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Supplementary Data from Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging

CCR Translation for the Article from Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials.Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab.Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12.Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)

Purpose: Multiple endocrine neoplasia type 1 (MEN1) is defined clinically by the combined occurrence of multiple tumors, typically of the parathyroid glands, pancreatic islet cells, and anterior pituitary gland. A mouse model with a heterozygous deletion of the Men1 gene recapitulates the tumorigenesis of MEN1. We wished to determine the role of vascular endothelial growth factor (VEGF)-A in the vascularization and growth of MEN1-associated tumors, with an emphasis on pituitary adenomas.Experimental Design: To investigate whether tumor growth in Men1+/− mice is mediated by VEGF-A dependent angiogenesis, we carried out a monotherapy with the anti–VEGF-A monoclonal antibody (mAb) G6-31. We evaluated tumor growth by magnetic resonance imaging and assessed vascular density in tissue sections. We also measured hormone levels in the serum.Results: During the treatment with mAb G6-31, a significant inhibition of the pituitary adenoma growth was observed, leading to an increased mean tumor doubling-free survival compared with mice treated with a control antibody. Similarly, the growth of s.c. pituitary adenoma transplants was effectively inhibited by administration of anti–VEGF-A mAb. Serum prolactin was lowered by mAb G6-31 treatment but not by control antibody, potentially providing a new therapeutic approach for treating the hormonal excess in MEN1 patients. Additionally, the vascular density in pancreatic islet tumors was significantly reduced by the treatment.Conclusions: These results suggest that VEGF-A blockade may represent a nonsurgical treatment for benign tumors of the endocrine system.

Supplementary Figures S1-S2 from Blocking Vascular Endothelial Growth Factor-A Inhibits the Growth of Pituitary Adenomas and Lowers Serum Prolactin Level in a Mouse Model of Multiple Endocrine Neoplasia Type 1

Aging is accompanied by altered intercellular communication, deregulated metabolic function, and inflammation. Interventions that restore a youthful state delay or reverse these processes, prompting the search for systemic regulators of metabolic and immune homeostasis. Here we identify MANF, a secreted stress-response protein with immune modulatory properties, as an evolutionarily conserved regulator of systemic and in particular liver metabolic homeostasis. We show that MANF levels decline with age in flies, mice and humans, and MANF overexpression extends lifespan in flies. MANF deficient flies exhibit enhanced inflammation and shorter lifespans, and MANF heterozygous mice exhibit inflammatory phenotypes in various tissues, as well as progressive liver damage, fibrosis, and steatosis. We show that immune cell-derived MANF protects against liver inflammation and fibrosis, while hepatocyte-derived MANF prevents hepatosteatosis. Liver rejuvenation by heterochronic parabiosis in mice further depends on MANF, while MANF supplementation ameliorates several hallmarks of liver aging, prevents hepatosteatosis induced by diet, and improves age-related metabolic dysfunction. Our findings identify MANF as a systemic regulator of homeostasis in young animals, suggesting a therapeutic application for MANF in age-related metabolic diseases.

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin., Highlights • A humanized bispecific antibody that selectively activates FGFR1/βKlotho complex was generated. • Anti-FGFR1/βKlotho agonist antibody induced sustained thermogenesis in brown fat and induced weight loss. • Anti-FGFR1/βKlotho agonist antibody improved insulin sensitivity even before the onset of weight loss.

The vascular endothelial growth factor (VEGF) family of cytokines are important regulators of angiogenesis that have emerged as important targets for the treatment of obesity. While serum VEGF levels rise during obesity, recent studies using genetic models provide conflicting evidence as to whether VEGF prevents or accelerates metabolic dysfunction during obesity. In the current study, we sought to identify the effects of VEGF-A neutralization on parameters of glucose metabolism and insulin action in a dietary mouse model of obesity. Within only 72 h of administration of the VEGF-A–neutralizing monoclonal antibody B.20-4.1, we observed almost complete reversal of high-fat diet–induced insulin resistance principally due to improved insulin sensitivity in the liver and in adipose tissue. These effects were independent of changes in whole-body adiposity or insulin signaling. These findings show an important and unexpected role for VEGF in liver insulin resistance, opening up a potentially novel therapeutic avenue for obesity-related metabolic disease.

Oxidation of LDL (oxLDL) is a crucial step in the development of cardiovascular disease. Treatment with antibodies directed against oxLDL can reduce atherosclerosis in rodent models through unknown mechanisms. We demonstrate that through a novel mechanism of immune complex formation and Fc-γ receptor (FcγR) engagement, antibodies targeting oxLDL (MLDL1278a) are anti-inflammatory on innate immune cells via modulation of Syk, p38 MAPK phosphorylation and NFκB activity. Subsequent administration of MLDL1278a in diet-induced obese (DIO) nonhuman primates (NHP) resulted in a significant decrease in pro-inflammatory cytokines and improved overall immune cell function. Importantly, MLDL1278a treatment improved insulin sensitivity independent of body weight change. This study demonstrates a novel mechanism by which an anti-oxLDL antibody improves immune function and insulin sensitivity independent of internalization of oxLDL. This identifies MLDL1278a as a potential therapy for reducing vascular inflammation in diabetic conditions.

