Target For Anti-angiogenic Therapy Research Articles

Mast cells density positive to tryptase correlates with angiogenesis in pancreatic ductal adenocarcinoma patients having undergone surgery.

Background. Literature data suggest that cells such as mast cells (MCs), are involved in angiogenesis. MCs can stimulate angiogenesis by releasing of several proangiogenic cytokines stored in their cytoplasm. In particular MCs can release tryptase, a potent in vivo and in vitro proangiogenic factor. Nevertheless few data are available concerning the role of MCs positive to tryptase in primary pancreatic cancer angiogenesis. This study analyzed MCs and angiogenesis in primary tumour tissue from patients affected by pancreatic ductal adenocarcinoma (PDAC). Method. A series of 31 PDAC patients with stage T2-3N0-1M0 (by AJCC for Pancreas Cancer Staging 7th Edition) was selected and then underwent surgery. Tumour tissue samples were evaluated by means of immunohistochemistry and image analysis methods in terms of number of MCs positive to tryptase (MCDPT), area occupied by MCs positive to tryptase (MCAPT), microvascular density (MVD), and endothelial area (EA). The above parameters were related to each other and to the main clinicopathological features. Results. A significant correlation between MCDPT, MCAPT, MVD, and EA group was found by Pearson's t-test analysis (r ranged from 0.69 to 0.81; P value ranged from 0.001 to 0.003). No other significant correlation was found. Conclusion. Our pilot data suggest that MCs positive to tryptase may play a role in PDAC angiogenesis and they could be further evaluated as a novel tumour biomarker and as a target of antiangiogenic therapy.

Identification Of Diffuse Large b-Cell Lymphoma (DLBCL) Subtypes Defined By The Expression Of Stromal Proteins and Their Association With Proangiomirs and Antiangiomirs

The identification of subtypes of DLBCL defined by expression of genes related to tumor stroma (Lenz et al, 2008) may determine new paths in the treatment of these diseases using anti-angiogenic drugs. The relationship of these genes with the pro- and antiangiomiRs is still unknown. Aims1) to classify DLBCL cases according to the signature stromal-1 and stromal-2 using tissue microarray (TMA) and immunohistochemistry; 2) to evaluate the expression of pro- and antiangiomiRs in paraffin embedded tissues affected by DLBCL and correlate them with the signatures of the tumor stroma. Methods111 DLBCL (NOS and variants, all HIV negative) admitted to the Hospital São Paulo between 2000 and 2010 were included in this study. Expression of CD68 (stromal-1 marker) was classified into four (0-3) levels. To analyze the expression of CD34 (MVD, stromal-2), we conducted a manual count of microvessels in the entire field of TMA. We performed miRNA extraction from paraffin samples using RecoverAll ™ Total Nucleic Acid Isolation Kit for FFPE Tissues (Applied Biosystems). Real-time quantitative PCR was performed using TaqMan® Small RNA kit Assays and normalized with U18 and RNU44. Pro-angiomirs Let-7f, miR-92a, miR-130a, miR-210, miR-296 and miR-378 and anti-angiomiRs miR-16, miR-20b, miR-221 and miR-328 were selected for analyses and were considered differentially expressed when tumor samples levels were 1.2 times higher or lower than normal samples. ResultsStromal-1 cases had high expression of CD68 (scores 2-3) and low expression of CD34 (quartiles I-II) (32.6% of cases), and as stromal-2 cases had low expression of CD68 (scores 0-1) and high expression of CD34 (quartiles III-IV) (11.6% of cases). The 40 cases that had high expression of both CD68 and CD34 (42.1%) were additionally considered as stromal-2, due to high MVD scores. Cases of low expression of CD68 and CD34 (13.7% of cases) were not scored. We observed a statistically significant relationship between the median expression of CD34 and Ann Arbor stage (I-II versus III-IV), with a predominance of high MVD expression in patients with advanced stage disease (III-IV). The median of vessels in stages I-II was 82.5 (range 9 to 240) and in stages III-IV was 111 (range 12-297) (p = 0.0276, Mann-Whitney). For the marker CD68 (stromal-1) and for the other variables, there were no statistically significant associations between groups. 101 of 111 initial cases were considered suitable for analysis of microRNAs. We observed overexpression of angiomiRs miR-92a, miR-296, miR-210, miR-378, Let-7f and miR-130a in 100%, 56.5%, 55,5%, 48.5%, 14.9% and 13.9% of cases, respectively. Among antiangiomiRs we found underexpression of miR-20b, miR-221, miR-16 and miR-328 in respectively 65.7%, 45.5%, 27.7% and 12.9%. We also observed a correlation between overexpression of miR-20b and low MVD (p = 0.0225, Mann-Whitney) and overexpression of miR-221 and low MVD (p = 0.0339, Mann-Whitney), suggesting a correlation between increased antiangiomiRs expression and low angiogenesis profile. For other variables, there were no statistically significant differences between groups. ConclusionStromal-2 signature was found in 53.7% of cases. The higher MVD in cases of DLBCL with advanced stage (Ann Arbor III-IV) shows a possible association between angiogenesis and more aggressive/disseminated disease, being a possible target for antiangiogenic therapy in cases that do not achieve a complete response with R-CHOP. Two angiomiRs emerge as potential new targets: proangiomiR miR-92a, overexpressed in 100% of DLBCL, and antiangiomiR miR-20b, underexpressed in 65.7% of cases. Our study provides new information about the importance of the role of microRNAs in the development of DLBCL, which can be exploited as a therapeutic target for patients with this malignant disease of high prevalence. (Supported by FAPESP 2010/17668-6). Disclosures:No relevant conflicts of interest to declare.

