ARTICLE
Auteur(s) : Marco Locatelli1, Leonardo
Boiocchi1,2, Stefano Ferrero2, Filippo
Martinelli Boneschi3, Mario Zavanone1,4,
Samantha Pesce5, Paola Allavena5, Sergio
Maria Gaini1,4, Lorenzo Bello1,4, Alberto
Mantovani5,6
1Neurosurgery, Fondazione IRCCS, Ca’Granda Ospedale
Maggiore Policlinico Mangiagalli e Regina Elena, Milan
2II Cattedra di Anatomia Patologica, Dipartimento di
Medicina, Chirurgia e Odontoiatria, University of Milan, A.O.
San Paolo Milan
3Institute of Experimental Neurology (INSPE),
Scientific Institute San Raffaele, Milan
4Department of Neurological Sciences, University
of Milan
5Istituto Clinico Humanitas IRCCS, Rozzano, Milan
6Department of Translational Medicine, University
of Milan, Italy
accepté le 8 Decembre 2009
Diffuse gliomas are the most common malignant tumors of the
brain. They include heterogeneous tumors that are classified,
according to their pathological characteristics, as astrocytomas,
oligodendrogliomas, and glioblastomas. The World Health
Organization (WHO) has defined a malignancy scale: usually
astrocytomas and oligodendrogliomas are grade II (or III in the
anaplastic form), while glioblastomas are grade IV and are
considered highly malignant [1-3]. In spite of optimal treatment,
patients with glioblastomas survive less than one year and
prognosis has not changed in the last two decades. These tumors
have a rapidly expanding nature and invade the normal brain by
active cell migration. The migratory ability of glioma cells has
been investigated by electron microscopy, and it was shown that
neoplastic cells easily adjust their shape and size to slip through
the narrow extracellular brain spaces, a process that requires
Cl- Channels [4].
It is now established that migrating malignant cells may exploit
chemokine receptors to invade surrounding tissues and leading to
distant metastasis [5, 6]. Chemokines are a large family of
chemotactic factors inducing cell motility in several cell types
[7, 8]. Chemokines have been mostly studied for their potent effect
on the recruitment of leukocytes at sites of inflammation; however,
it has become increasingly clear that tumors also express
functional chemokine receptors [5, 6, 9, 10]. In addition to cell
mobilization and metastatic ability, other important roles -
relevant to tumor biology - have been attributed to the chemokine
system, e.g. enhanced tumor cell proliferation, resistance to
apoptosis and regulation of angiogenesis [5].
A number of studies investigated the expression of chemokine
receptors in tumors, including gliomas. mRNA for receptors of the
CXC subfamily have been reported, with CXCR4 being the most
frequently expressed [5, 11-13]. Furthermore, the presence of CXCR4
has been associated with the most aggressive forms of gliomas and
with poor patient survival [14, 15]. Cancer stem cells isolated
from glioblastoma are positive for CXCR4 and treatment with
the specific ligand CXCL12 stimulates their proliferation
[15].
In this study, we have explored the expression of the chemokine
receptor CX3CR1 in human gliomas. Physiologically,
CX3CR1 is predominantly expressed by leukocytes such as
monocytes, NK and Th1 lymphocytes, and mediates adhesion and
migration through the endothelium, the latter expressing the
specific ligand CX3CL1 as a trans-membrane protein [16-19]. In
the brain, CX3CR1 is expressed by the microglia, while neurons
produce the ligand CX3CL1 (originally identified as
neurotactin or fractalkine) [20-22]. A few studies have
documented an exception to this rule: in different species and
conditions, neurons may also express the receptor [23, 24], while
positivity for CX3CR1 in glial cells was more controversial
[23-26].
The ligand CX3CL1 is one of the most expressed chemokines
in the brain [21, 22, 25]. Experimental evidence has established
that the CX3CR1/CX3CL1 axis plays a major role in the
neuron/microglia cross-talk, and in neuro-protection under
conditions of inflammation/injury [22, 27-33].
We show in this study, involving a large case list of human
gliomas, that neoplastic cells strongly express the CX3CR1.
Receptor expression already occurs in low-grade tumors, suggesting
that its up-regulation is an early event during malignant
transformation.
Methods and materials
Patients
Seventy consecutive patients with primary CNS tumors, who attended
the Neurosurgery Division of IRCCS Ospedale Maggiore Policlinico,
Mangiagalli and Regina Elena, Milan, Italy, between 2005 and
2007 were enrolled in this study. Tumor specimens were
diagnosed according to the last 2000 WHO classification:
olidodendrogliomas (n = 23); low-grade astrocytomas (n = 9),
high-grade astrocytomas or anaplastic astrocytomas (n = 10),
glioblastomas (n = 23), and oligoastrocytomas (n = 5). Informed
consent was obtained from all patients.
Immunohistochemistry of glioblastoma samples
for CX3CR1
All specimens were reviewed independently by two pathologists (SF
and LB) blinded to the diagnosis and clinical data. From each
block, three sections were selected, and deparaffinized in xylene.
Antigen retrieval was performed using sodium citrate buffer (pH
6.0) in a microwave oven, three times for five minutes and samples
were stained (Genomix i-6000, BioGenex, San Ramon CA, USA) with
rabbit polyclonal anti-human CX3CR1 antibody (Abcam,
Cambridge, UK; 1:350 dilution, overnight at 4°C). Reactions
were revealed using NovoLink Polymer Detection System (Novocastra),
according to the manufacturer’s instructions. After a
diaminobenzidine reaction (DAB; Liquid DAB + Substrate Chromogen
System, DakoCytomation), sections were counterstained with
hematoxylin (Mayer, DIAPATH). We evaluated the percentage of
positive tumor cells and the intensity of the staining.
