Judy Mak

Slit Proteins Prevent Midline Crossing and Determine the Dorsoventral Position of Major Axonal Pathways in the Mammalian Forebrain

We report that Slit proteins, a family of secreted chemorepellents, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 and, even more so, mice deficient in both Slit1 and Slit2 show significant axon guidance errors in a variety of pathways, including corticofugal, callosal, and thalamocortical tracts. Analysis of multiple pathways suggests several generalizations regarding the functions of Slit proteins in the brain, which appear to contribute to (1) the maintenance of dorsal position by prevention of axonal growth into ventral regions, (2) the prevention of axonal extension toward and across the midline, and (3) the channeling of axons toward particular regions.

Blocking Neuropilin-2 Function Inhibits Tumor Cell Metastasis

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.

Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.

If neuropilin-2 and the growth factor VEGF-C don’t come together, lymphatic vessels don’t branch apart.

Neuropilin-1 expression in cancer and development†

Neuropilin (NRP)‐1 is a co‐receptor for vascular endothelial growth factor (VEGF). Preclinical data suggest that blockade of NRP1 suppresses tumour growth by inhibiting angiogenesis, in addition to directly inhibiting tumour cell proliferation in certain models. A humanized monoclonal antibody to NRP1 is currently being evaluated as a potential anti‐cancer therapy in clinical trials. However, the expression of NRP1 in cancer and physiological angiogenesis has yet to be systematically described. Here we characterize the in situ expression of NRP1 in human cancer and during mammalian development. A monoclonal antibody to human NRP1 was generated and validated for immunohistochemistry by western blotting, use of formalin‐fixed cell pellets transfected with NRP1, immunofluorescence, and comparison with in situ hybridization. NRP1 expression was assessed in whole sections of 65 primary breast carcinomas, 95 primary colorectal adenocarcinomas, and 90 primary lung carcinomas. An additional 59 human metastases, 16 xenografts, and three genetically engineered mouse tumour models were also evaluated. Immunoreactivity for NRP1 was seen in vessels from normal tissues adjacent to cancer and in 98–100% of carcinomas. Tumour cell expression of NRP1 was also observed in 36% of primary lung carcinomas and 6% of primary breast carcinomas, but no colorectal adenocarcinomas. NRP1 was evaluated in mouse embryos, where expression was limited to the nervous system, endocardium, vascular smooth muscle, and, focally, endothelium on subsets of vessels. Moreover, in a model of VEGF‐dependent angiogenesis in the postnatal mouse trachea, blockade of NRP1 signalling resulted in defective angiogenesis and recapitulated the effects of anti‐VEGF treatment. These observations confirm NRP1 as a valid anti‐angiogenic target in malignancy, and as a potential direct anti‐tumour target in a subset of cancers. The data also confirm a role for NRP1 in physiological, VEGF‐mediated angiogenesis. Copyright

Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.

IFNγ-induced Chemokines Are Required for CXCR3-mediated T-Cell Recruitment and Antitumor Efficacy of Anti-HER2/CD3 Bispecific Antibody

Purpose: The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell–dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors. Experimental Design: The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models. Results: Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8+ T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB. Conclusions: In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment.

Abstract 1363: Inhibition of Neuropilin-1 by anti-Nrp1 results in reduced angiogenesis and inhibition of tumor growth

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Neuropilin-1 (NRP1) guides the development of the nervous and vascular systems and has been implicated in modulating various aspects of tumor cell function. We have previously shown that an antibody directed against NRP1 inhibits tumor growth and reduces tumor angiogenesis, as a single agent and more potently in combination with anti-VEGF. However, as NRP1 was also expressed in the tumor cells tested, the impact from inhibiting tumor cell NRP1 in these experiments was unclear. The objective of this study was to determine if inhibition of tumor cell NRP1 was necessary for the observed anti-tumor effects previously described for anti-NRP1. We tested the effects of anti-NRP1 in tumor lines that do not express NRP1, and where expression of NRP1 is restricted to the vasculature. Inhibition of tumor growth as a single agent, and in combination with anti-VEGF in these xenografted tumor lines, confirm that tumor expression of NRP1 is not necessary for the anti-tumor effects of anti-NRP1. We furthermore demonstrate that anti-NRP1 treatment of endothelial cells in in vitro sprouting assays results in potent inhibition of vascular sprout formation, confirming its anti-angiogenic activity. In vivo, in developmental models of angiogenesis, treatment results in reduced angiogenesis that is augmented by anti-VEGF. Finally, we demonstrate that treatment of primates with anti-NRP1 results in up-regulation of cytokines, such as PlGF, that are commonly increased with anti-angiogenic agent treatment. In total, these data confirm that anti-NRP1 mechanistically acts to inhibit tumor angiogenesis, thus resulting in reduced tumor growth and this tumor inhibition is most effective in combination with VEGF inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1363.

