Polysialic acid in Siglec- and chemokine-dependent responses of tumor-associated macrophages

肿瘤相关巨噬细胞的 Siglec 和趋化因子依赖性反应中的聚唾液酸

• 批准号: 432236295
• 负责人: Professor Dr. Herbert Hildebrandt
• 金额: --
• 依托单位: Institut für Klinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/432236295?language=en
• 关键词: Polysialic; acid; Siglec; chemokine; dependent

In humans, polysialic acid (polySia) is implicated in the regulation of microglia and macrophage activity through interactions with the opposing immune receptors Siglec-11 and Siglec-16. Due to an inactive pseudogene, Siglec-16 penetrance is below 40% and, because it has no counterpart in non-primates, its functional assessment in animal models is impeded. Previously, we demonstrated that proinflammatory activation of tumor-associated macrophages (TAM) by the polySia-Siglec-16 axis is linked to increased survival of glioblastoma (GB) patients. To gain insights into the underlying mechanisms, we established heterotypic tumor spheroid cultures consisting of polySia-positive GB cells that incorporate peripheral blood-derived monocytes of donors with known SIGLEC16 status and induce their differentiation into TNF-producing macrophages. In the proposed project, this model will be used to analyze the impact of Siglec-16 and polySia on TAM-GB cell interactions by single cell RNA-seq comparisons and by proteomic and glycomic approaches (in collaboration with P9 Büttner). This will be complemented by determining inflammatory TAM activity in relation to GB cell proliferation and apoptosis, as well as immunomodulatory effects of available Siglec-16 antibodies and of soluble polySia. In collaboration with P6 (Münster-Kühnel/Gerardy-Schahn), polySia fractions with defined degrees of polymerization (DP) were generated and we have established the DP required for interactions with specifically the gamma isoform of the chemokine CXCL12. CXCL12 and its receptors are promising therapeutic targets to interfere with glioblastoma progression and chemotactic recruitment of pro-tumorigenic TAM. Therefore, we will analyze CXCL12 isoform expression in GB and study interactions of CXCL12 with polySia in heterotypic tumor spheroids. We will investigate effects of polySia or polySia-degrading endosialidase on CXCL12 applications, determine the lengths of polySia chains presented by GB cells and search for soluble polysialylated proteins in supernatants of the heterotypic spheroid cultures and in liquor samples from GB patients. Furthermore, preliminary data indicate that high levels of 9-O-acetylated GD3 ganglioside (CD60b) are associated with significantly reduced survival of GB patients and that CD60b is highly enriched in GB spheroids. Based on reports that 9-O-acetylation suppresses the proapoptotic activity of GD3, we will collaborate with P7 (Mühlenhoff) to analyze effects of 9-O-acetylated GD3 on apoptosis and proliferation as well as on the susceptibility to the chemotherapeutic agent temozolomide in GB spheroids. Together, the proposed experiments may lead to novel therapeutic options in GB.

在人类中，聚唾液酸（polySia）通过与相反的免疫受体Siglec-11和Siglec-16相互作用参与小胶质细胞和巨噬细胞活性的调节。由于假基因失活，Siglec-16的突变率低于40%，并且由于它在非灵长类动物中没有对应物，因此其在动物模型中的功能评估受到阻碍。之前，我们证明了polySia-Siglec-16轴对肿瘤相关巨噬细胞（TAM）的促炎激活与胶质母细胞瘤（GB）患者的生存率增加有关。为了深入了解潜在的机制，我们建立了由polySia阳性GB细胞组成的异型肿瘤球状体培养物，该细胞包含具有已知SIGLEC 16状态的供体的外周血来源的单核细胞，并诱导其分化为产生TNF的巨噬细胞。在拟议项目中，该模型将用于通过单细胞RNA-seq比较以及蛋白质组学和糖组学方法（与P9 Büttner合作）分析Siglec-16和polySia对TAM-GB细胞相互作用的影响。这将通过确定与GB细胞增殖和凋亡相关的炎性TAM活性以及可用Siglec-16抗体和可溶性polySia的免疫调节作用来补充。与P6（Münster-Kühnel/Gerardy-Schahn）合作，生成了具有定义聚合度（DP）的polySia组分，并且我们已经确定了与趋化因子CXCL 12的γ亚型特异性相互作用所需的DP。CXCL 12及其受体是干扰胶质母细胞瘤进展和促肿瘤性TAM的趋化性募集的有希望的治疗靶点。因此，我们将分析GB中的CXCL 12同种型表达，并研究异型肿瘤球体中CXCL 12与polySia的相互作用。我们将研究polySia或polySia降解内切唾液酸酶对CXCL 12应用的影响，确定GB细胞呈递的polySia链的长度，并在异型球状体培养物的上清液和GB患者的液体样品中寻找可溶性聚唾液酸化蛋白。此外，初步数据表明，高水平的9-O-乙酰化GD 3神经节苷脂（CD 60 b）与GB患者的生存率显著降低相关，并且CD 60 b在GB球状体中高度富集。基于9-O-乙酰化抑制GD 3的促凋亡活性的报道，我们将与P7（Mühlenhoff）合作分析9-O-乙酰化GD 3对细胞凋亡和增殖的影响，以及对GB球状体中化疗药物替莫唑胺的敏感性。总之，拟议的实验可能会导致GB的新的治疗选择。