Maximal Resection of Gliomas Adjacent to the Corticospinal Tract Using 3-T Intraoperative Magnetic Resonance Imaging.

BACKGROUND: Gliomas adjacent to the corticospinal tract (CST) should be carefully resected to preserve motor function while achieving maximal surgical resection. Modern high-field intraoperative magnetic resonance imaging (iMRI) enables precise visualization of the residual tumor and intraoperative tractography. We prospectively evaluated the extent of resection and distance between the tumor resection cavity and CST using 3-T iMRI combined with motor evoked potentials (MEP) in glioma surgery. METHODS: Participants comprised patients who underwent surgery for solitary supratentorial glioma located within 10 mm of the CST. All cases underwent surgery using neuronavigation with overlaid CST under MEP monitoring. The correlation between distance from CST and transcortical MEP amplitude was calculated using Spearman rank correlation. RESULTS: Among the 63 patients who underwent surgery, 27 patients were enrolled in the study. Gross total resections were achieved in 26 of the 27 cases. Volumetric analysis showed the extent of resection was 98.6%. Motor function was stable or improved in 24 patients (Stable/Improved group) and deteriorated in 3 patients (Deteriorated group). All patients in the Deteriorated group showed motor deficit before surgery. Mean intraoperative minimal distance was significantly longer in the Stable/Improved group (7.3 mm) than in the Deteriorated group (1.1 mm; P < 0.05). MEP amplitude correlated with minimal distance between the resection cavity and CST (R = 0.64). CONCLUSIONS: Resection of gliomas adjacent to CST with a navigation system using 3-T iMRI could result in an ultimate EOR >98%. The combination of intraoperative tractography and MEP contributes to maximal removal of motor-eloquent gliomas.

Nonconvulsive status epilepticus due to pneumocephalus after suprasellar arachnoid cyst fenestration with transsphenoidal surgery: illustrative case.

BACKGROUND: Nonconvulsive status epilepticus (NCSE) requires prompt diagnosis and treatment, particularly after neurosurgical procedures for cerebral damage. Here, the authors reported an extremely rare case of suprasellar arachnoid cyst presenting with NCSE after cyst fenestration with transsphenoidal surgery, which was associated with pneumocephalus. OBSERVATIONS: A 61-year-old man presented with visual impairment and was diagnosed with a suprasellar arachnoid cyst on magnetic resonance imaging (MRI). The patient received cyst fenestration with endonasal transsphenoidal surgery. His visual symptoms improved immediately after the operation; however, on postoperative day 3, semicoma appeared and was prolonged. The patient was diagnosed with NCSE due to pneumocephalus based on MRI and electroencephalography (EEG) findings. The administration of antiepileptic drugs (AEDs) improved his clinical symptoms and the abnormal findings on MRI and EEG. LESSONS: This is the first case of NCSE with pneumocephalus after transsphenoidal surgery for a suprasellar arachnoid cyst. Pneumocephalus due to cerebrospinal fluid leakage can cause NCSE. Arterial spin labeling perfusion imaging and diffusion-weighted imaging are as useful for differentially diagnosing NCSE as EEG and AED tests.

EHF suppresses cancer progression by inhibiting ETS1-mediated ZEB expression.

ETS homologous factor (EHF) belongs to the epithelium-specific subfamily of the E26 transformation-specific (ETS) transcription factor family. Currently, little is known about EHF's function in cancer. We previously reported that ETS1 induces expression of the ZEB family proteins ZEB1/δEF1 and ZEB2/SIP1, which are key regulators of the epithelial-mesenchymal transition (EMT), by activating the ZEB1 promoters. We have found that EHF gene produces two transcript variants, namely a long form variant that includes exon 1 (EHF-LF) and a short form variant that excludes exon 1 (EHF-SF). Only EHF-SF abrogates ETS1-mediated activation of the ZEB1 promoter by promoting degradation of ETS1 proteins, thereby inhibiting the EMT phenotypes of cancer cells. Most importantly, we identified a novel point mutation within the conserved ETS domain of EHF, and found that EHF mutations abolish its original function while causing the EHF protein to act as a potential dominant negative, thereby enhancing metastasis in vivo. Therefore, we suggest that EHF acts as an anti-EMT factor by inhibiting the expression of ZEBs, and that EHF mutations exacerbate cancer progression.

An Adult Case of Sellar Atypical Teratoid/Rhabdoid Tumor Presenting with Lung Metastasis, Harboring a Compound Heterozygous Mutation in INI1.

