ICANCER RESEARCH 56. 2185-219«. May I. 19961

Concentration of Vascular Endothelial Growth Factor in the Serum and TumorTissue of Brain Tumor Patients1

Shingo Takano,2 Yoshihiko Yoshii, Shinichi Kondo, Hideo Suzuki, Tooru Maruno, Shizuo Shirai, and Tadao Nose

Department of Neuraxitriiery. Soltjinkai Hospital. 1467 Oka. Fitjishiro. Kitaxoumagun, Ibaraki 300-15 IS. T.. T. M., S. S.f; Division of Neurological Surgery. Tsitkubu UniversitàSchool of Medicine, 1-1-1 Tennoudai, Tsukuba. Ibaraki 305 ¡Y.Y.. T. N.I; and Tsukuba Research Lilhoralory. Toagosei Co., Ltd., 2 Ohkubo. Tsukuba. lharaki 300-33

¡S.K.. H. S.I, Japan

ABSTRACT

Vascular endothelial growth factor (VEGF) has been investigated as apotent mediator of Drain tumor angiogenesis, vascular permeability, andglioma growth. Using a VEGF ELISA, we determined the concentration ofVEGF in the sera and tumor extracts of 19 brain tumor patients includingglioblastoma, anaplastic astrocytoma, low grade astrocytoma, meningi-

oiiiii. malignant lymphoma, and metastatic brain tumor as well as normal

brain. VEGF concentration in the tissue of glioblastomas was significantlyhigher than that in other types of tumors as well as normal brain.Although VEGF concentration of the serum was not correlated with thatof the tissue, VEGF concentrations of glioblastoma cyst fluid were 200-300-fold higher than those of serum in the patients. VEGF concentration

in the tumors was significantly correlated with the vascularity measuredby counting vessels stained with von Willebrand factor antibody. VEGFprotein localized to the cytoplasm of tumor cells and vasculature ingliomas, predominantly in the peripheral microvessel "hot spots" as well

as around the necrosis in glioblastomas. VEGF immunopositivities werewell reflected with VEGF concentration determined by ELISA. VEGFELISA demonstrated time-dependent increase of the VEGF concentrationin the serum-free conditioned medium of various glioma cell lines. The

conditioned medium with high VEGF concentration induced endothelialcell migration. These observations suggest that VEGF represents a usefulmarker and measurable element of glioblastoma angiogenesis. The measurement of VEGF concentration by ELISA in tumor and tumor cyst fluidmay allow for the assessment of vascularity in gliomas.

INTRODUCTION

VEGF' is believed to play a bifunctional role in malignant brain

tumor biology leading to both angiogenesis and vasogenic edema(1-3). The role of VEGF in glioblastoma angiogenesis was confirmed

by blocking its activity in tumors by a monoclonal antibody (4) and byapplication of a dominant-negative VEGF receptor mutant (5). These

treatments led to a decrease in angiogenesis and to slower gliomagrowth.

There were only a few studies concerning the quantitation of VEGFprotein (4, 6-10). Recently, an ELISA for VEGF has been developed.

VEGF levels in the sera from uterine, ovarian, and lung cancerpatients were significantly higher than those in sera from the individuals with no sign of cancer (11, 12). To assess whether VEGF proteinis a measurable element for brain tumor angiogenesis. we directlymeasured the VEGF concentration by ELISA in the sera and tumorextracts, as well as tumor cyst fluid, of brain tumor patients andcorrelated the VEGF concentration with VEGF immunopositivitiesand vascular density by immunohistochemistry.

Received 9/12/95; accepted 3/1/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by Grant-in-Aid 06671372 from the Japanese

Ministry of Education. Science, and Culture do Y. Y.).~ To whom requests for reprints should be addressed.' The abbreviations used are: VEGF. vascular endothelial growth factor; bFGF. basic

fibroblast growth factor; CSF. cerebral spinal fluid; MAh. monoclonal antibody; WO.vesicular-vacuolar organelle.

