ORIGINAL PAPER

Cilengitide response in ultra-low passage glioblastoma cell lines:relation to molecular markers

Christina S. Mullins • Julia Schubert •

Bjorn Schneider • Michael Linnebacher •

Carl F. Classen

Received: 10 April 2013 / Accepted: 25 May 2013 / Published online: 8 June 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract

Purpose In glioblastoma multiforme (GBM), a tumor still

characterized by dismal prognosis, recent research focuses

on novel-targeted compounds, in addition to standard

temozolomide (TMZ) chemotherapy. One of these

emerging compounds is cilengitide (CGT), which by

binding to integrins (i.e., avb3 and avb5) may inhibit

angiogenesis and also is directly cytotoxic to tumor cells by

interfering with intracellular signaling pathways.

Methods A total of ten patient-derived ultra-low passage

GBM cell lines were treated with increasing doses of CGT,

TMZ, and a combination of both substances. Inhibitory

concentrations of 50 % (IC50) were determined for the

single agents and as a combination. Cell lines were stratified

according to MGMT promoter methylation. The expression

of relevant integrins was assessed by flow cytometry.

Results In monotherapy, all GBM cell lines showed

higher sensitivity to CGT than to TMZ, as determined by

IC50 values in relation to clinically relevant patient plasma

levels. MGMT promoter methylation correlated with a

significantly higher TMZ response, but tended to be

associated with a lower CGT response. Response to CGT

was not correlated with cell surface integrin expression as

measured by flow cytometry. Finally, addition of CGT to

TMZ enhanced growth inhibition, but only in those cell

lines with a methylated MGMT promoter.

Conclusions As suggested by this analysis, patients with

MGMT promoter-methylated GBM may benefit from

addition of CGT to the standard TMZ treatment, while

patients with MGMT promoter-unmethylated GBM may

better respond to CGT monotherapy.

Keywords Glioblastoma � Ultra-low passage cell lines �Response-prediction � Integrins � Cilengitide �Temozolomide

Abbreviations

CGT Cilengitide

DMSO Dimethyl Sulfoxide

GBM Glioblastoma multiforme

IC50 Inhibitory concentrations of 50 %

MGMT O6-Methylguanine-Methyltransferase

PBS Phosphate-Buffered Saline

RGD Arginine-glycine-aspartic acid

TMZ Temozolomide

VEGF Vascular endothelial growth factor

Background

High-grade gliomas primarily include glioblastoma multi-

forme (GBM; WHO grade IV) (Miller and Perry 2007).

These tumors have an extraordinarily poor prognosis.

Median progression-free survival of children treated in the

combination studies HIT-GBM A to D (surgery followed

C. S. Mullins � J. Schubert � C. F. Classen (&)

University Children’s Hospital, University Medicine,

Ernst-Heydemann-Straße 8, 18057 Rostock, Germany

e-mail: carl-friedrich.classen@med.uni-rostock.de

C. S. Mullins

e-mail: christina.mullins@uni-rostock.de

C. S. Mullins � M. Linnebacher

Department of Surgery, University Medicine,

Schillingallee 35, 18057 Rostock, Germany

B. Schneider

Department of Pathology, University Medicine,

Strempelstraße 14, 18057 Rostock, Germany

123

J Cancer Res Clin Oncol (2013) 139:1425–1431

DOI 10.1007/s00432-013-1457-6

by radio-chemotherapy) was 1.02 years (Wolff et al. 2008),

and data were even worse in adults (Stupp et al. 2005).

Thus, new treatment strategies are vital. One reason for

therapy failure may be the great heterogeneity of GBM. A

large spectrum of different molecular patterns may be

observed not only within a patient cohort but also on the

level of individual tumors (Bonavia et al. 2011). This wide

spectrum must also be considered in drug development and

preclinical testing. Patient-individual tumor models pro-

vide ideal material for such studies. Although, not explic-

itly shown for GBM, such individual models are likely to

allow the most accurate response and resistance prediction

outside the patient. The high precision of prediction with

such individual models was demonstrated in carcinomas by

Voskoglou-Nomikos and colleagues as well as by Fiebig

and co-workers with 90 % and even 97 % accuracy for

prediction of response and resistance, respectively (Vo-

skoglou-Nomikos et al. 2003; Fiebig et al. 2004).

One emerging strategy in cancer treatment is preventing

neo-angiogenesis. There are compounds directly targeting

specific molecules involved in new blood vessel formation

and such inhibiting endothelial cell function or response.

