fibrin and Glioblastoma

Fibrinogen-like protein 2 (Fgl2), a member of the fibrinogen family, can be expressed as a membrane-associated protein with coagulation activity or in a secreted form possessing unique immune suppressive functions. The biological importance of Fgl2 is evident within viral-induced fibrin depositing inflammatory diseases and malignancies and provides a compelling rationale for Fgl2 expression to not only be considered as a disease biomarker but also as a therapeutic target. This article will provide a comprehensive review of the currently known biological properties of Fgl2 and clarifies future scientific directives.

Topics: Animals; Biomarkers; Blood Coagulation; Costimulatory and Inhibitory T-Cell Receptors; Fibrin; Fibrinogen; Glioblastoma; Humans; Immunosuppressive Agents; Inflammation; Lymphocyte Activation; T-Lymphocytes, Regulatory

Purpose: Glioblastoma multiform (GBM) is the most aggressive glial neoplasm. Researchers have exploited the\ fact that GBMs are highly vascularized tumors. Anti-angiogenic strategies including those targeting VEGF pathway\ have been emerged for treatment of GBM. Previously, we reported the anti-inflammatory effect of atorvastatin on\ GBM cells. In this study, we investigated the anti-angiogenesis and apoptotic activity of atorvastatin on GBM cells.\ Methods: Different concentrations of atorvastatin (1, 5, 10μM) were used on engineered three-dimensional (3D)\ human tumor models using glioma spheroids and Human Umbilical Vein Endothelial cells (HUVECs) in fibrin gel\ as tumor models. To reach for these aims, angiogenesis as tube-like structures sprouting of HUVECs were observed\ after 24 hour treatment with different concentrations of atorvastatin into the 3-D fibrin matrix and we focused on it by\ angiogenesis antibody array. After 48 hours exposing with different concentrations of atorvastatin, cell migration of\ HUVECs were investigated. After 24 and 48 hours exposing with different concentrations of atorvastatin VEGF, CD31,\ caspase-3 and Bcl-2 genes expression by real time PCR were assayed. Results: The results showed that atorvastatin\ has potent anti-angiogenic effect and apoptosis inducing effect against glioma spheroids. Atorvastatin down-regulated\ the expression of VEGF, CD31 and Bcl-2, and induced the expression of caspase-3 especially at 10μM concentration.\ These effects are dose dependent. Conclusion: These results suggest that this biomimetic model with fibrin may provide\ a vastly applicable 3D culture system to study the effect of anti-cancer drugs such as atorvastatin on tumor malignancy\ in vitro and in vivo and atorvastatin could be used as agent for glioblastoma treatment.

Topics: Angiogenesis Inhibitors; Anticholesteremic Agents; Atorvastatin; Cell Culture Techniques; Cell Movement; Cells, Cultured; Fibrin; Gels; Glioblastoma; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Neovascularization, Pathologic; Spheroids, Cellular

Tumor-homing cytotoxic stem cell (SC) therapy is a promising new approach for treating the incurable brain cancer glioblastoma (GBM). However, problems of retaining cytotoxic SCs within the post-surgical GBM resection cavity are likely to significantly limit the clinical utility of this strategy. Here, we describe a new fibrin-based transplant approach capable of increasing cytotoxic SC retention and persistence within the resection cavity, yet remaining permissive to tumoritropic migration. This fibrin-based transplant can effectively treat both solid and post-surgical human GBM in mice. Using our murine model of image-guided model of GBM resection, we discovered that suspending human mesenchymal stem cells (hMSCS) in a fibrin matrix increased initial retention in the surgical resection cavity 2-fold and prolonged persistence in the cavity 3-fold compared to conventional delivery strategies. Time-lapse motion analysis revealed that cytotoxic hMSCs in the fibrin matrix remain tumoritropic, rapidly migrating from the fibrin matrix to co-localize with cultured human GBM cells. We encapsulated hMSCs releasing the cytotoxic agent TRAIL (hMSC-sTR) in fibrin, and found hMSC-sTR/fibrin therapy reduced the viability of multiple 3-D human GBM spheroids and regressed established human GBM xenografts 3-fold in 11 days. Mimicking clinical therapy of surgically resected GBM, intra-cavity seeding of therapeutic hMSC-sTR encapsulated in fibrin reduced post-surgical GBM volumes 6-fold, increased time to recurrence 4-fold, and prolonged median survival from 15 to 36 days compared to control-treated animals. Fibrin-based SC therapy could represent a clinically compatible, viable treatment to suppress recurrence of post-surgical GBM and other lethal cancer types.

