Brain Tumor Pathol
DOI 10.1007/s10014-013-0172-y

ORIGINAL ARTICLE

Characterizing invading glioma cells based on IDH1-R132Hand Ki-67 immunofluorescence
Hemragul Sabit • Mitsutoshi Nakada • Takuya Furuta •Takuya Watanabe • Yutaka Hayashi • Hiroshi Sato •Yukinari Kato • Jun-ichiro Hamada

Received: 22 August 2013 / Accepted: 12 December 2013
Ó The Japan Society of Brain Tumor Pathology 2013

Abstract Glioma, the most common primary brain
tumor, is characterized by proliferative-invasive growth.
However, the detailed biological characteristics of invading
glioma cells remain to be elucidated. A monoclonal antibody (clone HMab-1) that specifically and sensitively
recognizes the isocitrate dehydrogenase-1 (IDH1) protein
carrying the R132H mutation can identify invading glioma
cells by immunostaining. To investigate the degree ofinvasion in gliomas of distinct grades and the proliferative
capacity of the invading cells, immunofluorescent staining
was conducted using antibodies against IDH1-R132H and
Ki-67 on 11 surgical and autopsy specimens of the tumor
core and the invading area. Higher numbers of IDH1R132H-positive cells in the invading area correlated with ahigher tumor grade. Double staining for IDH1-R132H and
Ki-67 demonstrated that most invading cells that expressed
IDH1-R132H were not stained by the Ki-67 antibody, andthe ratio of Ki-67-positive cells among IDH1-R132Hpositive cells was significantly lower in the invasion area
than in the tumor core in all grades of glioma. These datasuggest that higher grade gliomas have a greater invasive

H. Sabit  M. Nakada (&)  T. Furuta  T. Watanabe Y. Hayashi  J. Hamada
Department of Neurosurgery, Division of Neuroscience,Graduate School of Medical Science, Kanazawa University,
13-1 Takara-machi, Kanazawa, Ishikawa 920-8641, Japane-mail: mnakada@med.kanazawa-u.ac.jp
H. Sato
Department of Molecular Virology and Oncology, Cancer
Research Institute, Kanazawa University, Kanazawa, Japan
Y. Kato
Department of Regional Innovation, Tohoku University
Graduate School of Medicine, Sendai, Japanpotential and that invading cells possess low proliferative
capacity.
Keywords Glioma  IDH1  Invasion  Proliferation Immunofluorescence
Abbreviations
IDH1 Isocitrate dehydrogenase1
GBM Glioblastoma

Introduction
One of the main characteristics of gliomas is that the tumor
cells exhibit infiltrative growth within the existing structureof the brain [1]. This invasive growth is a crucial factor thatmakes gliomas difficult to treat. Because the border
between gliomas and the surrounding normal brain tissue isindistinct, extracting the tumor surgically is extremely
challenging. To understand glioma biology, the mechanismof glioma invasion must be elucidated. However, one
major obstacle that hinders research on invasion is thatinvading glioma cells cannot be readily identified among
existing glial cells, especially when attempting to differentiate invading cells from reactive glial cells at tumor
margins.
Isocitrate dehydrogenase 1 (IDH1) mutation has been
identified as early and frequent genetic alterations in lowgrade glioma and secondary GBMs [2]. By contrast, IDH1
mutations are rarely found in primary GBMs. The IDH1
mutations are remarkably specific to a single codon in theconserved and functionally important Arg132 residue,
which is changed to a histidine [3]. Recently, an antiIDH1-R132H mouse monoclonal antibody that specifically123Brain Tumor Pathol

recognizes the IDH1-R132H protein was developed [4, 5],which has facilitated the detection of IDH1 mutation inglioma tissues by routine immunostaining. Immunohistochemical staining with the IDH1-R132H-specific antibody
has been shown to help distinguish single infiltrating neoplastic cells from reactive astrocytes [6].
The degree of invasion in gliomas of distinct grades has
not been defined to date, and the relationship between theinvasive and proliferative capacities of malignant glioma
cells is not entirely clear. Experimental evidence indicatesthat cell motility is inherently and inversely correlated withproliferation in a cellular population [7, 8]. However, to thebest of our knowledge, no in vivo experiment has demonstrated an association between invasion and proliferationin gliomas.
In this study, we clarified the degree of invasion ingliomas and analyzed the invasive and proliferative properties of glioma cells by immunostaining for IDH1-R132H
and Ki-67 in surgical and autopsy specimens of gliomas ofvarious grades. Our results suggest that invasion is moreaggressive in high-grade glioma and that invading glioma
cells proliferate poorly.

