RESEARCH ARTICLE
High Expression of Vimentin is Associated With
Progression and a Poor Outcome in Glioblastoma
Jiaxin Zhao, MD,* Liqiu Zhang, PhD,w Xingli Dong, PhD,z Lu Liu, MSc,y Linman Huo, MSc,y
and Huirong Chen, MD*
Abstract: Glioblastoma multiforme (GBM) has a high re-
currence and mortality rate. Because of a poor understanding of
the mechanism for this disease, treatment regimens have re-
mained limited. Vimentin, one of the major cytoskeletal pro-
teins, is associated with cellular structure. However, the function
of vimentin in GBM is still undened. In the present study, we
investigated the expression level of vimentin in 179 GBM tissues
using immunohistochemistry. We found that the vimentin ex-
pression level was associated with the time to progression
(P = 0.029). A Kaplan-Meier analysis revealed that patients
with high vimentin expression had a signicantly shorter overall
survival (P = 0.0002) and progression-free survival (P = 0.0001)
compared with those with low expression. Furthermore, in vitro
experiments showed that withaferin-A, a chemical inhibitor of
vimentin, could inhibit GBM cell migration and invasion ac-
tivity when its concentrations were <0.5 mM, and higher con-
centrations of withaferin-A could decrease the viability of
U251and U87 cells signicantly. In conclusion, our results in-
dicated that vimentin may play an important role in the pro-
gression of GBM.
Key Words: vimentin, glioblastoma, prognosis, withaferin-A
(WFA)
(Appl Immunohistochem Mol Morphol 2016;00:000000)
G lioma is the most common and deadly form of
primary tumor in the central nervous system.1
High-grade gliomas, such as anaplastic astrocytoma
or glioblastoma multiforme (GBM), have a diusely in-
ltrative nature, with a high recurrence and mortality rate.
Despite the best available therapeutic options, the life ex-
pectancy of GBM patients is approximately only 12 to 15
Received for publication April 7, 2016; accepted June 20, 2016.
From the *Department of Neurosurgery, The Fourth Aliated Hospital
of Harbin Medical University; Departments of wParasitology;
zBiochemistry and Molecular Biology; and yPharmacology, Harbin
Medical University, Harbin, Heilongjiang, China.
Supported by Youth Science Foundation of Heilongjiang Province
[grant number: QC2015109]. J.Z. and L.Z. contributed equally to the
work and should be considered corst authors.
The authors declare no conict of interest.
Reprints: Huirong Chen, MD, Department of Neurosurgery, The
Fourth Aliated Hospital of Harbin Medical University, Yiyuan
Road No. 37 of Nangang District, Harbin 150001, Heilongjiang,
China (e-mail: chenhuironghyd@126.com).
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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, 2016
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months after diagnosis.2 The reason for this poor prog-
nosis is that tumor cells in GBM are not only highly
proliferative, but also invade surrounding brain tissues.
Surgical resection of the tumor mass is performed, but the
remaining malignant cells eventually lead to tumor pro-
gression and patient death. Because of a poor under-
standing of the mechanism governing the invasive behavior
of GBM, viable treatment regimens have remained limited.
Hence, it is necessary to identify the molecular character-
istics responsible for the tumor invasion process and the
potential prognostic and therapeutic marker for GBM.
Intermediate lament proteins are the main cytoske-
letal proteins that play dierent roles under a variety of
conditions. Vimentin, a 57-kDa protein, belongs to the type
III of 6 dierent intermediate lament families.3 It is nor-
mally present from the nucleus to the plasma membrane in
cells of mesenchymal origin,4 where it is known to provide
cellular structural support and maintain tissue integrity.5
Moreover, accumulating data have shown that it is also
expressed in epithelial cells, and is associated with meta-
static disease,6 endothelial cell adhesion,7 acquisition of
migration, and/or invasive properties of cell signaling.8
Similar correlations are observed in cancer cell lines and in
most tumor types. Some studies have mentioned that vi-
mentin is expressed in lung cancers, and is signicantly
associated with a poorer dierentiation grade.911 In breast
cancer, vimentin overexpression is usually observed in
younger women and is related to the tumor size.12 Previous
researchers have detected the expression of vimentin in oral
squamous cell carcinoma patients or cell lines13; sub-
sequently, Liu et al14 rst reported the close relationship
between the vimentin expression level and local recurrence.
A process that transforms stationary epithelial cells into
migratory cells is termed as epithelial-mesenchymal tran-
sition,15 and thus should play a role in cell migration and
invasion.16 Vimentin is now regarded as a canonical marker
of epithelial-mesenchymal transition, and the upregulation
of vimentin is observed in a wide range of cancer types
undergoing this process, such as prostate cancer, breast
cancer, malignant melanoma, and tumors of the central
nervous system.17 On the basis of these ndings, further
evidence is needed to prove that vimentin is a proinvasive
molecular marker and a potential anticancer therapeutic
target for GBM.
In the current study, we investigated the relation-
ship between the expression of vimentin protein and the
clinicopathologic features and the prognosis of GBM
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Zhao et al Appl Immunohistochem Mol Morphol
patients. Subsequently, cytologic experiments were per-
formed to dene the role of vimentin in the biological
