Angiogenic Network Research Articles

Direct transfection of miR-137 mimics is more effective than DNA demethylation of miR-137 promoter to augment anti-tumor mechanisms of delphinidin in human glioblastoma U87MG and LN18 cells

Glioblastoma is the deadliest brain tumor in humans. Recent studies suggested that 5-aza-2-deoxycytidine (AzaC) could inhibit cell cycle progression in human glioblastoma stem cells by an indirect increase in expression of the tumor suppressor microRNA-137 (miR-137). Delphinidin (DPN), a new anthocyanidin, inhibits cell growth in different cancers. We investigated inhibition of glioblastoma growth after indirect or direct overexpression of miR-137 and then DPN treatment. The highest inhibition of cell growth occurred due to treatment with combination of 10μM AzaC and 50μM DPN in human glioblastoma U87MG and LN18 cells. The methylation sensitive-polymerase chain reaction (MS-PCR) results showed that AzaC inhibited methylation of miR-137 promoter region, which was hypermethylated in both glioblastoma cell lines, to cause indirect increase in miR-137 expression. Our results also indicated the highest miR-137 expression after direct transfection of miR-137 mimics and DPN treatment. Combination of miR-137 mimics transfection and DPN treatment caused the highest inhibition of cell invasion and prevented angiogenic network formation due to the least expression of angiogenic factor (VEGF) in human glioblastoma cells in co-culture with human microvascular endothelial cells. This combination strategy most effectively inhibited survival factors (p-Akt and NF-κB), angiogenic factors (VEGF and b-FGF), growth factor receptor (EGFR), and invasive factors (MMP-9 and MMP-2). Direct overexpression of miR-137 most effectively augmented efficacy of DPN to induce apoptosis with activation of extrinsic and intrinsic pathways. So, sequential miR-137 overexpression and DPN treatment could be a promising combination treatment to inhibit growth of human glioblastoma cells.

P0119 PTEN expression affects outcome of human hepatocellular carcinoma via modulation of the oncogenic phenotypes and angiogenic processes

Background Reduced PTEN expression is associated with overexpression of VEGF in various malignancies. We aimed to investigate the expression of PTEN and VEGF and their association in the development of hepatocellular carcinoma. Findings PTEN expression was reduced in 43% and VEGF was overexpressed in 31% of 113 resected hepatocellular carcinoma clinical specimens. VEGF overexpression is positively correlated with young age, male sex, hepatitis B viraemia, high α -fetoprotein levels, reduced PTEN expression, advanced stage hepatocellular carcinoma, and hepatocellular carcinoma dedifferentiation. Survival analysis revealed that reduced PTEN expression, microvessel density, and advanced tumour stage were independent poor prognostic factors for disease-free survival. Adenovirus-mediated restoration of PTEN -inhibited cell proliferation, clonogenic growth and invasive capability of PTEN-deficient human hepatocellular carcinoma cells in vitro . In a xenograft animal model of hepatocellular carcinoma, PTEN gene delivery could efficiently suppress the incidence and growth rate of implanted tumour cells and reduce microvessel density. Decreased expression of angiogenic-associated factors, including VEGF, IL-8, and HIF1 α , was found in PTEN-restored hepatocellular carcinoma cells in vitro . Angiogenic networks of human umbilical vein endothelial cells were also suppressed by the increased PTEN expression. Interpretation These results show that tumour microenvironment can be affected by PTEN expression through modulation of the oncogenic phenotypes of tumour cells and a tendency to interfere in angiogenic processes. In conclusion, expression of PTEN and VEGF affecting the outcome of human hepatocellular carcinoma might serve as good prognostic biomarkers.

Autocrine control of angiogenesis by endogenous acetylcholine in an in vitro model using human endothelial cells: evidence for an autocrine cholinergic system in endothelial cells.

We wanted to elucidate whether acetylcholine as the endogenous ligand at cholinoceptors (ChRs) may have effects on angiogenesis and whether they are transduced through muscarinic or nicotinic ChRs. Human umbilical vein endothelial cells were cultured until confluence and thereafter seeded in Matrigel in vitro angiogenesis assays for 18 hours. During the entire cell culture and angiogenesis period, cells were treated with vehicle, eserine (1 μM), in the absence or presence of additional atropine (1 μM) or mecamylamine (1 μM). Finally, the resulting angiogenetic network was investigated histologically. Eserine significantly enhanced acetylcholine formation. When acetylcholine acted through muscarinic ChRs (eserine + mecamylamine), we observed enhanced complexity of the angiogenic network pattern with increased tube length and cell number. In contrast, when acting through nicotinic ChRs (eserine + atropine), we found reduced complexity of pattern with less branches, shorter tubes, and reduced cell number. If acting on both types of ChRs (eserine alone), there were only very small effects. Using α-bungarotoxin, lobeline, and dihydro-β-erythroidine, we also could show that these effects to various degrees involve α7, α3/β2, and α4/β2 n-ChRs. In conclusion, our results support the hypothesis that human umbilical vein endothelial cells possess an autocrine nonneuronal cholinergic system regulating angiogenesic branch formation through the partially opposing effects of n-ChRs and m-ChRs.