VEGFRS IN NEOPLASTIC ANGIOGENESIS AND PROSPECTS FOR THERAPY ОF BRAIN TUMORS

Glioblastoma (GBM) is the most common type of primary brain cancer that characterized by poor prognosis due to the rapid progression, active angiogenesis, enhanced tumor cell invasion and the emergence of resistance toward conventional therapy. In this connection, nowadays, new approaches for selective inhibition of crucial steps in tumor progression are actively developing. The key feature of tumor growth and development is angiogenesis. VEGF and its receptor VEGFR2 play the pivotal role in regulation of tumor vessel formation. Therefore, VEGFR2, as the main receptor of VEGF's pro-angiogenic signal transducer, is a promising molecular target for anti-angiogenic therapy. There is evidence that inhibitors of VEGF and VEGFR2 reduce endothelial cell proliferation, migration and survival that lead to regression of vessel density and decrease vascular permeability, thereby slowing tumor growth. Currently, a number of VEGFR2 inhibitors are under clinical trials (ramucirumab, cediranib) and several were approved (sunitinib, sorafenib). Despite the promising results of preclinical studies, the efficacy of antiangiogenic drugs in the clinical practice is significantly lower, mainly, due to rapid adaptation of malignant cells that consists of alternative pro-angiogenic pathways activation, recruitment of endothelial progenitor cells from bone marrow and increasing of the invasive growth. Given the diversity of pro-angiogenic mechanisms, enhancement of the efficacy of tumor therapy could be achieved by specific inhibition of VEGFR2 functions that will be supplemented by other antiangiogenic drugs (anti-VEGF,-PIGF,-HIF1alpha). In addition, multitargeting therapy should focus on the combined inhibition of angiogenesis, invasion, metastasis, proliferation and survival of tumor cells.

PSS313 - The NADPH Oxidase Nox4 is a Novel Target for Anti-Angiogenic Therapy

PSS313 - The NADPH Oxidase Nox4 is a Novel Target for Anti-Angiogenic Therapy

Sprouting Vessels with Nrf2

Nrf2 enhances neovascularization by suppressing Delta-Notch signaling.

Angiogenic marker associated with resistance to neoadjuvant chemoradiotherapy in rectal cancer.

