Angiogenic Tumor Research Articles

Increased vascularity and spontaneous metastasis of breast cancer by hedgehog signaling mediated upregulation of cyr61

The Hedgehog (Hh) pathway is well known for its involvement in angiogenesis and vasculogenesis during ontogeny. The ligand, Sonic hedgehog (SHH), plays an important role in vascular formation during development. However, SHH expression is upregulated on tumor cells and can impact the tumor microenvironment. We have investigated the effects of autocrine as well as paracrine Hh signaling on tumor cells as well as on endothelial cells, respectively.Upon constitutive expression of SHH, breast cancer cells showed aggressive behavior and rapid xenograft growth characterized by highly angiogenic tumors that were spontaneously metastatic. In these cells, SHH caused activation of the Hh transcription factor, GLI1, leading to upregulated expression of the potent pro-angiogenic secreted molecule, CYR61 (cysteine rich angiogenic inducer 61). Silencing of CYR61 from these SHH-expressing Hh activated cells blunted the malignant behavior of the tumor cells and resulted in reduced tumor vasculature and limited hematogenous metastases. Thus, CYR61 is a critical downstream contributor to the Hh influenced pro-angiogenic tumor microenvironment. We also observed concomitant upregulation of SHH and CYR61 transcripts in tumors from patients with advanced breast cancer, further ratifying the clinical relevance of our findings. In summary, we have defined a novel, VEGF-independent, clinically relevant, pro-angiogenic factor, CYR61, that is a transcriptional target of Hh-GLI signaling.

Melatonin prevents human pancreatic carcinoma cell PANC‐1‐induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression

Melatonin is an important natural oncostatic agent, and our previous studies have found its inhibitory action on tumor angiogenesis, but the mechanism remains unclear. It is well known that vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis and has become an important target for antitumor therapy. Pancreatic cancer is a representative of the most highly vascularized and angiogenic solid tumors, which responds poorly to chemotherapy and radiation. Thus, seeking new treatment strategies targeting which have anti-angiogenic capability is urgent in clinical practice. In this study, a co-culture system between human umbilical vein endothelial cells (HUVECs) and pancreatic carcinoma cells (PANC-1) was used to investigate the direct effect of melatonin on the tumor angiogenesis and its possible action on VEGF expression. We found HUVECs exhibited an increased cell proliferation and cell migration when co-cultured with PANC-1 cells, but the process was prevented when melatonin added to the incubation medium. Melatonin at concentrations of 1 μm and 1 mm inhibited the cell proliferation and migration of HUVECs and also decreased both the VEGF protein secreted to the cultured medium and the protein produced by the PANC-1 cells. In addition, the VEGF mRNA expression was also down-regulated by melatonin. Taken together, our present study shows that melatonin at pharmacological concentrations inhibited the elevated cell proliferation and cell migration of HUVECs stimulated by co-culturing them with PANC-1 cells; this was associated with a suppression of VEGF expression in PANC-1 cells.

Stratégies anti-angiogéniques dans le glioblastome

Introduction Glioblastoma, a highly angiogenic tumor, has poor prognosis despite aggressive conventional therapies combining surgery, chemotherapy and radiotherapy. Anti-angiogenic strategies that have recently come to the clinic, are the most promising therapeutic approaches for these tumors. State of art Tumor hypoxia is the main trigger of angiogenesis processes driven primarily by vascular endothelial growth factor (VEGF). Clinical data indicate that inhibitors of VEGF such as bevacizumab or VEGF receptors such as tyrosine kinase inhibitors are of potential interest in the treatment of recurrent glioblastoma, with an acceptable toxicity. However, despite high rate of initial radiological response and rapid clinical improvement in about half of patients, therapeutic failure is the rule. Mechanisms of resistance remain poorly understood but an invasive phenotype and alternative angiogenesis factors may contribute to tumor escape. Conclusions and perspectives Anti-angiogenic strategies already play an important role in the management of recurrent glioblastoma. However, optimal combination and schedules of angiogenic inhibitors with radiotherapy and chemotherapy remain to be established. Important randomized clinical trials currently investigate therapeutic combinations for newly diagnosed glioblastoma.

