LABORATORY INVESTIGATION

Lack of evidence for PlGF mediating the tumor resistanceafter anti-angiogenic therapy in malignant gliomas

Kristin Schneider • Astrid Weyerbrock •

Soroush Doostkam • Karl Plate •

Marcia Regina Machein

Received: 22 March 2014 / Accepted: 26 October 2014

� Springer Science+Business Media New York 2014

Abstract Placenta growth factor (PlGF) is a member of

vascular endothelial growth factor family which can pro-

mote cancer growth by various mechanisms. Placenta

growth factor is upregulated in many neoplastic diseases

and serum levels of PlGF are increased in cancer patients

following anti-angiogenic therapy. However, its role in

glioma growth is yet not fully elucidated. In this study we

analyzed the expression of PlGF mRNA using real time

PCR in human gliomas of different WHO grades. Placenta

growth factor mRNA levels were highly variable and did

not correlate with WHO grades, arguing against a signifi-

cant role in glioma progression. The highest PlGF

expression was observed in anaplastic astrocytomas

whereas grade II astrocytomas and glioblastomas displayed

lower levels of expression. Immunohistochemical analysis

showed that PlGF was expressed by inflammatory and

endothelial cells in addition to tumor cells. Placenta growth

factor mRNA expression in 12 matched glioblastoma

samples before and after therapy, including bevacizumab

and cilengitide treatment was largely unaffected by the

aforementioned treatment modalities. In vitro, the exposure

of VEGFR-1 expressing glioma cells to bevacizumab did

not increase the expression levels of PlGF mRNA. In

summary, our results do not support the hypothesis that

PlGF plays a major role in the resistance of gliomas after

anti-angiogenic therapy.

Keywords Glioma � PlGF � Bevacizumab � VEGFR-1 �Angiogenesis � Anti-angiogenic therapy

Introduction

Placenta growth factor (PlGF) is an angiogenic protein of

the VEGF family that was first identified in the early 1990s

in the placenta. Like other members of the VEGF family,

PlGF is expressed in different isoforms (PlGF-1, PlGF-2,

PlGF-3 and PlGF-4), which arise through alternative

splicing of the PlGF gene. PlGF can form heterodimers

with VEGF and binds to VEGFR-1 (vascular endothelial

receptor 1, flt-1) with higher affinity than other members of

VEGF family [1, 2]. Furthermore, PlGF-2, like other iso-

forms of the VEGF family, can bind to Neuropilin-1 and to

Neuropilin-2 [3]. Placenta growth factor has been shown to

be modulated by hypoxic stimuli, although no hypoxia-

responsive elements (HRE) could be detected in the pro-

moter as in the VEGF-A and VEGFR-1 receptor [4].

Since the first description of PlGF, its spectrum of

biological activities has been broadened with activities not

only on endothelial cells but on also on different other cells

[5]. Placenta growth factor is expressed by placenta,

endothelial cells, inflammatory cells and tumor cells [6].

Knock-out models have shown that PlGF is redundant for

development and physiological angiogenesis but has an

important function in disease [7]. In cancer, PlGF mRNA

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11060-014-1647-3) contains supplementarymaterial, which is available to authorized users.

K. Schneider � A. Weyerbrock � M. R. Machein (&)

Department of Neurosurgery, University Medical Center

Freiburg, Breisacher Strabe 64, 79106 Freiburg, Germany

e-mail: Marcia.machein@uniklinik-freiburg.de

S. Doostkam

Department of Neuropathology, University Medical Center

Freiburg, Freiburg, Germany

K. Plate

Department of Neuropathology, Edinger Institute, University

of Frankfurt Medical School, Frankfurt, Germany

123

J Neurooncol

DOI 10.1007/s11060-014-1647-3

and protein levels correlate with tumor stage and metastasis

and inversely with survival in several tumor types [8, 9].

Placenta growth factor is over-expressed in some cancers

such as breast [10] and gastric carcinoma [11]. On the other

hand, expression of PlGF is down-regulated by hyperme-

thylation of the promotor in both colon and lung carcinoma

[12]. Placenta growth factor is required for the growth of

medulloblastoma [13] and is also involved in the progres-

sion of chronic myeloid leukemia [14].