11030 Background: The ability to achieve pathologic down staging after neoadjuvant chemoradiotherapy (CRT) is correlated with improved survival. However, there is no effective method of predicting which patients will response to neoadjuvant CRT. Neoadjuvant CRT can change the expression of angiogenic factors. However, little is known about its possible changes in response to preoperative CRT. We examined the expression of angiogenic factors in rectal cancer tissues before preoperative CRT and after surgery. Methods: Fifty five patients with locally advanced rectal cancer were studied. All patients were given preoperative CRT of 5040 cGy for 5-6 weeks with concurrent administration of 5-fluorouracil and leucovorin. Surgical resection was performed 6–8 weeks later in all patients. Immunohistochemical staining for angiogenenic markers (vascular endothelial growth factor [VEGF], placenta growth factor [PLGF], hypoxia inducible factor 1α [HIF 1α], stromal cell derived factor [SDF 1α]) were performed on specimens obtained before preoperative CRT and after surgery. A semiquantitative-immunohistochemical score established from the extension and intensity of the angiogenic factors was used for analysis. Results: The positive expression rate of VEGF, PLGF, SDF 1α, and HIF 1α was 56.4% (31/55), 65.5% (36/55), 70.9% (39/55), and 47.3% (26/55), respectively. The expression rate of VEGF, PLGF, SDF 1α, and HIF 1α was increased by 3.6% (2/55), 7.3% (4/55), 30.9% (17/55), and 1.8% (1/55) after neoadjuvant CRT, respectively. Expression of VEGF, PLGF, and HIF 1α protein was downregulated after neoadjuvant CRT in the rectal cancer tissues (P < 0.001, P = 0.001, P = 0.044, respectively). However, SDF 1α was upregulated after neoadjuvant CRT (P < 0.001). And also, upregulated expression of SDF 1α after neoadjuvant CRT was significantly associated with resistance to CRT (P = 0.035). However, SDF 1α showed no correlation with other clinical factors (age, sex, clinical stage). Conclusions: Expression of SDF-1α was increased in the rectal cancer tissue after neoadjuvant CRT, as well as has been associated with CRT resistance. Our data suggests that SDF 1α should be evaluated as new target for antiangiogenic therapy.

Abstract 5475: Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage.

Abstract Background: Endoglin (ENG, CD105) is a protein markedly overexpressed in tumor-associated endothelial cells, and a target for anti-angiogenic therapy. Recently we have noted it to be overexpressed in chemoresistant ovarian cancer cells as well. Our objective was to evaluate the effects and mechanisms of targeting endoglin specifically in ovarian cancer cells. Methods: Endoglin expression was assessed in multiple ovarian cancer lines by Western blot, immunohistochemistry, and flow cytometry. Anti-endoglin siRNAs were used to downregulate expression in ES2 and HeyA8MDR. In vitro, the effects of endoglin-knockdown individually and with chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation and qPCR array for mediators of DNA damage and repair. In an orthotopic murine model, mice inoculated with ES2 or HeyA8MDR cell lines were administered chitosan-encapsulated anti-ENG siRNA or control siRNA with and without carboplatin. PCNA, γH2AX, and 53BP1 IHC and the TUNEL assay were performed to examine biologic effects of endoglin knockdown. Results: Endoglin is expressed to varying degrees by multiple ovarian cancer cell lines. Expression was on the cell surface, consistent with its recognized role as a cofactor with TGF-beta receptor, in only 5% of cells, with most other cells having cytoplasmic expression. In ES2 and HeyA8MDR cell lines, endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Endoglin downregulation led to a decrease in expression of several DNA repair genes, including NBN, NTHL1, BARD1, H2AFX and SIRT1, and an increase in expression of DDIT3 and PPP1R15A. BARD1-specific downregulation, in turn, led to a significant decrease in BRCA1 expression, likely through ubiqutinylation. In vivo, anti-endoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared to control (35-41% reduction, p<0.05). Combination treatment with anti-Endoglin siRNA and carboplatin was associated with even greater inhibitory effect compared to control (58-62% reduction, p<0.001). Conclusions: Targeting endoglin improves platinum sensitivity of ovarian cancer cells both in vivo and in vitro. Endoglin downregulation induces DNA damage at multiple levels, one of which is through decreased expression of BARD1 and BRCA1. Anti-endoglin therapies should be pursued further as their development would allow dual treatment of both tumor angiogenesis and an aggressive subset of chemoresistant tumor cells. Citation Format: Angela J. Ziebarth, Somaria Nowsheen, Adam D. Steg, Monjri M. Shah, Ashwini A. Katre, Zachary C. Dobbin, Anil K. Sood, Michael G. Conner, Eddy S. Yang, Charles N. Landen. Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2013-5475 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Expression of Semaphorin3B protein in human hepatocellular carcinoma and its clinical significance