behavior of GBM cells. We found that vimentin may be
an independent adverse prognostic factor of survival in
GBM patients. In vitro experiments further illustrated the
function of vimentin in the proliferation and the invasion
of GBM cells.
MATERIALS AND METHODS
Patients and Tissue Specimens
GBM specimens were obtained from 179 Chinese
patients who underwent operation in the Department of
Neurosurgery, the Fourth Aliated Hospital of Harbin
Medical University, between January 2007 and July 2014.
Detailed clinicopathologic information was obtained
from patient records. The histologic diagnosis was con-
rmed by 2 pathologists according to the World Health
Organization criteria. All the patients had been surgically
treated with gross total resection of the primary tumors.
Follow-up was conducted regularly, and clinical records
of all patients in the study were obtained till the closing
date (October 1, 2015) or till death. A magnetic resonance
imaging scan was performed when tumor recurrence was
suspected. Patients with a survival of <1 month were
excluded. All protocols were reviewed and approved by
the Ethical Committee of Harbin Medical University.
Written informed consent was obtained from all patients.
Immunohistochemistry
Paran-embedded GBM tissues were cut into 4-
mm-thick slices and placed on poly-L-lysinecoated slides.
The slides were deparaffinized in xylene and rehydrated
through graded ethanol. Endogenous peroxidases were
blocked using 3% hydrogen peroxide for 10 minutes at
room temperature, and the slides were autoclaved in
0.01 mol/L citrate buffer (pH 6.0) at 1211C for 10 minutes
for antigen retrieval. This was followed by rinsing in
phosphate-buffered solution (PBS) 3 times; the slides were
incubated at 41C overnight with primary rabbit anti-
vimentin antibody (bs-8533 R, dilution 1:200; BIOSS,
China). After incubation, the slides were lavaged with
PBS and stained with the 2-step plus polymerized horse-
radish peroxidase anti-rabbit immunoglobulin G de-
tection system (ZSGB-Bio, Beijing, China) for 20 minutes
at room temperature. Finally, the slides were counter-
stained with hematoxylin and covered with glycerin gel,
followed by diaminobenzine staining. PBS replaced anti-
vimentin antibody in negative controls.
Immunoreactivity was evaluated on the basis of the
staining intensity and the percentage. The intensity score
was dened as 0 (negative), 1 (weak staining), 2 (moderate
staining), or 3 (strong staining). The percentage score was
determined on the basis of the total number of positive
cells, and was graded as 0 (0%), 1 (1% to 25%), 2 (26%
to 50%), 3 (51% to 75%), or 4 (76% to 100%). The sum
of the staining intensity and the percentage scores was
used as the nal vimentin expression score (0 to 7).
2 | www.appliedimmunohist.com
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 Volume 00, Number 00, 2016
Cell Preparation and Treatment
Human glioblastoma cell lines U251 and U87 were
obtained from the Chinese Academy of Sciences Cell
Bank (Shanghai) and maintained in Dulbecco-modied
Eagle medium (Gibco, Carlsbad, CA) containing 10%
fetal bovine serum (Invitrogen), 1% penicillin/strepto-
mycin, and 1% hydroxyethyl piperazine ethanesulfonic
acid buer solution in a humidied atmosphere contain-
ing 5% CO2 at 371C.
Withaferin-A (WFA), a chemical inhibitor of vi-
mentin protein, was purchased from J&K Chemical
(Beijing, China). It is dissolved in dimethyl sulphoxide
(DMSO) and stored at  201C. For in vitro experiments,
WFA groups of cells were incubated with increasing
concentrations of WFA (0.2, 0.4, 0.6, 0.8, and 1.0 mM),
respectively. DMSO treatment was used as the control
group, and mock groups were set up by applying the same
volume culture medium.
The Cell-Proliferation Assay
Mock, control, and WFA groups of cells were
seeded on 96-well culture plates (5000 cells/well) with
200 mL the culture medium. After culturing for 48 hours,
the cell proliferation was analyzed by an 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)
assay. In brief, cells were treated with 20 mL MTT for 4
hours, and then incubated with 150 mL DMSO for
10 minutes. The optical density was then measured at
490 nm.
The Wound-Healing Assay
Cells were placed in 24-well plates, and a 2.0-mm
wound was scraped with a 1-mL pipette tip. Mock, con-
trol, and WFA groups were treated as described above.
Cell motility was assessed by measuring the movement of
cells into the scraped area. The speed of wound healing
was monitored everyday and the ratio of the width of the
wound against the initial wound width was calculated.
The Transwell Invasion Assay
The upper transwell chambers (diameter 6.5 mm,
pore size 8 mm; Costar, Cambridge) were coated with
50 mL Matrigel (BD-356234). After the Matrigel solidied
at 371C, each group of cells (3  105, cultured as described
above) as added into the upper chamber with a serum-free
medium (100 mL), and the lower chamber received the
completed medium containing 10% fetal bovine serum
(600 mL). After 24 hours of incubation at 371C with 5%
CO2, membranes coated with Matrigel were swabbed
with a cotton swab and the cells that had migrated from
the Matrigel into the pores of the inserted lter were xed
with 100% methanol for 10 minutes. The membranes
with cells were soaked with crystal violet and dried
completely. The number of cells attached to the lower
surface of the lter was counted in 3 randomly selected
visual elds from the central and the peripheral portions
of the lter using an inverted microscope (  100 magni-
cation).
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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, 2016 Vimentin is Associated With Glioblastoma

