Tumor Vessel Normalization Research Articles

Abstract IA5: The role of nitric oxide in the tumor microenvironment

Abstract Tumor blood vessels exhibit abnormal organization, structure, and function characterized by heterogeneous and at times compromised blood flow, and hyperpermeability. Furthermore, intra-tumoral lymphatics are not functional even if they exist and peritumoral lymphatics are dysfunctional. Collectively, these abnormalities result in a hostile tumor microenvironment, which hinders the delivery and efficacy of therapeutic agents, and also promotes a more malignant phenotype. Nitric oxide (NO), a mediator of diverse physiological and pathological processes, may contribute towards these deleterious processes. NO and NO synthases (NOS) are ubiquitous in malignant tumors and have been shown to exert both pro- and anti-tumor activities depending on their localization and activity. In tumor blood vessels, NO, as endotheliumderived relaxing factor, dilates blood vessels and thus serves to maintain blood flow. NO can mediate the angiogenic process either directly, or both up- or downstream of various other angiogenic factors. Furthermore, NO mediates vessel remodeling and maturation via its effect on perivascular cell recruitment. We have found that endothelial NOS (eNOS)-derived NO is critical for angiogenesis and vessel maturation. Conversely, NO produced from non-endothelial sources (for example tumor cells) interferes with vessel maturation and hence contributes to the abnormal functioning of blood vessels. Similarly, NO influences the formation and function of lymphatic vessels. In particular, NO mediates the proliferation of lymphatic endothelial cells, in vivo lymphangiogenesis, and lymphatic metastasis – all of which are enhanced by vascular endothelial growth factor-C. We have found that eNOS-derived NO produced in lymphatic endothelial cells promotes efferent lymphatic flow by regulating lymphatic contraction. On the other hand, interstitial NO produced by inducible NOS (iNOS) in inflammatory cells or tumor cells inhibits the function of these lymphatics. Collectively, these studies suggest that NO can regulate the tumor vasculature both positively and negatively, and that its precise localization is an important determinant of its role. Based on these findings, we hypothesized that the modulation of tissue NO location would normalize tumor vessels and improve the efficacy of concomitantly administered cytotoxic therapy. To test this hypothesis, we blocked tumor-derived NO production from glioma (neuronal NOS) and breast cancer (iNOS) cells both pharmacologically and genetically. In vivo imaging showed that after inhibition of tumor cell NOS, NO was seen primarily in a vascular location (eNOS-derived). This redistribution of tissue NO gradients were associated with an improved structure and function of tumor blood vessels. We observed a reduction in hypoxia suggesting improved overall function of vessel networks, and a consequent enhancement of radiotherapy efficacy in both orthotopic tumor models. Taken together, our data suggest that aberrant production of NO from tumor cells interferes with vessel maturation, and that the restoration of tissue NO distribution by selective NOS modulation improves tumor vascular function, facilitating the effects of concomitantly administered cytotoxic therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA5.

Time-Course Imaging of Therapeutic Functional Tumor Vascular Normalization by Antiangiogenic Agents

We describe here new technology that enables noninvasive imaging of therapeutic functional normalization of tumor blood vessels by antiangiogenic agents. Noninvasive variable-magnification in vivo-fluorescence imaging as well as fluorescence tomography was used to visualize functional vessel normalization. Changes in the same vessel before and after drug treatment were imaged with high resolution in real time. Differences in vascular responses to the mTOR inhibitor rapamycin and to an anti-VEGF antibody were functionally imaged. Tumor vessel normalization was shown by significantly reduced leakiness and subsequent improved tumor delivery of Paclitaxel-BODPY as well as by normalized morphology. The tumor vascular pool agent, AngioSense(750), was retained only in tumors after either anti-VEGF antibody or rapamycin treatment, as visualized by noninvasive fluorescence tomography. The antiangiogenic therapy normalized vessels, which significantly enhanced the antitumor efficacy of paclitaxel because of increased drug penetration throughout the tumor. The optical imaging technology described here is thus a powerful, noninvasive, time-course imaging tool of functional tumor vessel normalization and its therapeutic consequences.