The connection between inflammation, autoimmunity, and atherosclerosis is long established. In this issue of Immunity, Lim et al. (2014) demonstrate that oxidized low-density lipoprotein is one of the key environmental factors driving the development of inflammatory T helper 17 cells in atherosclerosis.

Despite the availability of new targeted therapies, ductal pancreatic adenocarcinoma continues to carry a poor prognosis. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM)6 has been reported as a potential biomarker and therapy target for this malignancy. We have evaluated CEACAM6 as a potential therapy target, using an antibody-drug conjugate (ADC). Expression of CEACAM6 in pancreatic adenocarcinomas was determined using immunohistochemistry on tissue microarrays. The expression pattern in granulocytes and granulocytic precursors was measured by flow cytometry. Murine xenograft and non-human primate models served to evaluate efficacy and safety, respectively. Robust expression of CEACAM6 was found in > 90% of invasive pancreatic adenocarcinomas as well as in intraepithelial neoplastic lesions. In the granulocytic lineage, CEACAM6 was expressed at all stages of granulocytic maturation except for the early lineage-committed precursor cell. The anti-CEACAM6 ADC showed efficacy against established CEACAM6-expressing tumours. In non-human primates, antigen-dependent toxicity of the ADC consisted of dose-dependent and reversible depletion of granulocytes and their precursors. This was associated with preferential and rapid localization of the antibody in bone marrow, as determined by sequential in vivo PET imaging of the radiolabelled anti-CEACAM6. Localization of the radiolabelled tracer could be attenuated by predosing with unlabelled antibody confirming specific accumulation in this compartment. Based on the expression pattern in normal and malignant pancreatic tissues, efficacy against established tumours and limited and reversible bone marrow toxicity, we propose that CEACAM6 should be considered for an ADC-based therapy approach against pancreatic adenocarcinomas and possibly other CEACAM6-positive neoplasms.

Molecular imaging can allow the non-invasive assessment of biological and biochemical processes in living subjects. Such technologies therefore have the potential to enhance our understanding of disease and drug activity during preclinical and clinical drug development, which could aid decisions to select candidates that seem most likely to be successful or to halt the development of drugs that seem likely to ultimately fail. Here, with an emphasis on oncology, we review the applications of molecular imaging in drug development, highlighting successes and identifying key challenges that need to be addressed for successful integration of molecular imaging into the drug development process.

Tumor heterogeneity complicates the quantification of tumor microvascular characteristics assessed by dynamic contrast-enhanced MRI (DCE-MRI). To address this issue a novel approach was developed that combines DCE-MRI with diffusion-based multispectral (MS) analysis to quantify the microvascular characteristics of specific tumor tissue populations. Diffusion-based MS segmentation (feature space: apparent diffusion coefficient, T(2) and proton density) was performed to identify tumor tissue populations and the DCE-MRI characteristics were determined for each tissue class. The ability of this MS DCE-MRI technique to detect microvascular changes due to treatment with an antibody (G6-31) to vascular endothelial growth factor-A (VEGF) was evaluated in a tumor xenograft mouse model. Anti-VEGF treatment resulted in a significant reduction in K(trans) for the MS viable tumor tissue class (-0.0034 +/- 0.0022 min(-1), P < 0.01) at 24 hr posttreatment that differ significantly from the change observed in the control group (0.0002 +/- 0.0025 min(-1)). Viable tumor K(trans) for the anti-VEGF group was also reduced 62% relative to the pretreatment values (P < 0.01). Necrotic tissue classes were found to add only noise to DCE-MRI estimates. This approach provides a means to measure physiological parameters within the viable tumor and address the issue of tumor heterogeneity that complicates DCE-MRI analysis.