Topics: Animals; Brain Neoplasms; Cell Death; Cell Line, Tumor; Cell Movement; Disease Progression; Fibrin; Glioblastoma; Humans; Mice, Nude; Spheroids, Cellular; Stem Cell Transplantation; Tissue Scaffolds; Treatment Outcome

Glioblastoma multiform (GBM) is one of the most common and highly aggressive primary brain tumors that thought to be of glial cells origin. The new available therapy for glioblastoma is based on better understanding of molecular malignant progression in this tumor. It is better to identify key molecular targets stimulating signaling pathways that lead to initiation of apoptosis for treatment of glioblastoma. Tumorigenesis broadly is controlled by tumor microenvironment and design of best biomimetic culture systems dependency on these conditions allow for in vitro and in vivo tumor modeling for studies of cancer cells behavior to drugs. We engineered three-dimensional (3D) human tumor models using U87 glioma cells in fibrin gel that mimic microenvironmental feature of glioblastoma in vivo. In this study, atorvastatin was used as a kind of statins for induction of apoptosis, and inhibition of migration and invasion in glioma cells.. To reach for these aims, 3D model of glioma in fibrin gel was used with different concentrations of atorvastatin (1, 5, 10μM) to assay apoptotic genes expression by real time PCR and Tunel assay. After 24 and 48h exposing with different concentrations of atorvastatin, cell migration and invasion of tumor cells were investigated.. The results showed atorvastatin induced apoptosis of glioma spheroids dose- dependently. The most likely mechanisms are the induction of apoptosis by caspase-8- caspase-3 signaling pathway. The invasion and migration of U87 spheroid cells decreased after 48h especially with 10μM concentration of atorvastatin.. Finally these results suggest that this biomimetic model with fibrin may provide a vastly applicable 3D culture system to study the effect of anti-cancer drugs such as atrovastatin on tumor malignancy in vitro and in vivo and atorvastatin could be used as anticancer agent for glioblastoma treatment.

Topics: Antineoplastic Agents; Apoptosis; Atorvastatin; Cell Movement; Dose-Response Relationship, Drug; Fibrin; Gels; Glioblastoma; Humans; Spheroids, Cellular; Tumor Cells, Cultured

Glioblastoma multiforme (GBM) is the most aggressive central nervous system (CNS) tumor because of its fast development, poor prognosis, difficult control and terrible mortality. Poor penetration and retention in the glioblastoma parenchyma were crucial challenges in GBM nanomedicine therapy. Nanoparticle diameter can significantly influence the delivery efficiency in tumor tissue. Decreasing nanoparticle size can improve the nanoparticle penetration in tumor tissue but decrease the nanoparticle retention effect. Therefore, small nanoparticles with high retention effect in tumor are urgently needed for effective GBM drug delivery. In present study, a small nanoparticle drug delivery system was developed by conjugating fibrin-binding peptide CREKA to Polyamidoamine (PAMAM) dendrimer, where PEGylated PAMAM is used as drug carrier due to its small size and good penetration in tumor and CREKA is used to target the abundant fibrin in GBM for enhanced retention in tumor. In vitro binding ability tests demonstrated that CREKA can significantly enhanced nanoparticle binding with fibrin. In vivo fluorescence imaging of GBM bearing nude mice, ex vivo brain imaging and frozen slices fluorescence imaging further revealed that the CREKA-modified PAMAM achieved higher accumulation and deeper penetration in GBM tissue than unmodified one. These results indicated that the CREKA-modified PAMAM could penetrate the GBM tissue deeply and enhance the retention effect, which was a promising strategy for brain tumor therapy.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Dendrimers; Drug Delivery Systems; Fibrin; Glioblastoma; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Oligopeptides

Glioblastoma-targeted drug delivery systems facilitate efficient delivery of chemotherapeutic agents to malignant gliomas, while minimizing systemic toxicity and side effects. Taking advantage of the fibrin deposition that is characteristic of tumors, we constructed spherical, Cy7-labeled, targeting micelles to glioblastoma through the addition of the fibrin-binding pentapeptide, cysteine-arginine-glutamic acid-lysine-alanine, or CREKA. Conjugation of the CREKA peptide to Cy7-micelles increased the average particle size and zeta potential. Upon intravenous administration to GL261 glioma bearing mice, Cy7-micelles passively accumulated at the brain tumor site via the enhanced permeability and retention (EPR) effect, and Cy7-CREKA-micelles displayed enhanced tumor homing via active targeting as early as 1 h after administration, as confirmed via in vivo and ex vivo imaging and immunohistochemistry. Biodistribution of micelles showed an accumulation within the liver and kidneys, leading to micelle elimination via renal clearance and the reticuloendothelial system (RES). Histological evaluation showed no signs of cytotoxicity or tissue damage, confirming the safety and utility of this nanoparticle system for delivery to glioblastoma. Our findings offer strong evidence for the glioblastoma-targeting potential of CREKA-micelles and provide the foundation for CREKA-mediated, targeted therapy of glioma.