Epigenetic profile of human adventitial progenitor cells correlates with therapeutic outcomes in a mouse model of limb ischemia.

We investigated the association between the functional, epigenetic, and expressional profile of human adventitial progenitor cells (APCs) and therapeutic activity in a model of limb ischemia. Antigenic and functional features were analyzed throughout passaging in 15 saphenous vein (SV)-derived APC lines, of which 10 from SV leftovers of coronary artery bypass graft surgery and 5 from varicose SV removal. Moreover, 5 SV-APC lines were transplanted (8×10(5) cells, IM) in mice with limb ischemia. Blood flow and capillary and arteriole density were correlated with functional characteristics and DNA methylation/expressional markers of transplanted cells. We report successful expansion of tested lines, which reached the therapeutic target of 30 to 50 million cells in ≈10 weeks. Typical antigenic profile, viability, and migratory and proangiogenic activities were conserved through passaging, with low levels of replicative senescence. In vivo, SV-APC transplantation improved blood flow recovery and revascularization of ischemic limbs. Whole genome screening showed an association between DNA methylation at the promoter or gene body level and microvascular density and to a lesser extent with blood flow recovery. Expressional studies highlighted the implication of an angiogenic network centered on the vascular endothelial growth factor receptor as a predictor of microvascular outcomes. FLT-1 gene silencing in SV-APCs remarkably reduced their ability to form tubes in vitro and support tube formation by human umbilical vein endothelial cells, thus confirming the importance of this signaling in SV-APC angiogenic function. DNA methylation landscape illustrates different therapeutic activities of human APCs. Epigenetic screening may help identify determinants of therapeutic vasculogenesis in ischemic disease.

Novel human-derived extracellular matrix induces in vitro and in vivo vascularization and inhibits fibrosis

The inability to vascularize engineered organs and revascularize areas of infarction has been a major roadblock to delivering successful regenerative medicine therapies to the clinic. These investigations detail an isolated human extracellular matrix derived from the placenta (hPM) that induces vasculogenesis in vitro and angiogenesis in vivo within bioengineered tissues, with significant immune reductive properties. Compositional analysis showed ECM components (fibrinogen, laminin), angiogenic cytokines (angiogenin, FGF), and immune-related cytokines (annexins, DEFA1) in near physiological ratios. Gene expression profiles of endothelial cells seeded onto the matrix displayed upregulation of angiogenic genes (TGFB1, VEGFA), remodeling genes (MMP9, LAMA5) and vascular development genes (HAND2, LECT1). Angiogenic networks displayed a time dependent stability in comparison to current in vitro approaches that degrade rapidly. In vivo, matrix-dosed bioscaffolds showed enhanced angiogenesis and significantly reduced fibrosis in comparison to current angiogenic biomaterials. Implementation of this human placenta derived extracellular matrix provides an alternative to Matrigel and, due to its human derivation, its development may have significant clinical applications leading to advances in therapeutic angiogenesis techniques and tissue engineering.

Lipopolysaccharide (LPS)-mediated Angiopoietin-2-dependent Autocrine Angiogenesis Is Regulated by NADPH Oxidase 2 (Nox2) in Human Pulmonary Microvascular Endothelial Cells

