The strange Microenvironment of Glioblastoma.

Glioblastoma (GB) is the most common and aggressive primary brain tumor, with poor patient survival and lack of effective therapies. Late advances trying to decipher the composition of the GB tumor microenvironment (TME) emphasized its role in tumor progression and potentialized it as a therapeutic target. Many components participate critically to tumor development and expansion such as blood vessels, immune cells or components of the nervous system. Dysmorphic tumor vasculature brings challenges to optimal delivery of cytotoxic agents currently used in clinics. Also, massive infiltration of immunosuppressive myeloid cells and limited recruitment of T cells limits the success of conventional immunotherapies. Neuronal input seems also be required for tumor expansion. In this review, we provide a comprehensive report of vascular and immune component of the GB TME and their cross talk during GB progression.

CXCL4L1-fibstatin cooperation inhibits tumor angiogenesis, lymphangiogenesis and metastasis.

Anti-angiogenic and anti-lymphangiogenic drugs slow tumor progression and dissemination. However, an important difficulty is that a tumor reacts and compensates to obtain the blood supply needed for tumor growth and lymphatic vessels to escape to distant loci. Therefore, there is a growing consensus on the requirement of multiple anti-(lymph)angiogenic molecules to stop cell invasion efficiently. Here we studied the cooperation between endogenous anti-angiogenic molecules, endostatin and fibstatin, and a chemokine, the Platelet Factor-4 variant 1, CXCL4L1. Anti-angiogenic factors were co-expressed by IRES-based bicistronic vectors and their cooperation was analyzed either by local delivery following transduction of pancreatic adenocarcinoma cells with lentivectors, or by distant delivery resulting from intramuscular administration in vivo of adeno-associated virus derived vectors followed by tumor subcutaneous injection. In this study, fibstatin and CXCL4L1 cooperate to inhibit endothelial cell proliferation, migration and tubulogenesis in vitro. No synergistic effect was found for fibstatin-endostatin combination. Importantly, we demonstrated for the first time that fibstatin and CXCL4L1 not only inhibit in vivo angiogenesis, but also lymphangiogenesis and tumor spread to the lymph nodes, whereas no beneficial effect was found on tumor growth inhibition using molecule combinations compared to molecules alone. These data reveal the synergy of CXCL4L1 and fibstatin in inhibition of tumor angiogenesis, lymphangiogenesis and metastasis and highlight the potential of IRES-based vectors to develop anti-metastasis combined gene therapies.

The cytoprotective drug amifostine modifies both expression and activity of the pro-angiogenic factor VEGF-A.

BACKGROUND: Amifostine (WR-2721, delivered as Ethyol) is a phosphorylated aminothiol compound clinically used in addition to cis-platinum to reduce the toxic side effects of therapeutic treatment on normal cells without reducing their efficacy on tumour cells. Its mechanism of action is attributed to the free radical scavenging properties of its active dephosphorylated metabolite WR-1065. However, amifostine has also been described as a potent hypoxia-mimetic compound and as a strong p53 inducer; both effects are known to potently modulate vascular endothelial growth factor (VEGF-A) expression. The angiogenic properties of this drug have not been clearly defined. METHODS: Cancer cell lines and endothelial cells were used in culture and treated with Amifostine in order to study (i) the expression of angiogenesis related genes and proteins and (ii) the effects of the drug on VEGF-A induced in vitro angiogenesis. RESULTS: We demonstrated that the treatment of several human cancer cell lines with therapeutical doses of WR-1065 led to a strong induction of different VEGF-A mRNA isoforms independently of HIF-1alpha. VEGF-A induction by WR-1065 depends on the activation of the eIF2alpha/ATF4 pathway. This up-regulation of VEGF-A mRNA was accompanied by an increased secretion of VEGF-A proteins fully active in stimulating vascular endothelial cells (EC). Nevertheless, direct treatment of EC with amifostine impaired their ability to respond to exogenous VEGF-A, an effect that correlated to the down-regulation of VEGFR-2 expression, to the reduction in cell surface binding of VEGF-A and to the decreased phosphorylation of the downstream p42/44 kinases. CONCLUSIONS: Taken together, our results indicate that amifostine treatment modulates tumour angiogenesis by two apparently opposite mechanisms - the increased VEGF-A expression by tumour cells and the inhibition of EC capacity to respond to VEGF-A stimulation.

Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms.

To study possible functional differences of the 18-kD and high molecular weight forms of basic fibroblast growth factor (bFGF), we have examined the effect of endogenous production of different bFGF forms on the phenotype of NIH 3T3 cells. Cells transfected with cDNAs coding for either 18-kD bFGF (18-kD bFGF) or all four molecular forms (18, 22, 22.5, 24 kD; wild type [WT] bFGF) exhibit increased migration and decreased FGF receptor number compared to parental cells. However, migration and FGF receptor number of cells transfected with a cDNA coding only for high molecular weight bFGF (22, 22.5, and 24 kD; HMW bFGF) were similar to that of parental cells transfected with vector alone. Cells expressing HMW, 18 kD, or WT bFGF grew to high saturation densities in 10% serum. However, only cells expressing HMW or WT bFGF grew in low serum. Cell surface or metabolic labeling of the different cell types followed by immunoprecipitation with anti-bFGF antibody showed primarily cell surface-associated 18-kD bFGF. In addition, when cells expressing exclusively HMW bFGF were transfected with a cDNA coding for 18-kD bFGF, migration was increased, bFGF receptors were down-regulated, and 18-kD bFGF was found on the cell surface. Cells expressing 18-kD bFGF transfected with a cDNA encoding FGF receptor-2 lacking the COOH-terminal domain (dominant negative bFGF receptor) exhibited a flat morphology and decreases in migration and saturation density. Cells expressing HMW bFGF transfected with the dominant negative bFGF receptor continued to grow to a high saturation density, proliferated in low serum, and exhibited no morphological changes. These results indicate that increased cell migration and FGF receptor down-regulation are mediated by the extracellular interaction of 18-kD bFGF with its cell surface receptor. Growth in low serum may be stimulated by the intracellular action of HMW bFGF through mechanisms independent of the presence of a cell surface receptor. Thus, the different molecular forms of bFGF may act through distinct but convergent pathways.

A model of guided cell self-organization for rapid and spontaneous formation of functional vessels.

Most achievements to engineer blood vessels are based on multiple-step manipulations such as manual sheet rolling or sequential cell seeding followed by scaffold degradation. Here, we propose a one-step strategy using a microfluidic coextrusion device to produce mature functional blood vessels. A hollow alginate hydrogel tube is internally coated with extracellular matrix to direct the self-assembly of a mixture of endothelial cells (ECs) and smooth muscle cells (SMCs). The resulting vascular structure has the correct configuration of lumen, an inner lining of ECs, and outer sheath of SMCs. These "vesseloids" reach homeostasis within a day and exhibit the following properties expected for functional vessels (i) quiescence, (ii) perfusability, and (iii) contractility in response to vasoconstrictor agents. Together, these findings provide an original and simple strategy to generate functional artificial vessels and pave the way for further developments in vascular graft and tissue engineering and for deciphering the angiogenesis process.

Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR).

The chemokine receptor CXCR3 plays important roles in angiogenesis, inflammation and cancer. Activation studies and biological functions of CXCR3 are complex due to the presence of spliced isoforms. CXCR3-A is known as a pro-tumor receptor whereas CXCR3-B exhibits anti-tumor properties. Here, we focused on the conformational change of CXCR3-A and CXCR3-B after agonist or antagonist binding using Plasmon Waveguide Resonance (PWR). Agonist stimulation induced an anisotropic response with very distinct conformational changes for the two isoforms. The CXCR3 agonist bound CXCR3-A with higher affinity than CXCR3-B. Using various concentrations of SCH546738, a CXCR3 specific inhibitor, we demonstrated that low SCH546738 concentrations (≤1 nM) efficiently inhibited CXCR3-A but not CXCR3-B's conformational change and activation. This was confirmed by both, biophysical and biological methods. Taken together, our study demonstrates differences in the behavior of CXCR3-A and CXCR3-B upon ligand activation and antagonist inhibition which may be of relevance for further studies aimed at specifically inhibiting the CXCR3A isoform.