The importance of neovascularization and its inhibition for allogeneic hematopoietic stem cell transplantation

AbstractGVHD and tumor relapse are fundamental problems in allogeneic HSCT. Recent research has linked neovascularization to GVHD, tumor growth, and graft-versus-tumor (GVT) activity. Damage of the endothelium by the conditioning regimen provides the initiation stimulus for recruitment of donor-derived endothelial cells and their progenitors. During the early inflammatory phase of GVHD there is considerable neovascularization facilitating migration of inflammatory cells to target organs. In the course of GVHD, however, the vasculature itself becomes a target of alloreactive donor T cells. As a consequence, later stages of GVHD are characterized by fibrosis and rarefaction of blood vessels. Importantly, the inhibition of tumor-neovascularization by activated donor T cells that release antiangiogenic substances contributes to GVT and may be enhanced by pharmacologic inhibition of neovascularization. Furthermore, the therapeutic inhibition of neovascularization may improve immunotherapy for cancer by enhancing leukocyte infiltration in tumor tissue because of normalization of tumor vessels and stimulation of leukocyte–vessel wall interactions. These insights identify important mechanisms underlining the importance of neovascularization for allogeneic immune responses and move therapeutic approaches targeting neovascularization into the spotlight. This perspective covers current knowledge of the role of neovascularization during GVHD as well as GVT and its implications for HSCT.

Persistent vascular normalization as an alternative goal of anti‐angiogenic cancer therapy

Angiogenesis is recognized as one of the principal hallmarks of cancers. Cancers contain newly formed immature vessels devoid of firm coverage by pericytes. Several drugs targeting vascular endothelial growth factor signals are now in clinical use for anti-angiogenic cancer treatment. Those drugs transiently normalize tumor vessels and ultimately provoke vascular regression. This regression causes tumor hypoxia, which could trigger certain cancer cells to become more invasive and metastatic. Normalized vessels do not induce tumor hypoxia, and may protect from cancer cell intravasation and enhance anticancer treatment with chemotherapeutic agents, radiation, or immune therapy. Thus, persistent vascular normalization could be an alternative goal of anti-angiogenic cancer treatment.

Effect of a therapeutic sphingosine 1-phosphate antibody on intratumoral hypoxia and sensitivity to standard chemotherapy in prostate cancer animal model.

24 Background: Hypoxia promotes neovascularization, metastasis, growth and resistance to treatments. The activation of HIF-1α has been identified as the master mechanism of adaptation to hypoxia. We recently identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway as a new modulator of HIF-1α activity under hypoxia in multiple cancer cell models (Ader et al, Cancer Res, 2008). S1P elicits proliferation, survival, or angiogenesis, and is believed to exert most of its actions as a ligand for a family of specific GPCRs to elicit paracrine or autocrine signaling. We have suggested that inhibiting SphK1/S1P signaling, which is up-regulated under hypoxia, may help normalizing the tumor microenvironment and increase sensitivity to chemotherapy, in the broader concept of normalization of tumor vessels as tumor oxygenation is known to enhance response to chemotherapy (Ader et al., Cancer Res, 2009). Methods: Quantitation of hypoxia and angiogenesis, and treatment efficacy using an orthotopic (o.t) xenograft model of fluorescent HRPC cells. Results: We provide in vitro evidence that inhibiting the S1P exogenous signaling, through pharmacological inhibition of its receptors or by taking advantage of a monoclonal antibody neutralizing S1P, blocks HIF-1α accumulation and its activity in prostate cancer cells under hypoxia. Second, using an o.t model of prostate cancer, we show that an anti-S1P antibody inhibits intratumoral hypoxia and modifies vessel architecture within 5 days of treatment. Third, we demonstrate that an anti-S1P strategy sensitizes to docetaxel, the “gold standard” treatment for HRPC. A 5-day anti-S1P antibody pretreatment markedly sensitizes to docetaxel in an o.t. PC-3/GFP model established in nude mice. The combination anti-S1P antibodytogether withdocetaxel was not only accompanied by a smaller primary tumor volume compared to docetaxel alone, but also significantly reduced the occurrence and number of metastases. Conclusions: These data establish the proof-of-concept that blocking the exogenous action of S1P reduces intratumoral hypoxia and sensitizes to chemotherapy in prostate cancer animal model. [Table: see text]