A typical teratoid/rhabdoid tumors (AT/RT) are highly malignant embryonal tumors in children that are associated with inactivation of the integrase interactor 1 (INI1) gene. Several adult cases of AT/RT have been reported, which were characterized by the sellar occurrence and predominantly occurred in females with INI1 mutation variants. However, clinical and genetic features are poorly understood in this unusual entity. We experienced a case of a 45-year-old female with sellar AT/RT presenting diplopia, who underwent subtotal removal of the tumor by the endoscopic endonasal transsphenoidal approach. Pathological diagnosis was AT/RT with INI1 inactivation on immunohistochemistry. Subsequently, multiple lung metastases were confirmed on fluorodeoxyglucose positron emission tomography (FDG-PET). Although she received postoperative chemoradiotherapy, she died of cerebrospinal fluid dissemination. Autopsy revealed cerebrospinal dissemination and lung metastasis of AT/RT. Biallelic alterations in the INI1 gene were identified by direct sequencing, harboring on different alleles (compound heterozygous mutations) was observed, which is the potential genetic pattern in adult AT/RT. Literature review indicated that lung metastasis frequently occurs in sellar AT/RTs, which is accompanied by cavernous sinus invasion. These observations suggested that cavernous sinus invasion causes haematogenous metastasis to the lung in sellar AT/RT. We discuss clinical and pathological features in adult sellar AT/RT to improve understanding of this unique entity.

Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma.

BACKGROUND: The utility of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values. RESULTS: We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively). CONCLUSIONS: The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.

Expression of ZEBs in gliomas is associated with invasive properties and histopathological grade.

The invasiveness of glioma cells is the predominant clinical problem associated with this tumor type, and is correlated with pathological malignant grade. ZEB1 is highly expressed in glioma cells and associated with the aggressiveness of various types of cancer. In the present study, the expression of ZEB1 and ZEB2 was examined with the aim of determining the role of ZEBs in glioma. ZEB1 and ZEB2 were highly expressed in all glioma cells used in this study. Double knockdown of ZEB1 and ZEB2 suppressed tumor invasiveness more effectively than knockdown of either alone, in both in vitro and in vivo experiments. ZEB1 and ZEB2 were marginally expressed in grade II, but expressed at higher levels in grade IV. Importantly, ZEB-positive cells were more abundant in recurrent glioma with malignant transformation than in initial grade II tissue from the same case. These results indicate that the levels of ZEB1 and ZEB2 are positively correlated with histopathological grade and invasiveness of glioma, suggesting that δEF1 family proteins (ZEB1 and ZEB2) could be useful as prognostic markers and therapeutic targets in patients with glioma.

[Spontaneous Improvement in Visual Symptom after Delivery in a Case of Tuberculum Sellae Meningioma].

A 36-year-old woman presented with temporal visual field defect in the 27th week of her pregnancy. Magnetic resonance(MR)imaging demonstrated a tuberculum sellae meningioma compressing the optic chiasm. Although we planned surgical resection after her delivery, the symptom worsened in the late stage of her pregnancy. After administering a steroid medication, the symptom stabilized. In the 38th week of pregnancy, she delivered a child, and interestingly, her visual field defect improved, and MR imaging demonstrated tumor shrinkage. However, at the request of the patient, we performed the tumor resection by extended endonasal transsphenoidal surgery. The histological diagnosis was angiomatous meningioma, and the tumor cells were strongly positive for progesterone receptors. This is a very rare case of tuberculum meningioma wherein the patient showed a natural improvement in visual function after delivery. Regarding the appropriate surgical timing for meningioma, spontaneous shrinkage of meningioma after delivery would be considered an option.

Opening the ventricle during surgery diminishes survival among patients with newly diagnosed glioblastoma treated with carmustine wafers: a multi-center retrospective study.