Liposomes as carriers for colchicine-derived prodrugs: Vascular disrupting nanomedicines with tailorable drug release kinetics

Newly formed tumor vasculature has proven to be an effective target for tumor therapy. A strategy to attack this angiogenic tumor vasculature is to initiate local blood vessel congestion and consequently induce massive tumor cell necrosis. Vascular disrupting agents (VDAs) typically bind to tubulin and consequently disrupt microtubule dynamics. Colchicine and its derivatives (colchicinoids) are very potent tubulin binding compounds but have a narrow therapeutic index, which may be improved by employing a liposomal targeting strategy. However, as a result of their physicochemical properties, colchicinoids are problematic to retain in liposomes, as they are released relatively rapidly upon encapsulation. To overcome this limitation, two hydrolyzable PEGylated derivatives of colchicine were developed for encapsulation into the aqueous core of long-circulating liposomes: a moderately rapid hydrolyzing PEGylated colchicinoid containing a glycolic acid linker (prodrug I), and a slower hydrolyzing PEGylated colchicinoid with a lactic acid linker (prodrug II). Hydrolysis studies at 37°C and pH 7.4 showed that prodrug I possessed relatively rapid conversion characteristics (t1/2=5.4h) whereas prodrug II hydrolyzed much slower (t1/2=217h). Upon encapsulation into liposomes, colchicine was released rapidly, whereas both PEGylated colchicine derivatives were efficiently retained and appeared to be released only after cleavage of the PEG-linker. This study therefore demonstrates that, in contrast to colchicine, these novel PEGylated colchicine-derived prodrugs are retained within the aqueous interior after encapsulation into liposomes, and that the release of the active parent can be controlled by using different biodegradable linkers.

Regulation of non‐classical FGF1 release and FGF‐dependent cell transformation by CBF1‐mediated notch signaling

FGF1, a widely expressed proangiogenic factor involved in tissue repair and carcinogenesis, is released from cells through a non-classical pathway independent of endoplasmic reticulum and Golgi. Although several proteins participating in FGF1 export were identified, genetic mechanisms regulating this process remained obscure. We found that FGF1 export and expression are regulated through Notch signaling mediated by transcription factor CBF1 and its partner MAML. The expression of a dominant negative (dn) form of CBF1 in 3T3 cells induces transcription of FGF1 and sphingosine kinase 1 (SphK1), which is a component of FGF1 export pathway. dnCBF1 expression stimulates the stress-independent release of transduced FGF1 from NIH 3T3 cells and endogenous FGF1 from A375 melanoma cells. NIH 3T3 cells transfected with dnCBF1 form colonies in soft agar and produce rapidly growing highly angiogenic tumors in nude mice. The transformed phenotype of dnCBF1 transfected cells is efficiently blocked by dn forms of FGF receptor 1 and S100A13, which is a component of FGF1 export pathway. FGF1 export and acceleration of cell growth induced by dnCBF1 depend on SphK1. Similar to dnCBF1, dnMAML transfection induces FGF1 expression and release, and accelerates cell proliferation. The latter effect is strongly decreased in FGF1 null cells. We suggest that the regulation of FGF1 expression and release by CBF1-mediated Notch signaling can play an important role in tumor formation.

Deficiency of the macrophage growth factor CSF-1 disrupts pancreatic neuroendocrine tumor development

Tumor-associated macrophages have recently emerged as a key regulatory cell type during cancer progression, and have been found to promote tumor malignancy in the majority of studies performed to date. We show in this study that CD68(+) macrophages positively correlate with tumor grade and liver metastasis in human pancreatic neuroendocrine tumors (PNETs). To investigate the potential mechanisms whereby macrophages can promote PNET progression, we crossed the RIP1-Tag2 (RT2) mouse model of pancreatic islet cancer to colony-stimulating factor-1 (CSF-1)-deficient Csf1(op/op) mice, which have reduced numbers of tissue macrophages. Csf1(op/op) RT2 mice had a substantial reduction in cumulative tumor burden, which interestingly resulted from a significant decrease in angiogenic switching and tumor number, rather than an evident effect on tumor growth. In the tumors that did develop in CSF-1-deficient animals, however, there were no significant differences in tumor cell proliferation, apoptosis, angiogenesis or invasion. CSF-1 deficiency decreased macrophage infiltration by approximately 50% during all stages of RT2 tumor progression. Interestingly, several cytokines were upregulated in CSF-1-deficient RT2 tumors, and neutrophil infiltration was increased. These results show that macrophages are important for promoting PNET development and suggest that additional factors contribute to the recruitment and survival of myeloid cells in RT2 tumors in the absence of CSF-1.