For example, SU5416 inhibits vascular endothelial growth

factor (VEGF) receptor signaling, Bevacizumab is a

monoclonal antibody directed against VEGF, and Marim-

astat inhibits matrix metalloproteases (Rosen 2000).

Cilengitide (CGT), a cyclic RGD pentapeptide

(R = arginine, G = glycine, D = aspartic acid) antagonist

of the integrins avb3 and avb5, which are over expressed

both on GBM and on tumor invasive endothelial cells

(Mikkelsen et al. 2009), is a novel-promising compound for

the treatment of solid cancers, and various clinical trials

have been performed or are still ongoing (Nabors et al.

2007, 2012; Stupp et al. 2010).

Integrins are dimeric membrane proteins composed of

alpha and beta subunits (Hynes 2002; Humphries et al.

2006). The classic role of integrins is anchoring cells to the

extracellular matrix, but they also participate in a variety of

signaling pathways. They are involved in malignant

transformation, migration, and metastasis (Jin and Varner

2004). The integrins avb3 and avb5 play key roles in

different angiogenic pathways: avb3 integrins are involved

in the basic fibroblast growth factor/tumor necrosis factor

a-induced pathway, while avb5 integrins regulate the

VEGF/transforming growth factor a-dependent one (Weis

and Cheresh 2011). In addition to anti-angiogenesis, CGT

displays a broad anti-neoplastic effect which is not yet fully

understood, but likely involves both integrin-expressing

tumor cells and the surrounding stroma. In vitro, treated

cells detach from the surface and undergo cell death by

anoikis (or a similar mechanism) (Dechantsreiter et al.

2003; Nisato et al. 2003), and in vivo, CGT has strong anti-

GBM activity both as monotherapy (MacDonald et al.

2001) and in combination with the standard of care, ion-

izing radiation (Mikkelsen et al. 2009). Recent studies

showed beneficial effects of CGT in the treatment of GBM

either as monotherapy (Reardon et al. 2008) or as add-onto

standard irradiation plus temozolomide (TMZ) treatment

(Stupp et al. 2010; Nabors et al. 2012).

In the present study, we systematically analyzed in vitro

the response to CGT and TMZ on a collection of well-

characterized ultra-low passage patient-derived GBM cell

lines. Results were correlated with the molecular setup of

the cells in order to identify patient subgroups in which

best response can be expected.

Methods

Cell lines and culture

Cell lines were established from primary resection speci-

men of GBM tumors (Table 1). Briefly, tumor tissue was

minced (by crossed scalpels) in DMEM/Ham’s F12 cell

culture medium supplemented with 10 % FCS, 2 mM

L-glutamine, and penicillin–streptomycin, and passed

through a cell strainer (100 lm; Becton-Dickinson-Falcon,

Heidelberg, Germany) to obtain a single-cell suspension.

Table 1 Patient data and molecular characteristics of the tumors

Tumor ID Age

(years)

Gender Localization MGMT

promoter

HROG02 68 M Right hemisphere,

parietooccipital

m

HROG04 53 F Right hemisphere;

frontal

u

HROG05 60 F Left hemisphere;

temporal

m

HROG06 53 M Left hemisphere;

frontal

u

HROG10 74 M Right hemisphere;

temporal

u

HROG13 77 F Left hemisphere;

temporal

m*

HROG15 56 M Right hemisphere;

parietal

m

HROG17 70 M Left hemisphere;

parietooccipital

m

HROG36 80 F Right hemisphere;

parietal

del

HROG38 49 F Right hemisphere;

parietooccipital

u

Relevant clinical patient data concerning age (at time point of sur-

gery), gender (M = male; F = female), tumor localization and

methylation status (m = methylated; m* = borderline methylated;

u = unmethylated; del = deletion of the entire gene) of the MGMT

promoter are summarized

1426 J Cancer Res Clin Oncol (2013) 139:1425–1431

123

Cells were washed with PBS and seeded in six-well plates

coated with collagen. All cell culture plastics were from

Greiner Bio one, Frickenhausen, Germany, and cell culture

media and supplements were purchased from PAA, Colbe,

Germany.

In the present study, all experiments were performed

with cells not exceeding 30 passages.

MGMT promoter methylation

For analyzing the MGMT promoter methylation, the

MethyLight method was applied (Ogino et al. 2006).