MATERIALS AND METHODS

Subjects. Nineteen patients with brain tumors (5 glioblastoma. 5 anaplasticastrocytoma. 2 recurrent anaplastic astrocytoma. 3 low-grade astrocytoma. 2

meningioma. 1 malignant lymphoma, and 1 metastasis; 7 men and 12 women;mean age ±SD. 49.5 ±13.6 years) and 10 normal control subjects (8 men and2 women; 43.6 ±15.4 years) were included in the current study. The bloodwas taken from nine patients and each normal subject between 10 a.m. and 12a.m. After coagulation at 4°C.the blood was centrifuged at H).(KK)rpm tor 10min. and the serum was stored at —¿�70°Cuntil use for analysis. Informed

consent was obtained from all subjects involved in the current study.Tissue Preparation. Brain tumor tissues and normal brain tissues (nontu-

mor brain, 2 cases; brain distant far from tumor. 3 cases) were obtained duringroutine surgical procedures performed for diagnostic or therapeutic indications.A part of tissues was immediately fixed in 10% phosphate-buffered formalin

for 48 h, paraffin embedded, and used for routine pathological diagnosis andimmunohi.stochemistry. Other parts of tissues were immediately frozen withliquid nitrogen and stored at -70°C. The tissues were homogenated with a

motor-driven Teflon pestle for 5 min on ice in 1 ml of extraction buffer [25 niM

Tris (pH 7.4). 100 mM NaCl. 20 mM NH4HCO,; Ref. 13] per 100 mg tissue wetweight, and the tissue extract obtained after centrifugalion at 15.000 rpm for 20min at 4°Cwas aliquoted to a 200-/J.1 vial, stored at -70°C. and used for

ELISA. Protein concentrations were determined by the method of Bradfordusing BSA as a standard (14).

VEGF ELISA. A recombinanl human VEGF12I was purified from theculture medium of the transformed yeast (12). An anti-VEGF polyclonul

antibody was prepared from rabbit serum immunized with recomhinant humanVEGF,21-glutathione S-transferase fusion protein (II). Ninety-six-well micro-

liter plates (Microfluor black plate; Dynatech Laboratories. Chantilly. VA)were coated with 10 /xg/ml of the anti-VEGF antibody in 0. l M NaCl and 0.025

M carbonate buffer (pH 9.0) and then blocked with \% BSA. 0.2 M carbonatebuffer (pH 9.5). 0.1 M NaCl. and 0.1% NaN,. For the assay. 100 ^1 of samplesand serially diluted VECE,,, (standards; Ref. 12) were added to the wells andincubated for 1 h at 22°C.After washing the wells six times, 100 fil of alkalinephosphatase-conjugated Fab' of the anti-VEGF antibody was added to each

well and incubated for 1 h at 22°C.Wells were washed eight times, then theenzyme reaction was carried out at 37°Cfor 1 h with 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-l,2-dioxetane disodium salt (Lumi-

phos 530: Wako Pure Chemical Industries. Ltd., Osaka, Japan) as a substrate.The chemiluminescence of each well was measured by a plate luminometer,and the VEGF content of the samples was estimated from the standard curve

determined from the serially diluted VEGF|:| standard.Immunocytochemistry. The Dako LSAB kit for mouse primary antibody

(DAKO. Glostrup. Denmark) was used. Tissue sections were deparaffined andincubated with 10% normal goat serum in PBS for 20 min. Sections were thenincubated with a monoclonal anti-VEGF antibody. MV303. at a dilution of1:100 ( 100 /xg/ml) in PBS overnight at 4°Cor a monoclonal antihuman von

Willebrand factor antibody (DAKO) at dilution of 1:50 (254 ng/ml) in PBS for60 min at room temperature. The mouse antihuman VEGF monoclonal antibody. MV303, was prepared from a hybridoma cell line, which was obtainedby fusing mouse myeloma Sp2/O-Agl4 and B cells isolated from a mouse

immunized with recombinant human VEGF,,, (15). Chromalographicallypurified mouse IgG (Cappel, West Chester. PA) at the same protein concentration was used as negative control. Sections were incubated with biotin-

conjugated goat antimouse immunogloblin for 10 min. followed by washing inPBS for 10 min. The sections were then incubated with peroxidase-conjugated

streptavidin solution for 5 min. followed by washing in PBS for 5 min.Sections were then stained with freshly prepared aminoethylcarbazole solutionfor 10 min. followed by a 5-min wash in tap water. Sections were then

