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

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，参与调节小胶质细胞和巨噬细胞的活动。由于不活跃的假基因，Siglec-16的外显率低于40%，而且由于它在非灵长类动物中没有对应的基因，它在动物模型中的功能评估受到阻碍。在此之前，我们证明了通过PolySia-Siglec-16轴激活肿瘤相关巨噬细胞()的致炎活性与提高胶质母细胞瘤(GB)患者的存活率有关。为了深入了解其潜在的机制，我们建立了由PolySia阳性的GB细胞组成的异型肿瘤球体培养物，这些细胞融合了已知SIGLEC16状态的捐赠者的外周血单核细胞，并诱导他们分化为产生肿瘤坏死因子的巨噬细胞。在拟议的项目中，该模型将用于通过单细胞RNA序列比较以及蛋白质组和糖学方法(与P9 Büttner合作)分析Siglec-16和PolySia对-GB细胞相互作用的影响。此外，还将测定与GB细胞增殖和凋亡相关的炎症活性，以及可用的Siglec-16抗体和可溶性PolySia的免疫调节作用。在与P6(Münster-Kühnel/Gerardy-Schahn)的合作下，产生了定义聚合度(DP)的PolySia组分，我们已经确定了与趋化因子CXCL12的伽马亚型相互作用所需的聚合度。CXCL12及其受体是干扰胶质母细胞瘤进展和促癌基因趋化募集的有效治疗靶点。因此，我们将分析CXCL12亚型在GB中的表达，并研究CXCL12与PolySia在异型肿瘤球体中的相互作用。我们将研究PolySia或PolySia降解内唾液酸酶对CXCL12应用的影响，确定GB细胞呈现的PolySIA链的长度，并在异型球状细胞培养上清液和GB患者的酒样中寻找可溶性多唾液酸化蛋白。此外，初步数据表明，高水平的9-O-乙酰化GD3神经节苷脂(CD60b)与GB患者的存活率显著降低有关，并且CD60b在GB球体中高度丰富。基于9-O-乙酰化抑制GD3促凋亡活性的报道，我们将与P7(Mühlenhoff)合作分析9-O-乙酰化GD3对细胞凋亡和增殖的影响以及对化疗药物替莫唑胺的敏感性。总之，拟议的实验可能会在英国带来新的治疗选择。

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.