Briefly, genomic DNA was subject to bisulfite conversion

using the Epitect Bisulfite Kit (Qiagen, Hilden, Germany)

according to the manufacturer’s recommendations. A pri-

mer (TIB MOLBIOL, Berlin, Germany)/probe combination

specific for methylated MGMT promoter sequence was used

(forward: 50-GCGTTTCGACGTTCGTAGGT-30; reverse:

50-CACTCTTCCGAAAACGAAACG-30; probe: 50-6FAM-

CGCAAACGATACGCACCGCGA-TMR-30), with Sensi-

Fast Probe Kit (Bioline, Luckenwalde, Germany). CpG

Methylase (SssI)-treated DNA served as calibrator, since it

is considered to be fully methylated. The collagenase gene

2A1 (COL2A1) was used as endogenous control (forward:

50-TCTAACAATTATAAACTCCAACCACCAA-30; reverse:

50-GGGAAGATGGGATAGAAGGGAATAT-30; probe:

50-6FAM-CCTTCATTCTAACCCAATACCTATCCCAC

CTCTAAA-TMR-30). The percentage of methylated refer-

ence (PMR) value was calculated by dividing the MGMT/

COL2A1 ratio of the sample by the MGMT/COL2A1 ratio

of the SssI-treated DNA, and multiplying by 100. Samples

with a PMR value [4 were considered as methylated (Ogino

et al. 2006). All reactions were performed in triplicates.

Flow cytometry

The expression of integrins was assessed by flow cytome-

try. Cells were harvested, counted, and 5 9 105 cells were

stained with 50 lg/ml anti-mouse CD51 antibody labeled

with PE (clone RMV-7; eBioscience, Frankfurt, Germany).

Cells stained with an irrelevant anti-mouse-PE antibody of

the same isotype served as negative controls (Immunotools,

Friesoythe, Germany). Similarly, 5 9 105 cells were

stained with 10 lg/ml mouse anti-avb3 antibody (clone

LM609; Merck Millipore, Schwalbach/Ts., Germany),

washed with PBS, and stained with a secondary anti-mouse

PE-labeled antibody (DakoCytomation, Glostrup, Den-

mark). 5 9 105 cells were stained with 5 lg/ml rabbit anti-

avb5 antibody (clone EM09902; kindly provided by Merck

KGaA, Darmstadt, Germany) (Goodman et al. 2012),

washed with PBS, and stained with a secondary anti-rabbit

FITC-labeled antibody (DakoCytomation, Glostrup, Den-

mark). For the latter two stainings, cells handled the same

way with no primary antibody served as negative controls.

All incubations were performed in PBS on ice for 30 min.

Drug testing

Cells (5 9 103 cells) were plated in 150 ll medium (as

above) per well in triplicate in 96-well flat-bottom culture

plates and allowed to attach for 24 h. The following con-

centration ranges of TMZ and CGT were tested administered

as single agents (given as final concentrations in the exper-

imental wells): 2 mM—128 nM TMZ (Sigma Aldrich,

Schnelldorf, Germany), 40 lM—10 nM CGT (kindly pro-

vided by Merck KGaA, Darmstadt, Germany). For combi-

nation treatment, we decided on the following three TMZ

doses for subsequent analysis: a low concentration (5 lM),

comparable to that used in metronomic treatment (Stock-

hammer et al. 2010), an intermediate concentration (50 lM)

consistent with plasma levels in patients receiving standard

treatment (Agarwala and Kirkwood 2000), and a very high

concentration (500 lM) to study maximum effects. In case

of CGT, we also chose three doses: a low concentration

(1.4 lM), comparable to the IC50 value of the sensitive cell

lines; an intermediate concentration (7 lM), which is close

to the IC50 value of the majority of tested cell lines; and a

high concentration (10 lM), representing the average IC90

value. Cells treated with TMZ equal volumes of DMSO were

added to cells serving as live control. Cells were incubated

with the substances for 72 h; media were replaced, and

identical concentrations of test substances were added. After

another 72 h incubation period, cells serving as dead control

were incubated for 30 min with 70 % ethanol, and viability

was assessed using the viability dye calcein AM (eBio-

science) in a final concentration of 0.7 lM in fresh medium/

PBS (2:1). Cells were incubated at 37 �C in the dark for

60 min, and fluorescence intensity was assessed using the

micro-plate reader Infinite M200 (Tecan, Mennedorf,

Switzerland) with 485 nm excitation, 535 nm emission, and

a constant gain of 160. Values were normalized (1 = value

live control; 0 = value dead control).

Statistics

All statistical analyses (t tests) were performed using

SigmaStat 3.5; IC50 values were calculated using

SigmaPlot10.0.

