Effects of targeting the VEGF and PDGF pathways in diffuse orthotopic glioma models

Currently available compounds that interfere with VEGF-A signalling effectively inhibit angiogenesis in gliomas, but influence diffuse infiltrative growth to a much lesser extent. Development of a functional tumour vascular bed not only involves VEGF-A but also requires platelet-derived growth factor receptor-β (PDGFRβ), which induces maturation of tumour blood vessels. Therefore, we tested whether combined inhibition of VEGFR and PDGFRβ increases therapeutic benefit in the orthotopic glioma xenograft models E98 and E473, both displaying the diffuse infiltrative growth that is characteristically observed in most human gliomas. We used bevacizumab and vandetanib as VEGF(R) inhibitors, and sunitinib to additionally target PDGFRβ. We show that combination therapy of sunitinib and vandetanib does not improve therapeutic efficacy compared to treatment with sunitinib, vandetanib or bevacizumab alone. Furthermore, all compounds induced reduction of vessel leakage in compact E98 tumour areas, resulting in decreased detectability of these mostly infiltrative xenografts in Gd-DTPA-enhanced MRI scans. These data show that inhibition of VEGF signalling cannot be optimized by additional PDGFR inhibition and support the concept that diffuse infiltrative areas in gliomas are resistant to anti-angiogenic therapy.     

Human and Rat Glioma Growth,Invasion, and Vascularization in a Novel Chick Embryo Brain Tumor Model

The mechanisms that control the insidiously invasive nature of malignant gliomas are poorly understood, and their study would be facilitated by an in vivo model that is easy to manipulate and inexpensive. The developing chick embryo brain was assessed as a new xenograft model for the production, growth, and study of human and rat glioma cell lines. Three established glioma lines (U-87 MG, C6, and 9L) were injected into chick embryo brain ventricles on embryonic day (E) 5 and brains were examined after several days to two weeks after injection. All glioma lines survived, produced vascularized intraventricular tumors, and invaded the brain in a manner similar to that in rodents. Rat C6 glioma cells spread along vasculature and also invaded the neural tissue. Human U-87 glioma cells migrated along vasculature and exhibited slight invasion of neural tissue. Rat 9L gliosarcoma cells were highly motile, but migrated only along the vasculature. A derivative of 9L cells that stably expressed the cell surface adhesion molecule NgCAM/L1 was produced and also injected into chick embryo brain ventricles to see if this protein could facilitate tumor cell migration away from the vasculature into areas such as axonal tracts. 9L/NgCAM cells, however, did not migrate away from the vasculature and, thus, this protein alone cannot be responsible for diffuse invasiveness of some gliomas. 9L/NgCAM cell motility was assessed in vitro using sophisticated time-lapse microscopy and quantitative analysis, and was significantly altered compared to parental 9L cells. These studies demonstrate that the chick embryo brain is a successful and novel xenograft model for mammalian gliomas and demonstrate the potential usefulness of this new model for studying glioma tumor cell growth, vascularization, and invasiveness.     

Monitoring of tumor growth and post-irradiation recurrence in a diffuse intrinsic pontine glioma mouse model

Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy because of its diffuse infiltrative growth pattern. Translational research suffers from the lack of a representative DIPG animal model. Hence, human E98 glioma cells were stereotactically injected into the pons of nude mice. The E98 DIPG tumors presented a strikingly similar histhopathology to autopsy material of a DIPG patient, including diffuse and perivascular growth, brainstem- and supratentorial invasiveness and leptomeningeal growth. Magnetic resonance imaging (MRI) was effectively employed to image the E98 DIPG tumor. [(18) F] 3'-deoxy-3'-[(18) F]fluorothymidine (FLT) positron emission tomography (PET) imaging was applied to assess the subcutaneous (s.c.) E98 tumor proliferation status but no orthotopic DIPG activity could be visualized. Next, E98 cells were cultured in vitro and engineered to express firefly luciferase and mCherry (E98-Fluc-mCherry). These cultured E98-Fluc-mCherry cells developed focal pontine glioma when injected into the pons directly. However, the diffuse E98 DIPG infiltrative phenotype was restored when cells were injected into the pons immediately after an intermediate s.c. passage. The diffuse E98-Fluc-mCherry model was subsequently used to test escalating doses of irradiation, applying the bioluminescent Fluc signal to monitor tumor recurrence over time. Altogether, we here describe an accurate DIPG mouse model that can be of clinical relevance for testing experimental therapeutics in vivo.     

