Abstract
Glioblastoma is the most common primary brain tumor with a poor prognosis due to its aggressive invasiveness, inevitable recurrence, and therapy resistance. One hallmark of the disease is its whole brain dissemination via preexisting brain structures like blood vessels or white matter tracts. While intravital glioblastoma imaging has been limited to the superficial cortical layers, we developed a method of three photon microscopy allowing the investigation of glioblastoma invasion and colonization in the white matter up to a depth of 1.2 mm over extended time periods. Our method, combining adaptive optics 3P imaging, customized deep learning- based denoising and machine learning segmentation, enables longitudinal volumetric multicolor imaging, being able to distinguish GBM cell signal from myelin structures as well as blood vessel signal. Thus, GBM cells and their interactions with and influence on the microenvironment within the white matter can be investigated longitudinally. We found that compared to cortical invasion, a majority of GBM cells in the corpus callosum are aligned with myelinated axonal tracts, showing the influence of the tumor microenvironment on GBM cell directionality. Also, we found that most GBM cells enter the corpus callosum via a perivascular route. We identified two blood vessel-related mechanisms that allow invasion orthogonal to the myelinated axonal tract direction. We discovered that white matter tract directionality did not change significantly in the early stages of GBM colonization. This might explain limitations of modern clinical imaging to depict white matter glioma infiltration. However, we found that the shape of THG discontinuities differs between GBM cells and other resident cells, which could potentially serve as an imaging biomarker. To summarize, this tailored three-photon microscopy methodology allows unprecedented intravital investigation of GBM invasion in the gray and white matter, enabling the discovery of novel (sub-)cellular mechanisms in cancer and its microenvironment deep in the brain.