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Although angiogenesis inhibitors have provided substantial clinical benefit as cancer therapeutics, their use is limited by resistance to their therapeutic effects. While ample evidence indicates that such resistance can be influenced by the tumor microenvironment, the underlying mechanisms remain incompletely understood. Here, we have uncovered a paracrine signaling network between the adaptive and innate immune systems that is associated with resistance in multiple tumor models: lymphoma, lung and colon. Tumor-infiltrating T helper type 17 (T(H)17) cells and interleukin-17 (IL-17) induced the expression of granulocyte colony-stimulating factor (G-CSF) through nuclear factor κB (NF-κB) and extracellular-related kinase (ERK) signaling, leading to immature myeloid-cell mobilization and recruitment into the tumor microenvironment. The occurrence of T(H)17 cells and Bv8-positive granulocytes was also observed in clinical tumor specimens. Tumors resistant to treatment with antibodies to VEGF were rendered sensitive in IL-17 receptor (IL-17R)-knockout hosts deficient in T(H)17 effector function. Furthermore, pharmacological blockade of T(H)17 cell function sensitized resistant tumors to therapy with antibodies to VEGF. These findings indicate that IL-17 promotes tumor resistance to VEGF inhibition, suggesting that immunomodulatory strategies could improve the efficacy of anti-angiogenic therapy.

Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.

Measurements in tumor growth experiments are stopped once the tumor volume exceeds a preset threshold: a mechanism we term volume endpoint censoring. We argue that this type of censoring is informative. Further, least squares (LS) parameter estimates are shown to suffer a bias in a general parametric model for tumor growth with an independent and identically distributed measurement error, both theoretically and in simulation experiments. In a linear growth model, the magnitude of bias in the LS growth rate estimate increases with the growth rate and the standard deviation of measurement error. We propose a conditional maximum likelihood estimation procedure, which is shown both theoretically and in simulation experiments to yield approximately unbiased parameter estimates in linear and quadratic growth models. Both LS and maximum likelihood estimators have similar variance characteristics. In simulation studies, these properties appear to extend to the case of moderately dependent measurement error. The methodology is illustrated by application to a tumor growth study for an ovarian cancer cell line., (Copyright © 2012 John Wiley & Sons, Ltd.)

Angiogenesis plays a crucial role during tumorigenesis and much progress has been recently made in elucidating the role of VEGF and other growth factors in the regulation of angiogenesis. Recently, microRNAs (miRNAs) have been shown to modulate a variety of physiogical and pathological processes. We identified a set of differentially expressed miRNAs in microvascular endothelial cells co-cultured with tumour cells. Unexpectedly, most miRNAs were derived from tumour cells, packaged into microvesicles (MVs), and then directly delivered to endothelial cells. Among these miRNAs, we focused on miR-9 due to the strong morphological changes induced in cultured endothelial cells. We found that exogenous miR-9 effectively reduced SOCS5 levels, leading to activated JAK-STAT pathway. This signalling cascade promoted endothelial cell migration and tumour angiogenesis. Remarkably, administration of anti-miR-9 or JAK inhibitors suppressed MV-induced cell migration in vitro and decreased tumour burden in vivo. Collectively, these observations suggest that tumour-secreted miRNAs participate in intercellular communication and function as a novel pro-angiogenic mechanism.

Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Combinations of MAP/ERK kinase (MEK) and phosphoinositide 3-kinase (PI3K) inhibitors have shown promise in preclinical cancer models, leading to the initiation of clinical trials cotargeting these two key cancer signaling pathways. GDC-0973, a novel selective MEK inhibitor, and GDC-0941, a class I PI3K inhibitor, are in early stage clinical trials as both single agents and in combination. The discovery of these selective inhibitors has allowed investigation into the precise effects of combining inhibitors of two major signaling branches downstream of RAS. Here, we investigated multiple biomarkers in the mitogen-activated protein kinase (MAPK) and PI3K pathway to search for points of convergence that explain the increased apoptosis seen in combination. Using washout studies in vitro and alternate dosing schedules in mice, we showed that intermittent inhibition of the PI3K and MAPK pathway is sufficient for efficacy in BRAF and KRAS mutant cancer cells. The combination of GDC-0973 with the PI3K inhibitor GDC-0941 resulted in combination efficacy in vitro and in vivo via induction of biomarkers associated with apoptosis, including Bcl-2 family proapoptotic regulators. Therefore, these data suggest that continuous exposure of MEK and PI3K inhibitors in combination is not required for efficacy in preclinical cancer models and that sustained effects on downstream apoptosis biomarkers can be observed in response to intermittent dosing., (©2011 AACR.)