Multi-parametric assessment of the anti-angiogenic effects of liposomal glucocorticoids

Inflammation plays a prominent role in tumor growth. Anti-inflammatory drugs have therefore been proposed as anti-cancer therapeutics. In this study, we determined the anti-angiogenic activity of a single dose of liposomal prednisolone phosphate (PLP-L), by monitoring tumor vascular function and viability over a period of one week. C57BL/6 mice were inoculated subcutaneously with B16F10 melanoma cells. Six animals were PLP-L-treated and six served as control. Tumor tissue and vascular function were probed using MRI before and at three timepoints after treatment. DCE-MRI was used to determine Ktrans, ve, time-to-peak, initial slope and the fraction of non-enhancing pixels, complemented with immunohistochemistry. The apparent diffusion coefficient (ADC), T2 and tumor size were assessed with MRI as well. PLP-L treatment resulted in smaller tumors and caused a significant drop in Ktrans 48 h post-treatment, which was maintained until one week after drug administration. However, this effect was not sufficient to significantly distinguish treated from non-treated animals. The therapy did not affect tumor tissue viability but did prevent the ADC decrease observed in the control group. No evidence for PLP-L-induced tumor vessel normalization was found on histology. Treatment with PLP-L altered tumor vascular function. This effect did not fully explain the tumor growth inhibition, suggesting a broader spectrum of PLP-L activities.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-010-9198-5) contains supplementary material, which is available to authorized users.

Polarization of Tumor-Associated Macrophages: A Novel Strategy for Vascular Normalization and Antitumor Immunity

Vascular normalization is an emerging concept in cancer treatment. In this issue of Cancer Cell, Rolny etal. show that histidine-rich glycoprotein normalizes tumor vessels and promotes antitumor immunity by polarizing tumor-associated macrophages, leading to decreased tumor growth and metastasis. Placental Growth Factor deletion in macrophages phenocopies many of these effects.

Abstract P1-02-07: Real-Time Functional Imaging of Tumor Vascular Normalization by Anti-Angiogenic Agents

Abstract Background: We describe here new technology that enables non-invasive imaging of normalization of tumor blood vessels by anti-angiogenic agents in real time. Materials and Methods: Variable-magnification in vivo-fluorescence imaging as well as fluorescence tomography were used to visualize vessel normalization. Results: Morphological changes in the same vessel prior to and after drug treatment were imaged with high resolution in real time. Differences in vascular responses to the mTOR inhibitor rapamycin and to an anti-VEGF antibody were functionally imaged. Tumor vessel normalization was demonstrated by significantly reduced leakiness and subsequent improved tumor delivery of Paclitaxel-BODPY as well as by normalized morphology. The tumor vascular pool agent, AngioSense750, was retained in tumors after both anti-VEGF antibody and rapamycin treatment as visualized by noninvasive fluorescence tomography. The anti-angiogenic therapy normalized vessels, which significantly enhanced the antitumor efficacy of Paclitaxel due to increased drug penetration throughout the tumor. Discussion: The optical imaging technology described here is thus a powerful real-tool imaging tool of functional and morphological tumor vessel normalization and its therapeutic consequences. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-02-07.