Osteoprotegerin Ligand (OPGL) is a member of the tumor necrosis factor ligand superfamily and has been shown to be involved in interactions between T cells and dendritic cells. Its role in monocyte effector function, however, has not been defined. In the present study a role for OPGL in activating monocytes/macrophages has been characterized. OPGL was found to up-regulate receptor activator of NF-kappaB (RANK) receptor expression on monocytes, regulate their effector function by inducing cytokine and chemokine secretion, activate antigen presentation through up-regulation of co-stimulatory molecule expression, and promote survival. This activation is mediated through the MAPK pathway as evidenced by activation of p38 and p42/44 MAPK and up-regulation of BCL-XL protein levels. A physiological role for OPGL in monocyte activation and effector function was tested in a model of lipopolysaccharide-induced endotoxic shock. Administration of receptor activator of NF-kappaB (RANK)-Fc to block OPGL activity in vivo was able to protect mice from death induced by sepsis, indicating a hitherto undescribed role for OPGL in monocyte function and in mediating inflammatory response. This was further tested in an animal model of inflammation-mediated arthritis. Treatment with RANK-Fc significantly ameliorated disease development and attenuated bone destruction. Thus, our study strongly suggests that administration of receptor fusion proteins to specifically block OPGL activity in vivo may result in blocking development of monocyte/macrophage-mediated diseases.

Tumor heterogeneity complicates the quantification of a therapeutic response by MRI. To address this issue, a novel approach has been developed that combines MR diffusion imaging with multispectral (MS) analysis to quantify tumor tissue populations. K-means (KM) clustering of the apparent diffusion coefficient (ADC), T2, and proton density (M0) was employed to estimate the volumes of viable tumor tissue, necrosis, and neighboring subcutaneous adipose tissue in a human colorectal tumor xenograft mouse model. In a second set of experiments, the temporal evolution of the MS tissue classes in response to therapeutic intervention Apo2L/TRAIL and CPT-11 was observed. The multiple parameters played complementary roles in identifying the various tissues. The ADC was the dominant parameter for identifying regions of necrosis, whereas T2 identified two necrotic subpopulations, and M0 contributed to the differentiation of viable tumor from subcutaneous adipose tissue. MS viable tumor estimates (mean volume = 275 +/- 147 mm(3)) were highly correlated (r = 0.81, P < 0.01) with histological estimates (117 +/- 51 mm(3)). In the treatment study, MS viable tumor volume (at day 10) was 77 +/- 67 mm(3) for the Apo2L/TRAIL+CPT-11 group, and was significantly reduced relative to the control group (292 +/- 127 mm(3), P < 0.01). This method shows promise as a means of detecting an early therapeutic response in vivo.

Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24 -deficient mice ( Zmpste24 −/− ) have indicated a role for Zmpste24 in the processing of CAAX -type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24 −/− mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures—akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24 −/− mice. Zmpste24 −/− mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24 −/− mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.

Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.

Molecular imaging is becoming an indispensable part of clinical drug development. The presented review highlights few state-of-the-art examples that serve to illustrate specific points and discuss future directions of the use of positron emission tomography (PET) imaging in various phases of clinical drug development.

VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF(165) (rhVEGF(165)) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF(165) to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF(165) to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF(165) to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.

Contrast-enhanced magnetic resonance imaging was used to produce high-resolution activation maps reflecting local changes in cerebral blood volume after a simple sensory stimulus, Activation of the forelimb region of the somatosensory cortex was performed in α-chloralose—anaesthetized rats with an electrical stimulus (5 V, 3 Hz) delivered through needle electrodes placed subcutaneously on the left forelimb, A gradient echo magnetic resonance imaging sequence, sensitive to changes in the relative amount of deoxyhemoglobin within the cerebral vasculature, produced a 4.05% ± 1.69% increase in signal intensity. This effect was enhanced with an injection of an intravascular iron oxide contrast agent (Combidex, Advanced Magnetics), resulting in a 9.11% ± 1.52% decrease in signal intensity.

In this article, CSL112, an HDL-infusion therapy in clinical development has been incorrectly described as a synthetic reconstituted HDL mimetic currently in a Phase 2 IVUS plaque regression study. Correction : CSL112 is a novel formulation of apoA-I purified from human plasma and reconstituted with phospholipids to form HDL particles suitable for intravenous infusion [1]. CSL112 is currently in a Phase II study with safety and pharmacokinetic endpoints in patients with atherothrombotic disease (NCT01499420).

The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine. Here we investigate the connection between IL-22 and metabolic disorders. We find that the induction of IL-22 from innate lymphoid cells and CD4(+) T cells is impaired in obese mice under various immune challenges, especially in the colon during infection with Citrobacter rodentium. While innate lymphoid cell populations are largely intact in obese mice, the upregulation of IL-23, a cytokine upstream of IL-22, is compromised during the infection. Consequently, these mice are susceptible to C. rodentium infection, and both exogenous IL-22 and IL-23 are able to restore the mucosal host defence. Importantly, we further unveil unexpected functions of IL-22 in regulating metabolism. Mice deficient in IL-22 receptor and fed with high-fat diet are prone to developing metabolic disorders. Strikingly, administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. IL-22 shows diverse metabolic benefits, as it improves insulin sensitivity, preserves gut mucosal barrier and endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. In summary, we identify the IL-22 pathway as a novel target for therapeutic intervention in metabolic diseases.