Immunofluorescence staining
Briefly, 5-lm-thick histologic sections were incubated in
5 % skim milk/TBST at room temperature for 30 min toblock nonspecific immunoreactions. Next, sections wereincubated overnight at 4 °C with anti-IDH1-R132H
mouse monoclonal antibody (clone: HMab-1) 1:300
dilution and then with donkey anti-mouse IgG secondary
antibodies conjugated with Alexa Fluor-568 (1:1,000;
Molecular Probes, Eugene, OR, USA). After rinsing in
PBS, sections were incubated with a rabbit polyclonal Ki67 antibody (clone: MIB-1; Thermo Fisher Scientific, SanJose, CA, USA) at 1:500 dilution at room temperature for
1 h and then with donkey anti-rabbit IgG secondary
antibodies conjugated with Alexa Fluor-488 (1:1,000;Molecular Probes, Eugene, OR, USA) at room temperature for 1 h. Then, sections were mounted using Vectashield mounting medium containing DAPI andphotographed using a High Standard All-in-One Fluorescence Microscope (Biorevo BZ-9000; Keyence, Osaka,Japan).
Image analysis and quantification

Materials and methods
SamplesTumor specimens derived from surgical operations orautopsies conducted between 2000 and 2012 were selectedfrom the stock at the Kanazawa University hospital.
Pathological grading was performed according to theWorld Health Organization (WHO) classification [9]. We
selected IDH1-R132H-positive tumor cases whose H&Estained slides included both tumor cores and bordering
areas (Table 1).
Table 1 Summary of patients
Caseno.

Age(years)

Sex

Pathological diagnosis

WHOgrade

151

M

Diffuse astrocytoma

II258

M

Diffuse astrocytoma

II345539

M
F

Diffuse astrocytoma
Oligodendroglioma

II
II

540

F

Anaplastic oligodendroglioma

III

624

F

Anaplastic astrocytoma

III

743

M

Anaplastic astrocytoma

III

836

M

Anaplastic astrocytoma

III

936

M

Anaplastic oligodendroglioma

III

1045

M

Secondary glioblastoma

IV

1155

M

Glioblastoma

IV

123

Cells stained for IDH1-R132H and Ki-67 were examined under a Biorevo BZ-9000 microscope. Positively
stained cells in a representative region of the tumor
center and invading area were counted by a neuropathologist (H.S.) using the Dynamic Cell Count BZ-H1C
software (Keyence, Osaka, Japan). Tumor borders weredefined using H&E staining. When specimens had clear
borders, the region within 200 lm from the border line
was demarcated as the tumor margin. In specimens
showing diffuse invasive growth, regions with an*10 % reduction in cell density were determined torepresent the end of the tumor center, and regions
200 lm from the end of tumor center were demarcatedas tumor margins. Microscopic fields were analyzed at
209 magnification to identify cells positive for IDH1R132H. For each cell that stained for IDH1-R132H, thepresence or absence of Ki-67 immunoreactivity wasrecorded. The total number of cells counted per field
ranged from 500 to 1600.

Results
Immunofluorescent staining of gliomas
with IDH1-R132H and Ki-67 antibodies
In the tumor core, most glioma cells were positive for
IDH1-R132H (Fig. 1). In the border areas, invading glioma
cells were identified as IDH1-R132H-positive cells inBrain Tumor Pathol

IDH1

Ki - 67

Merge

GIVGIIIGIIHEFig. 1 H&E and double-immunofluorescent stain with IDH1-R132H
and Ki-67 antibodies. The upper right is the tumor core, and the lower
left is the invasion area. IDH1-R132H-positive cells (red), Ki-67
positive cells (green); inset showing double-positive cells (yellow).

Upper panel: oligodendroglioma. Middle panel: anaplastic oligoastrocytoma. Lower panel: primary glioblastoma. The double-positive
cell is increasing with upgrading of both the tumor core and invasion
area. Bars = 50 lm

gliomas of all grades. Furthermore, Ki-67 immunofluorescent staining performed to estimate proliferative activity
showed that few Ki-67-positive cells were present in theborder areas in all samples compared with the core area. Toanalyze the proliferative activity of invading cells, double
staining was performed for IDH1-R132H and Ki-67(Fig. 1).

Ki-67 immunoreactivity in IDH1-R132H-positive cells

IDH1-R132H-positive cells in the invasion areas
To compare the invasive potential of cells in gliomas ofvarious grades, the cells positive for IDH1-R132H werecounted in the invasion areas. The number of invading cells
in all cases of grade III gliomas exceeded the maximum
number in grade II gliomas, and all cases of grade IV
gliomas showed greater numbers of invading cells than allcases of grade II and III gliomas (Fig. 2a). The mean
number of cells positive for IDH1-R132H per mm2 washigher in grade IV gliomas (1554.2 ± 5.67; mean ± SE)
than in grade II (504.9 ± 96.67) and III (912.2 ± 66.65)
gliomas. These results suggest that higher grade gliomas
are more invasive.