Objective To investigate the expression of Semaphorin3B (SEMA3B) in hepatocellular carcinoma (HCC) and reveal its clinical significance.Methods Immunohistochemistry and Western blotting were performed to examine the expression of SEMA3B protein in 56 hepatocellular carcinoma samples,matched perineoplastic tissues and 14 normal liver samples.Microvessel density (MVD) was determined by counting CD34-positive endothelial cells.Statistical methods were performed to analyse the relationships between the expression of SEMA3B and clinicopathologic factors.Results The rate of the SEMA3B-positive in HCC tissues was 42.9％ and significantly lower than that in normal liver tissues and non-cancerous adjacent liver tissues(78.6％ and 85.7％,respectively)(P＜0.05).MVD in SEMA3B-positive HCC tissus was lower than that in SEMA3B-negative HCC tissus (P＜0.05).The expression of SEMA3B was closely related to tumor nodular number,tumor size,tumor capsulation and CLIP score (P＜0.05 for all).The recurrence and metastasis rate in SEMA3B-positive group was lower than that in the negative group(P＜0.05),and the survival rate in positive group was higher than that in the negative(P＜0.05).Conclusion The expression of SE-MA3B was decreased in HCC tissues,and its expression was closely associated with tumor progression,angiogenesis and prognosis,indicating that it might seve as a predictor of prognosis and a possible novel target of antiangiogenic therapy for patients with HCC. Key words: Hepatocellular carcinoma; Semaphorin3B; Microvessel density; Recurrence; Prognosis

Endocan Is Upregulated on Tumor Vessels in Invasive Bladder Cancer Where It Mediates VEGF-A–Induced Angiogenesis

Tumor-associated blood vessels differ from normal vessels and proteins present only on tumor vessels may serve as biomarkers or targets for antiangiogenic therapy in cancer. Comparing the transcriptional profiles of blood vascular endothelium from human invasive bladder cancer with normal bladder tissue, we found that the endothelial cell-specific molecule endocan (ESM1) was highly elevated on tumor vessels. Endocan was associated with filopodia of angiogenic endothelial tip cells in invasive bladder cancer. Notably, endocan expression on tumor vessels correlated strongly with staging and invasiveness, predicting a shorter recurrence-free survival time in noninvasive bladder cancers. Both endocan and VEGF-A levels were higher in plasma of patients with invasive bladder cancer than healthy individuals. Mechanistic investigations in cultured blood vascular endothelial cells or transgenic mice revealed that endocan expression was stimulated by VEGF-A through the phosphorylation and activation of VEGFR-2, which was required to promote cell migration and tube formation by VEGF-A. Taken together, our findings suggest that disrupting endocan interaction with VEGFR-2 or VEGF-A could offer a novel rational strategy to inhibit tumor angiogenesis. Furthermore, they suggest that endocan might serve as a useful biomarker to monitor disease progression and the efficacy of VEGF-A-targeting therapies in patients with bladder cancer.

PECAM1+/Sca1+/CD38+ Vascular Cells Transform into Myofibroblast-Like Cells in Skin Wound Repair

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1+/Sca1+ vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1+/Sca1+/CD38+ cells proliferate upon wounding and could give rise to α-SMA+ myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1+/Sca1+/CD38+ vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1+/Sca1+/CD38+ cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.

Close Association of Metastasis‐Associated Protein 1 Overexpression with Increased Angiogenesis and Poor Survival in Patients with Histologically Node‐Negative Gastric Cancer