Inhibition of fibroblast growth factor/fibroblast growth factor receptor activity in glioma cells impedes tumor growth by both angiogenesis-dependent and -independent mechanisms.

We undertook a series of systematic studies to address the role of fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) activity in tumor growth and angiogenesis. We expressed dominant-negative FGFR2 (FGFR2-DN) or FGFR1 (FGFR1-DN) in glioma C6 cells by using constitutive or tetracycline-regulated expression systems. Anchorage-dependent or independent growth was inhibited in FGFR-DN-expressing cells. Tumor development after xenografting FGFR-DN-expressing cells in immunodeficient mice or after transplantation in rat brain was strongly inhibited. Quantification of microvessels demonstrated a significant decrease in vessel density in tumors derived from FGFR-DN-expressing cells. Furthermore, in a rabbit corneal assay, the angiogenic response after implantation of FGFR-DN-expressing cells was decreased. In tumors expressing FGFR-DN, vascular endothelial growth factor expression was strongly inhibited as compared with control tumor. These results indicate that inhibition of FGF activity may constitute a dominant therapeutic strategy in the treatment of FGF-producing cerebral malignancies and may disrupt both angiogenesis-dependent and -independent signals required for glioma growth and invasion.

Simultaneous inhibition of glioma angiogenesis, cell proliferation, and invasion by a naturally occurring fragment of human metalloproteinase-2.

Angiogenesis, tumor cell proliferation, and migration are the hallmarks of solid tumors, such as gliomas. This study demonstrates that a fragment derived from the autocatalytic digestion of matrix metalloproteinase (MMP)-2, called PEX, acts simultaneously as an inhibitor of glioma angiogenesis, cell proliferation, and migration. PEX is detected in the cultured medium of various human glioma, endothelial, breast, and prostate carcinoma cell lines. PEX is purified from the medium of glioma cell lines by chromatography, where PEX is constitutively expressed as a free and a TIMP-2-bound form. In human glioma tissue, PEX expression correlates with histological subtype and grade and with alpha v beta 3 integrin expression to which it is bound. Systemic administration of PEX to s.c. and intracranial human glioma xenografts results in a 99% suppression of tumor growth with no signs of toxicity. Thus, PEX is a very promising candidate for the treatment of human malignant gliomas.

Low-dose chemotherapy combined with an antiangiogenic drug reduces human glioma growth in vivo.

This study evaluates the efficacy of the combination of an antiangiogenic drug and conventional chemotherapeutics for the treatment of experimental human gliomas. As an antiangiogenic, we used recombinant human PEX, a fragment of matrix metalloproteinase-2 that we have previously shown to have a significant antimitotic, anti-invasive, and antiangiogenic properties against human glioblastoma in vitro and in vivo. We used carboplatin and etoposide as the two chemotherapeutic drugs routinely used in our institution (Ospedale Maggiore de Milano) for the treatment of malignant gliomas. Conventional chemotherapeutic drugs were administered at high dose or at a low and semicontinuous regimen. Combined treatment of high-dose chemotherapy and PEX did not produce an improvement of survival in comparison with chemotherapy alone, but it was associated with a decrease in tumor volume, vascularity, and proliferative index and an increased apoptosis. All of these animals experienced severe side effects. The longest survival was documented in animals submitted to low and semicontinuous chemotherapy and antiangiogenic treatment. This regimen was associated with no side effects, marked decrease in tumor volume, vascularity, and proliferative index, and an increased apoptosis. Our data suggest that low-dose chemotherapy in combination with PEX can be successfully used against human malignant glioma in vivo.