Abstract 1362: Mechanistic evaluation of combination anti-angiogenic effects of anti-neuropilin-1 and anti-VEGF by in vivo imaging

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction. Anti-neuropilin 1 (anti-NRP1) has been shown preclinically to have an additive effect when used in combination with anti-VEGF. This has been hypothesized to be due in part to the inhibition of vascular maturation that results from anti-NRP1 treatment, which leaves vasculature in a highly anti-VEGF sensitive state. In this study, we examined the effects of sequencing these agents to evaluate this hypothesis. We investigated the effect of pre-treating tumors with anti-NRP1 before treating with anti-VEGF, and vice versa. To monitor tumor vasculature during ongoing treatment, in vivo magnetic resonance imaging was used to reveal information about therapeutic effects on tumor vasculature beyond simple tumor volume measurements. The vessel imaging MRI technique determines the fractional blood volume, mean vessel size, and Q (a dimensionless parameter related to vessel density) on a voxel-by-voxel basis. Methods. MRI scans were performed on a 4.7 T Varian MRI system (Varian Inc, Palo Alto, CA), for 25 mice with Calu6 human lung cancer tumors grown subcutaneously on the leg. Mice were randomized into three groups based on tumor volume caliper measurements: Group 1 (N=8) was treated with appropriate control antibodies twice a week for 3 weeks, Group 2 (N=8) was treated first with anti-VEGF twice a week for 1.5 weeks and then with anti-NRP1 twice a week for 1.5 weeks, and Group 3 (N=9) was treated first with anti-NRP1 twice a week for 1.5 weeks and then with anti-VEGF twice a week for 1.5 weeks. All mice were imaged after the first 10 days of treatment and again after the second 10 days of treatment. Vessel size index maps, blood volume maps, and Q maps were calculated voxel-by-voxel, and mean values of each parameter were calculated across the viable tumor tissue as determined using tissue segmentation analysis on the images. Results & Discussion. After 3 weeks, both treated groups had significantly reduced viable tumor volume relative to the control group. There was no difference in mean vessel size between the three groups. However, Group 3 had significantly reduced blood volume and Q (vessel density) relative to both Groups 1 & 2. This may indicate that initial treatment with anti-NRP1 makes tumor vasculature more susceptible to later treatment with anti-VEGF, while tumors that had been treated first with anti-VEGF did not benefit from later treatment with anti-NRP1. A repeat study with an additional group that was treated with both antibodies for the entire study duration confirmed these results and showed that the group pre-treated with anti-NRP1 showed equivalent reductions in blood volume and Q to the group treated with both antibodies for the full three weeks. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1362.

Abstract 1292: Broad vascular distribution of Neuropilin-1 and its ligands in human tumors

Neuropilin-1 (NRP1) is a multi-domain transmembrane receptor that is important for the development of the nervous and vascular systems and has been implicated in modulating various aspects of tumor biology. We have previously shown that an antibody directed against NRP1 inhibits tumor growth and reduces tumor angiogenesis, as a single agent and more potently in combination with anti-VEGF. Currently, anti-NRP1 is in evaluation in early clinical studies. Therefore, understanding the expression pattern of Nrp1 and its relevant ligands in human tumor samples is of critical importance. To this end, we first evaluated the binding of a number of putative ligands that have been suggested to interact with NRP1 in the literature, and furthermore to understand which of these are biochemically or functionally inhibited by our anti-NRP1. We found that members of the semaphorin and VEGF families, but not HGF bind to NRP1. However, only binding and function of specific ligands from the VEGF family are inhibited by anti-NRP1. Additionally, we evaluated the expression pattern of NRP1 in human tumor samples from a number of different tumor types, including breast, colorectal, lung, ovarian, pancreatic, prostate and renal cancer. NRP1 is broadly expressed in the vasculature of most cases of all these tumor types. Additionally, NRP1 is also variably expressed by tumor cells and other stromal elements in a more variable manner that is tumor type dependent. These results suggest that NRP1 is a potentially important therapeutic target in many different tumor types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1292.