The therapeutic potential of PlGF inhibition using an

anti-PlGF neutralizing antibody was studied in pre-clinical

models, including melanoma, colon carcinoma and pan-

creatic cancer [15]. Placenta growth factor plasma levels of

patients receiving anti-VEGF inhibition treatment have

been shown to be elevated, raising the hypothesis that PlGF

might contribute to the resistance against anti-VEGF

therapies [16–18].

In gliomas, there are very few reports regarding the

expression of PlGF. Nomura et al. [19] studied seven gli-

omas and reported expression of PlGF in hypervascular

tumors. In a phase II trial of glioblastomas patient with

aflibercept (dual inhibition of VEGF and PlGF), increased

PlGF plasma levels were detected 14 and 28 days after

therapy [20]. Similarly, Batchelor et al. showed that

AZD2171 (a pan VEGF receptor tyrosine kinase inhibitor)

increased PlGF plasma levels throughout treatment [21].

However, it is not clear whether PlGF sustains the angio-

genic response in patients receiving anti-VEGF therapies,

and by this contributes to tumor escape from anti-angio-

genic treatment.

In the current study we analyzed the expression of PlGF

mRNA in gliomas of different grade using real time PCR

and immunohistochemistry. Moreover, we compared PlGF

expression in glioblastoma samples before and after an

anti-angiogenic therapy with bevacizumab (monoclonal

humanized anti-VEGF antibody) and cilengitide (an alpha

beta 3 and alpha beta 5 integrin inhibitor). Finally, we

studied the impact of bevacizumab treatment on PlGF

expression levels in glioma cells in vitro. In summary, our

findings cannot confirm a major role of PlGF in the therapy

escape from anti-angiogenic therapies in gliomas.

Methods

Patient sample collection

Tumor samples of glioma patients were obtained according

to the Helsinki’s declaration and on approval of the ethical

committee of the University Medical Center Freiburg (Nr.

280/11). For the current retrospective study, samples were

selected based on the availability of tissue in our tumor

bank. Neuropathological diagnosis was made by board-

certified neuropathologists according to the World Health

Organization (WHO) Classification of Tumors of the

Central Nervous System [22]. Samples of 32 glioma

patients admitted for tumor resection between June 2010

and June 2013 were selected for this study. Among these

samples, matched tumor samples from primary and recur-

rent surgeries of 12 glioblastoma patients (six after stan-

dard therapy and six after anti-angiogenic therapy) were

available for analysis of PlGF expression. Four patients

with glioblastoma multiforme received bevacizumab plus

radiotherapy as primary therapy followed by bevacizumab

combined with irinotecan or bevacizumab as single agent

until recurrence. Two patients received Cilengitide along

with radiation and temozolomid during the primary therapy

and cilengitide as single agent until recurrence.

RNA isolation, reverse transcription and real time PCR

Total RNA was extracted from tumor tissue or human cell

lines using RNAeasy mini Kit (Qiagen, Hilden, Germany).

DNAse treatment was performed using TURBO DNA-

freeTM kit (Ambion, Darmstadt, Germany). RNA integrity

was verified by agarose gel electrophoresis. Two micro-

grams of total RNA were reverse transcribed using Tran-

scriptor High Fidelity cDNA Synthesis Kit Each (Roche

Applied Science, Mannheim, Germany). cDNA probes

were analyzed in duplicate using real-time TaqMan probes

encoding for human PlGF gene (HS 00182176-m1), for

human ß-Actin (HS 9999903-m1) and for human VEGFR-

1 (Hs 00176473-m1) (all from Applied Biosystems, Life

technologies, Darmstadt, Germany). Real Time PCR was

performed using LightCycler� 480 Probes Master (Roche

Applied Science, Mannheim). Relative quantification of

mRNA levels was performed using the Ct method with

ß-actin as reference gene and the formula DDCt-method.