Angiogenic factors are hematopoietic growth factors and vice versa.

Angiogenic factors are potent growth factors promoting proliferation and differentiation of vascular endothelial cells. Recent evidence suggest that these factors also promote hematopoietic cell growth. The major group of angiogenic growth factors is the fibroblast growth factor (FGF) family. Two prototypes, acidic FGF and basic FGF, have been demonstrated to interact with granulopoiesis and megakaryocytopoiesis. Basic FGF stimulates granulopoiesis in long term bone marrow cultures while acidic and basic FGF promote megakaryocytopoiesis. These effects are presumably mediated via specific FGF receptors, that have been identified in bone marrow and leukemia cell lines. Besides the FGF family, angiogenic inhibitors such as platelet factor-4 (PF-4) have been found to exhibit an inhibitory effect on megakaryocytopoiesis. In contrast, it has been demonstrated that hematopoietic growth factors including granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin promote angiogenesis in vivo and in vitro. In light of these recent observations and the common origin of endothelial cells and hematopoietic cells, it is suggested that angiogenic factors are hematopoietic growth factors and vice versa. However, these data must be interpreted with caution and a careful in vivo evaluation should be done before these observed in vivo effects are proven to be significant to the physiopathology of hematopoiesis or angiogenesis.

INHIBITION OF ERN1 SIGNALING ENZYME AFFECTS HYPOXIC REGULATION OF THE EXPRESSION OF E2F8, EPAS1, HOXC6, ATF3, TBX3 AND FOXF1 GENES IN U87 GLIOMA CELLS.

Hypoxia as well as the endoplasmic reticulum stress are important factors of malignant tumor growth and control of the expression of genes, which regulate numerous metabolic processes and cell proliferation. Furthermore, blockade of ERN1 (endoplasmic reticulum to nucleus 1) suppresses cell proliferation and tumor growth. We studied the effect of hypoxia on the expression of genes encoding the transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F), and HOXC6 (homeobox C6) in U87 glioma cells with and without ERN1 signaling enzyme function. We have established that hypoxia enhances the expression of HOXC6, E2F8, ATF3, and EPAS1 genes but does not change TBX3 and FOXF1 gene expression in glioma cells with ERNI function. At the same time, the expression level of all studied genes is strongly decreased, except for TBX3 gene, in glioma cells without ERN1 function. Moreover, the inhibition of ERN1 signaling enzyme function significantly modifies the effect of hypoxia on the expression of these transcription factor genes. removes or introduces this regulation as well as changes a direction or magnitude of hypoxic regulation. Present study demonstrates that fine-tuning of the expression of proliferation related genes depends upon hypoxia and ERN1-mediated endoplasmic reticulum stress signaling and correlates with slower proliferation rate of glioma cells without ERN1 function.

Target molecules for anti-angiogenic therapy: from basic research to clinical trials.

There is growing evidence that anti-angiogenic drugs will improve future therapies of diseases like cancer, rheumatoid arthritis and ocular neovascularisation. However, it is still uncertain which kind of substance, out of the large number of angiogenesis inhibitors, will prove to be a suitable agent to treat these human diseases. There are currently more than 30 angiogenesis inhibitors in clinical trials and a multitude of promising new candidates are under investigation in vitro and in animal models. Important therapeutic strategies are: suppression of activity of the major angiogenic regulators like vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF); inhibition of function of alphav-integrins and matrix metalloproteinases (MMPs); the exploitation of endogenous anti-angiogenic molecules like angiostatin, endostatin or thrombospondin. Given the wide spectrum of diseases which could be treated by anti-angiogenic compounds, it is important for today's clinicians to understand their essential mode of action at a cellular and molecular level. Here we give an in-depth overview of the basic pathophysiological mechanisms underlying the different anti-angiogenic approaches used to date based on the most recent fundamental and clinical research data. The angiogenesis inhibitors in clinical trials are presented and promising future drug candidates are discussed.

New insights in the biology of fibroblast growth factor-2.