FIGURE 1. Immunohistochemical evaluation and representative immunostaining of vimentin. A, Receiver-operating characteristic

curve analysis for OS based on vimentin expression. B, The cutoff value by immunohistochemistry (IHC) scores for a longer OS. C,
Low expression of vimentin in glioblastoma multiforme. D, High expression of vimentin in glioblastoma multiforme (original
magnification: big figures 200, small figures  400). AUC indicates area under the curve; OS, overall survival.

Statistical Analysis TABLE 1. The Correlation Between Vimentin Expression and

Receiver-operating characteristic (ROC) curves Various Clinicopathologic Features in GBM Patients
were constructed to determine the cuto scores of vi-
Vimentin Expression
mentin expression for overall survival (OS) >1 year.18
The Youden index denes the optimal threshold value: Variables No. Cases Low High P
the sum of sensitivity and the specicity reaches a max- Age (y) 0.759
imum at this point. The area under the curve analysis was < 60 102 34 68
Z60 77 24 53
used to measure the prognostic or predictive accuracy. Sex 0.436
The correlation of vimentin expression with patients Male 97 29 68
Female 82 29 53
clinicopathologic variables was analyzed by a w2 test or a Karnofsky (KPS) 0.775
Fisher exact test. The Kaplan-Meier method was used to r60 33 10 23
estimate the progression-free survival (PFS) and the OS. > 60 146 48 98
Time to progression (mo) 0.029
Survival dierences according to vimentin expression <3 60 13 47
were analyzed by the log-rank test. Z3 119 45 74
Data from the experiments on cultured cells were Mean tumor diameter (cm) 0.504
<5 99 30 69
expressed as mean SD, and statistical signicance be- Z5 80 28 52
tween the groups was analyzed with a one-way analysis of Cystic degeneration 0.382
Yes 75 27 48
variance, followed by the leastsignificant difference No 104 31 73
method for post hoc test comparisons. All the analyses Seizure 0.196
were performed with MedCalc 13.0, and significance was Yes 38 9 29
No 141 49 92
defined as P < 0.05.

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Zhao et al Appl Immunohistochem Mol Morphol  Volume 00, Number 00, 2016

FIGURE 2. The Kaplan-Meier analysis for OS and PFS based on vimentin expression in GBM patients. A, The Kaplan-Meier analysis
for OS based on vimentin expression in patients with GBM (log-rank test, P = 0.0002). B, The Kaplan-Meier analysis for PFS based
on vimemtin expression in patients with GBM (log-rank test, P = 0.0001). C, The mean OS time for patients with high and low
vimentin expressions. D, The mean PFS time for patients with high and low vimentin expressions (green: the high vimentin
expression group; red: the low vimentin expression group; **P < 0.01). GBM indicates glioblastoma multiforme; OS, overall
survival; PFS, progression-free survival.