Levels of transcripts were normalized against levels of

PlGF/ß-Actin and VEGFR-1/ß-Actin of human umbilical

vein endothelial cells (HUVEC).

Cell lines and culture conditions

Human glioblastoma cell lines (LN 229, U-87 MG, SNB75

und SNB-19) were kindly provided by Dr. M. Carro

(Department of Neurosurgery, University Medical Center

Freiburg). Primary glioblastoma cells (TG, PM) were

kindly provided by Dr. N. Osterberg (Department of

Neurosurgery, University Medical Center Freiburg). Cells

were maintained in Dulbecco’s modified Eagle’s medium

containing 10 % fetal bovine serum. HUVEC were pur-

chased from Promocell (Heidelberg, Germany) and main-

tained in EGM-2 medium.

J Neurooncol

123

Development of a Bevacizuma-adapted glioblastoma

cell line

Bevacizumab was purchased from Roche, Switzerland. The

human cell line LN229 was exposed to a clinically relevant

dose of 250 lg/ml. Mouse IgG was used as control. Cells

were exposed to bevacizumab for 3 weeks. 1 9 106 control

cells or bevacizumab-adapted LN229 (Bev-LN229) were

plated in a 10 cm-dish in DMEM with 5 % FBS. For

hypoxia induction, cells were placed in a hypoxia incubator

chamber (Becton–Dickinson Company, New Jersey, USA)

overnight. Total RNA from hypoxic and normoxic cells

was collected and analyzed for transcript levels of PlGF

and VEGFR-1 using real time PCR.

Expression of VEGFR-1 protein in glioma cells

Glioblastoma cell lines were resuspended in DMEM con-

taining 10 % FBS and incubated for 5 h at 4 �C for

receptor reconstitution after trypsinization. 106 cells were

incubated for 1 h with FC-blocking reagent (BD Biosci-

ences) on ice and then incubated with an anti-human

phycoerythrin labeled VEGFR-1 (R&D, Systems, Minne-

apolis, USA) overnight at 4 �C. Dead cells were excluded

using DAPI. Cells were measured for VEGFR-1 protein

expression on a LSR Fortessa Analyzer using FlowJo

software. HUVEC was used as positive control.

Glioblastomas cells were plated in cell chamber and

fixed with ice-cooled acetone/methanol after growth. Cells

were incubated with a monoclonal anti-human VEGFR-1

(kindly provided by Prof. Shibuya, University of Tokyo,

Japan) in a 1:100 dilution. Cells were incubated with sec-

ondary anti-mouse Alexa 488 (Life Technologies Carlsbad,

CA, USA). After several washing cycles cells were coun-

terstained with DAPI, mounted and analyzed on confocal

microscope.

Cell proliferation assay

In order to test whether PlGF induces the proliferation of

glioblastoma tumor cells, the cell lines LN229, SNB75 and

PM were seeded in 96-well (5 9 103/well) and starved in 1 %

FCS in DMEM for 12 h. Subsequently, adherent cells were

incubated for 3 days with 100 ng/ml recombinant PlGF-2

(Reliatech, Braunschweig, Germany). MTT test was con-

ducted in sixplicate at day 1 and day 3 according the protocol

provided by Wallert and Provos (http://web.mnstate.edu/pro

vost/mtt%20proliferation%20assay%20protocol.pdf).

Immunohistochemistry for PlGF

Placenta growth factor was detected in immersion-fixed

paraffin-embedded glioma sections using a monoclonal

antibody against human PlGF (Clone 358905, R&D Sys-

tems, Minneapolis, USA) at 25 lg/mL overnight (4 �C).

Sections were stained using the Anti-Rat HRP-DAB Cell &

Tissue Staining Kit (R&D systems) according to the

manufacturer’s instructions and counterstained with

hematoxylin. As positive control, we used human placenta

sections. Negative controls were performed using unspe-

cific rat IgG2a.

