Purpose Recent clinical trials of antivascular endothelial growth factor (VEGF) agents for glioblastoma showed promising progression-free and overall survival rates. However, available clinical imaging does not separate antitumor effects from antipermeability effects of these agents. Thus although anti-VEGF agents may decrease tumor contrast-enhancement, vascularity, and edema, the mechanisms leading to improved survival in patients remain incompletely understood. Our goal was to determine whether alleviation of edema by anti-VEGF agents alone could increase survival in mice. Methods We treated mice bearing three different orthotopic models of glioblastoma with a VEGF-targeted kinase inhibitor, cediranib. Using intravital microscopy, molecular techniques, and magnetic resonance imaging (MRI), we measured survival, tumor growth, edema, vascular morphology and function, cancer cell apoptosis and proliferation, and circulating angiogenic biomarkers. Results We show by intravital microscopy that cediranib significantly decreased tumor vessel permeability and diameter. Moreover, cediranib treatment induced normalization of perivascular cell coverage and thinning of the basement membrane, as mirrored by an increase in plasma collagen IV. These rapid changes in tumor vascular morphology and function led to edema alleviation—as measured by MRI and by dry/wet weight measurement of water content—but did not affect tumor growth. By immunohistochemistry, we found a transient decrease in macrophage infiltration and significant but minor changes in tumor cell proliferation and apoptosis. Systemically, cediranib increased plasma VEGF and placenta growth factor levels, and the number of circulating CXCR4+CD45+ cells. However, by controlling edema, cediranib significantly increased survival of mice in the face of persistent tumor growth. Conclusion Anti-VEGF agents may be able to improve survival of patients with glioblastoma, even without inhibiting tumor growth.

Here we integrated multiphoton laser scanning microscopy and the registration of second harmonic generation images of collagen fibers to overcome difficulties in tracking stromal cell-matrix interactions for several days in live mice. We show that the matrix-modifying hormone relaxin increased tumor-associated fibroblast (TAF) interaction with collagen fibers by stimulating β 1 -integrin activity, which is necessary for fiber remodeling by matrix metalloproteinases.Our current understanding of extracellular matrix remodeling derives from in vitro experiments 1,2 , which are difficult to interpret and relate to in vivo physiology. Multiphoton laser scanning microscopy and second harmonic generation (SHG) of fibrillar collagen allow visualization of the matrix of normal and tumor tissues in vivo 3 . Using these technologies, it is possible to study the real-time movement of cells through the collagen network on time scales of minutes to hours 4,5 . However, the slow rate of extracellular matrix remodeling makes it difficult to monitor matrix reorganization by stromal cells.To track stromal cell collagen fiber interactions in vivo, we grew tumors (human sarcoma HSTS26T) in transparent dorsal skinfold chambers of immunodeficient mice expressing GFP under the control of the Vegfa promoter (VEGF-GFP mice). The transparent tumor chamber facilitated the tracking of the same cells and fibers for several days (Supplementary Methods online). Also, previous data in VEGF-GFP mice 6 and our immunostaining results suggest that peritumor GFP-positive cells are tumor-associated fibroblasts (TAFs) ( Supplementary Fig. 1 online).To induce collagen remodeling in a reliable fashion, we treated tumors with the small hormone relaxin, which causes matrix remodeling and increases tumor invasion and progression 7 . In vitro, relaxin enhanced the invasion of fibroblasts through increased collagen I degradation but did not affect the HSTS26T tumor cells ( Supplementary Fig. 2 Temporal changes in tumors and the inability to place mice under the microscope in the same orientation between imaging sessions necessitated the development of a registration method to align image sequences. The SHG signal arising from fibrillar collagen provided a convenient registration landmark because of the relative stability and the wide distribution of collagen fibers in comparison with other potential landmarks, such as blood vessels or exogenous fluorescent beads. Blood vessels within tumors are porous to injected tracer particles, can have time-dependent perfusion fluctuations and are more sparsely distributed than the extracellular matrix, which forms a distinct network distributed throughout the tumor. Likewise, fluorescent beads pose the problem of potentially being unevenly distributed in the region of interest or being unstable because of photobleaching or cellular phagocytosis. We used an intensity-based registration approach (Turboreg) 8 to align subsequent imaged volumes with those at the initial time point (Supplementary Methods).After ima...