Although metastasis-associated protein 1 (MTA1) has been recently demonstrated as a potent angiogenesis-promoting factor in various malignant tumors, its angiogenic property in gastric cancer (GC) remains unclear. This study has detected the expression of MTA1 protein in surgically resected tissues of pathologic N0 (pN0) GC and further investigated its relation with other clinicopathologic factors and tumor angiogenesis and prognosis. MTA1 protein expression was detected immunohistochemically in 111 pN0 GC specimens. Its correlations with clinicopathologic factors and tumor prognosis were evaluated. The intratumoral microvessel density (MVD) was assessed based on CD105 antigen immunoreactivity and analyzed for correlation with MTA1 protein expression. Overexpression of MTA1 was detected in 36.04 % of patients and exhibited a significant association with tumor size and MVD. Survival analysis demonstrated that both overall (OS) and disease-free (DFS) survivals in patients overexpressing MTA1 were significantly poorer than those without MTA1 overexpression 5 years after the operation (both p < 0.001). Multivariate survival analysis demonstrated that MTA1 overexpression was an independent prognosticator for unfavorable OS and DFS (p < 0.001, respectively). MTA1 overexpression is frequently observed in pN0 GC patients and is significantly associated with increased angiogenesis and poor prognosis. Detection of MTA1 protein expression may help predict the relapse and prognosis of pN0 GC. Also, MTA1 protein may form a novel target for antiangiogenic therapy.

Angiopoietins as biomarker of disease activity and response to therapy in multiple myeloma

Tumor angiogenesis is a complex process involving interplay of several angiogenic regulators. In the present study, mRNA expression and circulating levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hypoxia inducible factor (HiF)-1α, circulating endothelial progenitor cells (cEPCs) and bone marrow microvessel density (MVD) were evaluated in multiple myeloma (MM). Compared to healthy controls, the levels of VEGF, bFGF, Ang-2, HiF-1α and cEPCs were significantly higher and Ang-1 and Ang-1/Ang-2 were lower in MM (p < 0.01). cEPC numbers correlated with Ang-1 (p = 0.03), Ang-2 (p = 0.01) and VEGF (p = 0.002). On multivariate analysis, reduced Ang-1/Ang-2 ratio (p = 0.005) at baseline was an independent predictor for response to therapy. After therapy, a decrease in Ang-2 (p < 0.001) and an increase in Ang-1/Ang-2 ratio (p = 0.003) were observed in responders. This study highlights the role of angiopoietins in MM which may, thus, be evaluated as potential targets for anti-angiogenic therapy in future.

A Novel Role for Mesenchymal Stem/Progenitor Cells As Hypoxia Sensors During Initiation of Neo-Vasculogenesis in Vivo

Abstract 613 Background:Hypoxia is a major stimulus of neo-vasculogenesis. Under hypoxic conditions endothelial colony-forming progenitor cells (ECFCs) arrange tubular structures, which can connect to the pre-existing vasculature forming functional perfused vessels. The current view is that mesenchymal stem and progenitor cells (MSPCs) or their pericyte progeny are recruited subsequently to stabilize vessels. So far, clinical applications of endothelial progenitors to restore tissue oxygenation after ischemia, cardiovascular disease or stroke largely failed to meet medical needs. Based on previous work demonstrating patent vessel formation after MSPC/ECFC co-transplantation in vivo (Blood 2009), we hypothesized that MSPCs have a decisive role in the vasculogenic response to hypoxia. Here we show for the first time that ECFCs in hypoxic conditions in vivo strictly require the presence of functional MSPCs not only to stabilize but primarily to initiate neo-vasculogenesis by a hypoxia-inducible transcription factor (HIF)-dependent mechanism. Methods:Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. Progenitor cell phenotype, long-term proliferation, HIF stabilization, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen (5% O2) and standard ambient air culture conditions (20% O2). ECFC and MSPC crosstalk in vivo was studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelet lysate gel). Cell type-specific chemical and genetic inhibition of HIF (YC-1, shRNA) was used to delineate the role of hypoxia sensing in MSPCs and ECFCs, respectively, during vasculogenesis in vivo. To determine if downstream target proteins of HIF-1α could substitute for MSPC presence during vasculogenesis, selected growth factors and cytokines were tested. Results:Progenitor proliferation and function in vitro were reduced with declining oxygen levels. ECFCs stabilized hypoxia-inducible factor-1α (HIF-1α) only at 1% O2, while MSPCs stabilized HIF-1α already at 5% O2. In an NSG mouse model, ECFCs transplanted into a hypoxic environment did not stabilize HIF-1α, while transplanted sole MSPCs or MSPCs in co-transplants showed strong nuclear HIF-1α stabilization 1 day after transplantation preceding any vessel formation or perfusion. In the absence of MSPCs, the majority of ECFCs underwent apoptosis within 24h in vivo. Inhibition of HIF-1α stabilization in MSPCs but not in ECFCs significantly abrogated vessel formation in vivo. Blocking the prominent HIF-1α down-stream target vascular endothelial growth factor (VEGF) resulted in the expected inhibition of neo-vasculogenesis. Interestingly, substitution of VEGF alone could not restore vessel formation, neither when injected together with sole ECFCs nor in a model where ECFCs were co-transplanted with HIF-depleted MSPCs. Substitution of a complex mixture of platelet-derived factors in vivo partly restored the vasculogenic function of HIF-depleted MSPCs. Conclusions:MSPCs react to a low oxygen environment by stabilizing HIF-1α earlier and more sensitively than ECFCs. MSPCs promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis in the initial phase of vasculogenesis by a HIF-dependent trophic mechanism. Surprisingly, therapeutic vasculogenesis can occur independently of endothelial HIF stabilization. These results argue in favor of MSPC/ECFC co-transplantation as a promising strategy for vascular regenerative therapy. The observation that VEGF alone could not compensate for the vasculogenic competence of pericyte precursors in vivo underlines the complexity of the hypoxia-induced cytokine network. The fact that hypoxia sensing in MSPCs but not in ECFCs is crucial to initiate vascular regeneration supports a shift of focus from endothelial cells to perivascular mesenchymal cells as a therapeutic target in anti-angiogenic therapy. Disclosures:No relevant conflicts of interest to declare.