Despite dramatic advances in standard of care, the risk of recurrent myocardial infarction early after an acute coronary syndrome (ACS) remains high. This period of elevated risk after a cardiovascular event is associated with an acute inflammatory response. While post-ACS inflammation correlates with the risk for recurrent events and is likely to play a causal role in this period, the precise pathophysiologic mechanisms have been unclear. Recent studies have proposed that the cardiac event itself activates the sympathetic nervous system to directly mobilize hematopoietic stem cells to differentiate into inflammatory monocytes, acutely infiltrate plaque, and lead to recurrent plaque rupture. Here, we summarize the existing and emerging evidence implicating post-ACS activation of systemic inflammation in the progression of atherosclerosis, and identify possible targets for therapeutic intervention. We highlight experimental therapies and ongoing clinical studies that will validate these targets.

Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastatic cells. This notion is supported by preclinical findings that inhibition of pro-lymphangiogenic signaling during tumor development reduces cell spread to sentinel lymph nodes (SLNs). However, it is unclear how lymphatics downstream of SLNs contribute to metastatic spread into distal organs, or if modulating distal lymph transport impacts disease progression. Utilizing murine models of metastasis, longitudinal in vivo imaging of lymph transport, and function blocking antibodies against two VEGF family members, we provide evidence that distal lymphatics undergo disease course-dependent up-regulation of lymph transport coincidental with structural remodeling. Inhibition of VEGF-C activity with antibodies against VEGF-C or NRP2 prevented these disease-associated changes. Furthermore, utilizing a novel model of adjuvant treatment, we demonstrate that antagonism of VEGF-C or NRP2 decreases post SLN metastasis. These data support a potential therapeutic strategy for inhibiting distant metastatic dissemination via targeting tumor-associated lymphatic remodeling.

Background The BRAF inhibitor, vemurafenib, has recently been approved for the treatment of metastatic melanoma in patients harboring BRAFV600 mutations. Currently, dual BRAF and MEK inhibition are ongoing in clinical trials with the goal of overcoming the acquired resistance that has unfortunately developed in some vemurafenib patients. FDG-PET measures of metabolic activity are increasingly employed as a pharmacodynamic biomarker for guiding single-agent or combination therapies by gauging initial drug response and monitoring disease progression. However, since tumors are inherently heterogeneous, investigating the effects of BRAF and MEK inhibition on FDG uptake in a panel of different melanomas could help interpret imaging outcomes. Methods 18 F-FDG uptake was measured in vitro in cells with wild-type and mutant (V600) BRAF, and in melanoma cells with an acquired resistance to vemurafenib. We treated the cells with vemurafenib alone or in combination with MEK inhibitor GDC-0973. PET imaging was used in mice to measure FDG uptake in A375 melanoma xenografts and in A375 R1, a vemurafenib-resistant derivative. Histological and biochemical studies of glucose transporters, the MAPK and glycolytic pathways were also undertaken. Results We demonstrate that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous BRAFV600 but ineffective in cells with acquired resistance or having WT BRAF status. However, combination with GDC-0973 results in a highly significant increase of efficacy and inhibition of FDG uptake across all twenty lines. Drug-induced changes in FDG uptake were associated with altered levels of membrane GLUT-1, and cell lines harboring RAS mutations displayed enhanced FDG uptake upon exposure to vemurafenib. Interestingly, we found that vemurafenib treatment in mice bearing drug-resistant A375 xenografts also induced increased FDG tumor uptake, accompanied by increases in Hif-1α, Sp1 and Ksr protein levels. Vemurafenib and GDC-0973 combination efficacy was associated with decreased levels of hexokinase II, c-RAF, Ksr and p-MEK protein. Conclusions We have demonstrated that 18 F-FDG-PET imaging reflects vemurafenib and GDC-0973 action across a wide range of metastatic melanomas. A delayed post-treatment increase in tumor FDG uptake should be considered carefully as it may well be an indication of acquired drug resistance. Trial registration ClinicalTrials.gov NCT01271803

Clinical use of recombinant fibroblast growth factor 21 (FGF21) for the treatment of type 2 diabetes and other disorders linked to obesity has been proposed; however, its clinical development has been challenging owing to its poor pharmacokinetics. Here, we describe an alternative antidiabetic strategy using agonistic anti-FGFR1 (FGF receptor 1) antibodies (R1MAbs) that mimic the metabolic effects of FGF21. A single injection of R1MAb into obese diabetic mice induced acute and sustained amelioration of hyperglycemia, along with marked improvement in hyperinsulinemia, hyperlipidemia, and hepatosteatosis. R1MAb activated the mitogen-activated protein kinase pathway in adipose tissues, but not in liver, and neither FGF21 nor R1MAb improved glucose clearance in lipoatrophic mice, which suggests that adipose tissues played a central role in the observed metabolic effects. In brown adipose tissues, both FGF21 and R1MAb induced phosphorylation of CREB (cyclic adenosine 5'-monophosphate response element-binding protein), and mRNA expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α) and the downstream genes associated with oxidative metabolism. Collectively, we propose FGFR1 in adipose tissues as a major functional receptor for FGF21, as an upstream regulator of PGC-1α, and as a compelling target for antibody-based therapy for type 2 diabetes and other obesity-associated disorders.

