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.

胶质母细胞瘤是最常见和最具侵袭性的原发性脑肿瘤，目前正在对全肿瘤裂解物脉冲树突细胞疫苗接种进行临床评估。胶质母细胞瘤细胞外囊泡（EV）可能代表肿瘤相关（新）抗原的丰富无细胞来源，可脉冲树突状细胞（DC）以启动抗肿瘤免疫反应。DC 对 EV 的捕获和摄取可能以受体介导且可能依赖聚糖的方式发生，但胶质母细胞瘤 EV 的聚糖组成尚不清楚。在这里，我们着手通过凝集素结合 ELISA 和综合免疫金透射电子显微镜 (immuno-TEM) 来表征胶质母细胞瘤 EV 的糖萼组成。人胶质母细胞瘤细胞系来源的 EV（50-200 nm）的表面聚糖谱主要由 α-2,3- 和 α-2,6 连接唾液酸封端的复合 N-聚糖和双触角N-聚糖组成。由于唾液酸可以触发免疫抑制性唾液酸结合 Ig 样凝集素 (Siglec) 受体，因此我们在 EV 上筛选了 Siglec 配体。胶质母细胞瘤 EV 与 Siglec-9 显着结合，Siglec-9 在 DC 上高表达。然而，令人惊讶的是，胶质母细胞瘤 EV 缺乏可以结合树突状细胞特异性细胞间粘附分子-3-抓取非整合素（DC-SIGN，CD209）的聚糖，这是一种介导摄取和诱导 CD4 + 和 CD8 + T 细胞^活化的^受体。因此，我们探讨了 EV 聚糖表面的修饰是否可以减少免疫抑制性 Siglec 结合，同时以 DC-SIGN 依赖性方式增强 DC 的 EV 内化。使用泛唾液酸水解酶进行去唾液酸化导致 EV 上唾液酸表达减少。此外，插入 DC-SIGN 的高亲和力配体 (Lewis ^Y ) 导致单核细胞来源的 DC 的摄取量增加了四倍。总之，我们表明 EV 的糖萼组成是有效 DC 靶向的关键因素，并且 EV 糖萼的修饰增强了 EV 作为抗癌疫苗的作用。