Prolyl hydroxylase‐2 (PHD2) exerts tumor‐suppressive activity in pancreatic cancer

Pancreatic cancer is 1 of the most common and poorly treated tumors. In search of new therapeutic approaches, the oxygen sensors prolyl hydroxylases (PHD) are potential targets. PHD2 is considered the key oxygen sensor-regulating hypoxia-inducible factor (HIF). Currently, there is conflicting evidence regarding the exact role of PHD2 in tumorigenesis. The objective of this study was to investigate the role of PHD2 in pancreatic cancer growth and progression. PHD2 expression was analyzed by quantitative real-time polymerase chain reaction analysis and immunohistochemistry in human tissue specimens and cell lines. Knockdown of PHD2 was done by using short-interfering RNAs (siRNAs) specific against PHD2, and PHD2 overexpression was achieved by stable combinational DNA transfection. In vivo, an orthotopic murine model was used. Angiogenic cytokines were assessed with enzyme-linked immunosorbent assays, and invasion was studied with Matrigel assays. PHD2 expression was not altered substantially in cancer tissues and their metastases. Lymph node-negative tissues had higher levels of PHD2 than lymph node-positive tissues. PHD2 was hypoxia-inducible in pancreatic cancer cell lines and regulated cell growth through cyclin D1 down-regulation samples with PHD2 suppression and through p21 up-regulation in samples with of PHD2 overexpression. In vivo, PHD2 caused tumor growth retardation and reduced tumor invasion by inhibiting angiogenesis. This observation was caused by the suppression of angiogenic cytokines and tumor invasion. The current results indicated that PHD2 plays an important role in pancreatic tumorigenesis. In summary, the authors concluded that PHD2 may function as a tumor suppressor gene in pancreatic cancer and, thus, may define a potential target for the treatment of pancreatic cancer.

Sensitive Angiogenesis Imaging of Orthotopic Bladder Tumors in Mice Using a Selective Magnetic Resonance Imaging Contrast Agent Containing VEGF121/rGel

To investigate the efficiency of magnetic resonance imaging (MRI) contrast agents employing vascular endothelial growth factor (VEGF121)/rGel conjugated MnFe2O4 nanocrystals for imaging of neovasculature using a bladder tumor model. VEGF121/rGel was conjugated to MnFe2O4 nanoparticles (MNPs). The targeting efficiency and detection capability of the VEGF121/rGel-MNPs were investigated in both KDR-deficient (253JB-V) and KDR-overexpressing (PAE/KDR) cells using MRI. The internalization of VEGF121/rGel-MNPs into cells was confirmed by electron microscopy. Their phosphorylation ability and cytotoxicity were compared with unconjugated VEGF121/rGel. The orthotopic tumor mice were established by implanting low KDR-expressing 253JB-V cells into the bladder dome. After tail-vein injection of VEGF121/rGel-MNPs, the MR signal enhancement of intratumoral vessels by VEGF121/rGel-MNPs was observed and inhibition test using VEGF121 was also conducted. Ex vivo MR imaging of tumor tissue, and a fluorescence immunostaining study was also performed. The water-soluble VEGF121/rGel-MNPs (44.5 ± 1.2 nm) were stably suspended in the biologic media and exhibited a high relaxivity coefficient (423 mMs). They demonstrated sufficient targeting capability against KDR-overexpressing PAE/KDR cells, as confirmed by dose-dependent MR images and VEGF121 inhibition tests. The phosphorylation activity of KDR and cytotoxicity of VEGF121/rGel-MNPs were evaluated. VEGF121/rGel-MNPs successfully targeted the tumor and provided accurate anatomic details through (i) acquisition of clear neoangiogenic vascular distributions and (ii) obvious enhancement of the MR signal in T2*-weighted images. Immunostaining and blocking studies demonstrated the specific targeting ability of VEGF121/rGel-MNPs toward intratumoral angiogenesis. Synthesized VEGF121/rGel-MNPs as targeted MR imaging contrast agents can be specifically delivered to tumors and bind to KDR-expressing angiogenic tumor vessels.

TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration

Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 μm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and β-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.

Circulating Angiogenic Factors, Tumor Cells, and Outcome after Resection of Metastatic Colorectal Cancer—What Does It Mean?

Circulating Angiogenic Factors, Tumor Cells, and Outcome after Resection of Metastatic Colorectal Cancer—What Does It Mean?

Sentinel Lymph Node Pressure in Breast Cancer

Leakiness of angiogenic tumor vessels results in elevated pressure in primary breast cancers and increased lymphatic flow to sentinel lymph node(s) (SLNs). We hypothesized that a similar pathophysiology in metastatic axillary SLNs would result in increased intranodal pressure (INP). SLNs were "hot" and "blue" after intramammary injection of dilute methylene blue and filtered Tc99 sulfur colloid. Intraoperative pressure was measured in SLNs by a noncoring needle and recording device in 114 breast cancer patients. Excised axillary SLNs were examined by standard pathological techniques and metastases measured, recorded, and compared with INP measurements for SLN #1 and sometimes #2. INP in 131 SLNs with no tumor (SLN #1, n = 93; SLN #2, n = 38) was 9.1 ± 6.2 (SD; range -2, 35) mmHg and 21.4 ± 15.4 mmHg (range 0-50) in 35 tumor-containing SLNs (SLN #1, n = 29; SLN #2, n = 6) (P = 0.0066). Elevated INPs significantly correlated with SLN tumor metastasis sizes (P = 0.0038; r = 0.4904). In two patients, tumor-laden SLNs with high INP were not blue or "hot" while a blue lymphatic bypassed these nodes and was traced to the next echelon tumor-free blue and "hot" nodes with low INP. Breast cancer metastasis in axillary SLNs was associated with significantly higher INP than in tumor-free lymph nodes. When "true" SLNs were replaced by tumor, and the INP levels were very high, lymph flow direction changed; lymphophilic particles (blue dye and radiocolloid) were redirected to the next echelon of nodes, where the pressures were much lower. Mechanical factors may increase the likelihood of metastasis to neighboring lymph nodes with lower INP.

A phase I study of TRC105 (anti-CD105 antibody) in patients with advanced solid tumors.

3073 Background: CD105, also known as endoglin, is an endothelial cell membrane receptor that is highly expressed on angiogenic tumor vessels but not normal mature vessels. CD105 is essential for a...

In-Vivo Visualization of Tumor Microvessel Density and Response to Anti-Angiogenic Treatment by High Resolution MRI in Mice

PurposeInhibition of angiogenesis has shown clinical success in patients with cancer. Thus, imaging approaches that allow for the identification of angiogenic tumors and the detection of response to anti-angiogenic treatment are of high clinical relevance.Experimental DesignWe established an in vivo magnetic resonance imaging (MRI) approach that allows us to simultaneously image tumor microvessel density and tumor vessel size in a NSCLC model in mice.ResultsUsing microvessel density imaging we demonstrated an increase in microvessel density within 8 days after tumor implantation, while tumor vessel size decreased indicating a switch from macro- to microvessels during tumor growth. Moreover, we could monitor in vivo inhibition of angiogenesis induced by the angiogenesis inhibitor PTK787, resulting in a decrease of microvessel density and a slight increase in tumor vessel size.ConclusionsWe present an in vivo imaging approach that allows us to monitor both tumor microvessel density and tumor vessel size in the tumor. Moreover, this approach enables us to assess, early-on, treatment effects on tumor microvessel density as well as on tumor vessel size. Thus, this imaging-based strategy of validating anti-angiogenic treatment effects has high potential in applications to preclinical and clinical trials.