Abstract LB-35: A monoclonal sphingosine 1-phosphate antibody inhibits intratumoral hypoxia and sensitizes to docetaxel in prostate cancer animal model

Abstract The reduction in the normal level of tissue oxygen tension or hypoxia is a characteristic of solid tumors that triggers the activation of signaling pathways promoting neovascularization, metastasis, increased tumor growth, and resistance to treatments. The activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α) has been identified as the master mechanism of adaptation to hypoxia. In a recent study (Ader et al, Cancer Res, 2008), we identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway, which elicits various cellular processes including cell proliferation, cell survival, or angiogenesis, as a new modulator of HIF-1α activity under hypoxic conditions Indeed, we have shown that SphK1 activity (which generates S1P) was rapidly stimulated under low oxygen conditions. The pharmacological inhibition and RNA silencing of SphK1 activity could prevent the accumulation of HIF-1α and its transcriptional activity in multiple cancer cell lineages - including prostate - suggesting a canonical pathway. We have suggested that inhibiting the SphK1/S1P signaling, that is up-regulated under low oxygen tension, might help normalizing the tumor microenvironment and increase tumor sensitivity to radiation and chemotherapy, in the broader concept of « normalization of tumor vessels » as tumor oxygenation is known to enhance response to chemotherapy and radiation (reviewed in Ader et al., Cancer Res, 2009). S1P is believed to exert most of its actions as a specific ligand for a family of five cognate G protein-coupled receptors to elicit paracrine or autocrine signaling cascades. We now provide evidence that inhibition of the S1P exogenous signaling, through pharmacological inhibition of its receptors or by taking advantage of a monoclonal antibody neutralizing S1P, blocks HIF-1α accumulation and its transcriptional activity in prostate cancer cells exposed to hypoxia. More importantly, in an orthotopic (o.t) xenograft model of prostate cancer, we show that a monoclonal antibody directed against S1P markedly reduces intratumoral hypoxia and modifies vessel architecture within 5 days of treatment. Finally, we demonstrate for the first time that a monoclonal antibody directed against S1P could sensitize to the antitumoral effects of docetaxel, the ‘gold standard’ treatment for hormone-refractory prostate cancer. Indeed, a 5-day anti-S1P antibody pretreatment markedly sensitizes to docetaxel in an o.t. PC-3/green fluorescent protein model established in nude mice. The combination anti-S1P antibody together with docetaxel was not only accompanied by a smaller primary tumor volume compared to docetaxel treatment but also markedly reduced the occurrence and number of metastases. In conclusion, these data establish the proof-of-concept that blocking the exogenous action of S1P, by reducing intratumoral hypoxia, can sensitize to docetaxel-based chemotherapy in prostate cancer animal model. 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 LB-35.

Abstract 3721: Critical role of Bevacizumab schedule in combination with chemo-radiotherapy in neo-adjuvant treatment of rectal cancer: Circulating endothelial cells and FDG-PET as markers for early prediction

Abstract Vascular endothelial growth factor (VEGF) has a crucial role in tumor angiogenesis, and its inhibition by bevacizumab (BEV) leads to normalization of tumor vessels increasing tumor oxygenation and drug delivery. However, the clinical benefits of current anti-VEGF treatments have thus far been rather modest, stimulating interest in developing novel effective ways to combine anti-VEGF drugs and chemotherapy (CT) and in the identification of valid predictive biomarkers of clinical benefit. We have previously shown that pre-operative oxaliplatin (OXA), raltitrexed (RTX), fluorouracil (5FU), and folinic acid (LFA) during pelvic radiotherapy (RT) have high rate of complete (TRG1) or subtotal (TRG2) tumor regression in locally advanced rectal cancer. In this study in order to demonstrate the relevance of the timing of BEV added to primary CT and RT we evaluate two different schedules of treatment. Changes of CECs and glucose metabolism, evaluated by flow cytometry and FDG-PET respectively, were used as early surrogate markers of tumor response. Thirty-two patients (inclusion criteria: cT4, cN+, cT3≤ 5 cm from the anal verge and/or +ve CRM, M1 resectable/initially unresectable) received 3 biweekly courses of OXA (100 mg/m2)/RTX (2.5 mg/m2) on day 1, and 5FU (800 mg/m2)/LFA (250 mg/m2) on day 2 during pelvic RT (45 Gy). In schedule A (16 pts) BEV (5 mg/kg) was given biweekly from day −14 for 4 courses, while in schedule B (16 pts) it was given from day −4 for 2 courses. Toxicity was graded with NCI-CTC version 3. According to the Simon's two-stage design, assuming an hypothesis of a 50% TRG1 (α error=0.05, β error=0.20), at least 6/16 TRG1 should be obtained (first stage) to continue pts accrual. Grade ¾ neutropenia was the most common adverse event with the schedule A (7 pts, 44%), but it was considerably lower with the schedule B (2 pts, 13%). Pre-treatment basal CEC amounts showed increased levels in the pts samples compared to the healthy controls. Notably, a significant difference of CEC levels, compared to basal levels, was observed 2 days after CT-RT between the two schedules of treatment (median +6% and −84% in schedule A and B, respectively, p<0.05). Likewise, a major reduction of median tumor metabolic volume was observed in schedule B compared to schedule A (−75% vs −50%, p<0.05). Furthermore, while in the schedule A only 2 (12%) pts obtained a TRG 1, in the schedule B the number of TRG 1 required by statistical design was already reached in the first 13 treated pts (7 pts; 54%). Overall these data suggest the relevance of the BEV schedule to optimize the feasibility and efficacy of combination treatment, as well as the potential role of CEC evaluation and FDG-PET for the early prediction of response to anti-angiogenesis therapeutical approaches. 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 3721.