Angiopoietin-2 and tumor angiogenesis

Tumor angiogenesis is an important way of rapid proliferation and metastasis for malignancies,and a variety of cellular and molecular factors involved in the process.Angiopoietin-2(Ang-2) is one of the important vascular endothelial growth factor,which influences tumor angiogenesis mainly through releasing vascular structures and damaging the vessel stability.Recent researches reveal that Ang-2 modulates tumor angiogenesis interacting with other vascular growth factors.With the new research progress,Ang-2 will not only be an important target for antiangiogenic therapy,but also further improve clinical efficacy of antitumor therapy combined with other signal pathways as common targets. Key words: Angiopoietins; Neoplasms; Neovascularization, pathologic

Abstract 5275: Biglycan is a specific marker and an autocrine angiogenic factor of tumor endothelial cells

Abstract Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels are morphologically different from their normal counterparts. We isolated tumor endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. Biglycan is overexpressed in inflammation and fibrosis. However, there has been no report about the expression or the function of biglycan in TEC. In this study, we investigated the expression and the role of biglycan in TECs. Real-time PCR, western blotting and immunocytochemistry revealed higher biglycan expression levels in TECs than in NECs. Furthermore, we confirmed that biglycan was secreted from TECs. Biglycan knockdown inhibited cell migration and tube formation in TECs. TLR2 and TLR4 are the biglycan receptors. TLR2 and TLR4 blocking antibodies suppressed biglycan mediated cell migration and tube formation. We isolated TECs from human renal cell carcinoma tissue and NECs from normal renal tissue in the same patients. TECs and NECs were obtained from six patients. Real-time RT-PCR revealed that the biglycan expression levels were significantly higher in four of the six TEC samples than in the corresponding NEC samples. Furthermore, immunostaining revealed strong biglycan expression in vivo in several human tumor vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers. Biglycan is an autocrine angiogenic factor stimulating tumor endothelial cell migration and tube formation. These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5275. doi:1538-7445.AM2012-5275

Abstract 5292: Identification of antiangiogenic small molecule natural products targeting anthrax toxin receptor 2