Focal adhesion kinase is required for KSHV vGPCR signaling

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, an angiogenic and inflammatory endothelial cell (EC) tumor that is common in areas of high KSHV prevalence. KSHV encodes a pro-angiogenic viral chemokine receptor (vGPCR) that promotes EC growth in vitro and KS-like tumors in mouse models. vGPCR is therefore considered a viral oncogene that plays a crucial role in the pathobiology of KS. In this study, we show that focal adhesion kinase (FAK) becomes activated upon vGPCR expression in primary ECs and that FAK is required for vGPCR-mediated activation of ERK1/2, NFκB, AP-1, and vGPCR-induced migration and inhibition of anoikis. FAK is crucial to cell motility and tumor invasiveness and is a potential therapeutic target in various malignancies. Our data show that via vGPCR, KSHV has evolved a way to constitutively activate FAK signaling.

Viruses as key regulators of angiogenesis

Angiogenesis is an important physiological process that is controlled by a precise balance of growth and inhibitory factors in healthy tissues. However, environmental and genetic factors may disturb this delicate balance, resulting in the development of angiogenic diseases, tumour growth and metastasis. During the past decades, extensive research has led to the identification and characterization of genes, proteins and signalling pathways that are involved in neovascularization. Moreover, increasing evidence indicates that viruses may also regulate angiogenesis either directly, by (i) producing viral chemokines, growth factors and/or receptors or (ii) activating blood vessels as a consequence of endothelial cell tropism, or indirectly, by (iii) modulating the activity of cellular proteins and/or (iv) inducing a local or systemic inflammatory response, thereby creating an angiogenic microenvironment. As such, viruses may modulate several signal transduction pathways involved in angiogenesis leading to changes in endothelial cell proliferation, migration, adhesion, vascular permeability and/or protease production. Here, we will review different mechanisms that may be applied by viruses to deregulate the angiogenic balance in healthy tissues and/or increase the angiogenic potential of tumours.

Repeated tumor oximetry to identify therapeutic window during metronomic cyclophosphamide treatment of 9L gliomas

Malignant gliomas are aggressive and angiogenic tumors with high VEGF content. Consequently, approaches such as metronomic chemotherapy, which have an anti-angiogenic effect, are being investigated. However, a lack of an appropriate technique that can facilitate the identification of vascular changes during antiangiogenic treatments has restricted therapeutic optimization. We have investigated the potential of tumor pO2 as a marker to detect vascular changes during metronomic chemotherapy. Electron paramagnetic resonance (EPR) oximetry was used to repeatedly assess tumor pO2 during metronomic cyclophosphamide treatment of subcutaneous 9L tumors. The 9L tumors were hypoxic with a pO2 of 5.6-8mmHg and a tumor volume of 247-300mm3 prior to any treatment. Tumor pO2 increased significantly to 19.7mmHg on day10 and remained at an elevated level until day33 during 4 weekly treatments with 140mg/kg cyclophosphamide. A significant decrease in the tumor volume on days21-31 occurred in the cyclophosphamide group, while the tumor volume of the control group significantly increased during measurements for two weeks. A significant tumor growth delay was achieved with two weekly treatments of cyclophosphamide plus radiotherapy (4Gyx5) as compared to control, cyclophosphamide and radiotherapy alone groups. The results indicate the potential of EPR oximetry to assess tumor pO2 during metronomic chemotherapy. The ability to identify the duration of an increase in tumor pO2, therapeutic window, non-invasively by EPR oximetry could have a significant impact on the optimization of antiangiogenic approaches for the treatment of gliomas. This vital information could also be used to schedule radiotherapy to enhance therapeutic outcome.

