Low magnification confocal microscopy of tumor angiogenesis.

Blood vessels are critical to normal mammalian development, tissue repair, and growth and treatment of cancer. Mouse research models enable mechanistic studies of blood vessels. We detail how to perfuse mice with fluorescent tomato lectin or the lipophilic fluorophore DiI.

Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis.

Plasminogen activator inhibitor-1 (PAI-1) paradoxically enhances tumor progression and angiogenesis; however, the mechanism supporting this role is not known. Here we provide evidence that PAI-1 is essential to protect endothelial cells (ECs) from FasL-mediated apoptosis. In the absence of host-derived PAI-1, human neuroblastoma cells implanted in PAI-1-deficient mice form smaller and poorly vascularized tumors containing an increased number of apoptotic ECs.

A non-invasive, in vivo technique for monitoring vascular status of glioblastoma during angiogenesis.

The growth of solid tumors dependent on the process of angiogenesis in which growth factors secreted by tumor and stromal cells promote endothelial cell proliferation, migration, and maturation. This process generates a tumor-specific vascular supply and enables small or dormant tumors to grow rapidly with exponential increases in tumor volume. Determination of tumor oxygenation at the microvascular level will provide important insight into tumor growth, angiogenesis, necrosis, and therapeutic response, and will facilitate to develop protocols for studying tumor behavior.

A noninvasive multimodal technique to monitor brain tumor vascularization.

Determination of tumor oxygenation at the microvascular level will provide important insight into tumor growth, angiogenesis, necrosis and therapeutic response and will facilitate to develop protocols for studying tumor behavior. The non-ionizing near infrared spectroscopy (NIRS) technique has the potential to differentiate lesion and hemoglobin dynamics; however, it has a limited spatial resolution. On the other hand, magnetic resonance imaging (MRI) has achieved high spatial resolution with excellent tissue discrimination but is more susceptible to limited ability to monitor the hemoglobin dynamics.

Effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth in nude mice.

Objective: To determine the effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth and associated angiogenesis in the brains of nude mice.

Methods: Human glioblastoma cells (10 U87MG cells) in 1 mul of medium were stereotactically injected during a 20-minute period into the caudate/putamen of nude mice. The mice were intraperitoneally treated daily with Cilengitide or solvent (control) beginning 5 days after tumor injection.

In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma.

In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit.

Longitudinal microPET imaging of brain tumor growth with F-18-labeled RGD peptide.

Purpose: EMD 121974, a potent cyclic RGD peptide inhibitor of alphav-integrins, demonstrated effectiveness in suppressing brain tumor growth in both preclinical models and phases I/II clinical trials. The ability to non-invasively evaluate alphav-integrin expression provides a novel and unique way to better understand brain tumor angiogenesis in relationship to alphav-integrin expression, and allow for direct assessment of anti-integrin treatment efficacy.

Procedures: We developed a F-18-labeled RGD peptide [F-18]FB-RGD and performed serial microPET imaging scans to follow brain tumor growth and angiogenesis as a function of time in an orthotopic U87MG glioblastoma xenograft model in athymic nude mice.

A method to accurately inject tumor cells into the caudate/putamen nuclei of the mouse brain.

Objective: To improve currently used techniques to implant tumor cells into the parenchyma of the mouse brain.

Materials And Methods: The stereotactic injection of 0.5 to 5 microl of indigo carmine over 5 to 40 minutes into the caudate/putamen nuclei of the mouse was done followed by sacrifice and examination of the brain injection site.

Endothelial apoptosis induced by inhibition of integrins alphavbeta3 and alphavbeta5 involves ceramide metabolic pathways.

Matrix ligation of integrins alphavbeta3/alphavbeta5 is critical for endothelial survival and angiogenesis. We have previously shown that ceramide, a proapoptotic lipid second messenger, increases during endothelial anoikis (detachment-induced apoptosis). We now show that RGDfV, an integrin alphavbeta3/alphavbeta5 cyclic function-blocking peptide, increased ceramide and decreased sphingomyelin in human brain microvascular endothelial cells (HBMECs) plated on vitronectin, suggesting that sphingomyelin hydrolysis contributes to RGDfV-induced ceramide increase.

Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.

Unlabelled: The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast.

Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

Background: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism.

Methods: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours.

MicroPET imaging of brain tumor angiogenesis with 18F-labeled PEGylated RGD peptide.

We have previously labeled cyclic RGD peptide c(RGDyK) with fluorine-18 through conjugation labeling via a prosthetic 4-[18F]fluorobenzoyl moiety and applied this [18F]FB-RGD radiotracer for alphav-integrin expression imaging in different preclinical tumor models with good tumor-to-background contrast. However, the unfavorable hepatobiliary excretion and rapid tumor washout rate of this tracer limit its potential clinical applications. The aims of this study were to modify the [18F]FB-RGD tracer by inserting a heterobifunctional poly(ethylene glycol) (PEG, M.

Species-specific urokinase receptor ligands reduce glioma growth and increase survival primarily by an antiangiogenesis mechanism.

Species-specific urokinase receptor (uPAR) ligands with improved pharmacokinetics were generated by site-specific mutagenesis and amino-terminal pegylation. These molecules were used to probe the role of uPAR in brain tumor progression and angiogenesis. The ligands blocked endothelial cell tube formation in Matrigel in a species-specific manner and reduced both baseline and uPA amino-terminal fragment-stimulated cell migration on vitronectin gradients.

18F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis.

Brain tumors are highly angiogenesis dependent. The cell adhesion receptor integrin alpha(v)beta(3) is overexpressed in glioma and activated endothelial cells and plays an important role in brain tumor growth, spread and angiogenesis. Suitably labeled alpha(v)beta(3)-integrin antagonists may therefore be useful for imaging brain tumor associated angiogenesis.

