Distribution pattern of tenascin-C in glioblastoma: correlation with angiogenesis and tumor cell proliferation.

Abstract

Tenascin-C (TN-C) is an extracellular matrix protein which participates in different processes like normal fetal development, wound healing, inflammation, keloids and rheumatoid arthritis. Furthermore, the immunostaining for TN-C is seen in the stroma of various malignant tumors as in glioblastoma multiforme (GBM), however, the significance of these findings is still not clear. In this study 62 GBM samples were analyzed immunohistochemically for distribution patterns of TN-C and correlated with angiogenesis and tumor cell proliferation. Tenascin-C in GBM localizes in two compartments, perivascular and intercellular space. Intercellular tenascin-C (TN-C ic) showed focal distribution in 66%, and diffuse one in 34% of cases. Perivascular tenascin-C (TN-C pv) showed strong correlation with microvascular density (MVD) and vascular endothelial growth factor (VEGF) expression. Moreover, it seems that TN-C pv enhanced the effect of VEGF. Intercellular TN-C did not correlate with MVD and VEGF expression, but showed strong correlation with proliferation index. Furthermore, tumors with diffuse TN-C ic expression had higher proliferation indices than tumors with focal TN-C expression. Our results indicate that TN-C plays a role in angiogenesis and tumor cell proliferation, but beside the intensity of expression, the distribution patterns are also important in these processes. This study also suggests that perivascular and intercellular TN-C compartments have probably different sources and different roles in GBM.

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Distribution Pattern of Tenascin-C in Glioblastoma: Correlation
with Angiogenesis and Tumor Cell Proliferation
Senija BEHREM, Kamelija ŽARKOVIC,1Neven EŠKINJA,2Nives JONJIC
Department of Pathology, Medical Faculty, University of Rijeka, 1Department of Neuropathology,
Clinical Medical Center Zagreb and 2Department of Neurosurgery, Clinical Medical Center Rijeka, Croatia
ARTICLE
© 2005 Arányi Lajos Foundation
PATHOLOGY ONCOLOGY RESEARCH Vol 11, No 4, 2005
Article is available online at http://www.webio.hu/por/2005/11/4/0229
Introduction
Glioblastoma multiforme (GBM) is the most common
and most aggressive primary brain tumor. Histologically,
it is an anaplastic, highly cellular tumor, which is distin-
guished from the anaplastic astrocytoma (grade III astro-
cytoma) by the presence of necrosis and a prominent vas-
cular proliferation.
GBM is characterized by a high level of expression of
ECM protein tenascin-C (TN-C), a glycoprotein that is
normally found in various tissues during embryogenesis.
In adult tissues it is either absent or only a faint expression
is detectable in kidney, skin, mammary gland, periosteum,
ligaments, tendons and myotendinous junctions. It reap-
Received: Oct 1, 2005; accepted: Nov 15, 2005
Correspondence: Nives JONJIĆ, MD, Department of Pathology,
Medical Faculty, University of Rijeka, Braće Branchetta 20, 51 000
Rijeka, Croatia. Fax: 00 385 51 325 810, e-mail: nives@medri.hr
Tenascin-C (TN-C) is an extracellular matrix protein
which participates in different processes like normal
fetal development, wound healing, inflammation,
keloids and rheumatoid arthritis. Furthermore, the
immunostaining for TN-C is seen in the stroma of
various malignant tumors as in glioblastoma multi-
forme (GBM), however, the significance of these
findings is still not clear. In this study 62 GBM sam-
ples were analyzed immunohistochemically for dis-
tribution patterns of TN-C and correlated with
angiogenesis and tumor cell proliferation. Tenascin-
C in GBM localizes in two compartments, perivascu-
lar and intercellular space. Intercellular tenascin-C
(TN-C ic) showed focal distribution in 66%, and dif-
fuse one in 34% of cases. Perivascular tenascin-C
(TN-C pv) showed strong correlation with microvas-
cular density (MVD) and vascular endothelial
growth factor (VEGF) expression. Moreover, it seems
that TN-C pv enhanced the effect of VEGF. Intercel-
lular TN-C did not correlate with MVD and VEGF
expression, but showed strong correlation with pro-
liferation index. Furthermore, tumors with diffuse
TN-C ic expression had higher proliferation indices
than tumors with focal TN-C expression. Our results
indicate that TN-C plays a role in angiogenesis and
tumor cell proliferation, but beside the intensity of
expression, the distribution patterns are also impor-