Sepsis-mediated endothelial Angiopoeitin-2 (Ang2) signaling may contribute to microvascular remodeling in the developing lung. The mechanisms by which bacterial cell wall components such as LPS mediate Ang2 signaling in human pulmonary microvascular endothelial cells (HPMECs) remain understudied. In HPMEC, LPS-induced Ang2, Tie2, and VEGF-A protein expression was preceded by increased superoxide formation. NADPH oxidase 2 (Nox2) inhibition, but not Nox4 or Nox1 inhibition, attenuated LPS-induced superoxide formation and Ang2, Tie2, and VEGF-A expression. Nox2 silencing, but not Nox4 or Nox1 silencing, inhibited LPS-mediated inhibitor of κ-B kinase β (IKKβ) and p38 phosphorylation and nuclear translocation of NF-κB and AP-1. In HPMECs, LPS increased the number of angiogenic tube and network formations in Matrigel by >3-fold. Conditioned media from LPS-treated cells also induced angiogenic tube and network formation in the presence of Toll-like receptor 4 blockade but not in the presence of Ang2 and VEGF blockade. Nox2 inhibition or conditioned media from Nox2-silenced cells attenuated LPS-induced tube and network formation. Ang2 and VEGF-A treatment rescued angiogenesis in Nox2-silenced cells. We propose that Nox2 regulates LPS-mediated Ang2-dependent autocrine angiogenesis in HPMECs through the IKKβ/NF-κB and MAPK/AP-1 pathways.

Printing cancer cells into intact microvascular networks: a model for investigating cancer cell dynamics during angiogenesis.

While cancer cell invasion and metastasis are dependent on cancer cell-stroma, cancer cell-blood vessel, and cancer cell-lymphatic vessel interactions, our understanding of these interactions remain largely unknown. A need exists for physiologically-relevant models that more closely mimic the complexity of cancer cell dynamics in a real tissue environment. The objective of this study was to combine laser-based cell printing and tissue culture methods to create a novel ex vivo model in which cancer cell dynamics can be tracked during angiogenesis in an intact microvascular network. Laser direct-write (LDW) was utilized to reproducibly deposit breast cancer cells (MDA-MB-231 and MCF-7) and fibroblasts into spatially-defined patterns on cultured rat mesenteric tissues. In addition, heterogeneous patterns containing co-printed MDA-MB-231/fibroblasts or MDA-MB-231/MCF-7 cells were generated for fibroblast-directed and collective cell invasion models. Printed cells remained viable and the cells retained the ability to proliferate in serum-rich media conditions. Over a culture period of five days, time-lapse imaging confirmed fibroblast and MDA-MB-231 cell migration within the microvascular networks. Confocal microscopy indicated that printed MDA-MB-231 cells infiltrated the tissue thickness and were capable of interacting with endothelial cells. Angiogenic network growth in tissue areas containing printed cancer cells was characterized by significantly increased capillary sprouting compared to control tissue areas containing no printed cells. Our results establish an innovative ex vivo experimental platform that enables time-lapse evaluation of cancer cell dynamics during angiogenesis within a real microvascular network scenario.

The Role of miR-378a in Metabolism, Angiogenesis, and Muscle Biology.

MicroRNA-378a (miR-378a, previously known as miR-378) is one of the small noncoding RNA molecules able to regulate gene expression at posttranscriptional level. Its two mature strands, miR-378a-3p and miR-378a-5p, originate from the first intron of the peroxisome proliferator-activated receptor gamma, coactivator 1 beta (ppargc1b) gene encoding PGC-1β. Embedding in the sequence of this transcriptional regulator of oxidative energy metabolism implies involvement of miR-378a in metabolic pathways, mitochondrial energy homeostasis, and related biological processes such as muscle development, differentiation, and regeneration. On the other hand, modulating the expression of proangiogenic factors such as vascular endothelial growth factor, angiopoietin-1, or interleukin-8, influencing inflammatory reaction, and affecting tumor suppressors, such as SuFu and Fus-1, miR-378a is considered as a part of an angiogenic network in tumors. In the latter, miR-378a can evoke broader actions by enhancing cell survival, reducing apoptosis, and promoting cell migration and invasion. This review describes the current knowledge on miR-378a linking oxidative/lipid metabolism, muscle biology, and blood vessel formation.

Multiscale homogenization for fluid and drug transport in vascularized malignant tissues

A system of differential equations for coupled fluid and drug transport in vascularized (malignant) tissues is derived by a multiscale expansion. We start from mass and momentum balance equations, stated in the physical domain, geometrically characterized by the intercapillary distance (the microscale). The Kedem–Katchalsky equations are used to account for blood and drug exchange across the capillary walls. The multiscale technique (homogenization) is used to formulate continuum equations describing the coupling of fluid and drug transport on the tumor length scale (the macroscale), under the assumption of local periodicity; macroscale variations of the microstructure account for spatial heterogeneities of the angiogenic capillary network. A double porous medium model for the fluid dynamics in the tumor is obtained, where the drug dynamics is represented by a double advection–diffusion–reaction model. The homogenized equations are straightforward to approximate, as the role of the vascular geometry is recovered at an average level by solving standard cell differential problems. Fluid and drug fluxes now read as effective mass sources in the macroscale model, which upscale the interplay between blood and drug dynamics on the tissue scale. We aim to provide a theoretical setting for a better understanding of the design of effective anti-cancer therapies.