RESULTS (CI), 0.538-0.702]. Using a threshold value of 3.88

Clinical Characteristics of Patients
Among the patients (97 male and 82 female, 1.18:1),
57.0% of them were younger than 60 years, Karnofsky
was 81.6% before surgery for >60 patients, and the OS
of 70 patients (39.1%) was >12 months. Tumor re-
currence occurred within 3 months after surgery in 60
(33.5%) patients, and 38 (21.2%) patients had seizures. In
addition, the tumor diameter of 80 patients was Z5 cm,
and 41.9% of the patients had cystic degeneration on
magnetic resonance imaging scan before surgery.
The Expression of Vimentin Protein in Human
GBM
To calculate the immunohistochemistry cuto
scores for OS over 12 months, an ROC curve was gen-
erated, which is shown in Figure 1A, and the resulting
area under the curve was 0.620 [95% condence interval
(Fig. 1B), 121 patients were classied as high expression
(Fig. 1D) and the remainder (n = 58) as low expression
(Fig. 1C). As indicated in Figure 1, protein staining was
identied mainly in the cytoplasm and the membrane of
GBM cells.
Correlations Between Vimentin Expression and
Various Clinicopathologic Features
Correlations between the vimentin expression status
and various clinicopathologic features are summarized
in Table 1. Vimentin expression was not associated with
the age, the sex, the Karnofsky, the mean tumor diameter,
the cystic degeneration status, and the seizure status (each
P > 0.05); however, it was signicantly associated with
the time to progression (P = 0.029). The vimentin ex-
pression percentage of patients with recurrence time <3
months (47/60, 78.3%) was higher than that of patients
with recurrence time >3 months (74/119, 62.2%).

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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, 2016 Vimentin is Associated With Glioblastoma

FIGURE 3. Morphologic evaluation of WFA-treated glioblastoma multiforme cells. A, The chemical structure of WFA (molecular
formula, C28H38O6; molecular mass, 470.60 g/mol). B, WFA (0.2 mM/24 h) induces morphologic transformation of glioblastoma
multiforme cells (original magnification:  200). WFA indicates withaferin-A.

FIGURE 4. The effect of withaferin-A (WFA) on the viability of glioblastoma multiforme cells. A, The OD value of MTT assays on
U251 cell proliferation of each group. B, The OD value of MTT assays on the U87 cell proliferation of each group (**P < 0.01,
compared with the control). OD indicates optical density.

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Zhao et al Appl Immunohistochem Mol Morphol  Volume 00, Number 00, 2016

FIGURE 5. WFA suppresses glioblastoma multiforme cell migration and invasion. A, The 48-hour wounding migration assay with
low WFA concentrations or DMSO control for U251 (upper panel) and quantification (lower panel). B, The 48-hour wounding
migration assay with low WFA concentrations or DMSO control for U87 (upper panel) and quantification (lower panel). C,
Representative micrographs (upper panel) and quantification (lower panel) depicting the invasion abilities of low concentration
WFA-treated U251 cells or DMSO control in the transwell assay. D, Representative micrographs (upper panel) and quantification
(lower panel) depicting the invasion abilities of low-concentration WFA-treated U87 cells or DMSO controls in the transwell assay
(original magnification: 100; *P < 0.05, **P < 0.01). WFA indicates withaferin-A.

High Vimentin Expression Correlates With a
Poorer Survival of GBM Patients
Our results showed that patients with high ex-
pression of vimentin had a shorter OS (P = 0.0002,
Fig. 2A) and PFS (P = 0.0001, Fig. 2B) than patients
having low vimentin expression. The mean OS time was
14.185 (95% CI, 12.326-16.043) against 10.186 (95% CI,
9.277-11.096) months (Fig. 2C), and the mean PFS time
was 11.041 (95% CI, 9.610-12.472) against 7.380 (95%
CI, 6.633-8.126) months (Fig. 2D) in the low-expression
and the high-expression patients, respectively. In addi-
tion, the percentage of patients with >1 year OS was
20.8% for the high-expression patients and 40.7% for the
low-expression patients.
WFA Induces GBM Cell Morphology Changes
Compared with the microscopic morphology of
mock and control cells, the indicated GBM cells cultured
with 0.2 mM WFA (Fig. 3A) for 24 hours displayed
smaller round cell bodies and looked a little thin
(Fig. 3B). These morphologic changes may indicate that
WFA has an eect on the cytoskeleton of glioma cells
through vimentin protein, which eventually alters the cell
structure.
WFA Leads to a Reduction of GBM Cell Growth
In Vitro
To explore the biological function of WFA on
glioma cell growth, 2 GBM cell lines were treated with
varying concentrations of WFA for 48 hours, and the cell
viability was measured by an MTT assay. As shown
in Figures 4A and B, WFA concentrations <0.5 mM had
a negligible eect on proliferation. Higher concentrations
of WFA decreased the viability signicantly compared
with mock and control groups in both U251and U87
cells. Thus, the results revealed that WFA exhibits a
potent antiproliferative eect by inhibiting growth.

