Imaging Glioma Cell Invasion in Vivo Reveals Mechanisms of Dissemination and Peritumoral Angiogenesis

Infiltration of cancer cells into normal tissue is a hallmark of malignant gliomas and compromises treatment options. A lack of appropriate models limits the study of this invasion in vivo, which makes it difficult to fully understand its anatomy and the role of dynamic interactions with structures of the normal brain. We developed a novel methodology by utilizing multiphoton laser scanning microscopy (MPLSM) to image the movement of glioma cells deep within the normal brain of live mice in real time. This allowed us to track the invasion of individual RFP-expressing GL261 cells in relation to perfused vasculature or GFP-labeled endothelial cells repetitively over days, up to a depth of 0.5 mm. Glioma cells moved faster and more efficiently when the abluminal site of a blood vessel was utilized for invasion. Cells that invaded perivascularly were frequently found next to (a) multiple capillary structures where microvessels run parallel to each other, (b) capillary loops or glomeruloid-like bodies, and (c) dilated capillaries. Dynamic MPLSM for more than 48 h revealed that single invasive glioma cells induced intussusceptive microvascular growth and capillary loop formation, specifically at the microvascular site with which they had contact. As the main tumor grew by cooption of existing brain vessels, these peritumoral vascular changes may create a beneficial environment for glioma growth. In conclusion, our study revealed new mechanisms of peritumoral angiogenesis and invasion in gliomas, providing an explanation for their interdependence.