Imaging of tumor microvasculature has become an important tool for studying angiogenesis and monitoring antiangiogenic therapies. Ultrasmall paramagnetic iron oxide contrast agents for indirect imaging of vasculature offer a method for quantitative measurements of vascular biomarkers such as vessel size index, blood volume, and vessel density (Q). Here, this technique is validated with direct comparisons to ex vivo micro-computed tomography angiography and histologic vessel measurements, showing significant correlations between in vivo vascular MRI measurements and ex vivo structural vessel measurements. The sensitivity of the MRI vascular parameters is also demonstrated, in combination with a multispectral analysis technique for segmenting tumor tissue to restrict the analysis to viable tumor tissue and exclude regions of necrosis. It is shown that this viable tumor segmentation increases sensitivity for detection of significant effects on blood volume and Q by two antiangiogenic therapeutics [anti-vascular endothelial growth factor (anti-VEGF) and anti-neuropilin-1] on an HM7 colorectal tumor model. Anti-vascular endothelial growth factor reduced blood volume by 36±3% (p0.0001) and Q by 52±3% (p0.0001) at 48 h post-treatment; the effects of anti-neuropilin-1 were roughly half as strong with a reduction in blood volume of 18±6% (p0.05) and a reduction in Q of 33±5% (p0.05) at 48 h post-treatment.

Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF–mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti–G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.

Dynamic contrast enhanced MRI contrast agent kinetics in malignant tumors are typically complex, requiring multicompartment tumor models for adequate description. For consistent comparisons among tumors or among successive studies of the same tumor, we propose to estimate the total contrast agent-accessible volume fraction of tumor, including blood plasma, v(pe), and an average transfer rate constant across all tumor compartments, K(trans.av), by fitting a three-compartment tumor model and then calculating the area under the tumor impulse-response function (= v(pe)) and the ratio area under the tumor impulse response function over mean residence time in tumor (= K(trans.av)). If the duration of dynamic contrast enhanced MRI was too short to extrapolate the tumor impulse-response function to infinity with any confidence, then conditional parameters v(pe)(*) and K(trans.av*) should be calculated from the available incomplete impulse response function. Median decreases of 33% were found for both v(pe)(*) and K(trans.av*) in glioblastoma patients (n = 16) 24 hours after the administration of bevacizumab (P < 0.001). Median total contrast-enhancing tumor volume was reduced by 18% (P < 0.0001). The combined changes of tumor volume, v(pe)(*), and K(trans.av*) suggest a reduction of true v(pe), possibly accompanied by a reduction of true K(trans.av). The proposed method provides estimates of a scale and a shape parameter to describe contrast agent kinetics of varying complexity in a uniform way.

An “uncorrected” version of the following article was published in the June 2010 issue of MRM. The corrected version of the article is provided here. The publisher regrets the error. Imaging of tumor microvasculature has become an important tool for studying angiogenesis and monitoring antiangiogenic therapies. Ultrasmall paramagnetic iron oxide contrast agents for indirect imaging of vasculature offer a method for quantitative measurements of vascular biomarkers such as vessel size index, blood volume, and vessel density (Q). Here, this technique is validated with direct comparisons to ex vivo micro-computed tomography angiography and histologic vessel measurements, showing significant correlations between in vivo vascular MRI measurements and ex vivo structural vessel measurements. The sensitivity of the MRI vascular parameters is also demonstrated, in combination with a multispectral analysis technique for segmenting tumor tissue to restrict the analysis to viable tumor tissue and exclude regions of necrosis. It is shown that this viable tumor segmentation increases sensitivity for detection of significant effects on blood volume and Q by two antiangiogenic therapeutics [anti-vascular endothelial growth factor (anti-VEGF) and anti-neuropilin-1] on an HM7 colorectal tumor model. Anti-vascular endothelial growth factor reduced blood volume by 36 ± 3% (p < 0.0001) and Q by 52 ± 3% (p < 0.0001) at 48 h post-treatment; the effects of anti-neuropilin-1 were roughly half as strong with a reduction in blood volume of 18 ± 6% (p < 0.05) and a reduction in Q of 33± 5% (p < 0.05) at 48 h post-treatment. Magn Reson Med 63:1637–1647, 2010. © 2010 Wiley-Liss, Inc.