Abstract 3267: Inhibition of angiogenic potential of neuroblastoma cells through regulation of microRNA expression profiles by tumor suppressor gene dipeptidyl peptidase IV

Abstract Neuroblastoma (NB), a childhood cancer of peripheral nervous system generally progress to highly angiogenic and therapy resistant fatal metastatic tumor. We have recently shown that a cell surface protease called dipeptidyl peptidase (DPPIV) is expressed in normal neural crest derived differentiated cells and its expression is lost in proliferating NB, the malignant counterpart. We also demonstrated that re-expression of DPPIV in NB cells leads to their differentiation, apoptosis, and suppression of their migratory and angiogenic potential. Importantly, DPPIV inhibits the tumorigenic potential of NB cells in a xenograft mouse model. To further identify DPPIV-responsive genes and mechanisms, we used Affymetrix microarray analysis. This study showed an association of DPPIV expression with up-regulation of thrombospondin-1 (THBS1), an angiogenesis inhibitor. Interestingly, some of the genes regulated by DPPIV are also altered by microRNA (miRNAs), indicating that at least some of the miRNAs might act as intermediate effectors in mediating DPPIV function. PCR array screening of cancer specific miRNAs, showed that DPPIV indeed alters miRNA profile in NB cells. In particular, DPPIV down-regulated miR-19a which is a part of the miR-17-92 cluster that is known to decrease THBS1 levels. Furthermore, bioinformatics data mining of Sangers MicroCosm Targets v5 predicted THBS1 as a potential target of miR-19a suggesting that down-regulation of miR19a leading to up-regulation of THBS1 was a possible mechanism involved in DPPIV tumor suppressor function. In support of this idea, silencing of DPPIV expression by RNA interference resulted in increased miR-19a and decreased THBS1 expression. Furthermore, transfection of NB cells with anti-miR-19a decreased miR19a and increased THBS1 levels as shown by real time quantitative RT-PCR, immunofluorescence and western blot analysis. Furthermore, DPPIV expression decreased the levels of cMYC, a transcription factor that up-regulates miR-19a expression. These effects were associated with decreased levels of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), two strong downstream pro-angiogenic factors. Taken together, our work identifies a novel and an important mechanism wherein DPPIV inhibits angiogenesis and growth of NB tumor cells through down regulation of miR-19a mediated c-MYC expression and increased expression of THBS1, an angiogenesis inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3267. doi:10.1158/1538-7445.AM2011-3267

Abstract 4272: Hexastatin is a potent therapeutic agent for targeting tumor angiogenesis and tumor growth

Abstract Peptides or short protein domains derived from the extracellular matrix are reported to possess anti-tumor properties. In the present study, one such molecule “Hexastatin or [α6(IV)NC1]” a non-collagenous domain of α6 chain of type (IV) collagen was derived from the carboxy terminal and was found to inhibit tumor growth, but the mechanism by which it inhibits the growth of solid tumors has not been reported yet. In the present study, we identified that the biological functions of hexastatin are attributed to its binding to different cell surface integrins. We identified that hexastatin binds to α3β1, αVβ3 and α5β1 integrins and inhibits p38-MAPK signaling. Hexastatin competes with human vascular endothelial cells in binding to α1β1 integrins on type IV collagen, and or with α5β1/αVβ3 integrins on fibronectin/vitronectin matrix, thus inhibiting endothelial migration and tube formation. Interestingly, p38-MAPK phosphorylation was not inhibited in α3 and β3 integrin null endothelial cells upon treatment with Hexastatin which confirms that the antiangiogenic functions of Hexastatin are possibly mediated through α3β1 and αVβ3 integrins. While both the integrins (α3β1 and α5β1) are required for the inhibition of tube formation by Hexastatin in endothelial cells, only α3β1 integrin was found to regulate endothelial cells migration. In addition we also demonstrate that Hexastatin inhibits tumor growth, tumor angiogenesis and circulating endothelial cells in-vivo. These in-vitro and in-vivo findings indicate that α3β1 and αVβ3 integrins are critical for Hexastatin mediated inhibition of angiogenic signaling and tumor progression. Collectively, our findings demonstrate that hexastatin is a potent therapeutic agent for targeting tumor angiogenesis and tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2011-4272

Abstract 1763: The discovery of novel non-RGD-containing αvβ3 ligands for molecular imaging

