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

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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.