Topics: Animals; Brain; Brain Neoplasms; Carbocyanines; Drug Carriers; Drug Delivery Systems; Fibrin; Glioblastoma; Male; Mice; Mice, Inbred C57BL; Micelles; Oligopeptides

For a nanoparticulate drug-delivery system, crucial challenges in brain-glioblastoma therapy are its poor penetration and retention in the glioblastoma parenchyma. As a prevailing component in the extracellular matrix of many solid tumors, fibrin plays a critical role in the maintenance of glioblastoma morphology and glioblastoma cell differentiation and proliferation. We developed a new drug-delivery system by conjugating polyethylene glycol-polylactic acid nanoparticles (NPs) with cysteine-arginine-glutamic acid-lysine-alanine (CREKA; TNPs), a peptide with special affinity for fibrin, to mediate glioblastoma-homing and prolong NP retention at the tumor site. In vitro binding tests indicated that CREKA significantly enhanced specific binding of NPs with fibrin. In vivo fluorescence imaging of glioblastoma-bearing nude mice, ex vivo brain imaging, and glioblastoma distribution demonstrated that TNPs had higher accumulation and longer retention in the glioblastoma site over unmodified NPs. Furthermore, pharmacodynamic results showed that paclitaxel-loaded TNPs significantly prolonged the median survival time of intracranial U87 glioblastoma-bearing nude mice compared with controls, Taxol, and NPs. These findings suggested that TNPs were able to target the glioblastoma and enhance retention, which is a valuable strategy for tumor therapy.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Drug Delivery Systems; Fibrin; Glioblastoma; Humans; Lactates; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Oligopeptides; Paclitaxel; Polyethylene Glycols; Random Allocation; Survival Analysis; Xenograft Model Antitumor Assays

Perillyl alcohol (POH) is a naturally occurring monoterpene with antiangiogenic and anti-tumoral properties. This chemotherapeutic agent has proven effectiveness in several clinical trials, including an ongoing phase I, comprising patients with recurrent glioblastoma multiform (GBM) under treatment with POH by intranasal administration. Proteomics offers tools to distinguish states of biological systems according to protein expression differences and therefore, can be used to gain pathological insights and to search for disease follow-up biomarkers. In this work, a differential gel electrophoresis (DIGE) proteomic approach was used to search for plasma proteins that correlated with the disease progression in 10 of these patients. Our results pointed antithrombin (down) and fibrinogen (up) regulated after a four months treatment deserving to be further verified as prognostic markers for this treatment. Possible links between tumor progression and anti-thrombin expression level are also discussed.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Central Nervous System Neoplasms; Disease Progression; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinogen; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mass Spectrometry; Monoterpenes; Proteomics; Recurrence

Directional migration of capillaries towards tumour implants is generally assumed to be regulated by chemotaxis. Preliminary evidence has also been presented for the existence of a reverse chemotactic signalling pathway, with capillaries attracting tumour cells via paracrine factors. By using a variety of endothelial cell types and tumour cell lines, this study has systematically investigated chemotaxis between endothelial cells and tumour cells in two- and three-dimensional systems. Checkerboard analysis revealed faint attraction of human umbilical vein endothelial cells (HUVECs), but not porcine aortic endothelial cells (PAECs), by tumour cells. In reverse, both PAECs and HUVECs potently induced chemotactic migration of tumour cells. Using a microcarrier-based fibrin gel assay, directional migration of endothelial cells towards tumour cells was not observed. In reverse, tumour cells were strongly attracted by endothelial cells. Identification of endothelium-derived chemotactic molecules may provide a valuable approach for the treatment of tumour metastasis.