tant in these processes. This study also suggests that
perivascular and intercellular TN-C compartments
have probably different sources and different roles
in GBM. (Pathology Oncology Research Vol 11, No 4,
229–235)
Key words: tenascin, glioblastoma multiforme, angiogenesis, proliferation
pears, however, in diverse reactive conditions such as
wound healing, inflammation, keloids and rheumatoid
arthritis.7,24Strong immunostaining is often seen in the
stroma of various malignant tumors. 4,14,22
This protein was at first called glioma mesenchymal
extracellular matrix antigen, then myotendinous antigen,
hexabrachion protein, cytotactin, neuroactin, and tenascin
(from the Latin verbs “tenere” – to hold and “nascere” – to
be born), a name that describes its presence in tendon, lig-
aments and developmental tissues.5TN-C molecule, a six-
armed glycoprotein, is composed of a radially arranged
hexamer. Each monomer consists of four structural
domains: a hydrophobic N-terminal domain, epidermal
growth factor-like repeats, fibronectin type III-like repeats
and a C-terminal fibrinogen-like domain.24Numerous
studies suggest that TN-C is involved in various processes
such as cell adhesion, migration and growth, but its role in
these processes is still controversial. In some circum-
stances it can be adhesive, while in others it is antiadhe-
’’

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sive;8,24it can stimulate or inhibit migration of the same cell
type (glial cells),9,18,31and it can induce cell proliferation in
cell cultures (fibroblasts, laryngeal carcinoma cells,
glioblastoma cells)11,13,22or inhibit it (fibroblasts).6,21
In most parts of the normal brain TN-C is not detected.
Only weak immunopositivity is shown in the white matter
of the telencephalic lobes and in the first layer of the gray
matter. A strong to moderate expression is present in the
temporal lobe, but there is no TN-C staining in the vessels
in these areas.29On the contrary, a strong TN-C expression
is present in most cases of GBM, especially around hyper-
plastic blood vessels, but the importance of these findings
is not yet clear.1,15Therefore, the aim of this study was to
analyze the distribution pattern of TN-C in GBM, and to
correlate these data with proliferative activity (investigat-
ed by the MIB-1 antibody against Ki-67), expression of
VEGF and microvessel density (MVD, determined by
anti-CD105 antibody). Endoglin or CD105 is a homod-
imeric membrane glycoprotein, a member of transforming
growth factor-beta 1 (TGF-β1) receptor complex.16
Endoglin is preferentially expressed in proliferating
endothelial cells and leukemia cells but weakly or not at all
in cells of most normal tissues. Thus, it is a specific mark-
er for tumor neovascularization, defined as a new prolifer-
ation-associated marker of endothelial cells.17,26
In this study we have demonstrated that distribution pat-
terns of TN-C in GBM, defined as perivascular and inter-
cellular, correlate with tumor cell proliferation and angio-
genesis. Moreover, we investigated the glomeruloid vascu-
lar pattern appearance in GBM with focal and diffuse TN-C
immunostaining, and found that TN-C distribution has a sig-
nificant influence on glomeruloid vascular arrangement.
Materials and methods
Tissue preparation
Surgical specimens from 62 cases of GBM were fixed in
10% formalin and embedded in paraffin. Serial 4-µm sec-
tions were prepared from each sample and used for routine
H&E staining and immunohistochemistry. Tumors were
histopathologically classified according to the WHO Clas-
sification of Brain Tumors.
Immunohistochemical analysis
For immunohistochemical staining of TN-C, endoglin, Ki-
67 and VEGF, streptavidin-biotin staining method was used
(LSAB, DakoCytomation Corporation, Carpinteria, USA).
TN-C immunostaining was detected by a mouse mAb (1:70
dilution; DakoCytomation). For endoglin immunostaining,
mouse anti-CD105 mAb (clone SN6h) was used (1:10 dilu-
tion; DakoCytomation). For assessment of proliferation
index we used the mouse mAb MIB-1 recognizing Ki-67
(1:50 dilution; DakoCytomation). For VEGF immunostain-
ing, a mouse anti-VEGF mAb (C-1) was used (1:100 dilu-
tion; Santa Cruz Biotechnology, Santa Cruz, USA).
Formalin-fixed and paraffin-embedded tissue sections
were deparaffinized in xylene, and rehydrated through
100%, 96% and 70% ethanol. For TN-C and endoglin
staining, digestion with proteinase K enzyme (DakoCy-
tomation) was performed for 10 min at room temperature.