Xenograft trials allow tumor growth in human cell lines to be monitored over time in a mouse model. We consider the problem of inferring the effect of treatment combinations on tumor growth. A piecewise quadratic model with flexible phase change locations is proposed to model the effect of change in therapy over time. Each piece represents a growth phase, with phase changes in response to change in treatment. Piecewise slopes represent phase-specific (log) linear growth rates and curvature parameters represent departure from linear growth. Trial data are analyzed in two stages: (i) subject-specific curve fitting (ii) analysis of slope and curvature estimates across subjects. A least-squares approach with penalty for phase change point location is proposed for curve fitting. In simulation studies, the method is shown to give consistent estimates of slope and curvature parameters under independent and AR (1) measurement error. The piecewise quadratic model is shown to give excellent fit (median R(2)=0.98) to growth data from a six armed xenograft trial on a lung carcinoma cell line.

Purpose: Inhibition of the vascular endothelial growth factor (VEGF) axis is the basis of all currently approved antiangiogenic therapies. In preclinical models, anti-VEGF blocking antibodies have shown broad efficacy that is dependent on both tumor context and treatment duration. We aimed to characterize this activity and to evaluate the effects of discontinuation of treatment on the dynamics of tumor regrowth., Experimental Design: We evaluated the effects of anti-VEGF treatment on tumor growth and survival in 30 xenograft models and in genetic mouse models of cancer. Histologic analysis was used to evaluate the effects of treatment on tumor vasculature. We used a variety of treatment regimens to allow analysis of the effects of treatment duration and cessation on growth rate, survival, and vascular density., Results: Preclinical tumor models were characterized for their varied dependence on VEGF, thereby defining models for testing other agents that may complement or augment anti-VEGF therapy. We also found that longer exposure to anti-VEGF monoclonal antibodies delayed tumor growth and extended survival in established tumors from both cell transplants and genetic tumor models and prevented regrowth of a subset of residual tumors following cytoablative therapy. Discontinuation of anti-VEGF in established tumors resulted in regrowth at a rate slower than that in control-treated animals, with no evidence of accelerated tumor growth or rebound. However, more rapid regrowth was observed following discontinuation of certain chemotherapies. Concurrent administration of anti-VEGF seemed to normalize these accelerated growth rates., Conclusions: In diverse preclinical models, continuous VEGF suppression provides maximal benefit as a single agent, combined with chemotherapy, or as maintenance therapy once chemotherapy has been stopped., ((c) 2010 AACR.)

It has been recently reported that treatment with an anti-placenta growth factor (PlGF) antibody inhibits metastasis and primary tumor growth. Here we show that, although anti-PlGF treatment inhibited wound healing, extravasation of B16F10 cells, and growth of a tumor engineered to overexpress the PlGF receptor (VEGFR-1), neutralization of PlGF using four novel blocking antibodies had no significant effect on tumor angiogenesis in 15 models. Also, genetic ablation of the tyrosine kinase domain of VEGFR-1 in the host did not result in growth inhibition of the anti-VEGF-A sensitive or resistant tumors tested. Furthermore, combination of anti-PlGF with anti-VEGF-A antibodies did not result in greater antitumor efficacy than anti-VEGF-A monotherapy. In conclusion, our data argue against an important role of PlGF during primary tumor growth in most models and suggest that clinical evaluation of anti-PlGF antibodies may be challenging., (Copyright 2010 Elsevier Inc. All rights reserved.)

Ricinus communis agglutinin I (RCA I), a galactose-binding lectin from castor beans, binds to endothelial cells at sites of plasma leakage, but little is known about the amount and functional consequences of binding to tumor endothelial cells. We addressed this issue by examining the effects of RCA I on blood vessels of spontaneous pancreatic islet-cell tumors in RIP-Tag2 transgenic mice. After intravenous injection, RCA I bound strongly to tumor vessels but not to normal blood vessels. At 6 minutes, RCA I fluorescence of tumor vessels was largely diffuse, but over the next hour, brightly fluorescent dots appeared as the lectin was internalized by endothelial cells. RCA I injection led to a dose- and time-dependent decrease in vascular endothelial growth factor receptor-2 (VEGFR-2) immunoreactivity in tumor endothelial cells, with 95% loss over 6 hours. By comparison, VEGFR-3, CD31, and CD105 had decreases in the range of 21% to 33%. Loss of VEGFR-2 was followed by increased activated caspase-3 in tumor vessels. Prior inhibition of VEGF signaling by AG-028262 decreased RCA I binding and internalization into tumor vessels. These findings indicate RCA I preferentially binds to and is internalized by tumor endothelial cells, which leads to VEGFR-2 down-regulation, endothelial cell apoptosis, and tumor vessel regression. Together, the results illustrate the selective impact of RCA I on VEGF signaling in tumor blood vessels.

Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.