Topics: Animals; Capillaries; Cell Communication; Cell Culture Techniques; Chemotaxis; Culture Media, Conditioned; Endothelium, Vascular; Fibrin; Glioblastoma; Humans; Melanoma; Microscopy, Phase-Contrast; Neoplasms; Neovascularization, Pathologic; Swine; Tumor Cells, Cultured

Hepatocyte growth factor activator inhibitor type-2/placental bikunin (HAI-2/PB) is a serine proteinase inhibitor that contains 2 Kunitz-domains and a presumed transmembrane domain. It has broad inhibitory spectra against various serine proteinases showing potent inhibitory activities not only to hepatocyte growth factor activator but also to plasmin, trypsin and kallikreins. In this study, we investigated the expression of HAI-2/PB in human gliomas in vivo and the effects of HAI-2/PB on the fibrinolytic and invasive capabilities of human glioblastoma cells in vitro. With RNA blot analysis, HAI-2/PB mRNA was expressed in normal brain and in low-grade astrocytomas, but was hardly detectable in anaplastic astrocytomas and glioblastomas, indicating that its expression levels were inversely correlated with the histological grade of human gliomas. To further explore the possible role of HAI-2/PB in glioma progression, cultured human glioblastoma cell lines (U251 and YKG-1) were transiently transfected with an expression vector harboring human HAI-2/PB cDNA. Subsequent analysis indicated that the expression of HAI-2/PB suppressed the fibrinolytic activities of both glioblastoma cell lines. Moreover, HAI-2/PB inhibited Matrigel invasion of U251 and YKG-1 cells by 30% and 64%, respectively. This anti-invasive effect appeared to be mediated primarily by the inhibitory activity of HAI-2/PB against the serine proteinase-dependent matrix degradation. These findings suggest that the reduced expression of HAI-2/PB is possibly involved in the progression of human gliomas.

Topics: Astrocytoma; Blotting, Northern; Brain Neoplasms; Collagen; DNA Primers; Drug Combinations; Fibrin; Glioblastoma; Hepatocyte Growth Factor; Humans; Immunoblotting; Laminin; Membrane Glycoproteins; Neoplasm Invasiveness; Proteoglycans; Proto-Oncogene Proteins c-met; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Serine Endopeptidases; Serine Proteinase Inhibitors; Transfection; Trypsin Inhibitor, Kunitz Soybean

The diffuse and extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modifications of the proteolysis of extracellular matrix components. A key molecule in regulating plasminogen-mediated extracellular proteolysis is the urokinase-type plasminogen activator (uPA). To investigate the role of uPA in the invasive process of brain tumors, we stably transfected a human glioblastoma cell line SNB19 with a vector capable of expressing an antisense transcript complementary to the 1020 bases at the 3' end of the uPA cDNA. Parental, vector-, and antisense construct-stably transfected cell lines were analyzed for uPA mRNA transcript by Northern blot analysis, for uPA enzyme activity by zymography, and for uPA protein levels by Western blotting. The levels of uPA mRNA, protein, and enzyme activities were significantly lower in antisense clones than in parental and vector controls. Radioreceptor binding studies demonstrated that uPA receptor levels remained the same in parental, vector-, and antisense-transfected cells. The antisense-transfected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Green fluorescent protein (GFP) expressing parental and antisense transfectants was generated for detection in mouse brain tissue without any posttreatment. Intracerebral injection of antisense stable transfectants significantly reduced tumor formation compared with that in controls. Our results suggested that down-regulation of uPA expression may be a feasible approach to reducing the malignancy and invasiveness of glial tumors.

Topics: Animals; Blotting, Northern; Blotting, Western; Brain; DNA, Antisense; Fibrin; Genetic Therapy; Glioblastoma; Humans; Mice; Mice, Nude; Microscopy, Confocal; Neoplasm Invasiveness; Protein Binding; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transfection; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Xenograft Model Antitumor Assays

The glycosaminoglycan hyaluronan, which supports tumor cell migration and metastasis, interferes with fibrin polymerization and leads to increased fiber size and porosity of fibrin clots. Here we have studied the proportionate effect of fibrin polymerization on hyaluronan-mediated migration of glioblastoma cells. The structural and physical properties of hyaluronan-containing fibrin gels were analyzed by turbidity measurement, laser scanning microscopy, compaction assay, and calculation of pore size by liquid permeation. When fibrin polymerized in the presence of hyaluronan or dextran, the resulting gels strongly stimulated cell migration, and migration significantly correlated with fiber mass-to-length ratios and pore diameters. In contrast, cell migration was not induced by addition of hyaluronan to supernatants of already polymerized gels. Hyaluronan-mediated migration was inhibited in fibrin gels by antibodies to alphav- and beta1integrins and the disintegrin echistatin, but not by antibodies to the hyaluronan receptor CD44 (up to 50 microg/ml). As a control, we show that anti-CD44 (10 microg/ml) inhibited cell migration on a pure hyaluronan matrix using a two-dimensional Boyden chamber system. In contrast to three-dimensional migration, the migration of cells on the surfaces of variably structured fibrin gels was not significantly different, indicating that increased gel permeability (porosity) may account for hyaluronan-mediated migration. We conclude that, in complex three-dimensional substrates, the predominant effect of hyaluronan on cell migration might be indirect and requires modulation of fibrin polymerization.