Seeds and plant parts of Tephrosia vogellii were investigated in order to provide systems for the study of prenyl to chromen transformation in rotenoids, as exemplified by the conversion of rot-2 -enonic acid into deguelin. No hydroxylated intermediate was found. A cell free preparation has been obtained from T. vogellii seedlings or seeds and shown to catalyse the reaction. The water soluble enzyme has been partially purified using ammonium sulphate precipitation, gel chromatography and ion exchange procedures. Data for the enzyme, named deguelin cyclase, are reported—optima for pH and temperature and Km (which indicates strong binding between enzyme and substrate). Results relevant to Mr determination are discussed. The enzyme has a requirement for oxygen, but not for cofactors. It is inhibited by chloride ion and chelating agents, particularly 1,10-phenanthroline. Deguelin cyclase can convert the 11-hydroxyrotenoid sumatrolic acid into α-toxicarol and lapachol into dehydro-α-lapachone, though the prenyl to chromen conversion is not general. It does not convert rot-2′-enonic acid into rotenone under the conditions studied. Deguelin cyclase seems not to belong to the P450 group and resembles more closely the non-heme iron protein isopenicillin N synthase.

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials. Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab. Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12. Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)

Imaging of the glial activation that occurs in response to central nervous system trauma and inflammation could become a powerful technique for the assessment of several neuropathologies. The selective uptake and metabolism of 2-18F-fluoroacetate (18F-FAC) in glia may represent an attractive strategy for imaging glial metabolism. Methods: We have evaluated the use of 18F-FAC as a specific PET tracer of glial cell metabolism in rodent models of glioblastoma, stroke, and ischemia–hypoxia. Results: Enhanced uptake of 18F-FAC was observed (6.98 ± 0.43 percentage injected dose per gram [%ID/g]; tumor-to-normal ratio, 1.40) in orthotopic U87 xenografts, compared with healthy brain tissue. The lesion extent determined by 18F-FAC PET correlated with that determined by MRI (R2 = 0.934, P = 0.007). After transient middle cerebral artery occlusion in the rat brain, elevated uptake of 18F-FAC (1.00 ± 0.03 %ID/g; lesion-to-normal ratio, 1.90) depicted the ischemic territory and correlated with infarct volumes as determined by 2,3,5-triphenyltetrazolium chloride staining (R2 = 0.692, P = 0.010) and with the presence of activated astrocytes detected by anti–glial fibrillary acidic protein. Ischemia–hypoxia, induced by permanent ligation of the common carotid artery with transient hypoxia, resulted in persistent elevation of 18F-FAC uptake within 30 min of the induction of hypoxia. Conclusion: Our data support the further evaluation of 18F-FAC PET for the assessment of glial cell metabolism associated with neuroinflammation.

Purpose To develop magnetic resonace imaging (MRI) methods for functional assessment of arteriogenesis in a murine model of peripheral artery disease to quantify the influences of vascular endothelial growth factor (VEGF), age, and atherosclerosis. Materials and Methods Reactive hyperemia (RH), which was induced using a device designed for remote and transient occlusion of the aorta and vena cava, was measured by blood-oxygen-level–dependent MRI. Twenty-eight days after femoral artery ligation, peak height (PH) and time to peak (TTP) of the RH response was compared with sham-operated animals in 10-week-old C57Bl6, 9-month-old C57Bl6, and 9-month-old Ldlr−/−Apobec−/− mice. The contribution of VEGF to functional recovery was assessed in young mice. Angiogenesis was quantified using an anti-PECAM1 radioimmunoassay. Results In young animals, angiogenesis was maximal 7 days after ligation, whereas functional recovery took 28 days. Inhibition of VEGF eliminated the angiogenesis seen at 7 days and reduced RH (PH, P < 0.05). At day 28, RH was altered in old (TTP, P < 0.05) and atherosclerotic (PH, P < 0.05; TTP, P < 0.05) animals. RH was different in young, old, and atherosclerotic sham animals. Old and atherosclerotic mice showed reduced angiogenesis. Conclusion The method presented herein can provide a sensitive assay for the functional assessment of arteriogenesis and highlights the contribution of VEGF, age, and atherosclerosis to this process. J. Magn. Reson. Imaging 2008;28:996–1004. © 2008 Wiley-Liss, Inc.

