Glycan modification of glioblastoma-derived extracellular vesicles enhances receptor-mediated targeting of dendritic cells

ABSTRACT

Glioblastoma is the most prevalent and aggressive primary brain tumour for which total tumour lysate-pulsed dendritic cell vaccination is currently under clinical evaluation. Glioblastoma extracellular vesicles (EVs) may represent an enriched cell-free source of tumour-associated (neo-) antigens to pulse dendritic cells (DCs) for the initiation of an anti-tumour immune response. Capture and uptake of EVs by DCs could occur in a receptor-mediated and presumably glycan-dependent way, yet the glycan composition of glioblastoma EVs is unknown. Here, we set out to characterize the glycocalyx composition of glioblastoma EVs by lectin-binding ELISA and comprehensive immunogold transmission electron microscopy (immuno-TEM). The surface glycan profile of human glioblastoma cell line-derived EVs (50–200 nm) was dominated by α-2,3- and α-2,6 linked sialic acid-capped complex N-glycans and bi-antennary N-glycans. Since sialic acids can trigger immune inhibitory sialic acid–binding Ig-like lectin (Siglec) receptors, we screened for Siglec ligands on the EVs. Glioblastoma EVs showed significant binding to Siglec-9, which is highly expressed on DCs. Surprisingly, however, glioblastoma EVs lack glycans that could bind Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), a receptor that mediates uptake and induction of CD4⁺ and CD8⁺ T cell activation. Therefore, we explored whether modification of the EV glycan surface could reduce immune inhibitory Siglec binding, while enhancing EV internalization by DCs in a DC-SIGN dependent manner. Desialylation with a pan-sialic acid hydrolase led to reduction of sialic acid expression on EVs. Moreover, insertion of a high-affinity ligand (Lewis^Y) for DC-SIGN resulted in a four-fold increase of uptake by monocyte-derived DCs. In conclusion, we show that the glycocalyx composition of EVs is a key factor of efficient DC targeting and that modification of the EV glycocalyx potentiates EVs as anti-cancer vaccine.

KEYWORDS:

• Glioblastoma
• glioma
• extracellular vesicles
• EV
• glycans
• dendritic cells
• DC
• cancer vaccine

Author contributions

SD, SK and NP performed experiments, HK and MA synthesized palmitoyl-Lewis^Y, SD and JG analysed the data, RN, MP, TW, YK and JG supervised experiments. SD and JG drafted the manuscript. All authors revised the article and gave their approval for publication.

Acknowledgments

We kindly acknowledge Jacques Neefjes for providing anti-CD63 antibody for ELISA and TEM experiments. We would like to thank Nicole van der Wel and Jan van Weering for advice for improvement of our TEM images. Rubina Baglio and Monique A. J. van Eijndhoven kindly advised us about EV isolation procedures for which we are very grateful. We thank Edwin van der Pol his assistance with NTA analysis. The authors thank Xandra O. Breakefield for critically reading the manuscript.

This work was financially supported by the Institute for Chemical Immunology (ICI0011), ERC Advanced Glycotreat 339977 and the Cancer Center Amsterdam (CCA2014-5-18).

Disclosure of interest

The authors report no conflict of interest.

Supplementary material

Supplemental data for the article can be accessed here.

Additional information

Funding

This work was supported by the European Research Council [ERC Advanced Glycotreat 339977]; Institute for Chemical Immunology [ICI0011]; Cancer Center Amsterdam [CCA2014-5-18].