Abstract PR14: Quantitative and multiparameter analysis of tumor cell intravasation

Abstract Multiple steps in cancer metastasis, including tumorigenesis, tumor angiogenesis, invasion and colonization, can be successfully examined with well-established models. However, due to deficiency in experimental model systems that would accurately recapitulate tumor cell entry into the host vasculature, intravasation remains the least studied step in the metastatic cascade. Our laboratory has developed several independent avian and murine model systems to investigate the complex process of intravasation in a quantifiable and mechanistic manner. Chick embryo models of intravasation are based on the ability of a highly vascularized tissue, the chorioallantoic membrane (CAM), to sustain primary tumor development and spontaneous metastasis. Furthermore, the CAM serves as a repository of newly intravasated cells, which are quantified by human-specific Alu-qPCR. By employing different CAM models, the early events in spontaneous metastasis that ultimately lead to intravasation can be dissected and their individual input to overall intravasation can be quantified. These early processes include cell escape from the primary tumor, establishment of intratumoral and extratumoral angiogenic networks, invasion of adjacent stroma by escaped tumor cells, and migration of escaped tumor cells along tumor-associated blood vessels. In conjunction with confirmatory models of cancer metastasis in mice, we have identified several molecular systems that functionally contribute to the intravasation process. To investigate the specific mechanisms whereby tumor cells enter the angiogenic blood vessels, we have developed a new high-resolution confocal microscopy model based on acquisition and multiparameter analysis of primary microtumors imaged as entire units. This model was adapted to primary tumors developing within the mesoderm of the CAM as well as primary tumors developing in the dermis of mouse ear. In both avian and murine models, the volume of microtumors, the number of tumor cells per tumor, and the total volume of tumor-associated vasculature can be determined from 3D-reconstructed Z-stacks. Most importantly, the volume of intravascular tumor cells can be quantified by colocalizing tumor-vascular signals with a stringent threshold of a minimum volume of an individual tumor cell so that the cells that are localized within the vessels could be discriminated from the cells adherent to the abluminal vessel surface. In addition, high-resolution imaging of whole microtumors allows to visualize and quantify the tumor cells that appear as entering into the vasculature or in close association with the abluminal surface of the vessels. This approach allowed us for the fist time to visualize the intravasation events in numbers rendering statistical significance. By employing unique cell and protein markers in this novel system, the vascular cells interacting with the intravasating tumor cells can be identified and the candidate molecules involved in the close tumor-vascular interactions can be probed by treating the developing microtumors with specific reagents and small molecule inhibitors. By using these novel high-resolution models for intravasation, we have documented a majority of intravasation events within the primary tumor or in the nearest proximity to the primary tumor border. These data challenge the widely accepted concept that tumor cell escape, stromal invasion, and entry of escaped tumor cells into blood vessels adjacent to the primary tumor, are prerequisites of tumor cell intravasation. In contrast, our findings indicate that the bulk of cell intravasation events can take place within the primary tumor and prior to significant stromal invasion and migration of escaped tumor cells towards and along tumor-associated blood vessels. Our data suggest that tumor cell intravasation and metastatic spread can occur much earlier than commonly acknowledged by oncologists based on histological examination of resected tumors. This abstract is also presented as Poster A24. Citation Format: Elena I. Deryugina, William B. Kiosses, James P. Quigley. Quantitative and multiparameter analysis of tumor cell intravasation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR14.

Amphiphilic suramin dissolves Matrigel, causing an ‘inhibition’ artefact withinin vitroangiogenesis assays

The field of study concerning promotion and/or inhibition of angiogenesis has gathered much attention in the scientific community. A great deal of work has been invested towards defining reproducible assays to gauge for promotion or inhibition of angiogenesis in response to drug treatments or growth conditions. Two common components of these assays were noted by our group to have an unexpected and previously unreported interaction. Suramin is a commercially available compound, commonly used as a positive control for in vitro angiogenic inhibition assays. Matrigel is a popular extracellular substrate that supports angiogenic network formation when endothelial cells are cultured on its surface. However, our group demonstrated that suramin alone (without the presence of cells) will actively dissolve Matrigel, causing the extracellular matrix to transition from the gel-like physical state to a more liquid state. This causes cells on the Matrigel to congregate and sink to the bottom of the well. Therefore, previous observations of inhibition of endothelial cell angiogenesis through the incubation with suramin (including previous observations made by our group) are, largely, an artefact caused by suramin and matrix interaction rather than suramin and cells interaction, as previously reported. Our results suggest that the presence of sulphate groups and amphiphilic properties of suramin are likely responsible for the disruption of the matrix layer. We believe that this information is of prime importance to anyone using similar in vitro models, or employing suramin in any therapy or drug development assays.

