Using MRI techniques, we show here that normalization of tumor vessels in recurrent glioblastoma patients by daily administration of AZD2171-an oral tyrosine kinase inhibitor of VEGF receptors-has rapid onset, is prolonged but reversible, and has the significant clinical benefit of alleviating edema. Reversal of normalization began by 28 days, though some features persisted for as long as four months. Basic FGF, SDF1alpha, and viable circulating endothelial cells (CECs) increased when tumors escaped treatment, and circulating progenitor cells (CPCs) increased when tumors progressed after drug interruption. Our study provides insight into different mechanisms of action of this class of drugs in recurrent glioblastoma patients and suggests that the timing of combination therapy may be critical for optimizing activity against this tumor.

The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors.VEGF has a crucial role in physiological and pathological angiogenesis 1-3 . Although VEGF blockade, alone or in combination with cytotoxic therapies, is being tested in a number of

A B S T R A C T PurposeGlioblastoma is an incurable solid tumor characterized by increased expression of vascular endothelial growth factor (VEGF). We performed a phase II study of cediranib in patients with recurrent glioblastoma. MethodsCediranib, an oral pan-VEGF receptor tyrosine kinase inhibitor, was administered (45 mg/d) until progression or unacceptable toxicity to patients with recurrent glioblastoma. The primary end point was the proportion of patients alive and progression free at 6 months (APF6). We performed magnetic resonance imaging (MRI) and plasma and urinary biomarker evaluations at multiple time points. ResultsThirty-one patients with recurrent glioblastoma were accrued. APF6 after cediranib was 25.8%. Radiographic partial responses were observed by MRI in 17 (56.7%) of 30 evaluable patients using three-dimensional measurements and in eight (27%) of 30 evaluable patients using twodimensional measurements. For the 15 patients who entered the study taking corticosteroids, the dose was reduced (n ϭ 10) or discontinued (n ϭ 5). Toxicities were manageable. Grade 3/4 toxicities included hypertension (four of 31; 12.9%); diarrhea (two of 31; 6.4%); and fatigue (six of 31; 19.4%). Fifteen (48.4%) of 31 patients required at least one dose reduction and 15 patients required temporary drug interruptions due to toxicity. Drug interruptions were not associated with outcome. Changes in plasma placental growth factor, basic fibroblast growth factor, matrix metalloproteinase (MMP) -2, soluble VEGF receptor 1, stromal cell-derived factor-1␣, and soluble Tek/Tie2 receptor and in urinary MMP-9/neutrophil gelatinase-associated lipocalin activity after cediranib were associated with radiographic response or survival. ConclusionCediranib monotherapy for recurrent glioblastoma is associated with encouraging proportions of radiographic response, 6-month progression-free survival, and a steroid-sparing effect with manageable toxicity. We identified early changes in circulating molecules as potential biomarkers of response to cediranib. The efficacy of cediranib and the predictive value of these candidate biomarkers will be explored in prospective trials.

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Tissue engineering requires formation of a de novo stable vascular network. Because of their ability to proliferate, differentiate into endothelial cells, and form new vessels, blood-derived endothelial progenitor cells (EPCs) are attractive source of cells for use in engineering blood vessels. However, the durability and function of EPC-derived vessels implanted in vivo are unclear. To this end, we directly compared formation and functions of tissue-engineered blood vessels generated by peripheral blood-and umbilical cord blood-derived EPCs in a model of in vivo vasculogenesis. We found that adult peripheral blood EPCs form blood vessels that are unstable and regress within 3 weeks. In contrast, umbilical cord blood EPCs form normalfunctioning blood vessels that last for more than 4 months. These vessels exhibit normal blood flow, perm-selectivity to macromolecules, and induction of leukocyte-endothelial interactions in response to cytokine activation similar to normal vessels.

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrowderived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.Intravital microscopy has provided unprecedented molecular, cellular, anatomical and functional insight into tumor biology and response to treatment 1 . This technique captures fluorescence from molecules that are injected into a host or expressed by cells 2,3 . Additionally, intrinsic signals such as second harmonic generation (SHG) emanating from collagen can be imaged using multiphoton microscopy 4,5 . Traditional fluorophores are prone to photobleaching, compromising the ability to image the same region repeatedly, and have relatively narrow excitation and broad emission spectra. Also, several excitation wavelengths may be required to excite all fluorophores and intrinsic signals, and overlapping emissions may obscure the delineation between multiple probes. Quantum dots, colloidal semiconductor nanocrystals 6 , have the potential to overcome these limitations: they are photostable, tunable to a desired narrow emission spectrum, relatively insensitive to the wavelength of excitation Correspondence should be addressed to R.K.J. (E-mail: jain@steele.mgh.harvard.edu). Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. light, and are especially bright fluorophores 7 . Recent studies exploit these optical properties for imaging of cells 8 or whole tumors 9 . The ability of quantum dots to show crucial information at the length scale between these two extremes has yet to be established 10 . Here, we present studies that highlight the synergy of quantum dots and multiphoton intravital microscopy for tumor pathophysiology studies: differentiating tumor vessels from both perivascular cells and matrix, assaying the ability of microparticles to access the tumor, and monitoring the trafficking of precursor cells. NIH Public Access RESULTS Customizing quantum dot emissionBecause quantum dot emissions are tunable by both size and chemical composition 6 , we prepared ...

Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart disease and cancer. However, data interpretation has often been difficult because of multiple definitions, methods and protocols used to evaluate and count these cells by different laboratories. Here, we propose a cytometry protocol for phenotypic identification and enumeration of CECs and CPCs in human blood using four surface markers: CD31, CD34, CD133 and CD45. This method allows further phenotypic analyses to explore the biology of these cells. In addition, it offers a platform for longitudinal studies of these cells in patients with different pathologies. The protocol is relatively simple, inexpensive and can be adapted for multiple flow cytometer types or software. The procedure should take 2-2.5 h, and is expected to detect 0.1-6.0% viable CECs and 0.01-0.20% CPCs within blood mononuclear cell population.

The specialized microenvironment or niche where stem cells reside provides regulatory input governing stem cell function. We tested the hypothesis that targeting the niche might improve stem cell-based therapies using three mouse models that are relevant to clinical uses of hematopoietic stem (HS) cells. We and others previously identified the osteoblast as a component of the adult HS cell niche and established that activation of the parathyroid hormone (PTH) receptor on osteoblasts increases stem cell number. Here we show that pharmacologic use of PTH increases the number of HS cells mobilized into the peripheral blood for stem cell harvests, protects stem cells from repeated exposure to cytotoxic chemotherapy and expands stem cells in transplant recipients. These data provide evidence that the niche may be an attractive target for drug-based stem cell therapeutics.