Abstract 3238: Imaging sunitinib-induced vascular changes by DCE-MRI to schedule chemotherapy in renal cell carcinoma

Abstract The treatment of metastatic renal cell carcinoma (RCC) remains a challenge. Due to the high vascularity of kidney tumors, RCC are ideal candidates for anti-angiogenic therapy. The drug sunitinib (SU11248), a receptor tyrosine kinase (RTK) inhibitor, has demonstrated anti-tumor and anti-angiogenic activities in pre-clinical models. Sunitinib targets both tumor cells and tumor vasculature by inhibiting VEGF and PDGF receptor signaling. Sunitinib was approved by the FDA for RCC treatment in 2006 and clinical trials have reported dramatic responses observed in metastatic sites of disease but its efficacy was limited by adverse events due to toxicity to normal tissues, including cardiotoxicity. The vasculature of RCC tumors consists of abnormal enlarged and leaky vessels causing impaired blood supply that compromises the delivery of chemotherapy drugs to tumor cells. Using an orthotopic KCI-18 model of human RCC xenografts in nude mice, we have recently demonstrated that dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was reliable to monitor vascular changes induced by various doses of sunitinib in kidney tumors and in normal kidney tissues. DCE-MRI showed that a daily sunitinib dose of 20mg/kg/day (SU20) mildly affected normal vessels but caused better tumor perfusion of the contrast agent and decreased vascular permeability, in agreement with histological observations of thinning and regularization of tumor vessels. This dose was selected for combination therapy with gemcitabine. Dose titration studies of gemcitabine in KCI-18 kidney tumor-bearing mice showed that doses of 10, 20 and 40 mg/kg given twice a week for two weeks were tolerated by the mice, whereas higher doses were toxic. Based on DCE-MRI findings, mice bearing established KCI-18 kidney tumors were pre-treated on day 10, for 3 days with SU20, and then gemcitabine was given twice a week for 2 weeks with continued daily administration of SU20 up to day 28. We found that gemcitabine at a dose of 20 mg/kg given in combination with SU20 was not toxic and resulted in almost complete tumor growth inhibition, which was much greater than that observed with SU20 or gemcitabine alone. These data were histologically confirmed in tumor sections, by observation of small residual tumor nodules consisting mostly of dying tumor cells and surrounded by normal kidney tissue. In survival studies, the dose and schedule of SU20 and 20 mg/kg gemcitabine, described above, caused a significant increase in mouse survival. Vascular changes induced by sunitinib combined with gemcitabine and monitored by DCE-MRI suggested normalization of tumor vessels, as confirmed by thinning of tumor vessels, which were observed histologically in tumor sections. These studies show that DCE-MRI can be used to select the dose and schedule of anti-angiogenic drugs which cause regularization of blood flow to schedule chemotherapy and improve its efficacy. 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 3238.

