JCANCER RESEARCH 51, 5760-5765, October 15, 1991]
Acidic and Basic Fibroblast Growth Factors Are Present in Glioblastoma
Multiforme1
David F. Stefanik,2 Laila R. Rizkalla, Anuradha Soi, Sidney A. Goldblatt, and Waheeb M. Rizkalla
Department of Radiation Oncology, Laurel Highlands Cancer Program [D. F. S.], and the Department of Pathology, Conemaugh Valley Memorial Hospital [L. R. R.,
A. S., S. A. G., W. M. R.J, Johnstown, Pennsylvania 15905
ABSTRACT
Immunohistochemistry was performed on paraffin sections from hu
man glioblastoma multiforme and normal brain tissue. Acidic fibroblast
growth factor (FGF) was abundantly present in astrocytes from all
glioblastomas studied. Basic FGF was found in the matrix surrounding
proliferating blood vessels in most of the glioblastomas. In contrast,
astrocytes from normal brain did not contain acidic FGF, and peri vascular
matrix staining was not demonstrated for basic FGF in the normal brain.
Both growth factors could be demonstrated in neurons, Purkinje cells,
capillary endothelium, and arterial walls in the normal brain. This study
implicates both growth factors in the pathogenesis of malignant glioma.
Both may be significant mediators of angiogenesis in glioblastoma.
INTRODUCTION
Glioblastoma multiforme is a uniformly fatal brain tumor.
Approximately 10,000 cases occur yearly in the United States
(1). The tumor arises from the astrocytic series of neuroglial
cells. It contains both poorly differentiated and pleomorphic
cellular elements; hence, the name glioblastoma multiforme.
Microscopically, these tumors are characterized by bizarre ap
pearing cells with hyperchromatic nuclei, giant cells, and fre
quent mitotic figures. Various descriptive terms have been given
to the pleomorphic cells. Plump distended astrocytes are gemistocytes; elongated fibril-producing cells are fibrillary astro
cytes; thin hair-like cells are pilocytic astrocytes. Protoplasmic
astrocytes are normal constituents of gray matter. They have
short processes which terminate on blood vessels. Most glio
blastomas are pleomorphic. They are mixtures of the various
cell types. Abundant neovascularization is a hallmark of these
tumors. Necrotic segments are common. Tumors of astrocytic
origin are assigned grades I-IV with grade IV being the most
anaplastic and clinically aggressive (2). Grade III and IV
gliomas are classified as "malignant" because of their rapid
progressive course. In this study we evaluated 12 high grade
(III and IV) gliomas using the general descriptive term, glio
blastoma multiforme.
Acidic and basic FGFs3 are members of the class of angiogenic factors with affinity for heparin. Both are capable of
stimulating proliferation and motility of endothelial cells. They
are the prototype molecules within each class of heparin affinic
growth factors and are believed to play an essential role in
angiogenesis (3). They are distinct but structurally related mol
ecules sharing sequence homology through 53% of their pri
mary structure (4). The genes for acidic FGF are found on
Received 2/25/91; accepted 8/2/91.
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
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1This work was supported by the Conemaugh Valley Memorial and Lee
Hospitals, both of which are members of the Laurel Highlands Cancer Program.
The Laurel Highlands Cancer Program is a member of the Pittsburgh Cancer
Institute's Community Network.
'To whom requests for reprints should be addressed, at Department of
Radiation Oncology, Laurel Highlands Cancer Program, 1086 Franklin Street,
Johnstown, PA 15905.
1The abbreviations used are: FGF, fibroblast growth factor; PBS, phosphatebuffered saline.
chromosome 5, while those for basic FGF reside on chromo
some 2 (5). Endothelial cell growth factor is the precursor of
acidic FGF (6). Both acidic and basic fibroblast growth factors
have been isolated from neural tissues (7).
MATERIALS
AND METHODS
To determine whether the fibroblast growth factors are present in
glioblastoma multiforme, we performed immunohistochemistry on par
affin-embedded tissue. Neutralizing polyclonal antibodies to acidic and
basic fibroblast growth factors were used (F.G.F. Co., La .lolla, CA).