Double immunofluorescence and confocal analysis

Glioblastoma sections were stained with the following

antibodies: rat anti-human PlGF (1:25, R&D Systems,

Europe), rabbit anti-human von Willebrand factor (1:100,

Dako, Denmark), mouse anti-human CD68 (1:100, DAKO,

Denmark), and anti-human GFAP. Appropriate Alexa

labeled secondary antibodies were used. Sections were

analyzed using a Leica TCS AOBS spectral confocal

microscope.

Statistical analysis

Results of the PlGF mRNA expression were compared by

Wilcoxon-Mann–Whitney-Test, since the results showed a

non-normal distribution (Mann–Whitney-Test: PlGF

expression in low-grade vs high grade; Wilcoxon-test: PlGF

expression in primary vs recurrence GBM; Wilcoxon-Test

PlGF expression before and after anti-angiogenic therapy in

GBM). p values \ 0.05 were considered statistically sig-

nificant. All analyses were carried out with SPSS 19.

Results

Patient collective and expression of PlGF in gliomas

We retrospectively analyzed tumor samples from patients

admitted for tumor resection at our Department between

June 2010 and June 2013. Patient characteristics are shown

in Table 1. Forty four tumor samples were processed for

evaluation of PlGF mRNA levels (Fig. 1). These cohort

comprised four astrocytomas WHO II, five oligo-astrocy-

tomas WHO II, six anaplastic astrocytomas WHO III, five

anaplastic oligo-astrocytomas WHO III and 24 glioblasto-

mas WHO IV. PlGF mRNA was detected in gliomas from

all grades with no correlation between tumor grade and

PlGF expression (p [ 0.05). The mean expression did not

increase with tumor grade, with similar mean levels

detected in astrocytomas WHO II and glioblastomas. The

highest mean level of PlGF was observed in anaplastic

tumors. No statistically significant difference in was noted

between mRNA levels of PlGF and tumor grade

(p [ 0.05).

J Neurooncol

123

PlGF is expressed by tumor cells but also by stroma

cells

Immunohistochemistry was performed to investigate the

localization of PlGF protein in tumors of different grades.

Corresponding to the findings of real time PCR, immuno-

histochemical analysis showed the highest expression of

PlGF protein in anaplastic astrocytoma. Faint staining for

PlGF was found in grade II astrocytomas. Expression of

PlGF in glioblastomas is heterogeneous with some areas

with focal moderate staining (Fig. 1). In malignant gliomas

PlGF expression was observed in tumor cells and in

endothelial cells (Fig. 2a). Immunoreactivity for PlGF was

also detected in areas with accumulation of mononuclear

infiltrating cells in malignant gliomas (Fig. 2b). Using

double immunofluorescence labeling, we confirmed stain-

ing for PlGF in vWF-labeled endothelial cells (Fig. 2e) in

CD68-labeled macrophages (Fig. 2f) and in GFAP-

expressing tumor cells (Fig. 2g). This finding supports the

hypothesis that in malignant tumors the microenvironment

is also a source of PlGF. No immunoreactivity was seen in

negative controls using unspecific immunoglobulin

(Fig. 2c, d and h). In order to evaluate whether macrophage

infiltration increase after anti-angiogenic therapy, we

Table 1 Patient collective. Age at diagnosis in years, time to recurrence in months