Fibroblast growth factor (FGF)-2 stimulates endothelial cell proliferation and is a potent angiogenic molecule in vitro and in vivo. In this review, we have focused on recent findings that relate to the mechanism of action and function of FGF-2. FGF-2 is expressed as four different isoforms: one 18 kDa FGF-2 form that is mainly cytoplasmic and three high molecular weight (HMW) FGF-2 forms that are preferentially localized in the nucleus. It has been demonstrated that these different isoforms lead to specific cellular phenotypes when expressed in cells. HMW FGF-2 controls proliferation by a receptor-independent mechanism, whereas 18 kDa FGF-2 stimulates migration by autocrine receptor activation. Intracellularly, HMW FGF-2 may directly associate with molecules that are involved in growth control. The action of FGF-2 at the cell surface may be altered by angiogenic inhibitors. Angiogenic inhibitors may directly interfere with FGF receptor activation or downstream signaling and thus inhibit FGF activity.

Significance of angiogenesis in tumour progression and metastasis.

Angiogenesis is defined as a vascular neoformation usually of capillary origin. This phenomenon is important during development and under several physiological and or pathological conditions. In recent years, progress has been made to understand this phenomenon at the molecular level. This includes the identification of potent angiogenic factors, the appreciation of the role of proteases, the importance of the extracellular matrix, and the emerging characterisation of signal transduction pathways in endothelial cells. Two important participants in angiogenesis are molecules from the fibroblast growth factor (FGF) and the transforming growth factor-beta (TGF-beta) family. In our laboratory, we have extensively studied the roles and mechanisms of action of the major FGF prototype, FGF-2 and of the TGF-beta member, TGF-beta 1. Different isoforms of FGF-2 have been previously described, a high molecular weight (HMW) form associated with the nucleus and 18 kDa bFGF that is cytoplasmic. These two forms of FGF-2 also exhibit different functions when expressed endogenously. TGF-beta is formed from a latent complex by plasmin-dependent and plasmin-independent pathways. With the exception of macrophages, the plasmin-dependent pathway requires coculture conditions, urokinase, and the concentration of TGF-beta on the cell surface by the mannose-6-phosphate receptor and transglutaminase. Other important angiogenic modulators include vascular endothelial growth factor (VEGF) and angiostatin. The nature of the tumour angiogenesis factor is not yet known with certainty, but several identified and not yet identified angiogenic factors may act in concert. It is hoped that an angiostatic treatment for cancer will be derived from these molecular studies.

Prelining of polytetrafluoroethylene grafts with cultured human endothelial cells isolated from varicose veins.

Prelining graft material with autologous functioning endothelial cells might be one of the ultimate requirements to obtain a biocompatible surface. Accordingly, endothelial cells from stripped varicose veins were enzymatically harvested and grown on a fibronectin matrix. Proliferation was investigated in defined medium supplemented with various concentrations of endothelial cell growth supplement (ECGS) (25, up to 150 micrograms/ml) and heparin (10(-8), up to 10(-5)mol/L): optimal growth required both 150 micrograms/ml of ECGS and 10(-5)mol/L heparin. Under these conditions, cell culture achieved cell densities at a confluence of 1.2 +/- 1.1 10(5) cells/cm2 with a doubling time of 1 day. During subcultivation cultured cells consistently exhibited characteristic cobblestone morphology and immunofluorescent staining for factor VIII-related antigen, whereas prostacyclin production determined by enzyme-linked immunosorbent assay for 6-keto-prostaglandin F1 alpha reached 21.1 +/- 1.2 ng/10(6) cells after 15-minute stimulation with 1 U/ml of thrombin. Heparin-containing culture medium-endothelial cell interactions were particularly studied, and with iodine 125-heparin, binding was demonstrated with an apparent dissociation constant (Kd) of 0.36 +/- 0.04 mumol/L. A cold storage technique at -80 degrees C was sought, and freezed cells were used to coat in vitro polytetrafluoroethylene grafts. Protein-treated material allowed cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy. These data validate the feasibility of prelining grafts in vitro with autologous functioning endothelial cells. This approach may be useful in improving the performance of small-caliber vascular grafts according to prostacyclin production and surface-bound heparin of these cells.