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GLIOBLASTOMA VEOF CONCENTRATION

Table 1 Concentration of VEGF by EUSA, immunohislochemistry, and vascular density

HistologyGlioblastomaAn.ipl.i-.il.

astrocytomaLow

gradeastrocytomaMeningiomaMalignant

lymphomaMetastatic

braintumorNormal

brainAge/Sex51/M77/F72/F56/F32/M47/M53/F22/F33/F52/M51/M

(ree)62/F(ree)48/M55/F29/M45/F52/F49/F55/F49/F72/F30/F48/M38/FTissue

ELISApg/mg

protein Mean ±SD8756.67729.0r7381.2''

628 1.6 ±3317.27085.2455.82527.6562.058.249.0

493.6±916.016.8141.6100.01690.273.8

600.4 ±944.037.21277.0916.0

1096.6 ±255.248.8

48.8332.0

332.051.028.020.8

25.8 ±15.014.814.6Serum

ELISApg/mlND*40.423.4NDND85.4NDNDNDNDND277.021.418.4NDND120.629.0142.8NDNDNDNDNDImmunohistochemistry"Tumor+

+++++++ND+

+ND+ND—NDNDND—-NDNDNDND-—ND—NDVessel-+

++—ND-ND—ND—NDNDND—-NDNDNDND-—ND—NDVascular

Density85.0

±30.392.0±32.159.3±10.596.0

±31.8ND117.0

±38.777.0±38.646.3±21.8ND36.7

±9.8ND60.3

±10.03

1.0±6.222.0±4.416.7±6.4NDND36.7

±6.724.7

±3.523.5

±3.518.0±2.5ND22.3

±7.4ND

" Immunohislochemistry: -, negative; + , moderate; + + , strong immunoreactivity.* ND. not determined.r Concentration of cyst fluid '

Concentration of cyst fluid '1VEGF by ELISA of 9044.6 pg/ml.I VEGF by ELISA of 7892.6 pg/ml.

counterstained with hematoxylin and mounted with aqueous mounting media.The intracellular VEGF immunostaining was assessed using a semiquantitativescale (-, not detected: + , moderate; + + , strong).

Tumor Vascular Density. Vascular density was scored using the vasopro-

liferative component of the Microscopic Angiogenesis Grading System thathas been used to quantify angiogenesis in a variety of tumors (16, 17). Thenumber of vessels at X200 field (1.0 mm2) was measured in microvessel "hotspots" (i.e., microscopic areas containing the most dense collections of mi-

crovessels that first were identified under low power magnification) with theuse of an Olympus microscope, AHBT3 (Olympus, Tokyo, Japan) on vonWillebrand factor-stained tissue sections. Vascular density was defined by

averaging the number of vessels in the three most vascular areas.Preparation of Glioma Cell Conditioned Medium and Endothelial Cell

Migration. Human glioblastoma cell lines, U251, U373, and A172, and ratglioma cell line, C6, were obtained from the American Type Culture Collection (Rockville, MD). Rat glioma cell line, 9L, was a gift from Y. Ushio

10000i

GBM AA LGA Normal MEN LYM METABrain

Fig. I. VEGF concentration of brain tumor tissues and normal brain. GBM, glioblastoma; AA. anaplaslic astrocytoma; LGA, low-grade astrocytoma; MEN, meningioma;LYM, malignant lymphoma; META, metastatic brain tumor. *, P < 0.01 compared to other

type of tumors and normal brain.