Patient number Age Tumor localization Tumor Type 1st line

Therapy

Interval between

1st and 2nd

surgery (months)Histology Who

grade

1 32 Temporal L Astro II – –

2 73 Temporal L Astro II – –

3 38 Temporal L Astro II – –

4 40 Temporal L Astro II – –

5 41 Frontal L OA II – –

6 43 Insular R OA II – –

7 44 Temporal R OA II – –

8 48 Fronto-temporal R OA II – –

9 34 Fronto-temporal L OA II – –

10 45 Frontal R AA III – –

11 41 Temporo-occipital R AA III – –

12 34 Temporo-occipital R AA III – –

13 49 Frontal R AA III – –

14 29 Temporal L AA III – –

15 74 Frontal L AA III – –

16 45 Temporal R AOA III – –

17 76 Frontal R AOA III – –

18 75 Frontal R AOA III – –

19 25 Fronto-parietal R AOA III – –

20 52 Frontal L AOA III – –

21 62 Parietal R GBM IV RT and TMZ 10

22 71 Frontal R GBM IV RT and TMZ 9

23 68 Frontal L GBM IV RT and TMZ 8

24 49 Temporal R GBM IV RT and TMZ 11

25 50 Temporal L GBM IV RT and TMZ 7

26 51 Temporal L GBM IV RT and TMZ 8

27 64 Temporal R GBM IV RT/TMZ and cilengitide 26

28 47 Temporal R GBM IV RT/TMZ and cilengitide 30

29 63 Temporal R GBM IV RT/irinotecan /Bev 14

30 70 Temporal R GBM IV RT/Irinotecan /Bev 12

31 55 Temporo-parietal L GBM IV RT /Bev 11

32 47 Frontal L GBM IV RT/irinotecan /Bev 10

Astro Astrocytoma, AA anaplastic astrocytoma, OA oligoastrocytoma, AOA anaplatic oligoastrocytoma, RT radiotherapy, TMZ temozolomid,

Bev bevacizumab

J Neurooncol

123

stained samples before and after angiogenic treatment (four

samples) for CD68 and for CD163. No major differences in

macrophage infiltration were found after treatment (data

not shown).

Expression of PlGF in glioblastomas after

anti-angiogenic therapies

To evaluate whether anti-angiogenic therapies like VEGF

blockade (bevacizumab) and integrin inhibition (cilengi-

tide) affect the expression of PlGF in human glioblastoma

tissues we analyzed tumoral PlGF mRNA levels prior to

primary therapy and after receiving anti-angiogenic ther-

apy upon first recurrence. At recurrence, tissue samples

were obtained 4 weeks after discontinuation of anti-

angiogenic treatment. A time frame of 4 weeks after dis-

continuation of anti-angiogenic therapy was necessary for

elective operation because of the risk of wound healing

impairment or bleeding associated with antiangiogenic

therapy. Furthermore, matched samples from six patients

with glioblastomas who had received standard therapy

(temozolomid and radiation) were tested before and after

treatment. There were no significant changes in the

expression of tumoral PlGF mRNA in matched glioblas-

toma samples after either standard (Fig. 3a) or anti-

angiogenic therapies (Fig. 3b and c), arguing against a

treatment-induced up-regulation of tumoral PlGF

(p [ 0.05) (supplemental data Figure S1).

Analysis of PlGF and VEGFR-1 expression

in glioblastoma cells

Unstimulated glioma cell lines expressed low levels of

PlGF transcripts (Fig. 4a). We further screened these cell

lines for the expression of VEGFR-1 in vitro. Normalized

against the expression in HUVEC, only LN299 cells

expressed detectable levels of VEGFR-1. The expression

of VEGFR-1 protein in this cell line was confirmed by

FACS analysis and immunofluorescence for VEGFR-1

(supplemental data, Figure S2). To study whether bev-

acizumab induces the expression of PlGF in glioblastoma

cells in vitro, we treated VEGFR-1-expressing LN229 cells

A

B

C

H

F I

D G

E

PlGF mRNA levels in astrocytoma WHO II

PlGF mRNA levels in oligoastrocytoma WHO II

PlGF mRNA levels in anaplastic astrocytomas WHO III

PlGF mRNA levels in anaplastic oligoastrocytomas WHO III

PlGF mRNA levels in Glioblastomas WHO IV

Fig. 1 Real-time PCR analysis of PlGF mRNA expression in

astrocytomas WHO II (a), oligoastrocytomas WHO II (b), astrocy-

tomas WHO III (c), oligoastrocytomas WHO III (d) and glioblasto-

mas (e). The relative amounts of RT-PCR products were determined

by normalizing them to the amounts of PlGF mRNA in HUVEC and

standardized with reference to the intensities of b-actin mRNA.

