Invasive glioma cells remodel their guiding blood vessel: in vivo microscopy reveals strategies for effective tumour dissemination

Dissemination of cancer cells determines the dim prognosis of many malignancies. Malignant gliomas are characterized by highly invasive cells that extensively infiltrate the brain along fibre tracts and blood vessels, compromising treatment options. Up to today it has been difficult to image infiltrating glioma cells deep in the living brain over time in high resolution, which would allow better understanding of the pathophysiology, molecular determinants, and effectiveness of therapeutics. Here we propose a novel methodology by combining in vivo multiphoton laser scanning microsopy (MPLSM) with the chronic cranial window model, which allowed imaging of GL261 glioma cells stably transfected with dsRed2. Taking advantage of this method, the time course of glioma cell invasion into normal brain of a living animal could be tracked over minutes, hours, and days, up to a depth of 500µm and millimeters away from the main tumour. Perfused vessels were highlighted by angiograms, and endothelial cells by expression of GFP under the Tie2 promotor. Quantification of 59 invasive glioma cells/clusters demonstrated increased velocities when the abluminal site of a vessel was exploited for movement within normal brain (mean velocity, 34.1±4.0 vs. 9.4±4.2µm/24h). The majority of invading glioma cells moved away from the tumor (84%), stretching out in a strict perivascular location (78%). Cells moving perivascularly in the brain parenchyma were frequently found next to (a) multiple capillary structures where microvessels run parallel to each other, (b) capillary loops organizing to glomeruloid-like bodies, and (c) dilated capillaries. Dynamic MPLSM over 48 hours revealed that single invasive glioma cells induced intussusceptive microvascular growth and capillary loop formation, specifically at the microvascular site they had contact to. Close glioma cell/endothelial cell contacts where typical in invading cells. In conclusion, our study demonstrates the importance of pre-existing brain microvessels for glioma invasion, but also that single invasive glioma cells are capable of remodeling the host vasculature, improving the conditions for further cell invasion and tumour growth. The novel in vivo method can help to decipher the effectiveness of various therapies on the single steps of glioma cell invasion.