To compare the proliferative potential of glioma cells in thetumor core and the border areas, double immunofluorescent
staining was performed for IDH1-R132H and Ki-67, andthe cells that were stained for both proteins were counted ineach area. As expected, the ratio of Ki-67-positive cells to
the total number of cells that were positive for IDH1-R132H
in the tumor core was elevated when the degree of malignancy was higher (Fig. 2b). The average Ki-67-positive
ratios were 5.98 ± 1.67 % (mean ± SE), 7.8 ± 2.53 %and 13.35 ± 1.27 % in gliomas of grade II, III and IV,respectively. Interestingly, the Ki-67-positive ratio in
IDH1-R132H-positive cells was lower in the border areas
than in the tumor core in all cases, regardless of the tumorgrade, suggesting that invading cells proliferate poorly.

Discussion
In this study, we aimed to determine the degree of invasionin gliomas of distinct grades and the proliferative activity of123

Brain Tumor PatholA
Mutated IDH1 positive cells/mm2
border area

Fig. 2 a The number of IDH1R132H-positive cells per 1 mm2in the border area in grade II(GII), grade III (GIII) and grade
IV (GIV) gliomas. GIV gliomas
showed greater numbers ofinvading cells than all cases ofgrade II and III gliomas. Graphshows the data of individualcases. b The positive rate of Ki67-positive cells in IDH1R132H-positive cells in thetumor core and invasion area. It
is lower in the border areas thanin the tumor core in all cases.
The data of individual cases areshown

1800
1600
1400
1200
1000
800
600
400
2000
GII(n=4)

B
16

Ki-67 positive ratio in IDH1
positive cells

14GIV(n=2)

GIII
(n=5)

tumor coreinvasion area

121086420GII(n=4)

invading glioma cells. Colocalization of IDH1-R132H andKi-67 detected in the cells by using double immunofluorescent staining enabled us to identify proliferating invasivecells. Our results showed that the number of invading cells
was greater when the tumor grade was higher and that
invading cells were rarely proliferative in vivo.
Accumulated evidence including our results demonstrates that proliferative activity in gliomas is greater inhigher grade gliomas [10–12]. By contrast, whether invasiveness is also greater in higher grade tumors in vivo has
not, to our knowledge, been shown before. This is because
methods have not yet been developed to distinguishinvading cells from reactive glial cells, and few studies
have quantified glioma cell invasion in clinical samples.
Invasion-promoting genes such as matrix metalloproteinases are upregulated in higher grade gliomas, suggestingthat higher grade gliomas are more invasive [13–15].
However, based on analyzing advanced MRI data, Denget al. [16] reported that higher grade gliomas do not always
exhibit greater invasiveness. Because IDH1-R132H wasreported to be a marker of invading glioma cells [6, 17–19],
we used immunofluorescent staining for IDH1-R132H inthe border areas of surgical specimens to identify invadingcells and demonstrated quantitatively for the first time that

123

GIII
(n=5)

GIV
(n=2)

higher grades gliomas exhibit greater invasive activity thanlower grade gliomas.
Previous cDNA-microarray data indicated that stimulated migration of cells is accompanied by a downregulation of genes responsible for proliferation in vitro [8].
Moreover, extracellular matrix proteins that activate themotility of glioma cells also lower the growth rate of glioma cells [7, 20]. Analysis of proliferation markers in cells
obtained from the trajectories of stereotactic glioma biopsies has indicated that invasive cells show a lower proliferation rate [21, 22]. These experimental findings indicate
an inverse correlation between motility and proliferation inglioma cells. To demonstrate this inverse relationshipin vivo, we conducted double immunofluorescent staining
for IDH1-R132H and Ki-67 in glioma surgical specimens.
Our data showed that the Ki-67-staining ratio was lower ininvading glioma cells than in cells at the tumor core,suggesting that invading cells have a lower proliferative
potential than the cells at the tumor core. According to ourresults presented here, we are currently analyzing themolecules related to invasion and proliferation signaling ininvading glioma cells.
In conclusion, using clinical samples, we have demonstrated a negative correlation between invasion andBrain Tumor Pathol

proliferation in glioma cells, although the results presented
here must be validated using a larger number of cases. The
novel framework provided by this quantitative and comparative analysis should facilitate future studies examining
the histopathology of invading glioma cells and the biologyof glioma invasion.
Acknowledgments The authors are grateful to Akiko Imamura forhelp with immunohistochemistry.

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