Representative immunostainings for PlGF: faint expression in astro-

cytomas WHO II (d), strong expression in anaplastic astrocytomas

(e) and moderate expression in glioblastomas (f). Corresponding

negative controls (g, h, i) where stained with a non-immune

immunoglobulin of the same isotype (IgG2A)

J Neurooncol

123

IsotypeDAPI

PlGFCD68

PlGFGFAP

vWFPlGFDAPI

A

A

G

FE

DC

B

H

J Neurooncol

123

with bevacizumab in a clinical relevant dose for 3 weeks.

There was no significant change in PlGF- and VEGFR-1

mRNA expression levels after a 3-week incubation with

bevacizumab. We conducted hypoxia experiments in cul-

tured LN299 human glioma to determine whether a

decrease in tissue oxygenation induces a regulation of

PlGF transcripts. We conducted hypoxia experiments in

cultured LN299 human glioma in order to determine

whether decrease in tissue oxygenation induce a regulation

of PlGF transcripts as have been described by Green et al.

[4]. Hypoxia induced a 2.5-fold upregulation of VEGFR-1

and PlGF mRNA levels (Fig. 4b and c). Furthermore, we

test the ability of PlGF to induce proliferation in VEGFR-1

expressing cell line LN229 and VEGFR-1 non-expressing

cell lines SNB75 and PM. In comparision to control, we

did not detect an increase in proliferation of glioma cells

upon addition of PlGF (supplemental data, Figure S3).

Discussion

Malignant gliomas belong to the most aggressive tumors

among human cancers. Whereas the role of VEGF in

malignant progression in gliomas is well documented, there

are only few reports about the role of PlGF in brain tumors

[19]. We found that PlGF mRNA levels were highly

variable and did not correlate with WHO grades, arguing

against a significant role in glioma progression. PlGF

protein expression is heterogenous in malignant gliomas

with accumulation in pathological blood vessels and in

areas with inflammatory cells. This finding is in line with

previous studies in pre-clinical models of bone metastasis

of breast cancer showing that tumor cells ‘‘educated’’

stroma cells to produce PlGF [23].

Because of the increased tumor vascularization, anti-

angiogenic treatment strategies were considered promising

in malignant gliomas [24]. However the clinical benefit of

anti-angiogenic drugs in glioblastomas is mostly temporary

[25]. The mechanisms mediating resistance to anti-angio-

genic therapies are still not clearly understood. Among the

growth factors that might contribute to therapy resistance

and tumor escape, PlGF has gained increasing attention in

the last years [18]. PlGF plasma levels are elevated under

anti-angiogenic regimes [17, 26, 27]. We report for the first

time the expression of PlGF transcripts before and after

anti-angiogenic therapy in human glioblastoma specimens.

Our study supports the hypothesis that the increased serum

levels of PlGF of patients treated with anti-angiogenic

therapies are not linked to an upregulation of PlGF in the

tumor microenvironment. Corroborating this finding, there

is evidence that anti-angiogenic therapies might elicit a

host response rather than a tumor cell response [28].