Tumor- or cancer-associated fibroblasts (TAFs or CAFs) from different tumors exhibit distinct angiogenic and tumorigenic properties. Unlike normal skin fibroblasts or TAFs from TIB6 tumors that are sensitive to anti-VEGF treatment (TAF-TIB6), TAFs from resistant EL4 tumors (TAF-EL4) can stimulate TIB6 tumor growth even when VEGF is inhibited. We show that platelet-derived growth factor C (PDGF-C) is upregulated in TAFs from resistant tumors. PDGF-C-neutralizing antibodies blocked the angiogenesis induced by such TAFs in vivo, slowed the growth of EL4 and admixture (TAF-EL4 + TIB6) tumors, and exhibited additive effects with anti-VEGF-A antibodies. Hence, our data reveal an additional mechanism for TAF-mediated tumorigenesis and suggest that some tumors may overcome inhibition of VEGF-mediated angiogenesis through upregulation of PDGF-C.

The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.

Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.

In the healthy colon, intestinal epithelial cells (IEC) form a physical barrier separating the myriad of gut Ags from the cells of the immune system. Simultaneously, IEC use several mechanisms to actively maintain immunologic tolerance to nonpathogenic Ags, including commensal bacteria. However, during inflammatory bowel disease (IBD), the line of defense provided by IEC is breached, resulting in uncontrolled immune responses. As IEC are a principal mediator of immune responses in the gut, we were interested in discerning the gene expression pattern of IEC during development and progression of IBD. Laser capture microdissection and microarray analysis were combined to identify the LY6 superfamily as strongly up-regulated genes in inflamed IEC of the colon in two models of murine colitis. Surface expression of LY6A and LY6C on IEC is induced by several cytokines present within the colitic gut, including IL-22 and IFN-gamma. Furthermore, cross-linking of LY6C results in production of a number of chemokines which are known to be involved in the immunopathogenesis of IBD. Increased chemokine production was cholesterol dependent, suggesting a role for lipid raft structures in the mechanism. As such, LY6 molecules represent novel targets to down-regulate chemokine expression in the colon and limit subsequent inflammation associated with IBD.

Purpose: The purpose of this study was to evaluate different methods of detecting and quantifying experimentally induced choroidal neovascularization (CNV) and vascular changes induced on CNV by an anti-VEGF-A monoclonal antibody., Methods: Choroidal neovascularization was induced by 532-nm diode laser in C57BL/6 mice. Ten days after the laser, the following methods were used to detect the new vessels: high-resolution angiography with fluorescein isothiocyanate-dextran; immunohistochemistry with biotinylated isolectin, rabbit anti-NG2, rat anti-CD31, rabbit anti-VWF, rat ani-CD105, rabbit anti-collagen IV, rat anti-ICAM-2, rabbit anti-desmin, and rat anti-MECA 32; and intravital injection of fluorescein-labeled Lycopersicon esculentum (tomato) lectin. To verify the validity of these staining methods in the quantification of treated CNV, the authors applied the most effective of these techniques to three groups of mice after laser induction of CNV and treatment with an anti-VEGF full antibody (G6-31)., Results: Fluorescein isothiocyanate-dextran angiography, rat anti-ICAM-2 immunostaining, and tomato lectin intravital injection resulted in the most effective means of identifying choroidal neovascularization. A certain amount of nonspecific fluorescence was detected in the area of CNV for each, Method: This fluorescence appeared more intense when fluorescein isothiocyanate-dextran was used. Tomato lectin injection and rat anti-ICAM-2 immunostaining were the methods that better recorded the antiangiogenic drug effect., Conclusions: Because of easy execution, low background fluorescence, and detailed visualization of new vessels, intravital injection of tomato lectin followed by a quantification based on threshold fluorescence represents the best technique for measuring CNV and the vascular changes induced by anti-VEGF-A monoclonal antibody in mice.

During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.

Psoriasis is a chronic inflammatory skin disease characterized by hyperplasia of the epidermis (acanthosis), infiltration of leukocytes into both the dermis and epidermis, and dilation and growth of blood vessels. The underlying cause of the epidermal acanthosis in psoriasis is still largely unknown. Recently, interleukin (IL)-23, a cytokine involved in the development of IL-17-producing T helper cells (T(H)17 cells), was found to have a potential function in the pathogenesis of psoriasis. Here we show that IL-22 is preferentially produced by T(H)17 cells and mediates the acanthosis induced by IL-23. We found that IL-23 or IL-6 can directly induce the production of IL-22 from both murine and human naive T cells. However, the production of IL-22 and IL-17 from T(H)17 cells is differentially regulated. Transforming growth factor-beta, although crucial for IL-17 production, actually inhibits IL-22 production. Furthermore, IL-22 mediates IL-23-induced acanthosis and dermal inflammation through the activation of Stat3 (signal transduction and activators of transcription 3) in vivo. Our results suggest that T(H)17 cells, through the production of both IL-22 and IL-17, might have essential functions in host defence and in the pathogenesis of autoimmune diseases such as psoriasis. IL-22, as an effector cytokine produced by T cells, mediates the crosstalk between the immune system and epithelial cells.

Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.