The antibodies were prepared in rabbits against bovine antigen and
purified by the manufacturer using protein A Sepharose chromatography. As a control, slides were probed with purified polyclonal rabbit
immunoglobulin from nonimmunized animals (Dako Corp., Carpin
terÃ-a,CA). To demonstrate the specificity of each antibody, additional
slides were processed after the antibody was absorbed with antigen.
Twelve tumors were probed. Brain tissues from five victims of trauma
served as a normal tissue control. The Vectastain avidin-biotin complex
peroxidase kit was used for detection (Vector Labs, Inc., Burlingame,
CA). Ten ^g of each polyclonal antibody was applied per slide. Five¿tmslides were prepared on a microtome. They were deparaffinized in
xylene, dehydrated in ethanol, rinsed for 5 min in PBS, incubated for
30 min in 0.3% H2O2 in methanol, washed in PBS for 10 min,
preincubated in 1.5% goat serum for 30 min, incubated for 30 min with
the primary antibody in PBS, 1.5% bovine serum albumin, washed for
10 min in PBS, incubated for 30 min with biotinylated sheep antibodies
to rabbit IgG, washed for 10 min in PBS, incubated for 30 min with
avidin-biotin complex, washed for 10 min in PBS, incubated for 4 min
with the chromagen, diaminobenzidine, washed for 5 min in tap water,
and counterstained with Mayer's hematoxylin. For those slides preabsorbed with antigen, the primary antibody was prepared by adding 5 ¿ig
of antigen to 10 Mgof antibody in 50 *ilof PBS, pH 7.2. The solution
was shaken gently for 4 h, incubated overnight at room temperature,
and centrifuged at 7000 rpm for 10 min in a microfuge. The supernatant
was utilized as the primary antibody.
RESULTS
The distribution of acidic and basic fibroblast growth factors
in normal brain tissue is demonstrated in Fig. 1. Acidic fibro
blast growth factor was detected in capillary endothelial cells
and within the walls of leptomeningeal arteries where it could
be seen strikingly in the adventitial, as well as the muscular,
layer. It was also detected in the cerebellar Purkinje cells and
cortical neurons. Astrocytes failed to stain for acidic FGF. Basic
FGF was found in fewer subpopulations of cells than acidic
FGF in the normal brain. Like acidic FGF, basic FGF could be
seen in the muscular layer of large blood vessels. However, the
adventitia was spared. Purkinje cells also demonstrated staining
for basic FGF. Sporadic neurons and capillaries demonstrated
staining which was less dramatic in comparison to acidic FGF.
These findings are summarized in Table 1.
Twelve high grade gliomas were evaluated (Fig. 2). The
pattern of staining was consistent. All 12 contained numerous
astrocytes whose cytoplasm stained for acidic FGF. The inten
sity of the stain was considerably greater in the malignant as
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ACIDIC AND BASIC FGF IN GLIOBLASTOMA
k.**
Fig. 1.
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ACIDIC AND BASIC FGF IN GLIOBLASTOMA
Fig. 2.
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ACIDIC AND BASIC FGF IN GLIOBLASTOMA
Table 1 Summary of findings
FGF
Glioblastoma
Acidic
Abundantly present in
gemistocytic astrocytes throughout tu
mor
Basic
Present in perivascular
matrix
of gemistocytic or protoplasmic astrocytes were unstained,
while focal stretches of interstitial matrix stained positively.
The inhomogeneity of distribution may account for the lack of
staining in three of the tumors.
The specificity of each antibody for the antigen in question
is demonstrated in Fig. 4. Absorption with antigen resulted in
inhibition of cytoplasmic staining for anti-acidic FGF and
perivascular matrix staining for anti-basic FGF.