NGF Promotes Hemodynamic Recovery in a Rabbit Hindlimb Ischemic Model Through trkA- and VEGFR2-dependent Pathways

Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter >500 μm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.

Abstract 4157: A role for miR-137 in enhancing efficacy of delphinidin in human glioblastoma.

Abstract Glioblastoma is the most lethal brain cancer in humans. Heterogeneity of cells in glioblastoma makes it extremely hard to control this tumor with a single therapeutic agent. Combination of therapeutic agents with diverse anti-cancer mechanisms must be used to manage this malignancy. Failures of conventional therapies and systemic toxicity have forced the search for newer agents, which may inhibit glioblastoma with less or no toxicity. Recently, microRNAs (miRs) are emerging as important regulators of different cellular processes such as proliferation, differentiation, and apoptosis in different cancers. Recent studies also suggest that the tumor suppressor miR-137 (TS miR-137) can induce differentiation and cell cycle arrest in human glioblastoma stem cells. In colorectal cancer, miR-137 is frequently silenced due to promoter hypermethylation. Use of 5-aza-2′-deoxycytidine (AzaC), an inhibitor of DNA methylation, may increase expression of miR-137 in glioblastoma with significant inhibition of proliferation of tumor cells. Delphinidin (DPN), which belongs to a class of natural chemicals from colored fruits, has been shown to inhibit cell growth and induce apoptosis in many different cancers. We hypothesized that combination therapy with miR-137 overexpression and DPN treatment in presence of AzaC could be an effective therapeutic strategy to control growth of glioblastoma. We used MTT assay to examine the doses (10 to 50 μM) of DPN for inhibition of cell proliferation in three glioblastoma cell lines (U87MG, T98G, and LN18). All cell lines demonstrated 50% inhibition of proliferation at 50 μM DPN. We performed Annexin V staining and flow cytometric analysis after treatment with AzaC, transfection of precursor miR-137 mimic, and treatment with DPN alone and in combination in all three glioblastoma cell lines. Overexpression of only miR-137 could induce apoptosis, which was dramatically increased when the cells were subjected to combination therapy with DPN in presence of AzaC. Then, we examined efficacy of combination of AzaC, miR-137, and DPN for inhibition of angiogenic network formation and invasion properties in all three glioblastoma cell lines. Co-culture of human microvascular endothelial (HMVE) cells with control U87MG, T98G, or LN18 cells prompted HMVE cells for capillary-like network formation, which was completely inhibited during co-culture of HMVE cells and glioblastoma cells treated with combination therapy. Following combination therapy, we observed suppression of expression of angiogenic factors (VEGF and b-FGF), growth factor receptor (EGFR), and invasive factors (MMP-2 and MMP-9) and also inhibition of Akt and NF-κB survival pathways in all three glioblastoma cell lines. So, combination of the methylation inhibitor AzaC, overexpression of the TS miR-137, and the relatively new flavonoid DPN could be a promising therapeutic strategy for inhibiting growth of different human glioblastoma cells. Citation Format: Mrinmay Chakrabarti, Naren L. Banik, Swapan K. Ray. A role for miR-137 in enhancing efficacy of delphinidin in human glioblastoma. [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 4157. doi:10.1158/1538-7445.AM2013-4157

New activities for the anti-tumor agent trabectedin: taking two birds with one stone