Early detection of human glioma sphere xenografts in mouse brain using diffusion MRI at 14.1 T

Glioma models have provided important insights into human brain cancers. Among the investigative tools, MRI has allowed their characterization and diagnosis. In this study, we investigated whether diffusion MRI might be a useful technique for early detection and characterization of slow‐growing and diffuse infiltrative gliomas, such as the proposed new models, LN‐2669GS and LN‐2540GS glioma sphere xenografts. Tumours grown in these models are not visible in conventional T₂‐weighted or contrast‐enhanced T₁‐weighted MRI at 14.1 T. Diffusion‐weighted imaging and diffusion tensor imaging protocols were optimized for contrast by exploring long diffusion times sensitive for probing the microstructural alterations induced in the normal brain by the slow infiltration of glioma sphere cells. Compared with T₂‐weighted images, tumours were properly identified in their early stage of growth using diffusion MRI, and confirmed by localized proton MR spectroscopy as well as immunohistochemistry. The first evidence of tumour presence was revealed for both glioma sphere xenograft models three months after tumour implantation, while no necrosis, oedema or haemorrhage were detected either by MRI or by histology. Moreover, different values of diffusion indices, such as mean diffusivity and fractional anisotropy, were obtained in tumours grown from LN‐2669GS and LN‐2540GS glioma sphere lines. These observations highlighted diverse tumour microstructures for both xenograft models, which were reflected in histology. This study demonstrates the ability of diffusion MRI techniques to identify and investigate early stages of slow‐growing, invasive tumours in the mouse brain, thus providing a potential imaging biomarker for early detection of tumours in humans.     

Utility of the F98 rat glioma model for photodynamic therapy.

A syngeneic rat brain tumor model consisting of F98 glioma cells in Fischer rats was investigated for its utility in PDT studies. Results of in vitro studies demonstrated that the F98 cell line was sensitive to ALA-PDT, especially at low light irradiances. Histological examination revealed that F98 tumors share many fundamental characteristics with human GBMs, including rapid growth and infiltrative behavior. ALA-PDT in normal brain showed that high light fluences (26 J) delivered at relatively low powers (10 mW) are capable of causing significant edema. These findings suggest that light irradiation parameters should be chosen carefully when treating tumor-bearing animals. Rats inoculated with F98 cells preincubated in ALA showed a significant survival advantage following light exposure. Taken together, the results suggest that the F98 rat glioma model is appropriate for PDT studies of malignant gliomas.     

Potentials and limitations of adenovirus-p53 gene therapy for brain tumors

We investigated the antineoplastic potentials of recombinant adenovirus containing wild-type p53 cDNA (Ad5CMV-p53) for malignant gliomas. In four human glioma cell lines (U-251 and LG expressing endogenous mutant p53, and U-87 and EFC-2 expressing wild-type p53) and two rat glioma cell lines (9L and C6, each expressing mutant and wild-type p53), gene transfer efficiency determined by X-gal staining and Western blotting was varied (10-99% at 10-500 multiplicity of infection, MOI). Growth inhibitory effect was drastic (>90% at 100 MOI) in U-251 cells and only moderate or minimal in other cell lines harboring wild-type p53 or low gene transfer efficiency. Ex vivo transduction of U-251 cells with Ad5CMV-p53 suppressed the in vivo tumorigenicity of the cells. Histopathologic examination for Ad5CMV-p53 toxicity to rat brains showed inflammatory reactions in half of the tested brains at 10(8) MOI. U-251 cells were inoculated intracerebrally in nude mice and injected Ad5CMV-p53 into the tumor, in which neither the tumor suppression nor the survival benefit was observed. In conclusion, heterogeneity of the cellular subpopulations of malignant glioma in p53 status, variable and insufficient gene delivery to tumor, and adenoviral toxicity to brain at higher doses may be limiting factors to be solved in developing adenovirus-p53 gene therapy for malignant gliomas.     

Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas

DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.     

Expression and immunohistochemical localization of cathepsin L during the progression of human gliomas

Recent studies suggest that cysteine proteinase cathepsin L is involved in the process of tumor invasion and metastasis. We examined cathepsin L activity in brain tumor tissue samples by an enzymatic assay, and cathepsin L protein content by enzyme-linked immunoadsorbent assays and Western blotting to determine whether increased levels of cathepsin L correlate with the progression of human gliomas. Native and acid-activatable cathepsin L activities were highest in glioblastomas followed by anaplastic astrocytomas and were lowest in low-grade gliomas and normal brain tissues. Significantly higher amounts of an M _r 29 000 cathepsin L were present in glioblastomas and anaplastic astrocytomas than in normal brain tissues and low-grade glioma tissue extracts. Using specific antibodies to cathepsin L, we also studied its cellular distribution by immunohistochemical procedures. Higher diffuse cathepsin L immunoreactivity was found in glioblastomas than in low-grade gliomas and normal brain tissue samples. Finally, the addition of cathepsin L antibody inhibits the invasion of glioblastoma cell lines through Matrigel invasion assay. These results suggest the expression of cathepsin L is dramatically upregulated in malignant gliomas and correlates with the malignant progression of human gliomas in vivo.     

CD133+胶质瘤干细胞在胶质瘤中的分布

恶性胶质瘤是人类最致命的肿瘤之一,因其血管丰富和弥漫浸润性生长,使得手术不能全部切除并极易复发,患者预后差.因胶质瘤干细胞(GSCs)是胶质瘤发生、发展的源头,因而越来越多的受到关注.干细胞标志物CD133广泛表达于人体多种肿瘤中,同时,它也足研究得最多且应用最广泛的胶质瘤干细胞表面标志物.了解CD133+胶质瘤干细胞在胶质瘤中的分布可为临床靶向治疗恶性胶质瘤提供依据.     

CD44 expression and hyaluronic acid binding of malignant glioma cells

The mechanisms leading to rapid invasive growth of malignant gliomas are poorly understood. Expression of the hyaluronic acid (HA) receptor CD44 and adhesion to HA are involved in invasive properties. Our previous studies have shown that malignant glioma cells are able to adhere to extracellular HA. Here we investigated expression of the hyaluronic acid receptor CD44 protein in five human (T98G, A172, U87MG, 86HG39, 85HG66) and two rat (C6, 9L) glioma cell lines. Influence of anti-CD44 antibody and hyaluronidase-preincubation on the HA-binding was determined using HA/BSA (bovine serum albumin)-coated culture plates. While all gliomas were highly positive for CD44 with no differences in the number of positive staining cells, median fluorescence intensity decreased as follows: C6>T98G>9L>85HG66> 86HG39>A172> U87MG. Using HA/BSA coated culture plates the relative levels of specific adhesion to HA were determined as T98G>A172>9L>86HG39>U87MG> 85HG66. C6 cells failed to bind HA specifically. Incubation with anti-human-CD44 MAb significantly decreased HA-adhesion of T98G, A172, 85HG66 and U87MG human glioma cells. However the binding capacity was completely blocked only in 85HG66 cells. The three other cell lines kept a specific HA-adhesion after saturation of the receptor. Hyaluronidase pretreatment markedly enhanced HA-adhesion of C6 and 9L rat glioma cells. These results suggest that (i) HA-adhesion of malignant glioma cells is mainly, but not only, mediated by CD44, (ii) expression of CD44 does not correspond with adhesion capacity and (iii) cell-bound glycosaminoglycans may influence glioma cell adhesion to extracellular HA.     