Normal brain
Not present in astrocytes
Present in Purkinje cells,
capillary endothelium,
neurons, and muscular
and adventitial layers of
large arteries
Not present in perivascular
matrix
DISCUSSION
Present in Purkinje cells,
muscular layer of large
arteries, and faintly in
sporadic neurons and
capillaries
•¿
Fig. 3. Basic FGF is demonstrated within the walls of a thick walled prolifer
ating blood vessel in glioblastoma multiforme (x 400).
opposed to normal tissue. The cytoplasm of gemistocytic and
protoplasmic astrocytes stained most intensely. In nine of the
12 tumors, staining was uniform throughout the tumor. The
vast majority of malignant astrocytes contained acidic FGF.
However, in three tumors the staining was heterogenous with
areas of solid unstained cells. Tumors contain multiple subpopulations of cells. Heterogenous staining has been reported in
the immunohistochemical analysis of other tumors (8). These
findings concur with a report that glioma cells in culture pro
duce acidic FGF (9).
Basic FGF was demonstrated in nine of the 12 tumors studied
(Fig. 2). Staining was intense in the matrix surrounding prolif
erating blood vessels. In some sections basic FGF could be seen
within the walls of the tumor vasculature (Fig. 3). Fibrillary
astrocytes stained for basic FGF. The distribution of basic FGF
was inhomogenous throughout the tumors. Typically, segments
This study adds to a growing list of reports which implicate
the fibroblast growth factors in the pathogenesis of human
glioma. The presence of RNA coding acidic and basic FGFs
was demonstrated by Takahashi et al. (10) using Northern blot
analysis. In their study, 17 of 18 gliomas expressed basic FGF,
while 13 of 18 expressed acidic FGF. One tumor was probed
for basic FGF using in situ hybridization and immunohistochemistry. Hybridization signal was detected in malignant cells
as well as within endothelial cells of blood vessels. Immunohistochemistry confirmed the presence of the basic FGF protein
in malignant cells. Other studies have demonstrated the pres
ence of acidic FGF, basic FGF, and receptors for basic FGF in
cultured human glioma cells (11-13).
Staining for acidic FGF was exclusively intracellular. Basic
FGF was demonstrated in the cytoplasm of fibrillary astrocytes.
It also appeared to be present in the matrix surrounding prolif
erating vessels. Conclusions cannot be reached from this study
regarding the mechanics of extracellular deposition. The fibro
blast growth factors lack classic secretory signal sequences (4).
Folkman and Klagsbrun (3) have speculated that release from
cells might occur after cellular damage or under tightly regu
lated conditions.
Basic FGF has been demonstrated in bovine corneal base
ment membrane and in developing retinal capillaries (14, 15).
It has been demonstrated in the subendothelial extracellular
matrix of cultured bovine corneal and endothelial cells (16).
Folkman et al. postulated that these structures may serve as
storage depots which regulate the accessibility of the molecule
to the vascular endothelium. They hypothesized that tissue
injury could release basic FGF, thereby triggering angiogenesis.
In this study we demonstrated the presence of both acidic and
basic FGF in the cytoplasm of neurons, endothelium, and
Purkinje cells. They may also serve as reservoirs for the vascular
mitogens.
There was a considerable difference in the staining of malig
nant as opposed to normal brain tissue for both acidic and basic
FGF. This is summarized in Table 1. In normal brain tissue
acidic FGF was not demonstrated in astrocytes. In contrast,
glioblastoma contains a large population of intensely staining
Fig. 1. Immunohistochemistry of normal brain tissue of a trauma victim. Affinity-purified rabbit polyclonal antibodies to acidic FGF (A-C) and basic FGF (D-F)
were used as the primary antibodies. The purified IgG fraction of rabbit serum from nonimmunized animals (G-l) was used as a control. A, acidic FGF is demonstrated
in capillary endothelial cells and cortical neurons. Astrocytes failed to stain (x 400). //. acidic FGF is demonstrated in cerebellar Purkinje cells and in the cytoplasm
of adjacent neurons (X 400). C, acidic FGF is demonstrated in the muscular layer and adventitia of a leptomeningeal artery (x 400). D, cortical neurons, capillary
endothelial cells, and astrocytes fail to stain for basic FGF (x 400). E, basic FGF is demonstrated in the cytoplasm of a cerebellar Purkinje cell. Weak staining is also
seen in a nearby capillary and neurons (x 400). F, the muscular layer of a leptomeningeal artery stains for basic FGF. The adventitial layer is relatively spared of the
stain (x 200). G-l, purified nonimmune rabbit serum demonstrates no staining (G and //, x 400; /, x 200).