(Kumamoto University, Kumamoto, Japan). Among them, U251 cells (18), C6cells (9), and 9L cells (19) have been known to secrete VEGF in theirconditioned medium. They were grown in DMEM supplemented with 10%FCS and 1% penicillin-streptomycin at 37°Cin an atmosphere of 5% CO2 and

95% air in a humidified incubator. Subconfluent cultures in 10-cm culturedishes were washed twice with PBS and then incubated in 4 ml of serum-free

medium. At the indicated time points, aliquots of medium were collected,centrifuged, stored at -70°C, and subjected to ELISA and endothelial cell

migration assay.Endothelial cell migration assays were performed as described previously

(20). Transformed fetal bovine aortic endothelial cells (GM 7373; HumanGenetic Mutant Cell Repository, Institute for Medical Research, Camden, NJ)were grown in DMEM supplemented with 10% FCS. Briefly, confluentmonolayers of the endothelial cells in 60-mm dishes were wounded, incubatedfor 16 h in serum-free conditions containing glioma cell conditioned medium

at the equivalent concentrations of protein. Cellular migration was quantitatedwith the image analysis system (Quantimet 570: Leica, Deerfield, IL). Thedistance of migration from the edge of the wound was measured for 10 fields,using the line made by the razor cut as the origin.

Statistical Analysis. VEGF concentration and vascular density were expressed as the means ±SD. Statistically significant differences between tumortypes were determined by using a one-way ANOVA and the Tukey test.

Correlations between VEGF concentration and vascular density and endothelial cell migration were determined by a Pearson correlation matrix, with theconfidence level determined by Bonferroni probabilities.

RESULTS

VEGF Concentration in the Sera, Tissue Extracts, and CystFluid. The concentration of VEGF in the serum, tissue extracts,and cyst fluid in each of the patients is shown in Table 1. VEGFconcentration in the tumor tissue was significantly higher in glio-blastomas (6281.6 ±3317.2 pg/mg protein) compared to othertumors (anaplastic astrocytoma, 493.6 ±916.0; low grade astrocytoma, 600.4 ±944.0; meningioma, 1096.6; malignant lymphoma, 48.8; metastatic brain tumor, 332) as well as normal brain

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GLIOBLASTOMA VEGF CONCENTRATION

•¿�f-%^'^. -:•:"•;•••...•i* »m

¿&¿t?*&¿?f: ..-•> * Õ?*¿ *'•¿�&.•¿�••-••'.'"•'.T •¿�-: -'-A: »' ** '

.-••-:-•:•A. r

%:i»

.', -, .'s. /• ~:' . *,..•:;"-.£*r,-••'-'^«.V:

. t t•¿�•w<••*r

^. . : . 'i &:••••>•**, «•¿�

n o

FFig. 2. Immunohistochemical staining of VEGF in the gliomas and normal brain. A. glioblastoma, X50; fi. glioblastoma, X200; C. glioblastoma. X50; D. anapla.stic astrocytoma.

X100; E, anaplastic astrocytoma, xlOO; F, normal brain, X100. Note the immunopositive staining in the tumor cell cystoplasm and vasculature (arrows) of glioblastoma at theperipheral area and the area adjacent to tumor (lower part of A',A and B). Also at the central area of the glioblastoma (C). immunopositive staining in the tumor cells are predominantly

around vasculature and near necrosis (n). In anaplastic astrocytomas, the case with high concentration of VEGF by ELISA reveals immunopositivities in many of the tumor cells (D),in contrast to the case with low concentration of VEGF dose immunopositivities in a few tumor cells (E). There are no immunopositivities in the normal brain (F).

(25.8 ± 15.0; P < 0.01; Fig. 1). Tumor cyst fluid, which wasobtained during surgery of two glioblastoma patients, containedhigh VEGF concentration (9044.6, 7892.6 pg/ml), which was at a

similar VEGF concentration of the conditioned medium of glioblastoma cells in vitro. The concentration of VEGF in the serafrom normal volunteers ranged between 28.4 and 184.4 pg/ml.

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(¡I.IOlìl.ASTOMAVEGF CONCENTRATION

120 ^

lOO-

3

60-

40

20

10 100 1000

Tissue VEGF (pg / mg protein)

10000

Fig. 3. Corrélationof VEGF concentration in the tumor with tumor vascular density.The VEGF concentration and the vascular density were deduced from Table I. Theregression curve is Y = 13.069 log X°-05C".and the correlation coefficient is r = 0.759

(n = 17).