For immunostaining with MIB-1 and anti-VEGF (C-1),
microwave antigen retrieval was performed (4 x 5 min in
10 mM citrate buffer). All immunostained sections were
counterstained with hematoxylin.
Counting and assessment of immunostaining
Two pathologists assessed all parameters. Perivascular
(TN-C pv) and intercellular (TN-C ic) immunostaining for
tenascin were evaluated separately, by using a semiquantita-
tive scale: weak (1), less than 10% of stromal blood vessels
or intercellular interstitium with TN-C positivity; moderate
(2), 10-50% of stromal blood vessels or intercellular intersti-
tium with TN-C positivity; strong (3), more than 50% blood
vessels or intercellular interstitium with TN-C positivity.
For quantification of MVD, the most vascular areas (the
so-called hotspots) were located in the tumor at low mag-
nification. Blood vessels visualized by anti-CD105 mAb
were counted at 400-fold magnification. Any positive
endothelial cell or group of cells in contact with a spot was
counted as an individual vessel. The mean of counts from
three areas was calculated and used in statistical analysis.
The MIB-1 nuclear staining was quantitated on the
Image Analysis System. For each case, 1000 cells were
counted and the percentage of stained cells was expressed
as the proliferation index.
VEGF immunostaining was also evaluated by using a
semiquantitative scale: score 1, less than 25% VEGF-pos-
itive tumor cells; score 2, 25-50% VEGF-positive cells;
score 3, more than 50% VEGF-positive cells.
Statistical analysis
The correlation between TN-C distribution patterns and
the other parameters determined was evaluated by using
the Spearman test and multivariate analysis (multiple
regression test). Mean differences in MVD counts and Ki-
67 were compared with the use of t test. The relationship
between TN-C distribution and glomeruloid vascular
arrangement was analyzed by using the χ2test.
Results
Expression of TN-C and VEGF, and MVD
In concordance with previous studies, we detected high
perivascular and intercellular tenascin expression in GBM.
Namely, only 9.7% (6/62) and 17.7% (11/62) of the
230BEHREM et al
PATHOLOGY ONCOLOGY RESEARCH

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tumors showed weak TN-C pv and TN-C ic expression,
respectively. Strong TN-C pv expression was observed in
56.5% (35/62) of GBM. TN-C distribution, especially in
the intercellular compartment, was very heterogeneous.
Approximately an equal number of tumors exhibited mod-
erate (28 tumors) and strong (23 tumors) TN-C ic
immunostaining, but most of them had focal distribution
pattern (41/62, 66%), while the rest had diffuse distribu-
tion pattern (21/62, 34%).
Due to prominent neovascularization in GBM, all 62
samples stained positive for VEGF. In only 6 of 62 cases
(9%), a minority of tumor cells showed cytoplasmic stain-
ing (weak VEGF expression, score 1 or VEGF 1), while
most tumors (91%) had moderate (VEGF 2, 24 cases) or
strong (VEGF 3, 32 cases) expression.
CD105-positive MVD ranged from 18-99 microvessels.
Most tumors (44/62, 71%) had channeled microvascular
spaces, without glomeruloid bodies. Glomeruloid vascular
proliferation appeared in 18 of 62 tumors (29%).
Comparison between TN-C pv, VEGF expression
and MVD
Expression of TN-C pv classified as weak, moderate and
strong was correlated with mean CD105-MVD, as shown
in Table 1. It is notable that tumors with strong TN-C pv
expression contained significantly more newly-formed
blood vessels (61.89±19.76) than tumors with moderate
(40.58±10.48) or weak (34.05±8.65) TN-C pv expression,
and the difference was significant (p<0.05). A significant
correlation was found between TN-C pv expression and
CD105-MVD (R=0.59, p<0.001).
VEGF expression was classified by using a semiquanti-
tative scale (score 1, 2 and 3), and it was correlated with
mean CD105-MVD. As it can be seen in Figure 1, a strong
correlation was found between these two parameters
(R=0.40, p=0.001). In our samples, most tumors with
strong TN-C pv expression exhibited a strong VEGF
expression (Figure 2). This correlation proved significant
(R= 0.37, p=0.003; Figure 3). Moreover, we compared
CD105-MVD in the group of tumors with strong VEGF-
(score 3) but weak to moderate TN-C pv expression, as
well as in tumors with strong VEGF- and strong TN-C pv
expression. In the former group, the mean CD105-MVD
was 39.99±8.40, while in the latter it was 66.54±20.09
(Figure 4), the difference was significant (p=0.001). These
results suggest that TN-C pv and VEGF collaborate in the
process of angiogenesis, and it seems that TN-C enhanced
the effect of VEGF.