Abstract Metastasis is the cause of 90% of cancer deaths. The detection of neoplasms before they metastasize using non-invasive molecular imaging approaches can have a significant impact on patient survival. For many years it has been known that αvβ3 integrin, which is over-expressed on angiogenic endothelium and many tumours, associates with the extracellular matrix through an RGD tripeptide motif. RGD-mediated targeting has been utilized for a wide variety of peptide and nanoparticle molecular imaging agents that are in various stages of development and in clinical trials. As RGD peptide decreases cell adhesion, concerns regarding its utilization as a molecular imaging agent have been raised (Kiessling et. al., 2009 Radiology). RGD peptides exert an anti-angiogenic effect through their inhibition of αvβ3 integrin, raising the concern given recent studies (Ebos et. al., 2009 Cancer Cell v15; Paez-Ribes et. al., 2009 Cancer Cell v15) that this anti-angiogenic activity may result in increased tumour invasion and metastasis. We sought, therefore, to discover novel αvβ3 integrin-targeted peptides that do not contain RGD for the development of new molecular imaging agents. Using a recently developed “beads on a bead” approach, we screened a linear octapeptide one bead one compound library using purified αvβ3 integrin. Over one hundred peptides were isolated and sequenced “on bead” using a novel MALDI-TOF/MS technique and a number of non-RGD containing peptides were identified. Two of these peptides had a higher binding affinity for purified αvβ3 integrin than the linear RGD peptide (LCE62, KD = 4.7 nM and LCE64, KD = 16.3 nM) as determined by surface plasmon resonance. In contrast to peptides containing RGD, these peptides did not impact the morphology and adhesion of αvβ3 integrin-expressing MDA-435 cells, nor did they inhibit angiogenesis. The uptake of both fluorescein-labeled LCE62 and LCE64 by αvβ3 integrin-expressing breast cancer cells were significantly higher compared with a control AGD peptide. We have also demonstrated that this uptake was effectively blocked by an excess of free unlabeled peptide. These novel αvβ3 integrin-binding peptides could provide a basis for a new and potentially safer generation of molecular imaging agents for the early diagnosis of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1763. doi:10.1158/1538-7445.AM2011-1763

Vascular normalization in orthotopic glioblastoma following intravenous treatment with lipid-based nanoparticulate formulations of irinotecan (Irinophore C™), doxorubicin (Caelyx®) or vincristine

BackgroundChemotherapy for glioblastoma (GBM) patients is compromised in part by poor perfusion in the tumor. The present study evaluates how treatment with liposomal formulation of irinotecan (Irinophore C™), and other liposomal anticancer drugs, influence the tumor vasculature of GBM models grown either orthotopically or subcutaneously.MethodsLiposomal vincristine (2 mg/kg), doxorubicin (Caelyx®; 15 mg/kg) and irinotecan (Irinophore C™; 25 mg/kg) were injected intravenously (i.v.; once weekly for 3 weeks) in Rag2M mice bearing U251MG tumors. Tumor blood vessel function was assessed using the marker Hoechst 33342 and by magnetic resonance imaging-measured changes in vascular permeability/flow (Ktrans). Changes in CD31 staining density, basement membrane integrity, pericyte coverage, blood vessel diameter were also assessed.ResultsThe three liposomal drugs inhibited tumor growth significantly compared to untreated control (p < 0.05-0.001). The effects on the tumor vasculature were determined 7 days following the last drug dose. There was a 2-3 fold increase in the delivery of Hoechst 33342 observed in subcutaneous tumors (p < 0.001). In contrast there was a 5-10 fold lower level of Hoechst 33342 delivery in the orthotopic model (p < 0.01), with the greatest effect observed following treatment with Irinophore C. Following treatment with Irinophore C, there was a significant reduction in Ktrans in the orthotopic tumors (p < 0.05).ConclusionThe results are consistent with a partial restoration of the blood-brain barrier following treatment. Further, treatment with the selected liposomal drugs gave rise to blood vessels that were morphologically more mature and a vascular network that was more evenly distributed. Taken together the results suggest that treatment can lead to normalization of GBM blood vessel the structure and function. An in vitro assay designed to assess the effects of extended drug exposure on endothelial cells showed that selective cytotoxic activity against proliferating endothelial cells could explain the effects of liposomal formulations on the angiogenic tumor vasculature.