Recombinant acidic human fibroblast growth factor (aFGF) stimulates murine megakaryocyte colony formation in vitro.

Recombinant acidic human fibroblast growth factor (aFGF) significantly stimulated the formation of megakaryocyte colonies and the size of MK colonies as well as individual MKs in vitro in mice. When aFGF was combined with recombinant mouse interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-6 (IL-6) at their optimal doses, a synergistic action was found between aFGF and IL-3. The activity of aFGF could be completely abrogated by a monoclonal antimouse IL-6 antibody which specifically neutralized the action of mouse IL-6 but not human IL-6. These data indicate that aFGF provides positive growth signals of megakaryocyte progenitor cells, which can give rise to a synergistic action in the presence of IL-3 and which can be abrogated by the antimouse IL-6 antibody.

Long-term inhibition of glioma growth by systemic administration of human PEX.

AIM: The growth of gliomas depends on the balance of factors stimulating or inhibiting angiogenesis, tumor cell invasion and proliferation. The administration of endogenous inhibitors to experimental human gliomas in animal models resulted in a significant inhibition of tumor growth. It is becoming apparent that resistance can develop over time to many types of endogenous inhibitors and seems to be influenced by the tumor type and system of delivery. METHODS: We recently isolated a potent endogenous inhibitor, called human PEX, from human glioma cells in culture. Human PEX is a potent inhibitor of angiogenesis, tumor and endothelial cell proliferation and migration. In this paper, we investigated the ability of human PEX to sustain inhibition of glioma growth for a prolonged period of time. We initially developed a recombinant form of the inhibitor and showed that this form had similar in vitro and in vivo activities to the natural one. Human PEX was then administered to nude mice intracranial human glioma model, in combination with metronomic chemotherapy, for a period of 185 days, starting 15 days after tumor cells implantation. RESULTS: Our data showed that the systemic administration of human PEX mantained a very prolonged inhibition of glioma growth (50% survival of animals treated with 2 mg/kg/days was 160 days vs 24 days of the control) and had a synergistic effect with low dose chemotherapy. Histological analysis of tumors, showed that treatment with PEX was associated with a decrease of vascularity, cell proliferation, and increase in apoptosis. CONCLUSION: These data indicate that human PEX controls tumor growth by separate mechanisms. In addition, treatment with PEX produced well delineated tumors, indicating the persistence of a direct anti-invasive effect of the molecule even after a prolonged period of treatment.

[Angiogenesis and cancer]. / Angiogenèse et cancer.

Tumor invasion and dissemination is tightly controlled by angiogenesis. In recent years, a number of angiogenic factors and inhibitors have been identified. However, the molecular mechanisms leading to this phenomenon are still incompletely understood. In this review, we focus on recent developments in the angiogenesis field. We will summarize our present knowledge about the molecular mechanisms involved in angiogenesis and about the factors that control this phenomenon. We will then shortly discuss the pharmacological modulation and the therapeutic and clinical implications for cancer biology.

[Angiogenesis and neoangiogenesis]. / Angiogenèse et néoangiogenèse.

Angiogenesis, the development of new capillary networks from the normal vasculature, is a fundamental process during embryogenesis. In adulthood, angiogenesis contributes to corpus luteum formation, placental implantation and wound healing and is also required in some pathological conditions such as several intraocular syndromes, growth of solid tumors, and metastasis. Many factors are involved in the regulation of neovascularisation among which FGF-2 (fibroblast growth factor-2) and VEGF (vascular endothelial growth factor) are considered as key inducers. Their biological activity is highly controlled by extracellular matrix components and angiostatic factors. Better understanding of the molecular mechanisms regulating angiogenesis should contribute to the development of new molecules to be used for the treatment of neovascularisation-linked diseases.