Carmustine wafers (CW) were approved in Japan for newly diagnosed and recurrent malignant gliomas during 2013. The ventricle is often opened during surgery to achieve maximum resection. While not generally recommended in such situations, CW might be safely achieved by occluding an opened ventricle using gelform or collagen sheets. However, whether CW implantation actually confers a survival benefit for patients who undergo surgery with an open ventricle to treat glioblastoma remains unclear. Clinical, imaging, and survival data were collected in this multicenter retrospective study of 122 consecutive patients with newly diagnosed glioblastoma to determine adverse events and efficacy. Overall, 54 adverse events of all grades developed in 35 (28.6%) patients, with the most common being new seizures (16%). Adverse events did not significantly differ between patients with opened and closed ventricles during surgery. The 10- and 21.7-month, median, progression-free (PFS) and overall survival (OS), respectively did not significantly differ according to resection rates. However, median PFS and OS were significantly longer among patients with closed, than open ventricles (12.8 vs. 7.4 months; p = 0.0039 and 26.9 vs. 18.6 months; p = 0.011, respectively). Implanting CW into the resection cavity during concomitant radiochemotherapy with temozolomide seems to yield better survival rates without increased adverse events. Occlusion of the ventricular opening during surgery might be safe for CW implantation, but less so for treating patients with newly diagnosed glioblastoma.

[Efficacy and Safety of Levetiracetam as an Add-on Therapy for Symptomatic Refractory Epilepsy Treated in a Department of Neurosurgery].

The number of patients who suffer from symptomatic partial epilepsy is significant, and those who undergo neurosurgery often experience refractory seizures. Levetiracetam (LEV) is a new-generation anti epileptic drug (AED). Previous studies have shown that LEV has favorable efficacy and a good safety profile with few drug interactions, as it has a unique pharmacological mechanism and acts on synaptic vesicle protein. This study aimed at estimate the efficacy and safety of 1,000 to 2,000 mg/day of LEV as an add-on therapy for refractory symptomatic seizures treated in the department of neurosurgery of our hospital. Twelve patients with >1 seizure attack per year who were administrated one to three AED(s) and had never used LEV were enrolled in this study. The follow-up period was at least 6 months. Seizure-free ratio, health-related quality of life (QOL), and tolerability were assessed. Fifty-eight percent (7/12) of patients were seizure-free, and their "overall QOL", "energy/fatigue" and "health status" distress-item scores in the QOLIE-31-P had improved. All patients were able to continue oral treatment with LEV without major side effects. Thus, the efficacy and safety of oral add-on LEV therapy were high for these patients. (Received January 19, 2016; Accepted September 2, 2016; Published December 1, 2016).

A case of complete clearance of chronic subdural hematoma accompanied by recurrent glioblastoma multiforme after administration of bevacizumab.

The efficacy of bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), as an adjuvant therapy against various malignant tumors was recently established. Its pharmacological effects in malignant tumors, including gliomas, were speculated to involve neovascularization inhibition and vascular permeability. Recently, it has been reported that the outer membrane of chronic subdural hematoma (CSDH) contains high levels of VEGF, which were implicated in neovascularization of the outer membrane. Furthermore, studies suggested that VEGF has the etiology in CSDH development, although its involvement is not fully understood. Here, we report the first case of chronic subdural hematoma that was improved by bevacizumab administration for recurrent glioblastoma. The present case could contribute to the hypothesis that VEGF may be associated with CSDH. We also discuss the pathogenesis and mechanism of CSDH recurrence from the viewpoint of VEGF function.

Synergistic antitumor effect with indoleamine 2,3-dioxygenase inhibition and temozolomide in a murine glioma model.

OBJECT Indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan (Trp) metabolism, is involved in tumor-derived immune suppression through depletion of Trp and accumulation of the metabolite kynurenine, resulting in inactivation of natural killer cells and generation of regulatory T cells (Tregs). It has been reported that high expression of IDO in cancer cells is associated with suppression of the antitumor immune response and is consistent with a poor prognosis. Thus, IDO may be a therapeutic target for malignant cancer. The authors have recently shown that IDO expression is markedly increased in human glioblastoma and secondary glioblastoma with malignant change, suggesting that IDO targeting may also have therapeutic potential for patients with glioma. The aim of this study was to investigate the antitumor effect of IDO inhibition and to examine the synergistic function of IDO inhibitor and temozolomide (TMZ) in a murine glioma model. METHODS Murine glioma GL261 cells and human glioma U87 cells were included in this study. The authors used 3 mouse models to study glioma cell growth: 1) a subcutaneous ectopic model, 2) a syngeneic intracranial orthotopic model, and 3) an allogenic intracranial orthotopic model. IDO inhibition was achieved via knockdown of IDO in GL261 cells using short hairpin RNA (shRNA) and through oral administration of the IDO inhibitor, 1-methyl-l-tryptophan (1-MT). Tumor volume in the subcutaneous model and survival time in the intracranial model were evaluated. RESULTS In the subcutaneous model, oral administration of 1-MT significantly suppressed tumor growth, and synergistic antitumor effects of 1-MT and TMZ were observed (p < 0.01). Mice containing intracranially inoculated IDO knockdown cells had a significantly longer survival period as compared with control mice (p < 0.01). CONCLUSIONS These results suggest that IDO expression is implicated in immunosuppression and tumor progression in glioma cells. Therefore, combining IDO inhibition with standard TMZ treatment could be an encouraging therapeutic strategy for patients with malignant glioma.