Purpose: Multiple endocrine neoplasia type 1 (MEN1) is defined clinically by the combined occurrence of multiple tumors, typically of the parathyroid glands, pancreatic islet cells, and anterior pituitary gland. A mouse model with a heterozygous deletion of the Men1 gene recapitulates the tumorigenesis of MEN1. We wished to determine the role of vascular endothelial growth factor (VEGF)-A in the vascularization and growth of MEN1-associated tumors, with an emphasis on pituitary adenomas. Experimental Design: To investigate whether tumor growth in Men1+/− mice is mediated by VEGF-A dependent angiogenesis, we carried out a monotherapy with the anti–VEGF-A monoclonal antibody (mAb) G6-31. We evaluated tumor growth by magnetic resonance imaging and assessed vascular density in tissue sections. We also measured hormone levels in the serum. Results: During the treatment with mAb G6-31, a significant inhibition of the pituitary adenoma growth was observed, leading to an increased mean tumor doubling-free survival compared with mice treated with a control antibody. Similarly, the growth of s.c. pituitary adenoma transplants was effectively inhibited by administration of anti–VEGF-A mAb. Serum prolactin was lowered by mAb G6-31 treatment but not by control antibody, potentially providing a new therapeutic approach for treating the hormonal excess in MEN1 patients. Additionally, the vascular density in pancreatic islet tumors was significantly reduced by the treatment. Conclusions: These results suggest that VEGF-A blockade may represent a nonsurgical treatment for benign tumors of the endocrine system.

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.

Purpose To quantify spontaneous and therapeutic arteriogenesis in vivo in a murine model of peripheral arterial disease using magnetic resonance angiography. Materials and Methods Male, 8–12-week-old, C57/BL6 mice underwent femoral artery ligation; 21 days later, 2 mg/kg recombinant murine VEGF165, formulated for slow release, was injected into the ipsilateral gastrocnemius. The spontaneous (following ligation) and therapeutic (following vascular endothelial growth factor (VEGF)) formation of collateral vessels was quantified using 3D magnetic resonance angiography on a small-bore 4.7T system. Therapeutically induced angiogenesis and blood flow were quantified using an in situ anti-platelet endothelial cell adhesion molecule (PECAM) 1 radioimmunoassay and radiolabeled microsphere deposition, respectively. Results Spontaneous arteriogenesis was visible in all animals five days after ligation. VEGF treatment doubled the arteriogenic response five days after treatment compared to vehicle (cross-sectional area of vessels: 0.96 vs. 0.46 mm2, P < 0.01). VEGF also induced angiogenesis (PECAM1 levels 191% of vehicle, P < 0.05) and increased blood flow specific to the injection site (57 vs. 7 mL/minute/100 g, P < 0.05). Conclusion The presented methodology allowed in vivo quantification of spontaneous arteriogenesis in a murine model of peripheral arterial disease and demonstrated that therapeutic enlargement of collateral vessels is possible with VEGF. J. Magn. Reson. Imaging 2006. © 2006 Wiley-Liss, Inc.

Objective Ex vivo and in vivo micro–computed tomography (micro-CT) combined with a novel image analysis algorithm were used to quantify cortical bone loss and periosteal new bone formation for therapeutic evaluation in a murine model of collagen-induced arthritis. Methods An automated algorithm was created to locate 5 metatarsophalangeal and 3 metacarpophalangeal joints in 3-dimensional micro-CT images of mouse paws for evaluation of joint cortical bone volume (JCBV) within close proximity of the joints as well as cortical bone mineral density and periosteal new bone formation within the paws. For validation, automated estimates of JCBV were compared with radiographic visual scores (RVS) in 4 treatment groups (n = 9 per group): rat anti-mouse CD11a monoclonal antibody, methotrexate (MTX), anti-CD11a plus MTX, and saline only. In a separate study, serial images of hind limbs were evaluated in 2 treatment groups: murine tumor necrosis factor receptor II–Fc fusion protein (mTNFRII; n = 10) and control antibody (n = 7). Results Automated estimates of the JCBV were significantly correlated with the RVS (hind paws R = −0.94, front paws R = −0.81, combined R = −0.87). The anti-CD11a group had significantly higher JCBV compared with controls. In the serial study, the automated estimate of JCBV detected significant treatment effects in the mTNFRII-Fc group compared with controls. Cortical bone mineral density was significantly higher in all treatment groups compared with controls. Conclusion Micro-CT combined with a novel image analysis technique (estimation of JCBV) provides a fully automated means to quantify bone destruction in a mouse model of rheumatoid arthritis.

A mouse model of non-necrotic vascular deficiency in the adult heart was studied using cine-magnetic resonance imaging (MRI) and other techniques. The mice lacked cardiomyocyte-derived vascular endothelial growth factor (VEGF) following a targeted knockout in the ventricular cardiomyocytes. Quantitative endothelial labeling showed that the capillary density was significantly reduced in the hearts of knockout mice. Gene expression patterns suggested that they were hypoxic. Semiautomated MR image analysis was employed to obtain both global and regional measurements of left ventricular function at 10 or more time points through the cardiac cycle. MRI measurements showed a marked reduction in ejection fraction both at rest and under low- and high-dose dobutamine stress. Regional wall thickness, thickening, and displacement were all attenuated in the knockout mice. A prolonged high-dose dobutamine challenge was monitored by MRI. A maximal response was sustained for 90 minutes, suggesting that it did not depend on endogenous glycogen stores.

