Etiology and Pathogenesis > Invasion | Overall Management > Methodology

Meeting Abstract

A novel, orthotopic model of glioblastoma growth and invasion for intravital microscopy

Emmanuelle Di Tomaso, Edward Brown, Yotaro Izumi, Rakesh K. Jain

Massachusetts General Hospital, Boston, MA; Keio University School of Medicine, Keio, Japan

Diffusely infiltrating astrocytomas are the most frequent intracranial neoplasms and account for more than 60% of all primary brain tumors. 
They show a tendency for progression toward a more malignant phenotype and are associated with sequential acquisition of genetic alterations. 
The most frequent (50-60%) of all astrocytic tumors, they are composed of poorly differentiated neoplastic astrocytes with areas of vascular proliferation and necrosis. 
Glioblastoma cells are notorious for their rapid and extensive invasion of the neighboring structures using the perivascular space as one route of infiltration without, however, intravasating into the vessels. 
Extreme invasiveness is the hallmark of these tumors rendering them particularly difficult to remove surgically. 
Average survival time is approximately eighteen months after symptom onset, and less than 3 months in about 50% of cases. 
Unfortunately, research into the mechanisms of glioblastoma invasion is hindered due to a lack of relevant, orthotopic and optically-accessible animal models. 
To overcome this problem, we have developed a novel in vivo model in which glioblastoma growth and invasion can be visualized within the brain of SCID mice to depths of 700 microns using multiphoton microscopy. 
Seven days after surgical implantation of a glass coverslip to replace a portion of the cranium (1), the coverslip is removed and the tumor is implanted as follows: the human glioma tumor source HGL21 or U87-VEGF-gfp (a transfected variant of U87 that expresses GFP under control of the VEGF promoter) is cut in small chunks less than 1 mm diameter. 
A 23 gauge needle is then used to create a 1mm deep space in parenchyma of the left hemisphere of the brain. 
A single chunk of tumor is then implanted in this pocket and a new coverslip is placed on the cranial window. 
This procedure results in ~80% tumor-take. 
Intravital microscopy is used to follow the growth, angiogenic profile and cellular organization of the gliomas as they grow and emanate from the implantation site. 
Multiphoton laser scanning microscopy allows us to image the entire tumor volume and measure anatomical, molecular, cellular, and physiological parameters in vivo with three-dimensional resolution and with greatly improved depth penetration. 
An invasive tumor front and individual migrating cells are easily and consistently identified, providing a reliable tool for studying potential changes in angiogenic phenotypes. 
We illustrate the relevance and utility of this model in identifying and characterizing contra-lateral metastases produced by cell migration from the primary site. 
This approach should prove invaluable for further characterizing the growth, invasion and angiogenic activity of brain tumors. 
1- Yuan, F., et al, Vascular permeability and microcir culation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows. Cancer Research, 1994. 54: p. 4564-4568

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