NEOGENIN KNOCKOUT STOPS THE INVASION AND DISSEMINATION OF DIFFUSE INTRINSIC PONTINE GLIOMAS IN VIVO

Abstract INTRODUCTION: Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating high-grade glioma localized in the brainstem that occurs almost exclusively in children. The significant mortality rates of DIPG stem largely from its capacity to invade adjacent normal brain. We have previously published studies identifying the axon guidance factor, neogenin as a key driver regulating brain tumor cell invasion in medulloblastoma, glioblastoma and DIPG in vitro. Here we expand this work with an in vivo model to validate neogenin as promising inducer of DIPG invasion. METHODS: CRISPR-Cas9 system was used to knock-out the neogenin gene in DIPG cells. After validation of the knockout (KO) in vitro by RTqPCR, Western blot, and immunofluroresence, the cells were orthotopically implanted in the pons of nude mice (n=8/group) with a stereotactic frame. Tumor growth and dissemination were monitored using the In Vivo Imaging System (IVIS). Then, brains and spinal cords were collected, and H&E staining was performed. RESULTS:In vitro studies confirmed the KO of neogenin in DIPG cells. In vivo studies revealed that the neogenin total KO group had no progression of the tumor without any dissemination after 4-6 weeks, in marked contrast to the WT group, which exhibited steady growth and widespread dissemination into the spine. Immunohistochemistry confirmed the IVIS results. CONCLUSION: Expanding on previous work, this data suggests that neogenin is a major contributor of DIPG cell invasion and dissemination. The blockade of neogenin in DIPG cells demonstrates the potential utility of neogenin as potential therapeutic target, capable of reducing tumor growth and stopping tumor cell dissemination.