It has been reported recently that very delayed damage can occur as a result of focal cerebral ischemia induced by vascular occlusion of short duration. With use of diffusion-, T2-, and contrast-enhanced dynamic magnetic resonance imaging (MRI) techniques, the occlusion time dependence together with the temporal profile for this delayed response in a rat model of transient focal cortical ischemia have been established. The distal branch of the middle cerebral artery was occluded for 20, 30, 45, or 90 minutes. Twenty minutes of vascular occlusion with reperfusion exhibited no significant mean change in either the apparent diffusion coefficient of water (ADC) or the T2 relaxation time at 6, 24, 48, or 72 hours after reperfusion ( P = 0.97 and 0.70, respectively). Ninety minutes of ischemia caused dramatic tissue injury at 6 hours, as indicated by an increase in T2 relaxation times to 135% of the contralateral values ( P < 0.01). However, at intermediate periods of ischemia (30 to 45 minutes), complete reversal of the ADC was seen at 6 hours after reperfusion but was followed by a secondary decline over time, such that a 25% reduction in tissue ADC was seen at 24 as compared with 6 hours ( P < 0.02). This secondary response was accompanied by an increase in cerebral blood volume (CBV), as shown by contrast-enhanced dynamic MRI (120% of contralateral values; P < 0.001), an increase in T2 relaxation time (132%; P < 0.01), together with clear morphological signs of cell death. By day 18, the mean volume of missing cortical tissue measured with high-resolution MRI in animals occluded for 30 and 45 minutes was 50% smaller than that in 90-minute occluded animals ( P < 0.005). These data show that ultimate infarct size is reduced after early reperfusion and is occlusion time dependent. The early tissue recovery that is seen with intermediate occlusion times can be followed by cell death, which has a delayed onset and is accompanied by an increase in CBV.

VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.

Quantifying oxygenation in viable tumor remains a major obstacle toward a better understanding of the tumor microenvironment and improving treatment strategies. Current techniques are often complicated by tumor heterogeneity. Herein, a novel in vivo approach that combines 19 F magnetic resonance imaging ( 19 F-MRI)R 1 mapping with diffusionbased multispectral (MS) analysis is introduced. This approach restricts the partial pressure of oxygen (pO 2 ) measurements to viable tumor, the tissue of therapeutic interest. The technique exhibited sufficient sensitivity to detect a breathing gas challenge in a xenograft tumor model, and the hypoxic region measured by MS 19 F-MRI was strongly correlated with histologic estimates of hypoxia. This approach was then applied to address the effects of antivascular agents on tumor oxygenation, which is a research question that is still under debate. The technique was used to monitor longitudinal pO 2 changes in response to an antibody to vascular endothelial growth factor (B20.4.1.1) and a selective dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor (GDC-0980). GDC-0980 reduced viable tumor pO 2 during a 3-day treatment period, and a significant reduction was also produced by B20.4.1.1. Overall, this method provides an unprecedented view of viable tumor pO 2 and contributes to a greater understanding of the effects of antivascular therapies on the tumor's microenvironment.

The BRAF inhibitor vemurafenib (marketed as zelboraf) has recently been FDA approved for the treatment of metastatic melanoma in patients harboring BRAF V600 mutations. PET imaging will be essential for guiding therapy in terms of gauging initial drug response, monitoring for disease relapse, and evaluating combination treatments in clinical trials. Herein, we assess 18F-FDG uptake across V600E mutant and vemurafenib resistant cells. We find that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous V600E BRAF but is ineffective in cells with acquired resistance or BRAF WT cells. Combination treatment with MEK inhibitor GDC-0973 results in a highly significant enhancement of efficacy across all twenty lines tested. We show that FDG uptake is driven by Glut-1, and that the membrane localization of the transporter paralleled with drug efficacy. Interestingly, we find that vemurafenib induced increased FDG uptake in cells harboring RAS mutations, as well as in tumors that have acquired vemurafenib drug resistance. Hexokinase II, CRAF, KSR and p-MEK protein levels in sensitive and drug resistant tumors were all associated with decreased measurements of FDG uptake in vivo. Overall, we have demonstrated that FDG PET imaging should be effective at monitoring the efficacy of vemurafenib in V600E melanoma mutants and that any detected increases in tumor uptake will likely be an indication of drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 911. doi:1538-7445.AM2012-911