There was no difference with the VEGF concentration in the serumbetween tumor-bearing patients (84.2 ±85.8 pg/ml) and normal

volunteers (78.2 ±49.6 pg/ml). There was no correlation in VEGFconcentration between the serum and tissue extract in each of thepatients.

VEGF Immunopositivities in Glioma Tissues Correlate withVEGF Concentration Determined by ELISA. Immunohistochem-

ical staining was performed on the glioma tissues from nine patients[4 glioblastoma (three of them contain brain adjacent to and distant farfrom tumors). 3 anaplastic astrocytoma, and 2 low grade astrocyto-

mas| and the normal brain from 1 patient. Immunopositivities(Table 1) were characteristic for glioma and endothelial cells withdiffuse cytoplasmic staining. In four glioblastomas that had highVEGF concentration by ELISA, almost all tumor cells in the peripheral area were immunopositive (Fig. 2. A and B). Vasculature in twoof four glioblastomas was also immunopositive. both in the peripheralarea and the area adjacent to tumor (Fig. 2A). However, the vascula-

ture in two other glioblastomas was negative. In the central area ofglioblastomas, immunopositivities were found in the tumor cells predominantly around the vasculature and the necrosis (Fig. 2C). Inanaplastic and low-grade astrocytomas, immunopositivities were well

correlated with VEGF concentration measured by ELISA. The casewith high concentration of VEGF by ELISA showed immunopositivities in a large number of tumor cells (Fig. 2D). whereas the casewith low concentration of VEGF by ELISA showed immunopositivities in very few tumor cells (Fig. IE). In the normal brain, which hadlow VEGF concentration by ELISA, there was no immunoreactivities(Fig. 2F).

VEGF Concentration Correlates with Microvascular Density inGlioma Tissues. The glioma tissues from 12 patients, malignantlymphomu from 1 patient, metastatic brain tumor from 1 patient, andthe normal brain from 3 patients were immunostained withvon Willebrand factor. Vascular density measured by counting von

Willebrand factor-positive vessels is shown in Table 1. In glioblastoma tissues, the microvessel "hot spots" were localized in the pe

ripheral area without exception. Vascular density was significantlycorrelated with the VEGF concentration measured by ELISA(P = 0.003; r —¿�0.76: Fig. 3). The same correlation between vascular

density and VEGF concentration was also obtained in the astrocytictumors alone, including glioblastoma, anaplastic astrocytoma, andlow-grade astrocytoma (P = 0.04; r = 0.70).

VEGF Concentration in the Conditioned Medium of GliomaCells Correlates with the Induction of Endothelial Cell Migration.All of the glioma cell lines examined released VEGF in the culturemedium in a time-dependent fashion. Among them. U251 and U373

cell lines secreted a large amount of VEGF in the culture medium(Fig. 4). The concentration of VEGF in the glioma-conditioned me

dium was correlated with the induction of endothelial cell migration(Table 2; r = 0.96; P < 0.02).

DISCUSSION

VEGF Quantitation and Glioma Angiogenesis. In this study, weobserved significantly elevated levels of VEGF determined by ELISAin the tissue and cyst fluid of glioblastomas. Gene expression ofVEGF and VEGF receptors are up-regulated in glioblastomas compared to low-grade astrocytomas and normal brain correlating withvascularity (1,6. 21-24). However, concentration of VEGF in those

tumors has not yet been investigated. Quantitation of VEGF concentration has been demonstrated using ELISA for 21 of 32 humaneffusions with cytology-documented malignant cells (7). ocular fluid

from the patients with ischemie retinal diseases, such as diabeticretinopathy and retinal vein occlusion (9. 10). synovial fluids from thepatients with rheumatoid arthritis (8). and using a radioreceptor assayfor the cyst fluids of brain tumors (6). In this study, we: («)confirmedthe significance of VEGF ELISA by demonstrating a time-dependent

increase of secreted VEGF from cultured alioblastoma cells. The

U25112000-

48 72

Timc(hr)

120

Fig. 4. VEGF concentration in the conditioned medium of gliohlastoma cells. VEGFconcentration was time-dependently increased in the serum-free conditioned medium ofvarious glioma cells.