Results

Determination of IC50 values for TMZ and CGT

In a first step, response of the GBM cell lines to increasing

doses of TMZ and CGT was assessed (IC50 values;

J Cancer Res Clin Oncol (2013) 139:1425–1431 1427

123

Table 2). Response to TMZ correlated with the methyla-

tion status of the MGMT promoter and was significantly

higher in hypermethylated cell lines (p = 0.016). In addi-

tion, we observed strong responses to CGT; IC50 values did

not exceed 20 lM in any of the assays (Tables 2 and 3),

while plasma levels in patients receiving CGT peak at

40–50 lM (Stupp et al. 2010). Contrary to TMZ, cell lines

with unmethylated promoter tended to respond better to

CGT (p = 0.066).

Integrin expression

The expression of integrins targeted by CGT was analyzed

by flow cytometry. In all cell lines, a high general

expression of av integrins was detectable (Fig. 1). The

degree of avb3 and avb5 expression varied between the

cell lines but was positive in all cases (Fig. 1). Strong

staining both for avb3 and avb5 integrins was detected in

the cell lines HROG02, HROG15, and HROG17. An

intermediate staining was seen in HROG05, HROG10, and

HROG36, followed by relatively weak staining in

HROG04, HROG06, HROG13, and HROG38. Somewhat

unexpected, the amount of integrin expression, by which

CGT is thought to inhibit angiogenesis and induce cyto-

toxicity, did not correlate with the response to CGT.

Combination treatment

Next, we studied potential additive or synergistic effects of

a combination of CGT and TMZ. A functional in vitro test

regimen was performed by combining the three CGT

concentrations with the three TMZ concentrations.

In all cases, CGT monotherapy was more effective than

TMZ monotherapy. In cell lines harboring, a methylated

MGMT promoter addition of TMZ had a beneficial effect

Table 2 Overview on drug response and integrin expression

IC50 values % integrin

expression

MFI (x fold)

CGT

(lM)

TMZ

(mM)

av avb3 avb5 av avb3 avb5

HROG02 7.00 0.50 88.7 51.0 72.8 5.8 3.6 6.2

HROG04 5.40 3.50 96.4 3.0 10.8 2.9 0.9 1.2

HROG05 6.00 0.50 90.1 14.8 23.8 2.8 1.4 2.8

HROG06 8.00 1.50 96.3 1.4 5.5 6.7 0.1 1.3

HROG10 5.40 1.50 83.5 10.6 9.2 3.6 0.6 1.4

HROG13 2.00 2.00 84.4 0.1 0.3 0.7 0.2 0.0

HROG15 10.00 0.80 94.5 31.1 83.2 9.1 2.2 7.2

HROG17 5.00 0.05 86.6 72.9 33.3 4.5 3.9 2.5

HROG36 20.00 0.80 94.2 13.4 12.6 4.5 1.6 1.8

HROG38 0.80 1.00 91.4 0.0 11.3 0.2 0.2 4.0

Calculated IC50 values (mean of three independent assessments in

triplicates) for temozolomide and Cilengitide are provided for all cell

lines. The expression of integrins was assessed by flow cytometry and

is given as % expressing cells and as MFI [=(fluorescence intensity of

probe - fluorescence intensity of control)/fluorescence intensity of

control]

Table 3 Overview on

monotherapy

The percentage of viable cells

(mean of three independent

assessments in triplicates;

standard deviation is given in

brackets) after TMZ or CGT

treatment is provided for all cell

lines. Cell viability was assessed

by calcein AM viability stain

CGT (lM) TMZ (lM)

1.4 (%) 7 (%) 10 (%) 5 (%) 50 (%) 500 (%)

HROG02 95 (±9) 45 (±3) 23 (±4) 77 (±4) 80 (±5) 62 (±5)

HROG04 73 (±8) 43 (±4) 23 (±4) 80 (±10) 72 (±13) 65 (±5)

HROG05 58 (±10) 39 (±5) 35 (±2) 61 (±5) 74 (±3) 48 (±4)

HROG06 42 (±8) 33 (±13) 35 (±14) 75 (±10) 80 (±5) 77 (±9)

HROG10 71 (±5) 24 (±4) 14 (±5) 90 (±11) 90 (±8) 71 (±3)

HROG13 35 (±7) 11 (±11) 12 (±12) 78 (±7) 80 (±6) 60 (±2)

HROG15 71 (±2) 57 (±8) 49 (±4) 91 (±9) 89 (±11) 71 (±8)

HROG17 55 (±20) 21 (±6) 24 (±12) 71 (±23) 63 (±23) 46 (±23)