Topics: Animals; Antigens, CD; Biopolymers; Cell Movement; Dextrans; Fibrin; Gels; Glioblastoma; Humans; Hyaluronan Receptors; Hyaluronic Acid; Integrin alphaV; Integrin beta1; Intercellular Adhesion Molecule-1; Neoplasm Invasiveness; Neoplasm Metastasis; Tumor Cells, Cultured

Glioblastomas extensively invade the surrounding normal brain tissue, with a concomitant expression of various proteolytic enzymes, in particular urokinase-type plasminogen activator (uPA). In this study we used cis-diamminedichloroplatinum (cisplatin) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), commonly used anti-cancer drugs for the treatment of glioblastomas, to study the expression of uPA in three human glioblastoma cell lines in vitro. Cells were treated with 25 microM cisplatin and 50 microM BCNU, and uPA levels were estimated by fibrin zymography during a 72-h time course. Treatment of glioblastoma cells with cisplatin resulted in significantly decreased levels of uPA in serum-free conditioned medium and cell extracts, compared to BCNU-treated and untreated cell lines. Quantitative levels of uPA enzyme activity assessed by scanning laser densitometry and uPA protein by ELISA using antibody against uPA showed decreased levels of uPA in cisplatin-treated glioma cell lines relative to BCNU and untreated cell lines. Our results suggest that anti-tumor compound, cisplatin, may exert its anti-neoplastic effects by inhibiting uPA in malignant glioblastomas.

Topics: Antineoplastic Agents; Antineoplastic Agents, Alkylating; Carmustine; Cisplatin; Culture Media, Serum-Free; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fibrin; Glioblastoma; Humans; Neoplasm Invasiveness; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Extravascular, intratumoral fibrin deposition is frequently observed within and around neoplastic tissue and has been implicated in various aspects of tumour growth. This is the first report on the presence and distribution of fibrinogen/fibrin in primary (14 glioblastomas) and metastatic (nine samples of lung cancer origin) human brain tumours detected by immunofluorescent techniques. All tissue samples showed specific staining for fibrinogen/fibrin. In glioblastomas fibrin deposits could be detected within and around tumour foci, while in metastatic brain tumours the tumour cell nodules were surrounded by fibrin deposits localized almost exclusively in the connective tissue compartment of tumours. Double-labelling reactions for von Willebrand factor and fibrinogen/fibrin has revealed that fibrin deposition occurred throughout the tumour stroma independently of tumour vasculature. The overlapping reactions for fibrinogen/fibrin and factor XIII subunit A, as well as the urea-insolubility of the deposits indicate the crosslinked, highly stabilized nature of fibrin both within and around tumours. Staining with Ki M7 monoclonal antibody specific for phagocytosing macrophages showed these cells to be scattered in the nonnecrotic areas in glioblastomas and to be accumulated at the interface of tumorous parenchyma and connective tissue in both primary and metastatic tumours. The close association between fibrin deposition and macrophage accumulation strongly suggests the active participation of tumour associated macrophages in the formation of stabilized intratumoral fibrin network in human brain neoplasms.

Topics: Blood Coagulation; Brain Neoplasms; Connective Tissue; Factor XIII; Fibrin; Fibrinolysis; Glioblastoma; Humans; Lung Neoplasms; Macrophages; Neoplasm Proteins

Topics: Animals; Antigens; Brain Neoplasms; Carcinoma, Ehrlich Tumor; Fibrin; Fibrinogen; Fluorescent Antibody Technique; Glioblastoma; Glioma; Humans; Immunodiffusion; Immunoelectrophoresis; Iodine Isotopes; Meningioma; Mice; Neoplasms, Experimental; Plasminogen; Rabbits; Radiometry; Radionuclide Imaging; Rats; Sarcoma, Yoshida