Table 2 Eiulolhelial ceil migration with fiiionm-comlitioneil medium

Glioma conditioned medium

U373 A172

VEGF concentration(pg/ml)Endothelial

cell migration (% control)Medium

alone0100

±9.524

h1255.2

±37.8136.8

±3.048

h8103.6

±1052.4185.4

±6.624

h155.6±

18.6106.1

±2.348

h1181.8

±82.21

14.7 ±3.3

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GLIOBI.ASTOMA VEOF CONCENTRATION

secreted VEGF by glioblastoma cells was contributed to the inductionof endothelial cell migration; and (b) measured directly VEGF concentration by ELISA for tumor tissue and tumor cyst fluid and foundsignificantly elevated levels of VEGF in the tissue and cyst fluid ofglioblastomas, suggesting that VEGF is a measurable element of braintumor biology. Neovascularization often correlates with biologicalaggressiveness and degree of malignancy of brain tumors as well asclinical recurrence, and inversely, with postoperative survival of patients with astrocytomas (3. 25). Among angiogenic growth factors,VEGF mRNA, but not bFGF, transforming growth factor a. andtransforming growth factor ßmRNA. expression has been shown tobe closely correlated with vascularity in both human gliomas andmeningiomas (24). Our study clearly demonstrated that VEGF concentration of brain tumors correlated with thier vascularity. The bFGFlevel is elevated in the serum of patients with renal cell carcinoma(26), in urine in a wide spectrum of cancers including brain tumors(27, 28), and in the CSF from brain tumor patients (29). Determinationof bFGF level in CSF from brain tumor patients correlated withmicrovessel count. It has been proposed that evaluation of CSF bFGF,along with microvessel quantitation in brain tumors, may provideimproved prognostic information (29). In our study, interestingly evenin the anaplastic astrocytomas diagnosed by routine histopathology,there were different immunoreactivities for VEGF correlating withdifferent VEGF concentrations by ELISA. Palte et al. (23) proposedthat the onset of angiogenesis which occurs during the progression oflow-grade astrocytoma to high-grade glioma (anaplastic astrocytoma

and glioblastoma) appears to be mediated by at least three distinctevents: up-regulation of platelet-derived growth factor receptor ßand

of VEGF receptor (KDR) in endothelial cells, and overexpression ofVEGF in tumor cells. The determination of VEGF concentration mayprovide a diagnostic aid, e.g., to solve the problem of grading in cases,in addition to histopathological diagnosis. In the future, it is possiblethat the measurement of VEGF and other angiogenic peptides intissue, cyst fluid, and CSF. together with measurement of neovascu-

larization in the brain tumor itself, may be used to improve theaccuracy of prognosis of patients with brain tumors.

VEGF Immunolocalization. Our immunohistological study confirmed the significance of VEGF concentration by ELISA. The tumorswith high concentration of VEGF by ELISA demonstrated strongimmunoreactivities for VEGF by immunohistochemistry. The tumorswith low concentration of VEGF demonstrated weak or no immunoreactivities. We observed that in human glioblastomas, VEGF immu-

nopositivities are abundant in tumor cells surrounding necrosis. Ourobservation is consistent with a previous ¡mmunohistochemical study(22, 23) and is supported by the fact that glioma cells surroundingnecrosis express VEGF mRNA and they were highly hypoxic but notyet necrotic (23). VEGF also immunolocalized particularly in thetumor cells and vasculature at the peripheral area and in the infiltrating area where VEGF mRNA is expressed (21). The abundant localization of VEGF in the tumor cells and vasculature at the peripheralarea (with no apparent adjacent necrosis), where microvessel "hotspots" were observed, shows a direct line between glioma angiogen

esis and VEGF protein. Also, these observations suggest that, inaddition to hypoxia. there may be other factors that result in VEGFup-regulation m vivo (21), e.g., glucose deprivation (30).