HROG36 69 (±14) 64 (±7) 57 (±16) 96 (±15) 78 (±6) 60 (±19)

HROG38 21 (±17) 12 (±8) 15 (±11) 86 (±11) 80 (±6) 75 (±9)

av avb3 avb5

% e

xpre

ssio

n

0

20

40

60

80

100

120

Fig. 1 Integrin expression. The percentage of cell lines expressing

pan-av, avb3, and avb5 integrins, as assessed by flow cytometry, is

illustrated in a box plot diagram

1428 J Cancer Res Clin Oncol (2013) 139:1425–1431

123

(Figs. 2, 3). The effects of the combined in vitro treatment

regimen ranged from almost additive (HROG17 and

HROG36) to synergistic (HROG02 and HROG15) (Figs. 2,

3). In the unmethylated promoter setting, CGT mono-

therapy had even greater effects on cell viability than in the

methylated setting, but the addition of TMZ showed no

further benefit (Figs. 2, 3). The beneficial effect of TMZ

and CGT treatment for cells with methylated MGMT

promoter as opposed to those without methylation was

significant (p = 0.0476).

Discussion

Current efforts at improving GBM treatment include the

addition of novel-targeted agents to the standard of care

regimen. In the present study, we tested whether the

addition of CGT to the standard chemotherapeutic agent

TMZ had a beneficial effect as published previously (Stupp

et al. 2010; Nabors et al. 2012) and whether such responses

to CGT alone or in combination with TMZ could be cor-

related with particular molecular characteristics of the

tumor cells. The observed strong in vitro reaction to CGT

monotherapy is in accordance with the positive in vivo

response of the randomized phase II study published by

Reardon and colleagues (Reardon et al. 2008). Further, we

observed a positive correlation of MGMT promoter

methylation with the response toward TMZ as described

earlier (Palanichamy et al. 2006; Martinez and Esteller

2010). Contrary to that, a trend indicating a better response

to CGT (monotherapy) in non-promoter-methylated cells

was found. This finding is somehow conflicting with data

of Maurer et al. (2009) who demonstrated complete lack of

influence of MGMT expression on response toward CGT.

However, this shall be clarified in the near future since

several clinical studies are currently addressing this ques-

tion, that is, the CORE study only recruiting GBM patients

with an unmethylated MGMT promoter, the CENTRIC

study including exclusively patients with a methylated

promoter, and the pediatric CilMetro study, including both.

Recently, two studies revealed a significant benefit of a

combination therapy with TMZ and CGT for patients with

a methylated MGMT promoter (Stupp et al. 2010; Nabors

et al. 2012). We would like to stress the fact that we did

also observe this effect with our ultra-low passage cell line

collection.

Conclusions

The present response analysis on a panel of ultra-low

passage GBM cell lines to treatment with TMZ and CGT

suggests the following: (1) There is a clear positive cor-

relation between a methylated MGMT promoter and

response to TMZ. (2) Addition of CGT resulted in an at

least additive effect. Future clinical studies should address

whether patients with MGMT promoter-methylated GBM

benefit from the addition of CGT to the standard treatment

with TMZ. (3) In contrast, it should be carefully analyzed,

whether patients suffering from GBM with unmethylated

MGMT promoter may benefit most from CGT

monotherapy.

vita

l cel

ls

0.00

0.20

0.40

0.60

0.80

1.00

1.20

HROG02 HROG05 HROG15 HROG17 HROG36 HROG04 HROG06 HROG10 HROG13 HROG38

50 µM TMZ 1.4 µM CGT 1.4 µM CGT + 50 µM TMZ

uMGMTmMGMTFig. 2 Combination treatment.

Viability of cells treated with

50 lM TMZ (dark gray bars),

with 1.4 lM CGT (light gray

bars), or the combination of

both substances (black bars) is

displayed. The fluorescence

intensity of the calcein AM

viability dye is normalized to:

1 = untreated cells (indicated

by the vertical black bar);

0 = alcohol treated/dead cells.

Results for cell lines with

methylated MGMT promoter

(mMGMT) are displayed on the

left side and results for cell lines

with unmethylated MGMT

promoter (uMGMT) on the

right side

J Cancer Res Clin Oncol (2013) 139:1425–1431 1429

123

Acknowledgments The authors kindly thank Anne Lehmann for

her excellent technical assistance and Merck KGaA for making cil-

engitide and EM09902 available.

Conflict of interest CFC discloses a Consultancy Services Agree-

ment with Merck KgaA (2008). Otherwise, the authors declare that no

competing interests exist.

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