Abstract 386: Real-time noninvasive optical imaging of angiogenesis and vasculogenesis inhibition and normalization

Abstract We have developed and optimized non-invasive in vivo imaging to continuously visualize and characterize tumor angiogenesis and vascular response to anticancer therapies in real time in live mice. Tumor vessels could be monitored longitudinally undergoing vasculogenesis and angiogenesis the same mouse. Morphometric changes of the same vessel prior to and after drug treatment were captured with high resolution. Differences in vascular responses to the mTOR inhibitor rapamycin and to an anti-VEGF antibody were imaged. The multi-channel imaging capability of the OV100 small animal imaging system (Olympus) enabled visualization of the functional integrity (permeability) of tumor vessels before and after anti-angiogenic therapies using the near-infrared blood pool reagent Angiosense750. We observed real-time tumor vessel normalization as demonstrated by significantly reduced leakiness and subsequent improved delivery of BODPY-Paclitaxel upon administration of low-dose and short-term anti-angiogenic therapy. Further, alterations in Angiosense750 tumor retention in response to different treatments were confirmed and quantified using noninvasive fluorescent molecular tomography (VisEN FMT). Histological, and immunofluorescence staining (CD31, lectin, SMA, NG2) along with confocal microscopy, confirmed the in vivo imaging observations. Thus, imaging is a powerful tool for investigating tumor angiogenic progression and for preclinical drug screening of anti-angiogenic agents. 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 386.

TSU68, an antiangiogenic receptor tyrosine kinase inhibitor, induces tumor vascular normalization in a human cancer xenograft nude mouse model

Combination therapy using antiangiogenic and cytotoxic agents is a useful strategy for advanced cancer, but the mechanism has not yet been elucidated. Moreover, there is a persistent paradox that destroying tumor vasculature with antiangiogenic agents disturbs the delivery of cytotoxic agents. It has been hypothesized that antiangiogenic agents can lead to normalization of tumor vessels that are structurally and functionally abnormal. The normalization means enhancing the deliver of cytotoxic agents. Our purpose was to investigate whether TSU68, a multiple receptor tyrosine kinase inhibitor that targets vascular endothelial growth factor receptor-2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), would induce the normalization of tumor vessels. TSU68 was administered for 7 days to mice with xenografted tumors. Tumors of interstitial fluid pressure (IFP) were measured before and after administration of agents. Immunofluorescence double staining for CD31 and alpha-SMA was performed, and a medical video endoscopy system with narrowband illumination (NBI) was used to visualize the vascular pattern. TSU68 treatment decreased IFP significantly. Immunofluorescence double staining showed a significant increase in the fraction of pericyte coverage in the TSU68-treated group. NBI endoscopy showed that many tumor vessels in TSU68-treated mice were pruned and the diameters of remaining vessels were reduced. The data supported our hypothesis of tumor vascular normalization by the antiangiogenic agent TSU68.

Contrasting Actions of Selective Inhibitors of Angiopoietin-1 and Angiopoietin-2 on the Normalization of Tumor Blood Vessels

Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have complex actions in angiogenesis and vascular remodeling due to their effects on Tie2 receptor signaling. Ang2 blocks Ang1-mediated activation of Tie2 in endothelial cells under certain conditions but is a Tie2 receptor agonist in others. We examined the effects of selective inhibitors of Ang1 (mL4-3) or Ang2 (L1-7[N]), alone or in combination, on the vasculature of human Colo205 tumors in mice. The Ang2 inhibitor decreased the overall abundance of tumor blood vessels by reducing tumor growth and keeping vascular density constant. After inhibition of Ang2, tumor vessels had many features of normal blood vessels (normalization), as evidenced by junctional accumulation of vascular endothelial-cadherin, junctional adhesion molecule-A, and platelet/endothelial cell adhesion molecule-1 in endothelial cells, increased pericyte coverage, reduced endothelial sprouting, and remodeling into smaller, more uniform vessels. The Ang1 inhibitor by itself had little noticeable effect on the tumor vasculature. However, when administered with the Ang2 inhibitor, the Ang1 inhibitor prevented tumor vessel normalization, but not the reduction in tumor vascularity produced by the Ang2 inhibitor. These findings are consistent with a model whereby inhibition of Ang2 leads to normalization of tumor blood vessels by permitting the unopposed action of Ang1, but decreases tumor vascularity primarily by blocking Ang2 actions.

Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

Antiangiogenesis Targeting Tumor Microenvironment Synergizes Glucuronide Prodrug Antitumor Activity

This study is aimed at investigating the in vivo antitumor activity of a novel cell-impermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating the synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment. We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101 on human tumor xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high performance liquid chromatography, immunohistochemistry, and immunofluorescence assays. We developed a nontoxic and cell-impermeable glucuronide prodrug, 9ACG, which can only be activated by extracellular beta-glucuronidase to become severely toxic. 9ACG possesses potent antitumor activity against human tumor xenografts in BALB/c nu/nu nude mice but not for tumors implanted in NOD/SCID mice deficient in macrophages and neutrophils, suggesting that these cells play an important role in activating 9ACG in the tumor microenvironment. Most importantly, antiangiogenic monoclonal antibody DC101 potentiated single-dose 9ACG antitumor activity and prolonged survival of mice bearing resistant human colon tumor xenografts by providing strong beta-glucuronidase activity and prodrug delivery through enhancing inflammatory cell infiltration and normalizing tumor vessels in the tumor microenvironment. We also show that inflammatory cells (neutrophils) were highly infiltrated in advanced human colon cancer tissues compared with normal counterparts. Our study provides in vivo evidence that 9ACG has potential for prodrug monotherapy or in combination with antiangiognesis treatment for tumors with infiltration of macrophage or neutrophil inflammatory cells.

Modulation of G Protein Signaling Normalizes Tumor Vessels

G protein-coupled biological processes are important for an ever-increasing number of human diseases and require fine-tuning through accessory molecules such as the regulators of G protein signaling (RGS). RGS5, a marker for tumor-resident pericytes, was recently established as playing a pivotal role in vascular maturation and vessel remodeling during carcinogenesis. Remarkably, tumors arising in a RGS5-deficient background display vessels with normalized morphology and an overall improved blood flow. Furthermore, these morphologic changes also lead to dramatic improvements in lymphocyte access to tumors and success of antitumor immunotherapy. Here, we consider the implications of these findings, and how they contribute to enhancing our understanding of remodeling angiogenic vessels as means for improving anticancer therapies.

Abstract LB-247: A multidisciplinary phase II study of AZD2171 (cediranib), an oral pan-VEGF receptor tyrosine kinase inhibitor, in patients with recurrent glioblastoma

Abstract Targeted agents are promising new therapeutics that have entered clinical trials for glioblastoma. AZD2171 (cediranib) is a potent, oral pan-VEGF receptor tyrosine kinase inhibitor with a half-life of 20 hours compatible with once-daily dosing. A primary target of AZD2171, VEGFR2, is expressed on glioblastoma endothelium. We have demonstrated normalization of tumor vessels in recurrent glioblastoma patients administered AZD2171 on a daily basis. Normalization has rapid onset, is reversible, and is associated with alleviation of brain edema (Batchelor TT, et al. Cancer Cell 2007;11:83-95). In this Phase II study of 31 recurrent glioblastoma subjects we now report clinical outcomes, including the proportion of patients alive and progression-free at 6 months (APF6) as the primary endpoint. Secondary endpoints include radiographic response proportion; progression-free survival; and overall survival and toxicity. At this time we are presenting radiographic response data, APF6, PFS, and OS on 31 patients. Thirty patients have experienced disease progression and one patient remains in follow-up without progression. The primary and secondary endpoints are tabulated below: Radiographic Responses Partial Response (more than 50% decrease in volume) in 16/30 (56%) APF6 [95% CI] 25.8% [14.7%, 46.9%] PFS [95% CI] 117 days [88, 145] OS [95% CI] 221 days [172, 285] Only one of the first 16 patients was removed from the study due to toxicity (fatigue). Dose limiting toxicities were observed in 9/16 patients with hypertension; fatigue and diarrhea were encountered most often. There have been no intracerebral hemorrhages. AZD2171 alleviated brain edema, a major cause of morbidity in glioblastoma patients, and had a steroid-sparing effect in many of the patients enrolled. Blood biomarkers were serially assessed and it was observed that plasma VEGF, PlGF, and SDF1α were increased after treatment. Plasma PlGF and VEGF decreased upon cediranib discontinuation. Plasma bFGF and SDF1α and viable circulating endothelial cells increased when tumors escaped treatment with AZD2171. AZD2171 is a promising therapy for glioblastoma and an attractive therapeutic partner for chemotherapy or radiation.