In the last decade the neoplastic cell-centric paradigm is now changing in a stroma-centric paradigm, focusing on the tumor micro-environment. This is populated by inflammatory leukocytes, activated fibroblasts and newly formed vessels, which influence each other in a complex and dynamic cross-talk with neoplastic cells. It is now established that in most cancers Tumor-Associated Macrophages (TAM) display pro-tumor functions (e.g. increase tumor cell proliferation and survival, aid tumor cell dissemination, promote angiogenesis and matrix remodelling) [1-3]. Particular emphasis in cancer therapy has been put in combination strategies to attack both tumor cells and the stroma. We have recently reported in a paper published in Cancer Cell in February 2013 that the anti-tumor agent trabectedin has important modulatory effects on the tumor micro-environment [4]. Trabectedin is an anti-tumor compound registered in Europe and in several other countries, for the second line treatment of soft tissue sarcoma (STS) and for ovarian cancer in combination with liposomal doxorubicin [5-7]. This compound was originally extracted from a marine organism, the Tunicate Ecteinascidia, and is now synthetically produced by the Spanish company PharmaMar. Trabectedin is a minor groove DNA binder able to efficiently block cancer cell proliferation. However, this is only part of its several mechanisms of action; further studies, revealed inhibition on the activity of selected transcription factors, which translate – among other effects – in the reduced production of several inflammatory mediators like the chemokines CCL2 and CXCL8, the cytokine IL-6 and the angiogenic factor VEGF [8]. An effect on the micro-environment was suspected by the clinical evidence of atypical patterns of response: for instance, in patients with liposarcoma, a decrease in tumor density may occur without tumor shrinkage for several courses, eventually resulting in size decrease. In other patients, clinical responses take place only after several cycles of therapy. Indeed, STS patients have less than 10% responses (by RECIST criteria) but up to 50% stable disease and long overall survival [6-7]. A particularly high susceptibility to trabectedin has been observed in translocated-related soft tissue sarcoma. In myxoid liposarcoma, whose pathogenesis is related to a balanced translocation most commonly t(12;16)(q13;q12) fusing the FUS gene with the CHOP gene, trabectedin induces a prolonged response with a decrease in the vascular network and adipocytic differentiation in a large fraction of patients [7]. The FUS-CHOP chimera alters the transcription of several genes including some encoding for proteins involved in pro-inflammatory and angiogenic networks and in the repression of adipocytic maturation; indeed, the exquisite antitumor activity of trabectedin seems to be related to its ability to inhibit the aberrant transcription factor FUS-CHOP [5]. In the Cancer Cell paper [4] we have used trabectedin in different mouse tumor models and investigated its effects on the tumor micro-environment, more specifically on leukocytes, angiogenesis, and on the expression of inflammatory mediators. We found that in treated mice trabectedin significantly decreased the number of blood monocytes and of tumor macrophages, having direct cytotoxic effects on this lineage, but not on other leukocyte subsets, such as neutrophils and lymphocytes (Fig. ​(Fig.1).1). We further demonstrated that treated tumors had a reduced vessel network. Similar results were found in human STS samples from patients receiving trabectedin as neo-adjuvant therapy: a marked decrease in blood vessels and in the tumor infiltrating macrophages. Using human liposarcoma xenografts grown in nude mice, we observed also a significant decrease in the expression of relevant mediators such as CCL2 and CXCL8 [8]. Figure 1 The anti-tumor agent trabectedin has direct effects on tumor cells, blocking cell cycle and proliferation Overall, this compound has two major targets: it directly inhibits the neoplastic compartment but also affects the tumor micro-environment, in particular the macrophages and their pro-tumoral functions. Trabectedin is currently used to treat a limited number of tumor types, and as second line therapy. Our findings that trabectedin has wider mechanism of action and strikes the whole micro-environment may stimulate its use in less advanced neoplastic settings and in tumors of different histology.

Photofrin Based Photodynamic Therapy and miR-99a Transfection Inhibited FGFR3 and PI3K/Akt Signaling Mechanisms to Control Growth of Human Glioblastoma In Vitro and In Vivo

Glioblastoma is the most common malignant brain tumor in humans. We explored the molecular mechanisms how the efficacy of photofrin based photodynamic therapy (PDT) was enhanced by miR-99a transfection in human glioblastoma cells. Our results showed almost similar uptake of photofrin after 24 h in different glioblastoma cells, but p53 wild-type cells were more sensitive to radiation and photofrin doses than p53 mutant cells. Photofrin based PDT induced apoptosis, inhibited cell invasion, prevented angiogenic network formation, and promoted DNA fragmentation and laddering in U87MG and U118MG cells harvoring p53 wild-type. Western blotting showed that photofrin based PDT was efficient to block the angiogenesis and cell survival pathways. Further, photofrin based PDT followed by miR-99a transfection dramatically increased miR-99a expression and also increased apoptosis in glioblastoma cell cultures and drastically reduced tumor growth in athymic nude mice, due to down regulation of fibroblast growth factor receptor 3 (FGFR3) and PI3K/Akt signaling mechanisms leading to inhibition of cell proliferation and induction of molecular mechanisms of apoptosis. Therefore, our results indicated that the anti-tumor effects of photofrin based PDT was strongly augmented by miR-99a overexpression and this novel combination therapeutic strategy could be used for controlling growth of human p53 wild-type glioblastomas both in vitro and in vivo.

