Characterization and application of two RANK-specific antibodies with different biological activities.

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools

NG2 glia are required for vessel network formation during embryonic development.

The NG2(+) glia, also known as polydendrocytes or oligodendrocyte precursor cells, represent a new entity among glial cell populations in the central nervous system. However, the complete repertoire of their roles is not yet identified. The embryonic NG2(+) glia originate from the Nkx2.1(+) progenitors of the ventral telencephalon. Our analysis unravels that, beginning from E12.5 until E16.5, the NG2(+) glia populate the entire dorsal telencephalon. Interestingly, their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain. NG2(+) glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls. Absence of NG2(+) glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18.5. By revealing a novel and fundamental role for NG2(+) glia, our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains

Regulation of multiple angiogenic pathways by Dll4 and Notch in human umbilical vein endothelial cells

The Notch ligand, Dll4, is essential for angiogenesis during embryonic vascular development and is involved in tumour angiogenesis. Several recent publications demonstrated that blockade of Dll4 signalling inhibits tumour growth, suggesting that it may constitute a good candidate for anti-cancer therapy. In order to understand the role of Dll4 at the cellular level, we performed an analysis of Dll4-regulated genes in HUVECs. The genes identified included several angiogenic signalling pathways, such as VEGF, FGF and HGF. In particular we identified downregulation (VEGFR2, placenta growth factor PlGF) of VEGF pathway components resulting in the overall effect of limiting the response of HUVEC to VEGF. However extensive upregulation of VEGFR1 was observed allowing continued response to its ligand PlGF but the soluble form of the VEGFR1, sVEGFR1 was also upregulated. PlGF enhanced tubulogenesis of HUVEC suggesting that downregulation of PlGF and upregulation of VEGFR1 including sVEGFR1 are important mechanisms by which Dll4 attenuates PlGF and VEGF signalling. Dll4-stimulated HUVECs had impaired ERK activation in response to VEGF and HGF indicating that Dll4 signalling negatively regulates these pathways. Dll4 expression reduced vessel sprout length in a 3D tubulogenesis assay confirming that Dll4 signalling inhibits angiogenesis. Altogether, our data suggest that Dll4 expression acts as a switch from the proliferative phase of angiogenesis to the maturation and stabilisation phase by blocking endothelial cell proliferation and allowing induction of a more mature, differentiated phenotype. The regulation of sVEGFR1 provides a novel mechanism for Dll4 signalling to regulate cells at distance, not just in adjacent cells

Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth

Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis in vitro. Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics

Delta-like 4 Notch Ligand Regulates Tumor Angiogenesis, Improves Tumor Vascular Function, and Promotes Tumor Growth \u3ci\u3eIn vivo\u3c/i\u3e

The vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. However, clinical trials targeting the VEGF pathway are often ineffective, suggesting that other factors/pathways are also important in tumor angiogenesis. We have previously shown that the Notch ligand Delta-like 4 (DLL4) is up-regulated in tumor vasculature. Here, we show that DLL4, when expressed in tumor cells, functions as a negative regulator of tumor angiogenesis by reducing the number of blood vessels in all five types of xenografts, but acts as a positive driver for tumor growth in two of them (human glioblastoma and prostate cancer). The growth of in vivo models was not related to the effects on growth in vitro. DLL4 expressed in the tumor cells activated Notch signaling in host stromal/endothelial cells, increased blood vessel size, and improved vascular function within tumors. The promotion of tumor growth was, to some extent, due to a reduction of tumor hypoxia and apoptosis. DLL4-expressing tumor cells responded to anti-VEGF therapy with bevacizumab. A soluble form of DLL4 (D4ECD-Fc) blocked tumor growth in both bevacizumab-sensitive and bevacizumab-resistant tumors by disrupting vascular function despite increased tumor vessel density. In addition, we show that DLL4 is up-regulated in tumor cells and tumor endothelial cells of human glioblastoma. Our findings provide a rational basis for the development of novel antiangiogenic strategies via blockade of DLL4/Notch signaling and suggest that combined approaches for interrupting both DLL4 and VEGF pathways may improve antiangiogenic therapy. [Cancer Res 2007;67(23):11244–53