Expression of indoleamine 2,3-dioxygenase and correlation with pathological malignancy in gliomas.

BACKGROUND: : Indoleamine 2,3-dioxygenase (IDO) is a tryptophan catabolic enzyme involved in immune tolerance and tumor immune escape processes. Recently, IDO expression has been found to correlate with the prognosis of malignant tumors, but the implication of IDO in glioma progression remains unknown. OBJECTIVE: : To investigate the relationship between IDO expression and histological malignancy in gliomas. METHODS: : IDO expression was examined in a total of 75 surgical specimens obtained from 68 patients with glioma using immunohistochemical staining. The 75 specimens included 15 diffuse astrocytomas, 21 anaplastic astrocytomas, and 39 glioblastomas. Six of 39 glioblastomas were secondary glioblastomas, transforming from grade II or III gliomas that had been determined at the first surgery. IDO expression rate was compared in each histological grade, and patient survival was analyzed. RESULTS: : Expression of IDO was found in 72 of 75 gliomas at varying intensities. Stronger expression of IDO was more likely to be observed in malignant gliomas compared with low-grade gliomas. IDO expression in the 6 cases of secondary glioblastoma was stronger than in the initial low-grade glioma. Survival analysis using the Kaplan-Meier method revealed that grade IV patients with strong IDO expression had significantly worse overall survival rates (P = .04) than patients with weak IDO expression. CONCLUSION: : IDO is expressed more strongly in both primary and secondary glioblastoma tissue than low-grade glioma and may affect clinical outcome. If IDO promotes glioma cells to escape from the immune system, IDO may be a crucial therapeutic target for malignant gliomas.

A cortical dysembryoplastic neuroepithelial tumor initially occurring in the periventricular white matter.

In this report, a rare case of dysembryoplastic neuroepithelial tumor (DNET) initially presented as a small white matter lesion with calcification adjacent to the lateral ventricle and extending to the frontal cortex after 7 years. This 1-year-old boy initially suffered from partial seizures. Initial CT revealed a small, low-density area surrounding a tiny calcified mass in the deep white matter of the left frontal lobe. Seven years later, his seizures had become intractable to antiepileptic agents, and MR imaging demonstrated a relatively large mass extending from the calcified lesion up to the adjacent cortical surface. He underwent surgery and the tumor was subtotally removed. Histological examination of the tumor verified it as a DNET consisting of clusters of small oligodendrocytes with floating neurons in the mucoid background. The pattern of the tumor progression in this case suggests that a DNET in the cortex originates from the subependymal germinal layer near the ventricle.

Clear cell meningioma causing Castleman syndrome in a child.

This 11-year-old boy presented with a rare case of Castleman syndrome caused by a clear cell meningioma manifesting as persistent fever of unknown origin, 2 years after glomerulonephritis. Laboratory investigation of the patient showed an increased inflammatory reaction, as well as elevated polyclonal gamma globulin titer and serum level of C-reactive protein. Magnetic resonance imaging revealed a tumor at the cerebellopontine angle. Neurosurgical intervention was performed under the presumptive diagnosis of Castleman syndrome caused by intracranial tumor. Histological examination of the tumor verified that it was clear cell meningioma with infiltration of lymphoplasma cells, and surgical removal resulted in complete resolution of the patient's symptoms and biochemical abnormalities. The present case of clear cell meningioma manifesting as Castleman syndrome shows that the possibility of a brain tumor should be considered in patients presenting with fever of unknown origin, anemia, hypergammaglobulinemia, or other systemic illness.

Anaplastic ganglioglioma with malignant features in both neuronal and glial components--case report.