升压联合化疗对大鼠脑胶质瘤形态学变化的影响

目的:通过体外细胞化疗和体内对大鼠胶质瘤升压联合化疗后, 观察体外细胞形态变化和体内肿瘤抑制作用, 观察变压联合化疗对胶质瘤的治疗效果。方法:采用胶质瘤体外培养、化疗。体内采用脑定位注射接种法, 复制大鼠脑胶质瘤动物模型。体内进行联合升压化疗。结果: 体外培养瘤细胞, 使用化疗药后, 可见细胞体积增大、胞质呈颗粒样变、胞内容物外溢等形态学变化;体内化疗后肿瘤体积变小, 瘤内可见坏死区等病理变化;升压化疗组肿瘤内血流增加、药物浓度增加;动物生存期延长。 结论:升压联合化疗对大鼠脑胶质瘤具有明显抑制作用, 可使动物生存期延长。     

Repetitive photodynamic therapy of malignant brain tumors.

The probability of achieving local control with current single-shot, intraoperative photodynamic therapy (PDT) treatments of intracerebral gliomas seems improbable due to the length of time required to deliver adequate light fluences to depths of 1-2 cm in the resection margin. Additionally, due to the short doubling time of many malignant gliomas, the kill rate per cell doubling indicates that it seems unlikely that a single treatment would be sufficient to prevent tumor recurrence. Multiple repetitive treatments would therefore seem required. In this publication we primarily review our work examining the effects of repetitive PDT on malignant brain tumor cells both in vitro and in vivo. The in vitro therapy response of human and rat glioma spheroids to 5-aminolevulinic acid (ALA)-mediated PDT in repetitive form was investigated. The results indicated that PDT repeated at relatively long intervals (weeks) was more effective at inhibiting spheroid growth than either daily fractionated PDT or single-treatment regimes. The in vivo response to repetitive treatment was evaluated in a rodent glioma model where BT4C cell line tumors were established in the brains of inbred BD-IX rats. Microfluorometry of frozen tissue sections showed that PpIX is produced with a 10-20:1 tumor to normal tissue selectivity ratio 4 hr after ALA injection. Preliminary evidence of increased efficacy of repetitive PDT and low fluence rate treatment is presented.     

Immunotherapy of Diffuse Gliomas: Biological Background, Current Status and Future Developments

Despite aggressive multimodal treatment approaches, the prognosis for patients with diffuse gliomas remains disappointing. Glioma cells often extensively infiltrate in the surrounding brain parenchyma, a phenomenon that helps them to escape surgical removal, radiation exposure and chemotherapy. Moreover, conventional therapy is often associated with considerable local and systemic side effects. Therefore, the development of novel therapeutic approaches is essential to improve the outcome of these patients. Immunotherapy offers the opportunity to specifically target residual radio—and chemoresistant tumor cells without damaging healthy neighboring brain tissue. Significant progress has been made in recent years both in understanding the mechanisms of immune regulation in the central nervous system (CNS) as well as tumor-induced and host-mediated immunosuppression elicited by gliomas. In this review, after discussing the special requirements needed for the initiation and control of immune responses in the CNS, we focus on immunological phenomena observed in glioma patients, discuss different immunological approaches to attack glioma-associated target structures and touch on further strategies to improve the efficacy of immunotherapy of gliomas.     