VEGF, HIF-1α Expression and MVD as an Angiogenic Network in Familial Breast Cancer

Angiogenesis, which plays an important role in tumor growth and progression of breast cancer, is regulated by a balance between pro- and anti-angiogenic factors. Expression of vascular endothelial growth factor (VEGF) is up-regulated during hypoxia by hypoxia-inducible factor-1α (HIF-1α). It is known that there is an interaction between HIF-1α and BRCA1 carrier cancers, but little has been reported about angiogenesis in BRCA1-2 carrier and BRCAX breast cancers. In this study, we investigated the expression of VEGF and HIF-1α and microvessel density (MVD) in 26 BRCA1-2 carriers and 58 BRCAX compared to 77 sporadic breast cancers, by immunohistochemistry. VEGF expression in BRCA1-2 carriers was higher than in BRCAX cancer tissues (p = 0.0001). Furthermore, VEGF expression was higher in both BRCA1-2 carriers and BRCAX than the sporadic group (p<0.0001). VEGF immunoreactivity was correlated with poor tumor grade (p = 0.0074), hormone receptors negativity (p = 0.0206, p = 0.0002 respectively), and MIB-1-labeling index (p = 0.0044) in familial cancers (BRCA1-2 and BRCAX). The percentage of nuclear HIF-1α expression was higher in the BRCA1-2 carriers than in BRCAX cancers (p<0.05), and in all familial than in sporadic tumor tissues (p = 0.0045). A higher MVD was observed in BRCA1-2 carrier than in BRCAX and sporadic cancer tissues (p = 0.002, p = 0.0001 respectively), and in all familial tumors than in sporadic tumors (p = 0.01). MVD was positively related to HIF-1α expression in BRCA1-2 carriers (r = 0.521, p = 0.006), and, in particular, we observed a highly significant correlation in the familial group (r = 0.421, p<0.0001). Our findings suggest that angiogenesis plays a crucial role in BRCA1-2 carrier breast cancers. Prospective studies in larger BRCA1-2 carrier series are needed to improve the best therapeutic strategies for this subgroup of breast cancer patients.

Locus Chemotherapy in Cancer Angiogenesis

A cancer angiogenesias is a complex system. Its robustness is supported by the signaling elements from cancer hypoxic microenvironment. The signaling angiogenic networks have nodal organization of its elements. The AKT is crucial macromolecule in the nodal angiogenic system of solid cancers, leukemia, lymphoma, plasmocytoma and myeloma. Targeting AKT locus by the locus chemotherapy, angiogenesis and all cancer system will fail. The AKT dephosphorylation phenomenon is induced by the redox balance change, causing conversion of the positive loops into normal feedback loops and leading to abolishment of cancer angiogenesis and cancer progression.

RAIN-Droplet: a novel 3D in vitro angiogenesis model

Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion, there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed not only as outgrowths from endothelial cells on the surface of the droplets, but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof of principle for the utility of the model showing significant inhibition of sprout formation (P<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay, we also demonstrate a novel dose-dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (P<0.05) more endothelial sprouts compared with controls over a 48-h period. Higher doses of sorafenib (5 μM) resulted in a significant (P<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib, which may impact the clinical application and dosing regimen of that drug.

Abstract 3472: Molecular mechanisms of growth inhibition of human brain tumor stem cells and glioblastoma LN18 and U138MG cells by combination of curcumin and paclitaxel

