CNSC-32. SIMULTANEOUS INVESTIGATION OF TUMOR CELL BEHAVIOR AND CONNECTIVITY WITH CORRELATIVE IN VIVO TWO-PHOTON AND EX VIVO EXPANSION MICROSCOPY

Abstract

Abstract Glioblastomas are the most aggressive primary brain tumors, characterized by whole-brain colonization, high therapeutic resistance, and inevitable recurrence. It has previously been shown that glioblastoma cells can form functionally gap-junction connected tumor cell networks with other tumor cells and/or astrocytes, which play a crucial role in tumor progression and treatment response. Furthermore, there is increasing evidence that synaptic neuron-glioma networks promote tumor growth. However, methodologies to investigate the direct link between synaptic, astrocytic and tumor cell connectivity together with tumor cell behavior including invasion, proliferation and therapeutic resistance are yet lacking. We aimed to characterize neuron-tumor and tumor-tumor/astrocyte cell interactions on a single cell level by using correlative in vivo two-photon and ex vivo expansion microscopy with near synaptic-resolution. We studied glioblastoma cells with intravital two-photon microscopy using patient-derived glioblastoma mouse models and the fluorescent functional imaging dye Sulforhodamine 101 allowing to investigate intratumoral and astrocytic connectivity over an extended period up to weeks. Subsequently, these functional tumor cell subpopulations could be directly correlated ex vivo to identify neuron-glioma synaptic connectivity. We utilized machine learning based segmentation and a semi-automated analysis pipeline to explore the connection of different glioblastoma morphological and behavioral subtypes to direct and indirect neuronal input in multiple xenograft models. With this new correlative method, we were able to simultaneously evaluate tumor cell behavior, synaptic and tumor-tumor/astrocyte connections on a single cell level. In addition, we demonstrated that this pipeline can be used for patient samples, revealing its clinical-translational potential for cancer neuroscience. In summary, we have established a new correlative technology that interlinks two key aspects of brain cancer neuroscience: cancer cell behavior and brain tumor network connectivity. This methodology promises a more thorough understanding of glioblastoma biology, facilitating the discovery of cancer biology, including the role of brain tumor networks in therapeutic resistance.