HAUSP promoted the growth of glioma cells in vitro and in vivo via stabilizing NANOG

ObjectiveTo investigate the role and mechanisms of HAUSP (Herpesvirus Associated Ubiquitin Specific Protease) and NANOG in pathogenesis of malignant human gliomas progression.MethodsLentivirus-mediated HAUSP over-expression and RNAiHAUSP mediated HAUSP down-regulation were established in the glioma cells (U87 and U251 cell lines). Firstly, Real-time qPCR, western-blot (WB) and immunofluorescence staining were performed to detect mRNA levels, protein expressions and deposition of HAUSP and NANOG in the glioma cells, respectively. Then cell proliferation, invasion, apoptosis and xenograft tumor growth in nude mice were assessed by using cell counting kit-8 (CCK-8) assay, transwell assay, flow cytometry (FCM) and Hematoxylin-Eosin (HE) staining.ResultsWe first demonstrated HAUSP was significantly increased in lentivirus- mediated HAUSP over-expression cells compared to the Control group. HAUSP over-expression could upregulate genes involved in proliferation and invasion such as NANOG. However, the mRNA of NANOG had no significant changes. Similarly, in RNAiHAUSP mediated HAUSP down-regulation group, HAUSP were significantly decreased compared to the Control group. Simultaneously, NANOG protein were decreased significantly, which decreased the proliferation and invasion, increased the apoptosis rate of glioma cells. Finally, low expression of HAUSP could suppress xenograft tumors growth in nude mice in different periods.ConclusionThis study revealed that HAUSP-NANOG pathway is a key target to inhibit glioma cells proliferation, and NANOG play important role in the formation and evolution of glioma cells. The regulation of HAUSP could change the biological activity of glioma cells through regulate NANOG expression.     

αB-Crystallin Is Elevated in Highly Infiltrative Apoptosis-Resistant Glioblastoma Cells

We have previously established two distinct glioma phenotypes by serial xenotransplantation of human glioblastoma (GBM) biopsies in nude rats. These tumors undergo a gradual transition from a highly invasive nonangiogenic to a less-invasive angiogenic phenotype. In a protein screen to identify molecular markers associated with the infiltrative phenotype, we identified α-basic-crystallin (αBc), a small heat-shock protein with cytoprotective properties. Its increased expression in the infiltrative phenotype was validated by immunohistochemistry and Western blots, confirming its identity to be tumor-derived and not from the host. Stereotactic human GBM biopsies taken from MRI-defined areas verified stronger αBc expression in the infiltrative edge compared to the tumor core. Cell migration assays and immunofluorescence staining showed αBc to be expressed by migrating cells in vitro. To determine αBc function, we altered its expression levels. αBc siRNA depletion caused a loss of migrating tumor cells from biopsy spheroids and delayed monolayer wound closure. In contrast, glioma cell migration in a Boyden chamber assay was unaffected by either αBc knockdown or overexpression, indicating that αBc is not functionally linked to the cell migration machinery. However, after siRNA αBc depletion, a significant sensitization of cells to various apoptotic inducers was observed (actinomycin, tumor necrosis factor α, and TNF-related apoptosis-inducing ligand [TRAIL]). In conclusion, αBc is overexpressed by highly migratory glioma cells where it plays a functional role in apoptosis resistance.Glioblastoma multiforme (GBM), the most common primary intracranial malignancy, is a morphologically diverse neoplasm with dismal prognosis despite multimodal therapy. At present, two-year survival of GBM patients with optimal therapy is less than 30%.¹ A major problem for effective glioma treatment is their highly invasive nature, where tumor cells can be identified several centimeters away from the main tumor mass. They are therefore incurable by local therapies such as surgery and radiotherapy.² Ninety-five percent of gliomas recur within 2.5 cm of the resection margin, which contains invasive cells that act as a “disease reservoir” and make current treatment options inefficient.³ The mechanism of glioma cell invasion has been addressed in different studies and experimental settings, yet there is a need for novel markers characterizing the invasive phenotype.^4–6 The invasive phenotype is often poorly reflected in experimental model systems where GBM cell lines are xenotransplanted into the brain of immunodeficient animals. Generally, such tumors show a well-circumscribed growth with little single cell infiltration into the brain parenchyma.⁵ To avoid this problem, we have developed a xenograft model where human brain tumor biopsy spheroids are transplanted into the nude rat brain.^7,8 The tumors derived from such spheroids show a highly infiltrative behavior in the central nervous system, reflecting the invasive characteristics of the human tumors in situ (low generation tumors). On serial transplantation in vivo, the tumors will develop a less invasive and more angiogenic phenotype (high-generation tumors).⁹In a search of novel markers associated with the infiltrative phenotype, we performed comparative 2D protein electrophoresis of tumor samples obtained from low- versus high-generation tumors and identified several differentially expressed proteins. One of the proteins found to be highly expressed in the invasive phenotype compared to the less-invasive angiogenic phenotype was αB-crystallin (αBc), a small heat shock protein with chaperone and autokinase activity.^10–13 This protein is found in the central nervous system where it represents a major constituent of Rosenthal fibers (RF), which are intracytoplasmic inclusions frequently found within astrocytes in brain tissue of patients with Alexander disease.¹⁴ αBc can associate with intermediate filaments and their soluble subunits such as vimentin and glial fibrillary acidic protein.^15–17 Previous studies have shown expression of αBc in astrocytic tumors, schwannomas, hemangioblastomas, chordomas, and renal cell carcinomas as well as in some glioma cell lines.^18,19 In the present study we show that αBc is mainly expressed by the infiltrative glioma cells both in experimental xenograft models as well as in human gliomas in situ. Moreover, we show that the molecule is not directly functionally linked to cell migration but plays a prominent role in apoptosis resistance.     