Abstract Glioblastoma is the deadliest human brain tumor that responds poorly to conventional chemotherapeutic agents and continues to form tumor mass because of existence of highly chemoresistant human brain tumor stem cells (HBTSC). There is an urgent need for new therapeutic strategy that can target HBTSC as well as other glioblastoma cells. We explored the synergistic efficacy of a low dose of curcumin (CCM) and a low dose of paclitaxel (PTX) to induce apoptosis and inhibit cell survival, proliferation, invasion, and angiogenesis in HBTSC and human glioblastoma LN18 (p53 mutant and PTEN proficient) and U138MG (p53 mutant and PTEN mutant) cells. Western blotting indicated the highest expression of the cancer stem cell markers aldehyde dehydrogenase 1 (ALDH1) and CD133 in HBTSC when compared with LN18 and U138MG cells. Further, confocal laser scanning immunofluorescence microscopy confirmed more expression of ALDH1, presently considered as a novel cancer stem cell marker, in HBTSC than that in LN18 and U138MG cells. Our MTT assay revealed that combination of 20 µM CCM and 10 nM PTX worked synergistically and more effectively decreased the cell viability than the single drug treatments in HBTSC, LN18, and U138MG cells. Our in situ Wright staining and light microscopy showed that combination of CCM and PTX was highly effective in inducing morphological features of apoptosis in these cells. We also confirmed the induction of the highest amounts of apoptosis by this combination therapy in the cells after Annexin V/PI staining. The molecular mechanisms of induction of apoptosis required activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, and mitochondrial release of cytochrome c, Smac, and apoptosis-inducing factor (AIF). Further, increase in phosphorylation of Bcl-2 (p-Bcl-2) following combination therapy could promote Bax homodimerization for enhancing mitochondrial release of pro-apoptotic factors into the cytosol. Increased expression of calpain and caspase-3 and their proteolytic activities confirmed the completion of apoptotic process in all cell lines. Our in vitro Matrigel invasion assay showed efficacy of the combination therapy in inhibiting invasion of cells. Efficacy of this combination therapy was also evident in reduced expression of cell survival and proliferation factors (p-Akt, NF-κB, survivin, and hTERT), invasion factors (MMP-2 and MMP-9), and angiogenic factors (VEGF, b-FGF, and CD31) most prominently in HBTSC and very clearly in glioblastoma LN18 and U138MG cells. Moreover, our in vitro angiogenic network formation assay indicated that this combination therapy significantly reduced the angiogenic capability of HBTSC, LN-18, and U138MG cells. Taken together, our results clearly show that combination of CCM and PTX is a promising therapy for controlling malignant growth of HBTSC and other glioblastoma cells. 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 3472. doi:1538-7445.AM2012-3472

Abstract LB-16: DACH1 inhibited prostate cancer cellular proliferation and interleukin-6 signaling

Abstract The Retinal Determination Gene Network (RDGN), consisting of dachshund (dac), eyes absent (eya), and sin oculis (so), is best known for its role in eye specification. Recently, coordinate abnormal expression of the DACH1/Eya/Six complex has been observed in human tumors supporting a tumor promoting role of Six and Eya family members, and a tumor-suppressor function of DACH. Oncogenes sustain host-cell interactions, promoting invasiveness, recruiting inflammatory mediators and developing angiogenic networks. Ras and Myc promote tumor angiogenesis through up-regulation of VEGF and the inflammatory mediator, interleukin-8(IL-8) and interleukin-6 (IL-6). Altered expressions of the IL-6 and IL-8 genes are seen in many cancers including prostate cancer. Hormone-resistant prostate carcinoma cell lines produce large amounts of IL-6 and IL-8 compared to hormone responsive prostate cancer cell lines. The expression of IL-8 and IL-6 are linked to poor prognosis and metastatic phenotype in prostate cancer. Recent finding demonstrated that IL-6 regulates the dynamic equilibrium between cancer stem cells and non-stem cancer cells in human breast and prostate cancer. Moreover, IL-6 alone can transform non-tumorigenic benign prostate epithelial cells and further progress to invasive phenotype. We had previously reported that the expression of DACH1 was reduced in human prostate cancer and DACH1 inhibited androgen signaling transduction. To explore the role of DACH1 in androgen resistant prostate cancer, PC3 cells stably expressing DACH1 were established. DACH1 inhibited cellular proliferation evaluated by MTT assay and growth curve. Microarray analyses demonstrated DACH1 inhibited mRNA expression of GRO, IL-6, IL-8 by 75%, 80% and 90%, respectively. However, there was no change of mRNA expression for IL-6 and IL-8 receptor. Ectopic expression of DACH1 in PC-3 cells significantly decreased tumor growth compared with vector control group (p&amp;lt;0.001), and DS domain was required. It has been reported that stable expression IL-6 in SV40T immortalized non-tumorigenic prostate epithelial cell line p69 induced EMT and acquired malignant phenotypes through autocrine loop IL6 and IGF-1R signaling. To further test the functional interaction of DACH1 with IL-6, a table cells expressing IL-6 alone or IL-6 with DACH1 were deployed. Ectopic expression of DACH1 attenuated activation of IGF-1R by IL-6 as evaluated by phosphorylation of IGF-1R. Subcutaneous implantation study showed an in vivo tumor growth initiated by IL-6 in p69 was inhibited by more than 60% with the expression of DACH1. Together, our studies suggest that DACH1 is a potential tumor suppressor of prostate through repression of IL-6 signaling. 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 LB-16. doi:1538-7445.AM2012-LB-16