DU145 cells are blotted with the indicated antibodies. The micrographs show DU145 short hairpin RNA control and DU145 DIAPH3-silenced (short hairpin RNA) cells stained with CTxB, revealing large oncosomes produced by DIAPH3 silencing. C: MDEC and TEC migration induced by incubation with LNCaP/MyrAkt1-derived vesicles. The image depicts a heat map of a 24-well plate showing an increase in CellTracker fluorescence (red) from migrated cells, in response to treatment with LNCaP/MyrAkt1 vesicles or vehicle. Experiments (Exp.) are performed in technical duplicate and biological triplicates (Exp. A–C). Treatment with SVs increases migration rates in both cell lines significantly: P = 0.038 and P = 0.028, respectively. Ctrl, control. D: Fold change in CellTracker fluorescence of DU145 incubated with SV or vehicle; SV induces significantly higher migration than vehicle in DU145 prostate cancer cells (P = 0.011). E: Fold change in CellTracker fluorescence of DU145 cells migrating in response to WPMY-1 stromal cells as attractant. WPMY-1 cells are incubated with SV or vehicle, and DU145 cell migration toward the stromal cells is monitored, as in D (P = 0.021). F: Protein extracts from SV (lane 1), cells (lane 2), and cells + SV (lane 3) are blotted with the indicated antibodies. Lane 1, protein extracts from LNCaP/MyrAkt1-derived SVs; lane 2, protein extracts from WPMY-1 cells before exposure to SVs; and lane 3, protein extracts from WPMY-1 cells after exposure to LNCaP/MyrAkt1-derived SVs. G: Wild-type mouse prostate fibroblasts are incubated with SVs and analyzed by quantitative RT-PCR (RT2 profiler PCR array; Qiagen, Valencia, CA) for brain-derived neurotrophic factor (BDNF), CXCL12, osteopontin, and IL-6 mRNA levels. Exposure to SVs results in a significant up-regulation of these prometastatic factors compared with vehicle (Ctrl). Changes are shown as relative units of gene expression compared with a pool of five housekeeping genes.

Figure 3

Analysis of shed vesicles and detection of large oncosomes by FACS and microscopy. A: Purified vesicles are derived from LNCaP/MyrAkt1 cells, stained with an FITC-conjugated HA antibody, and plotted by setting a forward scatter signal (FSC) versus a PW signal on a linear scale (left panel). Right panels, a schematic representation of two signal pulses from the flow cytometer detector, corresponding to single particles (bottom panel) and doublet particles (top panel). Gated single events (within the red dotted line on the left panel) are visualized at the microscope and considered for further analysis. Signal pulses generated from aggregates (outside of the red dotted line) are excluded. B: Fluorescence and electron micrographs of MyrAkt1-positive particles sorted using size beads ≤1 and >1 μm. Arrowhead, lipid bilayer structure of the vesicle-encapsulating membrane.

Figure 4

Identification of large oncosomes in vivo. A: Tumor growth and tumor take in LNCaP/MyrAkt1 and LNCaP/LacZ xenografts (growth, P < 0.01; s.c. sites free of tumor, P = 0.01). B: Akt1 immunostaining of paraffin sections of the indicated xenografts. There is prominent Akt membrane staining in the MyrAkt1 tumors, in contrast to its diffuse cytosolic staining in the LacZ tumor sections. C: Left panel, FACS analysis of 1- to 10-μm MyrAkt1-positive vesicles purified from the plasma of mice carrying LNCaP/MyrAkt1 and LNCaP/LacZ xenografts. Dot plot, forward scatter signal (FSC) and FL1 (MyrAkt1); red gate, positive events. D: Quantitative evaluation of MyrAkt1-positive events. A cutoff corresponding to the 99th percentile of vesicles isolated from the plasma of mice with LNCaP/LacZ tumors is chosen to segregate negative and positive events. E: In mice with LNCaP/MyrAkt1 tumors, the percentage of MyrAkt1-positive vesicles correlates with tumor weight. *P = 0.007. F: MDECs exhibit increased migration when exposed to oncosomes compared with vehicle. Left panel, the vesicles are isolated from the plasma of mice with MyrAkt1 tumors. Right panel, the vesicles are derived from the medium of LNCaP/MyrAkt1 cells. Ctrl, control; SV, shed vesicle. G: Tumor sections of LNCaP/MyrAkt1 xenografts are stained with H&E. Arrowheads, large vesicles, similar in appearance to large oncosomes. Sections are also immunostained with an Akt1 antibody, which identifies Akt1 at the plasma membrane and allows visualization of large vesicles. H: Tumor sections of LNCaP/MyrAkt1 xenografts are imaged by transmission electron microscopy. Membrane blebs protruding from tumor cells near blood vessels are highlighted.

Figure 5

Large oncosome-like vesicles in metastatic prostate cancer. A: Representative paraffin section of human core biopsy specimens of patients with prostate cancer (Gleason score, 4 + 3) are immunostained with CK18 antibody. Arrowheads, structures resembling large oncosomes. B: Quantitative analysis of the distribution of ARF6-positive large oncosome-like vesicles among the diagnostic categories, showing vesicles are significantly more abundant in those with a Gleason score of >7 than a Gleason score of ≤7 (P = 0.020) and in metastases (Mets) than in organ-confined tumors [prostate cancer (PCa)] (P = 0.001). C: Representative sections of an additional prostate cancer TMA stained with ARF6, showing the absence (left panel) and presence (right panel) of large oncosome-like vesicles (arrowheads). D: The presence of structures resembling large oncosomes significantly discriminates between normal and tumor samples (P < 0.0001) and between organ-confined disease and metastasis (P = 0.0008).

Figure 6

Large oncosome-like structures containing Cav-1 identify aggressive prostate cancer. A: FACS analysis of 1- to 10-μm Cav-1–positive vesicles are purified from the plasma of TRAMP (n = 15) and non-transgenic littermates (WT) (n = 10), plotted in a forward scatter signal (FSC) histogram, and analyzed with respect to the diagnostic categories. Mets, metastases; PCa, prostate cancer; PIN, prostate intraepithelial neoplasia. B: Large oncosome-like structures, isolated from the plasma of TRAMP and WT mice, are analyzed by Western blot analysis. C: The abundance of Cav-1–positive large oncosome-like structures (1 to 10 μm) is significantly increased in tumor-bearing mice than in controls (P = 0.0007), and dramatically correlates with disease progression (almost 30-fold difference between mice with organ-confined tumors and mice with lung metastases) (P = 0.0002). D: The abundance of Cav-1–positive events <1 μm does not reflect changes across the diagnostic categories. E: Representative paraffin section of a TRAMP metastatic tumor is stained with ARF6 antibody. Arrowheads, large oncosome-like structures.

Figure 7

Model of potential sites of oncosome bioactivity. Our results suggest a working model in which oncosomes modify the microenvironment by inducing stromal reaction, ECM degradation, and migration of endothelial cells. Through these processes, circulating tumor-derived particles may promote endothelial leakage, allowing extravasation and colonization of distant sites. LO, large oncosome.