Abstract Bacillus anthracis protective antigen (PA), the B-subunit of the binary anthrax toxin, binds to the cellular receptors capillary morphogenesis gene protein 2 (CMG2) and tumor endothelial marker 8 (TEM8) with high affinity. Both CMG2 and TEM8 are expressed on endothelial cells during angiogenesis. We have shown that wild-type PA inhibits both VEGF-induced and bFGF-induced angiogenesis at moderate but statistically significant levels. Structure-activity studies identified a PA mutant that exhibited markedly enhanced inhibition of angiogenesis and also inhibited tumor growth in vivo. This mutant, PASSSR, is unable to undergo normal cellular processing and thus, remains bound to the surface receptor. Further mutation of PASSSR so that it does not bind to these cell surface receptors abolished its ability to inhibit angiogenesis. We conclude that high affinity anthrax receptor ligands, such as PA and PASSSR, are angiogenesis inhibitors, and that anthrax toxin receptors are useful targets for anti-angiogenic therapy. The long-term goal of our research is to determine the extent to which antrax toxin receptor directed therapeutics can control tumor growth and metastasis. Here we show that in addition to inhibition of subcutaneous lung tumors, PASSSR can inhibit the growth of orthotopically implanted breast cancer cells and extend survival in this mouse model. We also sought to identify small molecule natural products that can inhibit the interaction with the anthrax toxin receptor CMG2 and to determine their antiangiogenic and antitumor capacity in vivo. This work was based on the hypothesis that small molecule natural products can inhibit CMG2-ECM interactions required for breast cancer angiogenesis. This work leverages the wide variety of chemical entities generated by endophytic fungi, and the ability of other fungal compounds such as fumagillin to provide interesting insights into angiogenesis inhibitors. Following a high-throughput screen of &amp;gt;10,000 natural product extracts, we identified 48 extracts with significant inhibitory activity, of which ∼0.5% exhibited measurable activity. Of these, 9 exhibited 1:1 binding to CMG2 with IC50 &amp;lt;100 ug/ml total solids. Purification and chemical and in vitro characterization resulting in the identification of six compounds with anti-CMG2 and endothelial cell inhibitory, one of which exhibits antiangiogenic activity. Thus, we have identified several small molecule natural product inhibitors of CMG2 and demonstrated that CMG2 inhibitors can inhibit angiogenesis and breast cancer growth. While none of these molecules is currently suitable as a lead for the development of a breast cancer therapeutic, we believe that these studies confirm that the approach that we have taken is suitable for the development of such a therapeutic and propose to dramatically expand the number of molecules screened for such activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5292. doi:1538-7445.AM2012-5292

Biglycan is a specific marker and an autocrine angiogenic factor of tumour endothelial cells

Background:We isolated tumour endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. No report is available on biglycan expression or function in TECs.Methods:The NEC and TEC were isolated. We investigated the biglycan expression and function in TECs. Western blotting analysis of biglycan was performed on sera from cancer patients.Results:Biglycan expression levels were higher in TECs than in NECs. Biglycan knockdown inhibited cell migration and caused morphological changes in TECs. Furthermore, immunostaining revealed strong biglycan expression in vivo in human tumour vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers.Conclusion:These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy.

Abstract 5142: VEGFA gene amplification and protein expression in breast cancer

Abstract Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression. Methods: VEGFA gene amplification was evaluated by fluorescence in situ hybridization (FISH) on a multitumor tissue microarray (MTMA) comprising 3417 tissue samples from more than 100 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence immunoassay. In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers. Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 70) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p&amp;lt;0.0001). Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies. 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 5142. doi:10.1158/1538-7445.AM2011-5142

Abstract 1596: Human choriocarcinomas: Placental growth factor-dependent preclinical tumor models