DLL4-Notch Signaling Mediates Tumor Resistance to Anti-VEGF Therapy \u3ci\u3eIn Vivo\u3c/i\u3e

Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a ?-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic. Cancer Res; 71(18); 6073–83. ©2011 AACR

The Lantern Vol. 37, No. 1, Fall 1970

• Circumstance • Advice • For What You Do For Me • Blink • Love • Love II • Magic • To Be a Child • A Year Later • A Poem in February • The Crystal Brick Road • Ephemera • Life • Whiskers • Thoughts On Being Sick • A Non-Poem • A Gruk Anthology • Moon • A Thought • Dwarf in an Existential Dawn • Corridors To My Mind • Sadness • The Enzyme Song • Creatures of Sandhttps://digitalcommons.ursinus.edu/lantern/1098/thumbnail.jp

Tipping the Balance: Robustness of Tip Cell Selection, Migration and Fusion in Angiogenesis

Vascular abnormalities contribute to many diseases such as cancer and diabetic retinopathy. In angiogenesis new blood vessels, headed by a migrating tip cell, sprout from pre-existing vessels in response to signals, e.g., vascular endothelial growth factor (VEGF). Tip cells meet and fuse (anastomosis) to form blood-flow supporting loops. Tip cell selection is achieved by Dll4-Notch mediated lateral inhibition resulting, under normal conditions, in an interleaved arrangement of tip and non-migrating stalk cells. Previously, we showed that the increased VEGF levels found in many diseases can cause the delayed negative feedback of lateral inhibition to produce abnormal oscillations of tip/stalk cell fates. Here we describe the development and implementation of a novel physics-based hierarchical agent model, tightly coupled to in vivo data, to explore the system dynamics as perpetual lateral inhibition combines with tip cell migration and fusion. We explore the tipping point between normal and abnormal sprouting as VEGF increases. A novel filopodia-adhesion driven migration mechanism is presented and validated against in vivo data. Due to the unique feature of ongoing lateral inhibition, ‘stabilised’ tip/stalk cell patterns show sensitivity to the formation of new cell-cell junctions during fusion: we predict cell fates can reverse. The fusing tip cells become inhibited and neighbouring stalk cells flip fate, recursively providing new tip cells. Junction size emerges as a key factor in establishing a stable tip/stalk pattern. Cell-cell junctions elongate as tip cells migrate, which is shown to provide positive feedback to lateral inhibition, causing it to be more susceptible to pathological oscillations. Importantly, down-regulation of the migratory pathway alone is shown to be sufficient to rescue the sprouting system from oscillation and restore stability. Thus we suggest the use of migration inhibitors as therapeutic agents for vascular normalisation in cancer

Optoacoustic detection of early therapy-induced tumor cell death using a targeted imaging agent

Purpose: The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU).Experimental Design: C2Am was labeled with a near infrared (NIR) fluorophore and injected intravenously into mice bearing human colorectal TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 xenografts that had been treated with a potent agonist of TRAILR2 and in Colo205 tumors treated with 5-FU.Results: Three dimensional MSOT images of probe distribution showed development of tumor contrast within 3 h of probe administration and a signal-to-background ratio in regions containing dead cells of >10 after 24 h. A site-directed mutant of C2Am that is inactive in PS binding showed negligible binding. Tumor retention of the active probe was strongly correlated (R2=0.97, P valueConclusions: The rapid development of relatively high levels of contrast suggests that NIR fluorophore-labeled C2Am could be a useful optoacoustic imaging probe for detecting early therapy-induced tumor cell death in the clinic.The work was supported by a Cancer Research UK Programme grant (17242) and a Project grant from MedImmune to KMB and by the CRUK-EPSRC Imaging Centre in Cambridge and Manchester (16465)