A 34-year-old man presented with a case of anaplastic ganglioglioma with malignant features in both neuronal and glial components manifesting as seizure episodes over 11 months. The tumor was subtotally removed, followed by irradiation and chemotherapy. The histological diagnosis was anaplastic ganglioglioma. Atypical cells were morphologically estimated as glial and neuronal cells. Though these cells were weakly positive for synaptophysin and glial fibrillary acidic protein, the neural stem cell marker nestin was extremely expressed in both these cells. The MIB-1 index was 15%. Two months later, the tumor recurred with more pleomorphic appearance and higher cellularity with increased nestin expression level. Mitotic cells and multinucleated cells were found in the neuronal components. Cytological examination found dissemination to the leptomeningeal space. The patient died 6 months after the first surgery. This rare case of anaplastic ganglioglioma with both neuronal and glial components, which were extremely positive for nestin, showed progressive worsening of the clinical course. The expression of nestin may suggest that the origin or malignant transformation in anaplastic gangliogliomas is related to the undifferentiated neural stem cells.

Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin.

Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.

Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1), 10 (alpha5beta1gamma1).

Laminins, a family of large heterotrimeric (alphabetagamma) proteins, are major components of basement membranes implicated in a variety of cellular functions. Different commercial laminin preparations isolated from human placenta have been widely used in functional studies but their molecular properties are poorly known. In the present study, we characterized several of these preparations by ELISA, silver staining and Western blotting, in comparison to mouse laminin 1 (alpha1beta1gamma1), and recombinant human laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). The cell migration-promoting activity of different batches was also tested. The placenta laminin preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. Major functional differences between batches were also observed, reflecting molecular heterogeneity. Previous data obtained in functional studies using these preparations need to be interpreted with caution and may require revision, and future functional studies demand prior molecular characterization of the laminins, particularly their alpha-chain.

Establishment and partial characterization of five malignant glioma cell lines.

Five malignant glioma cell lines (YMG1, 2, 3, 4, and 5) were established from surgical specimens obtained from patients with glioblastoma or anaplastic astrocytoma, and these lines were partially characterized. Three glioma cell lines (YMG1, 3, and 5) were weakly positive for GFAP by Western blot analysis and two cell lines were negative. S-100 protein was positive in all glioma cell lines. The expression of p53, p16, p15, cyclin-dependent kinase 4 (CDK4), and EGF receptor (EGFR) proteins was examined by Western blotting. YMG1 and 2 cell lines showed accumulation of p53 protein and loss of p16 and p15 expression. YMG3 and 4 showed accumulation of p53 protein and expression of p16 and p15 proteins. YMG5 revealed weak expression of p53 protein, suggesting wild-type p53, and loss of p16 and p15 expression. All cell lines expressed various levels of CDK4 protein. YMG1, 2, and 3 showed higher EGFR protein expression and YMG4 and 5 showed lower EGFR expression compared to U251 glioblastoma cells, which express high levels of EGFR. Fluorescence in situ hybridization analysis for EGFR gene expression did not show any amplification in the glioma cell lines. Immunohistochemical studies revealed that the patterns of p53 and EGFR expressions in the original tumor tissues were mostly correlated with those in the malignant glioma cell lines. These results suggest that the characteristics of p53 and EGFR expression in the malignant glioma cell lines were passed over from the original tumor tissues. These newly established malignant glioma cell lines can be used for further analysis of the mechanisms of tumor growth and progression.

Cell density regulates thrombospondin-1 production in malignant glioma cells.

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein implicated in cancer cell adhesion, migration, and invasion, inhibition of angiogenesis, and activation of latent transforming growth factor-beta (TGF-beta). The effect of cell density was investigated on the production of TSP-1, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) by two glioblastoma cell lines. The effect of TGF-beta was also examined. The amount of intracellular TSP-1 protein decreased significantly as the cell density increased in cultures of both T98G and A172 cells. The amount of intracellular TSP-1 was highest in sparse tumor cell cultures and lowest in densely confluent tumor cell cultures. The maximum reduction of TSP-1 protein production was 56.8% and 44.6% in T98G and A172 cells, respectively. The cell density did not affect the production of bFGF or VEGF. TGF-beta2 treatment did not affect the production of TSP-1, bFGF, or VEGF proteins. Treatment with excess TGF-beta2 resulted in a slight but significant decrease (22%; P < 0.02) of TGF-beta2 production by A172 cells, but not by T98G cells. The present results indicate that the production of TSP-1 protein is regulated by cell density of glioblastoma cells, while that of angiogenic factors is not affected by tumor cell density. This suggests that high tumor cell density may tilt the angiogenic balance in favor of angiogenesis.