Fig. 2. Distribution of acidic and basic fibroblast growth factors in glioblastoma multiforme. The primary antibody is anti-acidic FGF (A, D, and G), anti-basic
FGF (B, E, and //), and nonimmune polyclonal rabbit immunoglobulin (C, F, and /). A-C, glioblastoma 1: acidic FGF is demonstrated in the cytoplasm of astrocytes
clustered about a blood vessel. Basic FGF is seen outlining the lumina of numerous blood vessels. It can be seen within some of the vascular walls. The control
antibody demonstrates no staining (A, x 100; B and C, x 200). D-F, glioblastoma 2: astrocytes stain deeply for acidic FGF. Basic FGF is demonstrated along fibrillary
astrocytes and the perivascular matrix. The gemistocytic astrocytes which stain for acidic FGF are unstained for basic FGF. Control polyclonal antibodies show no
staining (D, x 200; E and F, x 100). G-I, glioblastoma 3: heterogenous staining for acidic FGF is demonstrated. Basic FGF is demonstrated intensely in the matrix
surrounding numerous proliferating blood vessels. The control slide demonstrates no staining ((¡I, x 200).
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ACIDIC AND BASIC FGF IN GLIOBLASTOMA
Fig. 4. Immune staining is inhibited by absorption of the antibody with antigen..-( and B, a glioblastoma is immunohistochemically probed with anti-acidic FGF
(A) and anti-basic FGF (B). Note presence of staining for both antibodies. C and D, adjacent sections from the same tumor are probed with anti-acidic FGF absorbed
with acidic FGF (Q and anti-basic FGF absorbed with basic FGF (D). Inhibition of staining in comparison with .I and B demonstrates that both antibodies recognize
their respective antigens.
gemistocytic astrocytes. Often they were clustered about blood
vessels. In gliobliostoma basic FGF was abundantly present in
the matrix surrounding proliferating blood vessels. This was
not seen in the perivascular matrix from any section of normal
brain examined.
Neovascularization is a necessary phenomenon for both nor
mal and neoplastic growth. Tumor growth is dependent upon
the ability of the tumor to recruit a vasculature. Glioblastoma
multiforme is a lethal brain tumor characterized by aberrant
neovascularization. Thick walled proliferative vascular channels
are common. Both acidic and basic fibroblast growth factors
are mitogenic for endothelial, as well as glial, cells. Both are
expressed abundantly in glioblastoma. Each has a unique pat
tern of distribution. The gemistocytic and protoplasmic astro
cytes which stained for acidic FGF failed to stain for basic
FGF. Fibrillary astrocytes produced basic but not acidic FGF.
This indicates that each growth factor is produced by a separate
subpopulation of cells. Acidic FGF was seen in the cytoplasm
of astrocytes, often in proximity to blood vessels (Fig. 2). Basic
FGF was consistently demonstrated in the matrix around pro
liferating blood vessels (Fig. 2). It could also be seen within
some vessel walls (Fig. 3). Close proximity to the vasculature
suggests a role for both fibroblast growth factors in the angiogenic process. The abundant presence of both growth factors
suggests that each has a role in the pathogenesis of glioblastoma
multiforme. In particular, the presence of basic FGF in peri-
vascular matrix and within tumor vasculature suggests that this
molecule plays a role in the vascular proliferation which accom
panies glioblastoma multiforme.
ACKNOWLEDGMENTS
The authors thank Brad Goldblatt for technical assistance, Drs. Paul
Monteleone and John Verger for critical review, and Cindy Miller,
Karen Ryan, and Diane McLachlan for assistance in preparing the
manuscript.
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Acidic and Basic Fibrolast Growth Factors Are Present in
Glioblastoma Multiforme
David F. Stefnik, Laila R. Rizkalla, Anuradha Soi, et al.
Cancer Res 1991;51:5760-5765.
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