Comparison between TN-C ic, VEGF expression,
MVD and proliferation index
Intercellular expression of tenascin-C (TN-C ic) did not
correlate with either VEGF expression (R=0.23, p=0.11)
or CD105-MVD (R=0.23, p=0.06). A positive correlation
was observed between TN-C ic expression and the prolif-
eration index (R=0.40, p=0.001). Namely, tumors with
weak TN-C ic expression had a lower proliferation index
(percentage of Ki-67-positive cells; 15.88±8.22) than
those with moderate TN-C ic expression (22.64±9.22), and
tumors with strong TN-C ic immunostaining had the high-
est proliferative activity (29.93±13.26) (Table 2). In the
next step, distributions of TN-C ic immunostaining were
distinguished as focal (heterogeneous) and diffuse (homo-
geneous). Ki-67 count was 21.15±12.66 in the former and
30.19±10.18 in the latter group. This difference was sig-
nificant (p=0.003), indicating a role of TN-C ic in tumor
cell proliferation (Figure 5).
By investigating the appearance of glomeruloid vascular
pattern in cases of GBM with focal and diffuse TN-C ic
immunostaining, we found that tumors with focal TN-C ic
expression were more prone to have glomeruloid vascular
arrangements (16/41, 39%) than those with diffuse expres-
sion (2/21, 9.5%) where the channeled vascular spaces pre-
dominated (Figures. 2,6). This finding indicates that TN-C
distribution is important in the process of modulation of ves-
sels, and that a diffuse distribution is accompanied by a rare
231Tenascin-C Expression in Glioblastoma
Vol 11, No 4, 2005
Table 1. Perivascular expression of tenascin-C (TN-C pv)
and CD105-positive microvessel density (CD105-MVD)
in patients with glioblastoma multiforme
No. of
patients
TN-C pv
CD105-MVD
(mean ± SD)
6weak
moderate
strong
34.05 ± 8.65
40.58 ± 10.48
61.89 ± 19.76
21
35
Figure 1. Correlation between VEGF expression (score 1, 2, 3)
and CD105-MVD (R=0.40, p=0.001)
CD105-MVD
70
65
60
55
50
45
40
35
30
25
20
123
VEGF
Mean Mean±SEMean±1.96*SE

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glomeruloid microvascular proliferation. On the other hand,
the intensity of TN-C pv expression did not correlate with the
appearance of glomeruloid vessels (there was no significant
difference in the occurrence of glomeruloid pattern between
tumors with weak, moderate or strong TN-C pv expression).
Discussion
Although the adhesion-modulatory ECM protein TN-C
appears in many injuries and diseases, its role in neo-
plasms is of particular interest. TN-C plays a certain role
in modulating cell adhesion, migration and growth.
Numerous studies have been conducted on this subject, but
the results are controversial and the functional role of TN-C
is still unclear.
It is well known that TN-C immunopositivity is present
in the ECM in astrocytic brain tumors, and the intensity of
TN-C staining correlates with the tumor grade.11,12,19Many
researchers found a correlation between TN-C expression,
angiogenesis and tumor cell proliferation in GBM,11,12,33
but opposite results have also been reported.2
In the present study a high level of perivascular and
intercellular TN-C expression was observed in GBM. Fur-
thermore, a very heterogeneous TN-C distribution was
detected, especially with regard to different level of stain-
ing intensity in the intercellular space. Moreover, in the
intercellular compartment two different distribution pat-
terns were observed, most tumors exhibiting focal (inho-
mogeneous) TN-C ic expression, and a minority express-
ing diffuse (homogenous) staining.
The process of angiogenesis requires a direct interaction
of endothelial cells with their surrounding matrix. The
results of this study demonstrated that tumors with strong
TN-C pv expression have a significantly higher CD105-
MVD (newly formed blood vessels). These results suggest
that TN-C is synthesized not only by glioma cells but also
by endothelial cells, and thus concentrates around blood
vessels.28,33Its possible role could be explained by the find-
ings of some authors who showed that TN-C disrupts focal
adhesions and the actin-based cytoskeleton, and induces
cellular rounding and detachment, which is required for
the first step of angiogenesis (disconnecting endothelial
232 BEHREM et al
PATHOLOGY ONCOLOGY RESEARCH
ab
cd
Figure 2. Immunohistochemical staining for tenascin-C (TN-C) and VEGF in glioblastoma multiforme (GBM) (x100). Serial sec-
tions from the same sample of GBM with strong intercellular and perivascular TN-C expression (a) and strong cytoplasmic VEGF
expression in tumor cells (b). Tumors with focal intercellular TN-C expression (TN-C ic) are more prone to have glomeruloid vas-
cular arrangements (c), while in tumors with diffuse TN-C ic expression channeled vascular spaces predominate (d).

