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Abstract

The mucin-like sialoglycoprotein podoplanin (PDPN) is widely expressed throughout the human and rodent body. Although numerous studies have revealed its essential function in development, especially of the lymphatic system, the lungs and heart, the overall picture of its physiologic function is still incomplete. Emerging evidence of the past decade has associated PDPN de novo or overexpression with numerous cancer entities including glioblastoma, and in particular with the invasive behavior of tumor cells. As the infiltrative growth of tumor cells is one major challenge in glioblastoma therapy, the identification of novel candidates in tumor cell migration remains an essential pre-requisite for the development of new and effective therapeutic means. However, the postulated pro-tumorigenic and pro-invasive function of PDPN in glioblastoma has never been validated in vivo. Moreover, the underlying mechanism of a potential malignant effect of PDPN has not been addressed. Thus, the aim of this study was to close this gap of knowledge by the combination of correlative and functional assays. Descriptive in vivo approaches involving patient-derived xenografts were primarily taken to confirm the previous correlations of PDPN expression and malignant progression and to establish a model that enables the investigation of underlying mechanisms. For the functional validation of the hypothesis that PDPN is a major driver of glioblastoma progression and especially invasion, the gene was deleted by the novel CRISPR/Cas9 technology. Xenotransplantations of control and knockout cells indicated the dispensability of PDPN for glioblastoma growth and progression. The reliable analysis of the postulated pro-invasive function of PDPN required the optimization of a three-dimensional invasion assay based on organotypic brain slice cultures. The usage of adult murine brain slices and a red emitting fluorescent membrane dye significantly improved the assay quality. The application of this advanced technique identified PDPN as a non-rate limiting component in glioblastoma cell invasion. These data and the detailed analysis of further malignant features including proliferation, apoptosis and angiogenesis have rebutted the previous assumption of a tumor promoting effect of PDPN. Despite the dispensability of PDPN for tumor development and tumor cell invasion, the obtained results suggest PDPN as a marker for malignant glioblastoma cells. In conclusion, this study represents an important contribution in the process of preclinical drug development, as the results object the frequently suggested development of a PDPN blocking therapeutic agent. Instead, this work suggests PDPN as a marker for prognosis or targeted delivery of cytotoxic compounds into glioblastoma tumor cells.