Abstract Choriocarcinomas are an aggressive form of cancer that develops in the uterus from tissue that would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and the tumors are highly angiogenic. Human placental growth factor (hPlGF) is an angiogenic growth factor that promotes the growth of blood vessels in the placenta. hPlGF levels can be higher under pathological conditions such as cancer. Some human tumor specimens have higher PlGF levels than normal tissue, thus hPlGF is a target for anti-angiogenic therapy. The human choriocarcinoma cell lines BeWo, JAr, and JEG-3 were evaluated as preclinical models of hPlGF overexpression. The levels of hVEGF-A and hPlGF secreted by the choriocarcinomas and 11 human and mouse cancer cell lines in culture were measured by ELISA. The choriocarcinomas secreted higher levels of hPlGF and lower levels of hVEGF-A than 10/11 other cancer cell lines. The hPlGF serum levels in mice bearing subcutaneous choriocarcinoma tumors correlated with tumor volume and reached &amp;gt;2,000 pg/ml. By comparison, no hPlGF was detected in the serum of mice bearing H460 non-small lung carcinoma, HT29 colon carcinoma or MOLM-13 acute myeloid leukemia xenograft tumors. Although choriocarcinoma cells secreted both hPlGF and hVEGF-A in culture, serum levels of hVEGF-A in mice bearing choriocarcinoma xenografts were undetectable or &amp;lt;50 pg/ml at the time points serum was collected indicating that angiogenic factor secretion by the cells in culture was not the same in vivo. JEG-3 tumors grew faster than JAr tumors following the subcutaneous implantantion of 1×106 cells into nude mice. BeWo tumors grew the slowest and had the highest serum PlGF levels. Immunohistochemical methods confirmed expression of hPlGF in the choriocarcinoma xenografts. Studies involving double immunofluorescence on the xenograft tumors are ongoing to investigate endothelial cell and pericyte interactions and stromal involvement in the tumors. sFLT01, a novel fusion protein that neutralizes PlGF and VEGF-A, was added to choriocarcinoma cells in culture and administered to mice bearing choriocarcinoma xenografts in efficacy studies. Exposure of JAr and JEG-3 cells in culture to sFLT01 resulted in a phenotypic change whereby the cells produced less hPlGF and no significant hVEGF-A. In vivo, sFLT01 (25 mg/kg, IP injection, 2x per week) slowed tumor growth in JAr and JEG-3 tumors and increased median survival by 5-30 days compared to vehicle controls. These results indicate that human choriocarcinoma xenograft models are a valuable research tool for evaluating anti-angiogenic agents that target hPlGF. 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 1596. doi:10.1158/1538-7445.AM2011-1596

Abstract 5145: Nestin as a novel angiogenic marker and target for anti-angiogenic therapy in human pancreatic cancer

Abstract Background: Tumor angiogenesis is an important factor in the proliferation and metastasis of pancreatic ductal adenocarcinoma (PDAC). CD34, CD31 and factor VIII-related antigen are commonly used as endothelial cell markers for tumor vessels. However, these markers identify not only newly formed small tumor vessels, but also pre-existing large blood vessels. Nestin, a class VI intermediate filament protein, has been reported to be up-regulated in endothelial cells accompanying the process of angiogenesis. In an acute pancreatitis murine model, we have previously reported that nestin is strongly expressed in proliferating endothelial cells. Furthermore, we have reported that nestin-positive blood vessels correlated with a poor prognosis in colorectal cancer. In this study, we examined the effectiveness of nestin as an angiogenic marker and potent target for anti-angiogenic therapy in PDAC. Methods: Tissues from 45 patients with PDAC were immunostained with nestin and other common vascular endothelial markers including CD34, CD31 and factor VIII-related antigen. We measured the number and dimension of the nestin- and CD34-positive blood vessels using image analyzing software. In addition, we compared proliferation activity between nestin- and CD34-positive vessels as determined by PCNA-labeling indices. To clarify the roles of nestin in endothelial cells, we transfected siRNA targeting nestin transcripts to mouse endothelial TKD2cells, and performed cell growth and migration assays. Results: Immunohistochemically, CD34, CD31 and factor VIII-related antigen were localized in blood vessels of all sizes, while nestin was localized only in the small blood vessels in PDAC tissues. Nestin was also expressed in myofibroblasts and nerve fibers, but not in lymphatic vessels. In image analyzing software, nestin-positive vessels were small in number, and they formed small lumen compare with CD34-positive blood vessels. Nestin-positive vessels showed higher PCNA-labeling indices than those of CD34-positive vessels. Nestin was expressed in small and proliferating blood vessels in PDAC tissues, suggesting that nestin plays important roles in tumor angiogenesis in cancer. Knock down of nestin in mouse endothelial cells using siRNA inhibited the cell growth, but not cell migration in vitro. Conclusion: Nestin was specifically expressed in small and proliferating blood vessels in pancreatic cancer tissues, indicating that nestin is a useful angiogenic marker in cancer. Furthermore, nestin may be a novel therapeutic target for inhibition of tumor angiogenesis in pancreatic cancer patients. 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 5145. doi:10.1158/1538-7445.AM2011-5145