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cells from the “mother” vessels).24Since TN-C possesses
antiadhesive and adhesive activities, it may induce reattach-
ment of endothelial cells to ECM, and migration. These dif-
ferent TN-C effects on endothelial cells are possible through
its interaction with different endothelial integrins (α2β1)
that has a pro-migratory function and av-containing integ-
rin with anti-migratory tendency).28αvβ3 is selectively
expressed on growing neovasculature but not on quiescent
blood vessels.1Schenk et al. found that TN-C plays an
important role in early angiogenesis by modulating the
action of bFGF on endothelial cells, and the fibrinogen
globe of TN-C induces detachment of endothelial cells,
cytoskeletal reorganization and endothelial sprouting.25
Furthermore, our study has not only shown a significant
correlation between TN-C pv expression and the expres-
sion of VEGF, one of the strongest angiogenic factors, but
it has also demonstrated that VEGF is much more efficient
if it is supported by perivascular TN-C: tumors with strong
VEGF and strong TN-C pv expression contained more
newly formed blood vessels (higher CD105-MVD) than
tumors with strong VEGF but weak to moderate perivas-
cular TN-C expression (significantly lower CD105-MVD).
These results are supported by other authors who showed
that TN-C enhances the proangiogenic effects of various
growth factors (VEGF, TGF-β, P1GF).3 The conclusion
was that the upregulation of VEGF and TN-C is spatially
and temporally related to neovascularization, as they are
both found at the site of neovascularization and detected at
the peak of angiogenesis, but not when angiogenesis had
ceased.34Finally, Tanaka et al reported that TN-C regulates
tumor angiogenesis through the regulation of VEGF.27
It seems that the TN-C ic compartment is not involved in
the process of microvascular hyperplasia since it did not
correlate with CD105-MVD, but we observed a strong cor-
relation between intercellular TN-C expression and prolif-
eration index. Namely, proliferation index was significant-
ly higher when the intercellular TN-C immunopositivity
was stronger. This finding was corroborated by the obser-
vation that tumors with a diffuse distribution pattern had a
significantly higher proliferation index than those with a
focal TN-C ic distribution. Some studies reported that the
mitogenic activity of tumor cells is associated with the
interaction of TN-C fibronectin type III-like domains and
av-containing integrins.24Moreover, Huang et al. showed
that TN-C enhances proliferation of some tumor cell lines,
and that it might increase tumor mass by elevating the
number of tumor cells.13They found that TN-C blocks cell
adhesion to fibronectin, and thus prevents syndecan-4 acti-
vation (coreceptor in integrin signaling) and inhibits cell
adhesion and spreading. This prevents the suppressive
effect of fibronectin on tumor cell proliferation.
The hallmark of glioblastoma is microvascular prolifer-
ation, in particular glomeruloid vascular proliferation, and
233Tenascin-C Expression in Glioblastoma
Vol 11, No 4, 2005
Figure 3. Correlation between perivascular tenascin-C (TN-C
pv; weak – 1, moderate – 2, strong – 3) and VEGF expression
(score 1, 2, 3) in glioblastoma multiforme. All results are
grouped between the dotted lines, while the full line shows an
association between increased expression of TN-C pv and
increased expression of VEGF (R=0.37, p=0.003).
VEGF expression
3
2
1
01234
TN-C pv expression
Figure 4. Perivascular tenascin-C (TN-C pv) expression
enhances the effect of VEGF. Tumors with strong VEGF (VEGF
3) and strong TN-C pv expression have more newly formed
blood vessels (CD105-MVD) than tumors with strong VEGF
but weak to moderate TN-C pv expression.
CD105 MVD
80
75
70
65
60
55
50
45
40
35
30
TN-C pv weak to moderate
VEGF strong
TN-C pv strong
VEGF strong
Mean Mean±SEMean±1.96*SE
Table 2. Intercellular expression of tenascin-C (TN-C ic)
and proliferative activity (Ki-67 labeling) in patients
with glioblastoma multiforme
No. of
patients
TN-C ic
Ki-67 (%)
(mean ± SD)
11
28
23
weak
moderate
strong
15.88 ± 8.22
22.64 ± 9.22
29.93 ±13.26