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. 2025 Sep 12;20(9):e0329014.
doi: 10.1371/journal.pone.0329014. eCollection 2025.

A novel sialylation pathway mediated by extracellular vesicles in aggressive prostate cancer

Camila A Bach  1   2   3   4 , Md Niamat Hossain  1   2 , Ishan J Chaudhari  1   2 , Cecilia E Verrillo  1   2 , Nicole M Naranjo  1   2 , Isabella Amoroso  1   2 , Anna Testa  1   2 , Samuel Sey  1   2 , William K Kelly  5 , Susan L Bellis  6 , Aurelio Lorico  7 , Ada G Blidner  3 , Gabriel A Rabinovich  3   4   8 , Lucia R Languino  1   2
Affiliations

A novel sialylation pathway mediated by extracellular vesicles in aggressive prostate cancer

Camila A Bach et al. PLoS One. .

Abstract

Altered cell surface glycosylation is a hallmark of cancer; among aberrant glycan structures, hypersialylated proteins contribute to disease progression. The enzyme ST6 β-galactoside α2,6-sialyltransferase 1 (ST6GAL1) mediates α2,6-linked sialylation of N-glycosylated proteins and is upregulated in many cancers, including prostate cancer (PrCa). We propose that ST6GAL1 may be released by cancer cells in small extracellular vesicles (sEVs) in the PrCa tumor microenvironment to potentially modulate cell surface sialylation in recipient cells. We isolated sEVs from PrCa cells by density gradient separation and characterized them by nanoparticle tracking analysis using ZetaView and immunoblotting analysis. We identified ST6GAL1 in both its membrane-bound and soluble forms, both active, in circulating sEVs from healthy donors and patients with PrCa. ST6GAL1 is also expressed in human PrCa cells (PC3, DU145, and C4-2B), and in murine cells (TRAMP-C2 and RM1) at different levels, which correlate with aggressive cell phenotypes. In addition to classic sEV markers, such as CD9, TSG101 and Syntenin, sEVs isolated from PrCa cell lines express PDL1, an immune checkpoint ligand. The soluble ST6GAL1 form is present in the sEVs released from DU145 and PC3 cells and can be transferred via sEVs to recipient PrCa cells. This transfer is prevented by expression of Nogo-66 receptor homolog 2 (NgR2) and β3 integrin, which are elevated in the aggressive neuroendocrine phenotype of the disease. The soluble form is absent in the sEVs released from the bone metastatic line C4-2B, which only contains the membrane-bound form. Our results suggest that ST6GAL1 in sEVs derived from PrCa cells may potentially play a role in promoting bone metastasis by facilitating the formation of the pre-metastatic niche.

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Conflict of interest statement

Competing interests N.M. Naranjo and C.E. Verrillo are currently employees of private companies, which has had no influence on this work. No disclosures were reported by other authors.

Figures

Fig 1
Fig 1. Size distribution analysis of small extracellular vesicles (sEVs) from patients with invasive prostate cancer (PrCa) by ZetaView.
Fractions 1-10 were analyzed using ZetaView after iodixanol density gradient (IDG) separation.
Fig 2
Fig 2. ST6GAL1 expression in sEVs from plasma of patients with PrCa.
(A) Immunoblotting (IB) analysis of ST6GAL1, CD9 and Syntenin in lysates of sEVs isolated by IDG separation from plasma of patients with invasive (top, Prep# 1) or noninvasive (bottom, Prep# 2) PrCa; the total volume (30 μL) of each fraction was used. (B) IB analysis of ST6GAL1, CD9, TSG101 and Calnexin (CNX) in sEVs from plasma of healthy donors isolated by IDG and in PC3 total cell lysate (TCL); the total volume of each fraction and 40 µg of PC3 TCL were used. (C) IB analysis of CD41 and the αVβ3 integrin (β3) in platelet (PLT) lysate and sEVs from the plasma of healthy donors and patients with PrCa; 10 µg of TCL and 20 µg of sEV lysates were used. (m) indicates the membrane-bound ST6GAL1 form and (s) the soluble form.
Fig 3
Fig 3. ST6GAL1 expression in human PrCa cell lines and their derived sEVs.
IB analysis of ST6GAL1 at lighter (left panel) and darker exposures (middle panel), CNX, TSG101, Syntenin, PDL1 and CD81 in PC3, DU145 and C4-2B TCL and sEVs isolated by IDG separation (fractions 1-5 pooled; right panel); 85 µg of TCLs and 17 µg of sEV lysates were used.
Fig 4
Fig 4. ST6GAL1 expression in murine PrCa cell lines.
(A) IB analysis of ST6GAL1 and actin in PC3 and TRAMP-C2 TCL using 1 μg/mL (right panel) or 2 μg/mL (left panel) of ST6GAL1 antibody; 40 µg of TCLs were used. (B) IB analysis of ST6GAL1, CNX and PDL1 in TRAMP-C2, RM1 and NIH3T3 TCLs; 85 µg of TCLs were used. A lane loaded with non relevant sample is included (Non relevant).
Fig 5
Fig 5. Transfer of ST6GAL1 carrying sEVs to recipient PrCa cells.
(A) IB analysis of ST6GAL1, CNX and TSG101 in TRAMP-C2 TCL collected 24 hours after incubation with sEVs derived from PC3 cells isolated via IDG or PBS (untreated); 60 µg of TCLs were used. (B) IB analysis of ST6GAL1 and total focal adhesion kinase (tFAK) as loading control in DU145 exogenously expressing NgR2 (NgR2 transfectants) TCL, DU145 TCL collected 24 hours after incubation with sEVs from DU145 cells exogenously expressing NgR2 (NgR2 sEVs) or control Mock-DU145 sEVs (Mock sEVs) isolated via IDG or PBS (untreated); 40 µg of TCLs were used.
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