Micro-MRI at 11.7 T of a murine brain tumor model using delayed contrast enhancement.

In vivo imaging methodologies allow for serial measurement of tumor size, circumventing the need for sacrificing mice at given time points. In orthotopically transplanted murine models of brain tumors, cross-section micro-MRI allows for visualization and measurement of the physically inaccessible tumors. To allow for long resident times of a contrast agent in the tumor, intraperitoneal administration was used as a route of injection for contrast-enhanced micro-MRI, and a simple method for relative tumor volume measurements was examined.

Determination of drug synergism between the tyrosine kinase inhibitors NSC 680410 (adaphostin) and/or STI571 (imatinib mesylate, Gleevec) with cytotoxic drugs against human leukemia cell lines.

The primary growth factor receptors involved in angiogenesis and lymphomagenesis can be grouped into the vascular endothelial growth factor (VEGF) receptors and related families. Inhibition of VEGF and other growth factors, including c-Abl, c-Kit, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and insulin-like growth factor (IGF), or their receptors containing tyrosine kinase domains by antiangiogenesis drugs disrupts cell survival signal transduction pathways and may contribute to the proapoptotic pathways in malignant cells. However, clinical trials suggest that signal transduction inhibitors have considerable antitumor activity when used as single agents only for a short time, most likely due to the development of drug resistance by the host or by the tumor cells.

Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice.

In vivo bioluminescence imaging is becoming increasingly popular. Quantification of bioluminescence signals requires knowledge of the variability and reproducibility of this technique. The objective of this study was to analyze the time course of luminescent signal emitted from firefly luciferase-expressing tumors in two locations, following luciferin injection and at different times after tumor cell implantation.

Increased plasminogen activator inhibitor-1 in keloid fibroblasts may account for their elevated collagen accumulation in fibrin gel cultures.

Proteolytic degradation of the provisional fibrin matrix and subsequent substitution by fibroblast-produced collagen are essential features of injury repair. Immunohistochemical studies revealed that although dermal fibroblasts of normal scars and keloids expressed both urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), keloid fibroblasts had a much higher PAI-1 expression. In long-term three-dimensional fibrin gel cultures (the in vitro fibroplasia model), normal fibroblasts expressed moderate and modulated activity levels of uPA and PAI-1.

In vitro and in vivo evaluations of the tyrosine kinase inhibitor NSC 680410 against human leukemia and glioblastoma cell lines.

Purpose: NSC 680410, the novel adamantyl ester of AG957, an inhibitor of the p210bcr/abl tyrosine kinase (CML, Ph(+)) and possibly other kinases, was tested for antitumor activity in ten human leukemia and human glioblastoma cell lines.

Methods: CEM/0, seven ara-C- and/or ASNase-resistant clones, Jurkat/0, the myelomonocytic line U937 and U87 MG glioblastoma cell lines were used for these studies. The drug-resistant leukemic clones lack p53, express bcl-2 and VEGF-R1, and thus are refractory to apoptosis.

Soluble interleukin 6 receptor (sIL-6R) mediates colonic tumor cell adherence to the vascular endothelium: a mechanism for metastatic initiation?

The mechanisms by which surgery increases metastatic proliferation remain poorly characterized, although endotoxin and immunocytes play a role. Recent evidence suggests that endothelial adherence of tumor cells may be important in the formation of metastases. Soluble receptors of interleukin-6 (sIL-6R) shed by activated neutrophils exert IL-6 effects on endothelial cells, which are unresponsive under normal circumstances.

alpha v-Integrin antagonist EMD 121974 induces apoptosis in brain tumor cells growing on vitronectin and tenascin.

Orthotopic brain tumor growth is inhibited in athymic mice by the daily systemic administration of the alpha v-integrin antagonist EMD 121974. This compound, a cyclic RGD-penta-peptide, is a potent inhibitor of angiogenesis, which induces apoptosis of growing endothelial cells through inhibition of their alpha v-integrin interaction with the matrix proteins vitronectin and tenascin. Here we show that EMD 121974 also induces apoptosis in the alpha v-integrin-expressing tumor cell lines U87 MG and DAOY by detaching them from vitronectin and tenascin, matrix proteins known to be essential for brain tumor growth and invasion.

Plasminogen activator inhibitor-1 promotes angiogenesis by stimulating endothelial cell migration toward fibronectin.

Increased expression of plasminogen activator inhibitor-1 (PAI-1) in cancer patients is associated with unfavorable outcome, and the reason for this paradox has been poorly understood. We have previously reported elevated levels of PAI-1 in primary tumors of advanced neuroblastomas (Y. Sugiura et al.

Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

Background: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively.

Preferential susceptibility of brain tumors to the antiangiogenic effects of an alpha(v) integrin antagonist.

Objective: Brain tumors are highly angiogenic, and their growth and spread depend on the generation of new blood vessels. We examined the effect of the cyclic peptide antagonist pentapeptide EMD 121974, an antiangiogenic agent, on orthotopic and heterotopic brain tumor growth.

Methods: The human brain tumor cell lines DAOY (medulloblastoma) and U87 MG (glioblastoma) were injected into either the forebrain (orthotopic) or the subcutis (heterotopic) of nude mice, and daily systemic treatment with the active peptide was initiated after tumors were established.

The plasminogen-plasminogen activator (PA) system in neuroblastoma: role of PA inhibitor-1 in metastasis.

Proteases of the plasminogen-plasminogen activator (PA) system play an important role in cancer metastasis. We have examined the expression of these proteases and their cell surface receptors and inhibitors in neuroblastoma, a tumor that originates in cells of the neural crest and is the second most common solid tumor in children. This analysis was performed in seven established human cell lines and 20 primary tumor specimens.