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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, 2016
WFA Inhibits the Motility of GBM Cells In Vitro
Next, to evaluate the inuence of WFA on the mi-
gration ability of GBM cells, U251 and U87 cells were
treated with low doses of WFA, which were <0.5 mM,
and a scratch wound-healing assay was conducted. As
illustrated in Figures 5A and B, the WFA group of cells
displayed an apparently slower migration in comparison
with control cells. Similarly, transwell invasion studies
revealed that the WFA restrained GBM cell invasion
activity signicantly in a dose-dependent manner
(Figs. 5C, D). On the basis of these results, WFA inhibits
GBM cell migration and invasion at micromole doses that
have a weak eect on cell proliferation.
DISCUSSION
GBM is usually microscopically impossible to be
removed completely because of its remarkable migration
ability within the brain tissue. In this study, the ex-
pression of vimentin, which is extracellular matrix com-
ponents, was investigated in GBM patients. According to
the ROC curve, 4.0 was actually selected as the nal
cuto score, leading to the greatest number of patients
with OS <1 year being classied as high expression.
Survival analysis showed that a high expression of vi-
mentin was associated with poor OS and PFS (Fig. 2).
Moreover, this is the rst report showing a strong cor-
relation between vimentin expression and the time to
progression in GBM (Table 1). These ndings suggest
that vimentin acts as an independent prognostic factor for
GBM patients.
On the basis of the data summarized from dierent
laboratories, it is clear that vimentin plays an important
role in the invasive process of tumor cells.1921 In the
present study, our experiments further conrmed the
functions of vimentin in glioblastoma cells. WFA
(Fig. 3A), a highly oxygenated C-28 ergostane-type ster-
oidal lactone isolated from Withania somnifera,22 binds
covalently to the cysteine residue on vimentin and pre-
vents vimentin assembly into the intermediate lament.23
For U251 and U87 cells, WFA had a weak cytotoxic
eect in vitro at concentrations <0.5 mM (Fig. 4), but
retained potent anti-invasive activity at these low doses
(Fig. 5). Helfand et al24 reported that serine 38 phos-
phoryation of vimentin could control the formation of
lamellipodia, a process associated with cell polarization
and cytoskeletal remodeling. Increasing evidence also
suggest that the functions of vimentin extend beyond its
mechanical and structural properties to include demon-
strable roles in motility.25 Studies conducted in our re-
search showed that GBM cells treated with low doses of
WFA resulted in marked morphologic changes, including
cell rounding and nuclear condensation (Fig. 3B). Eects
of WFA occurred as early as 4 hours after treatment in-
itiation. In addition, wound healing and transwell assays
were conducted at low concentrations that were necessary
to inhibit the migration and the invasion of GBM cells.
Several previous studies have found that vimentin con-
trols cellular growth signaling pathways, acting as a
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Vimentin is Associated With Glioblastoma
chaperon for several protein kinases.26,27 For instance,
vimentin phosphorylation may be involved in focal ad-
hesion kinase as it links to downstream targets, and def-
icits in vimentin can downregulate focal adhesion kinase
and extracellular regulated protein kinases expression,
causing cell impairment.28 According to the cell growth
analysis, we showed that >0.5 mM WFA had a remark-
able inhibitory eect on GBM cell proliferation as against
lower concentrations.
Taken together, vimentin plays a key role in cell
shape and migration; WFA-induced vimentin degrada-
tion leads to a decline in the invasion and the pro-
liferation of GBM cells. Meanwhile, these research
ndings oer convincing evidence for the rst time that
the vimentin expression level is correlated with tumor
recurrence and is a marker of a poor outcome in glio-
blastoma patients. Thus, it is reasonable to speculate that
the suppression of vimentin by WFA possibly contributes
to its anti-GBM eect in vivo.
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