Bayley et al. [29] showed that administration of sFlt-1

resulted in elevated serum levels of PlGF both in tumor-

bearing and non-tumor-bearing mice. Similarly, a tumor-

independent increase in serum levels of PlGF was observed

in mice treated with sunitinib [30]. However, a number of

limitations must be considered in our study: first, the cohort

comprised only a small number of matched pre- and post-

treatment samples. Second, as anti-angiogenic therapy had

to be discontinued 4 weeks prior to reoperation, we cannot

rule out that an upregulation of PlGF in tumor tissue might

b Fig. 2 Immunostaining for PlGF in glioblastomas. a Tumor vessel

showing positive immostaining for PlGF (arrows). b Immunostaing

for PlGF in mononuclear cells (arrows) with corresponding negative

controls (c and d). Double immunofluorescence for PlGF and the

endothelial cell marker von Willebrandt Factor (e), and the macro-

phage marker CD68 (f) and Glial Faser Acid Protein (g) showing the

expression of PlGF not only in GFAP positive tumor cells but also in

macrophages (arrows in f indicate macrophages with strong expres-

sion of PlGF in the cytoplasm) and endothelial cells (arrows in

e indicate endothelial cells stained for PlGF). Isotype control (e) with

DAPI nuclear counterstaining

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

rela

�ve

PlGF

mRN

A ex

pres

sion

primary glioblastomarecurrent glioblastoma

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

rela

�ve

PlGF

mRN

A ex

pres

sion

primary glioblastomarecurrent glioblastoma

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

rela

�ve

PlGF

mRN

A ex

pres

sion

primary glioblastomarecurrent glioblastoma

A B CGBM treated with standard chemoradiation

GBM treated with 1st line bevacizumab

GBM treated with 1st line cilengititide

Fig. 3 Real-time PCR analysis of PlGF expression in primary and

recurrent glioblastomas. Primary glioblastomas and corresponding

recurrent tumors treated with standard radiation and temozolomide

(a), or with radiation and bevacizumab (b), or chemoradiation with

cilengitide (c) were analyzed. The relative amounts of RT-PCR

products were determined by normalizing them to the amounts of

PlGF mRNA in HUVEC and standardized with the intensities of

b-actin mRNA. Bars represent the mean of two measurements

J Neurooncol

123

be no longer detectable at the time of tissue collection.

Finally, since this was a retrospective study, corresponding

PlGF plasma levels could not be evaluated at different time

points and therefore we could not rule of the possibility that

resistance to anti-angiogenic therapies might be related to

circulating PlGF from non-tumor sources.

Assessing the effect of bevacizumab treatment on tumor

microenvironment is complex as different cells—tumor

cells, endothelial cells, pericytes, macrophages—express

VEGFRs and might therefore be affected by bevacizumab

treatment. In addition, some studies suggest a role of VEG-

FR-1 signaling in the survival and proliferation of certain

human cancers cells like colorectal, pancreatic tumors,

breast carcinoma and melanoma [31–35]. We therefore

analyzed the expression of VEGFR-1 mRNA in different

glioma cells in vitro and found detectable VEGR-1 tran-

scripts only in LN229 cells. Stimulation of these cells with

recombinant human PlGF and human VEGF-A led to

phosphorylation of AKT (Reiser, unpublished data) sug-

gesting a functional VEGFR-1 in this cell line. We treated the

VEGFR-1-expressing LN229 with a clinical relevant bev-

acizumab regime. Treatment with bevacizumab did not

induce the expression of PlGF or VEGFR-1 transcripts after

3 weeks of chronic exposure. It is also noteworthy, that

stimulation of glioma cells with PlGF did not increase pro-

liferation in vitro. These results are in contrast with a study in

colorectal cancer where bevacizumab treatment led to an

induction of PlGF and VEGFR-1 [36]. It is conceivable that

the effect of bevacizumab on the expression phenotype of

tumor cells might be restricted to certain cancers.

Taken together, our results show that gliomas of all grades

express PlGF and this expression occurs not only in tumor

cells but also in host cells in the tumor microenvironment.

The PlGF expression in the tumor microenvironment is not

modified by chemoradiation or anti-angiogenic therapy in

glioblastomas. Therefore, the proposed role of PlGF in the

resistance to anti-angiogenic therapy in glioblastoma

patients could not be confirmed by our date. However, we

could not rule out that an alternative upregulation of PlGF by

non-tumor sources might contribute to evasion to anti-

angiogenic approaches. Further prospective investigations

are warranted to clarify the mechanisms by which glioblas-

tomas escape current anti-angiogenic approaches.

Acknowledgments Our work was supported by grants from the

Deutsche Krebshilfe (Project Number 109410). We thank Dr. Carro

and Dr. Osterberg, Department of Neurosurgery, University Medical

Center Freiburg for providing the glioma cells, Prof. Shibuya (Uni-

versity of Tokio) for providing the monoclonal anti-VEGFR-1 anti-

body, S. Reiser for screening of the glioma cells, Ms. Eva Bug for

processing the tumor tissue samples and C. El Gaz, K. Strasser and V.

Sverdlick for help with the immunohistochemistry analysis.

Conflict of interest The authors have no conflicts of interest to

declare.

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