Direct orthotopic transplantation of fresh surgical specimen preserves CD133+ tumor cells in clinically relevant mouse models of medulloblastoma and glioma

Recent identification of cancer stem cells in medulloblastoma (MB) and high-grade glioma has stimulated an urgent need for animal models that will not only replicate the biology of these tumors, but also preserve their cancer stem cell pool. We hypothesize that direct injection of fresh surgical specimen of MB and high-grade glioma tissues into anatomically equivalent locations in immune-deficient mouse brains will facilitate the formation of clinically accurate xenograft tumors by allowing brain tumor stem cells, together with their non-stem tumor and stromal cells, to grow in a microenvironment that is the closest to human brains. Eight of the 14 MBs (57.1%) and two of the three high-grade gliomas (66.7%) in this study developed transplantable (up to 12 passages) xenografts in mouse cerebellum and cerebrum, respectively. These xenografts are patient specific, replicating the histopathologic, immunophenotypic, invasive/metastatic, and major genetic (analyzed with 10K single nucleotide polymorphism array) abnormalities of the original tumors. The xenograft tumor cells have also been successfully cryopreserved for long-term preservation of tumorigenicity, ensuring a sustained supply of the animal models. More importantly, the CD133(+) tumor cells, ranging from 0.2%-10.4%, were preserved in all the xenograft models following repeated orthotopic subtransplantations in vivo. The isolated CD133(+) tumor cells formed neurospheres and displayed multi-lineage differentiation capabilities in vitro. In summary, our study demonstrates that direct orthotopic transplantation of fresh primary tumor cells is a powerful approach in developing novel clinical relevant animal models that can reliably preserve CD133(+) tumor cell pools even during serial in vivo subtransplantations. Disclosure of potential conflicts of interest is found at the end of this article.     