In our study, immunopositivities for the vasculature were observedonly at the peripheral area of two glioblastomas. whereas immunopositivities for the tumor cells were consistent. Previous immunohisto-

chemical studies showed strong immunostaining predominantly in themicrovasculature of gliomas (22, 23). The discrepancy of the vasculature immunostaining could be explained by the difference of MAbused. Four different molecular species of VEGF. i.e.. VEGF121,VEGF|6V VEGFIX<),and VEGF-,,,,,, are generated by alternative splic

ing of mRNA (31). The MAb used in the previous study was raisedagainst human recombinant VEGF,65 and recognizes three isoformsof VEGF12I, VEGFI65, and VEGF,89 (23). The MAb used in thisstudy, MV303, was raised against human recombinant VEGFm.Although MV303 recognizes at least two isoforms of VEGF,21 andVEGF,65 (15), each of four variants of the VEGF family inevitablyretains different reactivity in the immunohistochemistry. VEGFm isentirely soluble; VEGF]K9 is virtually all bound to putative heparin-

containing proteoglycans in the cell surface or in the basement membrane; and VEGFI6S is intermediary, with 50-70% bound (32).MV303 MAb could be less sensitive to the heparin-binding form of

VEGF|65 and VEGF^g. Therefore, in our study, the vasculaturestaining was less prominent compared to a previous study. Finally,previous studies (22, 23) used frozen sections, whereas we usedparaffin sections, which could have caused degradation of the proteinand be less sensitive. Since the freely soluble VEGF121 may beespecially important during periods of active angiogenesis (32), theimmunohistochemical findings consistent with VEGF concentrationdetermined by ELISA are noteworthy.

Failure of Detection of VECF in the Serum from Brain TumorPatients. Initially, failure to detect VEGF in the sera from the patients with several types of cancer, including lung, breast, ovarian,cervix, colon, lymphoma, and stomach cancer, was explained by rapidbinding of VEGF to cell receptors or to extracellular matrix, resultingin the clearance of VEGF from the circulation (7). With modificationof the antibodies in ELISA, we observed that VEGF levels in the serafrom the patients with several types of cancers, including uterine,ovarian, and lung cancer, were significantly higher than those in thesera from the individuals with no sign of cancer (11). However, in thesera from brain tumor patients, VEGF levels were within normalrange, even in the case with extremely high levels of VEGF intumor tissue and tumor cyst fluid, possibly related to blood-brainor blood-tumor barrier. Although the capillaries of malignant gli

omas are hyperpermeable, they are morphologically and essentiallydiscontinuous without fenestration, whereas those in other thanbrain tumors sometimes were fenestrated and continuous (33, 34).Ultrastructural immunohistochemistry has localized tumor mi-crovessel-bound VEGF more precisely to the abluminal surface of

the endothelial cell plasma membrane and to VVOs (33), thefunction of which is up-regulated in the tumor vessels with recent

tracer study (35). They demonstrated passage through VVOs, likethat through simpler chains of vesicles and vacuoles, was directional, i.e., from the luminal to the abluminal side (35). Therefore,VEGF localized in the abluminal surface of the endothelial cellplasma membrane, and VVOs might not penetrate into the vascularlumen and into the general circulation.

Conclusion. VEGF may represent a useful marker and measurableelement of glioblastoma biology, allowing for the assessment ofvascularity. It is possible that measurement of VEGF and otherangiogenic peptides in tissue, cyst fluid, and CSF, together with themeasurement of neovascularization in the brain tumor itself, may beused to improve the management of patients with brain tumors.

ACKNOWLEDGMENTS

We thank Dr. Steven Brem for a critical review of the manuscript.

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GLIOBLASTOMA VEGF CONCENTRATION

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1996;56:2185-2190. Cancer Res   Shingo Takano, Yoshihiko Yoshii, Shinichi Kondo, et al.   Serum and Tumor Tissue of Brain Tumor PatientsConcentration of Vascular Endothelial Growth Factor in the

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