Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells

Abstract

The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVβ3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from αVβ3-expressing cells affect tumour growth differently than sEVs from control cells that do not express αVβ3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVβ3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVβ3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.

Keywords: Prostate cancer; aurora kinase A; iodixanol density gradient; synaptophysin; tumour growth.

Figure 1.

Characterization of αVβ3 sEVs and Mock sEVs isolated by iodixanol density gradient. (a) αVβ3 sEVs and Mock sEVs were isolated by differential centrifugation and iodixanol density gradient from the culture medium of C4-2B β3 and C4-2B Mock cells. Then, the sEVs were analysed by IB for β3, and sEV markers CD63, CD81. IB analysis was performed under non-reducing conditions. Calnexin (CANX), which is supposed to be absent in sEVs, was also analysed. (b) Fractions 1–5 for αVβ3 sEVs, and 1–6 for Mock sEVs from the iodixanol density gradients shown in (a) were pooled and further characterized to confirm expression of Androgen Receptor (AR), β3, TSG101, CD81, CD9 (left panel); ALIX and calnexin (right panel). IB analysis was performed under reducing conditions. (a and b, TCL = total cell lysate). Different gels were used to separate samples under reducing or non-reducing conditions. (c) NTA analysis of the pooled fractions characterized in (b).

Figure 2.

Digital detection of αVβ3 on sEVs using SP-IRIS (Single Particle Interferometric Reflectance Imaging Sensor). sEVs were isolated by differential centrifugation and iodixanol density gradient and analysed by SP-IRIS. (a) Left panel, C4-2B β3 sEVs captured on the chip by the Ab indicated on the x-axis were visualized using a fluorescent β3 integrin Ab labelled with CF555. The normalized number of particles is shown in the bar graph. Right panels, the αVβ3 integrin positive sEVs captured on the chip were visualized. Representative images of the sEVs captured on the chip are shown; the top right panel shows sEVs captured on the chip using CD81 Ab; the bottom right panel shows the sEVs captured on the chip by the isotype control. The bar represents 5 μm. (b) The normalized number of particles positive for αVβ3 integrin from LNCaP β3 sEVs is shown. (a-b) The number of particles was normalized as described in the Material and Methods section.

Figure 3.

Uptake by DU145 cells of αVβ3 positive and negative sEVs isolated using iodixanol density gradients. Immunofluorescence analysis of αVβ3 transfer to DU145 cells. DU145 cells (150,000) incubated with 20 μg (corresponding to ~6 × 10¹⁰ particles) sEVs derived from C4-2B β3-GFP cells (left panel) or C4-2B Mock-GFP (right panel) were plated on vitronectin-coated coverslips. Arrows indicate GFP. The Z-stack analysis confirmed the intracellular colocalization of a red fluorescent signal (AP3, β3 Ab) and a green fluorescent signal corresponding to GFP, indicating the internalization of β3-GFP from sEVs. DAPI was used to detect cell nuclei (blue). The bar represents 10 μm. Seventy-eight cells were examined for each treatment group; 15–16 cells were positive for GFP in both treatments.

Figure 4.

Stimulation of anchorage-independent growth and induction of synaptophysin by αVβ3 sEVs in vitro. (a) Quantification of anchorage-independent growth expressed as log₁₀ of the average colony area. sEVs (10,000 cells/3.3 × 10⁶ sEVs) were incubated with LM609 Ab, mouse IgG or with PBS for 24 h and then, added to DU145 (left panel) or PC3 (right panel) cells for 16 h. The cells then were embedded in 0.3% agar-containing complete medium and were allowed to grow for 3 weeks. The average colony area was measured as described in the Materials and Methods section. Mock+609 and β3+609 indicate that the sEVs used to treat DU145 and PC3 cells were incubated with the monoclonal Ab LM609 against αVβ3 Integrin, as described in the Material and Method section. Mock+IgG and β3+IgG indicate that the sEVs used to treat DU145 and PC3 cells were incubated with mouse IgG used as negative control. The first 3 groups were incubated with PBS, Ab LM609 or mouse IgG without sEVs; the remaining groups were incubated with sEVs. (b) Representative images of the data quantified in (a). The bar represents 0.5 mm. (c) IB analysis of synaptophysin (SYP) expression in DU145, PC3, and LNCaP recipient cells after 16 hour treatment with either αVβ3 or Mock sEVs (16 h) and 21 days after treatment (21dd). LNCaP lysates were also tested for AR expression. Actin was used as loading control.

Figure 5.

Tumour growth increase and induction of NE markers (AURKA, SYP, NSE) by αVβ3 sEVs in vivo. (a) αVβ3 sEV and Mock sEV-treated DU145 cells (2.5 × 10 ⁷cells/10 × 10¹⁰ sEVs) were injected subcutaneously in nude mice; the xenografts were collected 74 days after injection. Time course of tumour growth was measured as tumour volume (left panel) or tumour weight (right panel) 74 days after cell injection, as described in the Material and Methods section. P-values are indicated in the figure. (b) Left panel, IB analysis for αVβ3, and NE markers: aurora kinase A (AURKA), synaptophysin (SYP), and neuron specific enolase (NSE) of the xenografts. Lanes 1–4 are representative tumour lysates from the Mock sEV treatment, whereas lanes 5–8 are tumour lysates from the αVβ3 sEV treatment group. Right panel, IB analysis of NE markers, AURKA and SYP, of the sEVs used to treat DU145 cells. All IB analysis was performed under reducing conditions. (c) Left panel, immunohistochemical analysis of αVβ3-positive areas (0.075 mm²) of the sEV-treated DU145 xenografts shown above. Right panels, representative images of the data quantified in the left panel; IgG was used as negative control. The bar represents 10 μm.

Figure 6.

Upregulation of αVβ3 integrin in NEPrCa patients. (a) Bioinformatic analysis of public datasets. cBioPortal OncoPrint graphic [33,34] showing the genomic alterations in ITGB3, AURKA, SYP, ITGAV and ITGB6 genes across the NEPrCa dataset containing 44 NEPrCa patient samples [19]. Glyphs and colour coding are used to summarize genomic alterations such as amplification (red). Grey colour coding indicates no alterations. Benign tissues and blood samples were used as controls. (b) Representative image of the immunohistochemical analysis for SYP and αVβ3 in PrCa bone (stern) metastasis. A total of 10 specimens from eight patients were analysed. IgG was used as negative control. The bar represents 10 μm.

Figure 7.

Schematic representation of the findings described in this study. The schematic diagram shows that the transfer of sEVs from prostate cancer cells (C4-2B) expressing αVβ3 induces NED in the recipient cells (DU145, PC3, or LNCaP), whereas transfer of Mock sEVs does not. The sEVs used in this study may have multiple origins (e.g., Multi Vesicular Body, MVB, or membrane budding).