Photochemical internalization of bleomycin for glioma treatment

We study the use of photochemical internalization (PCI) for enhancing chemotherapeutic response to malignant glioma cells in vitro. Two models are studied: monolayers consisting of F98 rat glioma cells and human glioma spheroids established from biopsy-derived glioma cells. In both cases, the cytotoxicity of aluminum phthalocyanine disulfonate (AlPcS2a)-based PCI of bleomycin was compared to AlPcS(2a)-photodynamic therapy (PDT) and chemotherapy alone. Monolayers and spheroids were incubated with AlPcS(2a) (PDT effect), bleomycin (chemotherapy effect), or AlPcS(2a)+bleomycin (PCI effect) and were illuminated (670 nm). Toxicity was evaluated using colony formation assays or spheroid growth kinetics. F98 cells in monolayer/spheroids were not particularly sensitive to the effects of low radiant exposure (1.5 J/cm(2) @ 5 mW/cm(2)) AlPcS(2a)-PDT. Bleomycin was moderately toxic to F98 cells in monolayer at relatively low concentrations-incubation of F98 cells in 0.1 μg/ml for 4 h resulted in 80% survival, but less toxic in human glioma spheroids respectively. In both in vitro systems investigated, a significant PCI effect is seen. PCI using 1.5 J/cm(2) together with 0.25 μg/ml bleomycin resulted in approximately 20% and 18% survival of F98 rat glioma cells and human glioma spheroids, respectively. These results show that AlPcS(2a)-mediated PCI can be used to enhance the efficacy of chemotherapeutic agents such as bleomycin in malignant gliomas.     

Angiopoietin-1 promotes tumor angiogenesis in a rat glioma model

Angiopoietins have been implicated in playing an important role in blood vessel formation, remodeling, maturation, and maintenance. However, the role of angiopoietins in tumor angiogenesis remains uncertain. In this study, expression of human angiopoietin-1 (hAng-1) and angiopoietin (hAng-2) was amplified in the rat glioma cell line GS9L by stable transfection using an inducible tet-off system. Transfected cells were implanted intracerebrally into syngenic Fischer 344 rats. We demonstrated by means of magnetic resonance imaging that increased hAng-1 expression promoted a significant in vivo growth of intracerebral gliomas in rats. Overexpression of hAng-1 resulted in more numerous, more highly branched vessels, which were covered by pericytes. On the other hand, tumors derived from hAng-2-overexpressing cells were smaller than empty-plasmid control tumors. The tumor vasculature in these tumors was composed of aberrant small vascular cords, which were associated with few mural cells. Our results indicate that in the presence of hAng-1, tumors induce a more functional vascular network, which led to better tumor perfusion and growth. On the other hand, overexpression of hAng-2 led to less intact tumor vessels, inhibited capillary sprouting, and impaired tumor growth.     

Platelet-derived growth factor receptor-beta is induced during tumor development and upregulated during tumor progression in endothelial cells in human gliomas.

BACKGROUND: Endothelial cells proliferate during brain development, are quiescent in normal adult brain but proliferate again under pathologic conditions such as glioma growth. The vascular phenotype of low grade glioma is comparable to normal brain, however high grade gliomas are focally highly vascularized and there is associated prominent endothelial cell proliferation. The mechanisms of this change in vascular phenotype are unknown but there is evidence that growth factors play an important role in this process as well as in normal angiogenesis and vascular differentiation. EXPERIMENTAL DESIGN: To investigate whether endothelial cells become activated during tumorigenesis and progression of human gliomas by a platelet-derived growth factor (PDGF) dependent pathway, we analyzed platelet-derived growth factor receptor-beta (PDGFR-beta) expression by in situ hybridization and immunocytochemistry in normal human brain, astrocytoma (grade II), anaplastic oligo-astrocytoma (grade III), and glioblastoma multiforme (grade IV). RESULTS: PDGFR-beta mRNA was not detectable in the vessels of normal human brain, but was expressed in the vasculature of low and high grade gliomas, particularly in endothelial cell proliferations in glioblastomas. The expression of the receptor in the tumor microvessels, was confirmed by double immunofluorescence in which the staining appeared to be in the endothelial cells. Primary cultures of endothelial cells derived from glioblastoma multiforme maintained receptor expression for 2 days in vitro, whereas it was not detectable in vitro in endothelial cells derived from normal brain. Tumor cells in all grades of glioma expressed very little PDGFR-beta mRNA in situ. CONCLUSIONS: Our results indicate that the malignant phenotype in human glial tumors is associated with an upregulation of the PDGFR-beta on endothelial cells of vessels which vascularize the tumor. These findings may contribute to our understanding of the mechanisms that regulate vessel growth and differentiation in normal and pathologic states.