Anti‐tumor activity and tumor vessel normalization by the vascular endothelial growth factor receptor tyrosine kinase inhibitor KRN951 in a rat peritoneal disseminated tumor model

We assessed the antitumor efficacy of KRN951, a novel tyrosine kinase inhibitor of vascular endothelial growth factor receptors, using a rat colon cancer RCN-9 syngeneic model in which the tumor cells are transplanted into the peritoneal cavity of F344 rats. KRN951 treatments that commenced 4 days after tumor transplantation (day 4) significantly inhibited tumor-induced angiogenesis, the formation of tumor nodules in the mesenteric windows, and the accumulation of malignant ascites. Moreover, KRN951 treatments initiated on day 14, by which time angiogenesis and malignant ascites have already been well established, resulted in the regression of newly formed tumor vasculatures with aberrant structures and also in the apparent loss of malignant ascites by the end of the study period. Quantitative analysis of the vessel architecture on mesenteric windows revealed that KRN951 not only regressed, but also normalized the tumor-induced neovasculature. Continuous daily treatments with KRN951 significantly prolonged the survival of rats bearing both early stage and more advanced-stage tumors, compared with the vehicle-treated animals. The results of our current study thus show that KRN951 inhibits colon carcinoma progression in the peritoneal cavity by blocking tumor angiogenesis, ascites formation, and tumor spread, thereby prolonging survival. Moreover, these studies clearly demonstrate the therapeutic effects of KRN951 against established tumors in the peritoneal cavity, including the regression and normalization of the tumor neovasculature. Our findings therefore suggest that KRN951 has significant potential as a future therapeutic agent in the treatment of peritoneal cancers with ascites.

Non-Uniform Plasma Leakage Affects Local Hematocrit and Blood Flow: Implications for Inflammation and Tumor Perfusion

Vessel leakiness is a hallmark of inflammation and cancer. In inflammation, plasma extravasation and leukocyte adhesion occur in a coordinated manner to enable the immune response, but also to maintain tissue perfusion. In tumors, similar mechanisms operate, but they are not well regulated. Therefore, blood perfusion in tumors is non-uniform, and delivery of blood-borne therapeutics is difficult. In order to analyze the interplay among plasma leakage, blood viscosity, and vessel geometry, we developed a mathematical model that explicitly includes blood cells, vessel branching, and focal leakage. The results show that local hemoconcentration due to plasma leakage can greatly increase the flow resistance in individual vascular segments, diverting flow to other regions. Similarly, leukocyte rolling can increase flow resistance by partially blocking flow. Vessel dilation can counter these effects, and likely occurs in inflammation to maintain blood flow. These results suggest that potential strategies for improving perfusion through tumor networks include (i) eliminating non-uniform plasma leakage, (ii) inhibiting leukocyte interactions, and (iii) preventing RBC aggregation in sluggish vessels. Normalization of tumor vessels by anti-angiogenic therapy may improve tumor perfusion via the first two mechanisms.