A semiquantitative four-grades scoring system was used for the
evaluation of the percentage of positive neoplastic cells. Score 0:
no immunoreactivity; score 1: < 10% of neoplastic cells were
immunoreactive; score 2: immunoreactivity between > 10% and <
50%; score 3: immunoreactivity > 50%. Staining intensity was
scored: 0: for no staining, 1: faint staining, 2: moderate and 3:
strong. We multiplied these two scores (positive cells % ×
intensity) to obtain a final score with a continuous distribution.
RNA extraction and quantitative real-time RT-PCR (Q-PCR)
for CX3CR1 mRNA
Total RNA was isolated from the following frozen tissue specimens:
oligodendroglioma (n = 4); astrocytoma (n = 2); glioblastoma (n =
3). RNA was exctracted using TRI Reagent (Ambion), as previously
described [34], and quantified using a Nanodrop Spectrophotometer
ND-1000. Ten μg of RNA were treated with Turbo DNA-free (Ambion) to
eliminate genomic DNA contamination. Two μg of total RNA were
reverse-transcribed using the High Capacity cDNA Archive Kit
(Applied Biosystems) according to the manufacturer’s instruction.
CX3XR1 mRNA expression was analyzed using SYBR green-based
quantitative real time RT-PCR (Q-PCR) as previously described [34].
18S was used as an internal control to normalize samples. Specific
primers were computer-designed:
18S: Forward: 5′ CGC CGC TAG AGG TGA AAT TC 3′ Reverse: 5′ CTT
TCG CTC TGG TCC GTC TT 3′ CX3CR1 :
Forward: 5′ GGG ACT GTG TTC CTG TCC AT 3′ Reverse: 5′ GAC ACT
CTT GGG CTT CTT GC 3′
The amount of CX3CR1 mRNA relative to the housekeeping gene
18S was calculated as 2-ΔCt, where ΔCt =
CtCX3CR1 - Ct18S. The threshold cycle Ct was
automatically given by the SDS2.2 software package (Applied
Biosystems).
Loss of heterozygosity at chromosomes 1p and 19q
Tumor DNA was extracted from paraffin-embedded tissues using the
DNeasy Tissue Kit (QIAGEN, Inc. Milano, Italy) according to the
manufacturer’s protocol. Following DNA extraction, all tumor
samples were subjected to control gene (PGK) amplification to
assess DNA integrity. Constitutional DNA from peripheral blood
leukocytes was isolated using the standard phenol/chloroform
extraction method with ethanol precipitation. Constitutional DNA/
tumor DNA pairs were evaluated by standard PCR-based LOH assays, as
described. [35]
Methylation of the MGMT (O 6-methylguanine-DNA
methyltransferase) promoter
The methylation status of the MGMT promoter gene was determined
using methylation-specific PCR. Tumor DNA from paraffin embedded
tissues (10 μm sections) was modified by sodium bisulfite,
which converts unmethylated, but not methylated, cytosine to
uracil, as described. [36, 37] Modified DNA was submitted to
methylation-specific polymerase chain reaction (MSP)
after a nested-polymerase chain reaction protocol. The following
primers were used:
MGMT
Forward: 5′ GGATATGTTG GGATAGTT 3′; Reverse: 5′
CCAAAAACCCCAAACCC 3′.
The PCR products were separated on 4% agarose gels.
Statistical analysis
Statistical analysis was performed using the following
non-parametric test: 1) the Kruskall-Wallis test and the Wilcoxon
rank sum test for the comparison of the CX3CR1 scores
respectively, in four categories of brain tumor severity
(low-grade, low-grade recurrent, high-grade and high-grade
recurrent), and in two categories based on the histopathological
diagnosis (WHO grade II versus grade III); 2) The Wilcoxon rank sum
test was used to compare the CX3CR1 score in brain biopsies
with or without loss of heterozygosity (LOH) at chromosomes 1p and
19q, and with or without MGMT methylation. Moreover, a multivariate
logistic regression model was built to assess the potential effect
of demographic variables (age and sex), and brain biopsy-related
variable (CX3CR1 score; presence of LOH; presence of MGMT
methylation), on the risk of having a high-grade tumor. The
coefficient of determination (Nagelkerke Pseudo R2) was
used as a measure of the percentage of the total variance explained
by the different models. The strength of the association between
predictors and the dependent variables was assessed by means of
odds ratio (OR) and relative 95% confidence intervals (CI).
Results
Seventy patients affected by cerebral tumors, including low-grade
or type II WHO severity (oligodendroglioma; astrocytoma) and high
grade or type III-IV WHO severity (glioblastoma and anaplastic
oligodendroglioma) were involved in this study. Patients
(40 males and 30 females) had a mean age of
42.9 years (SD 12.8 years). Thirty-nine out of the
70 (55.7%) were affected by low-grade tumors, and the
remaining (44.3%) had high-grade tumors. Comparison of clinical
characteristics between high- and low-grade tumors revealed that
the male:female ratio was similar in the two groups
(1.60 versus 1.56; Chi-square analysis: p = 0.95), while age
was greater in high-grade tumors (49.5 versus 40.2; p =
0.003).
Immunohistochemical evaluation of CX3CR1 was performed with
a specific anti-CX3CR1 antibody on tumor sections obtained at
surgery. The results are shown in figure 1 and are summarized
for all cases studied in figure 2. We found that
immunoreactivity for CX3CR1 in normal brain was faint in
scattered cells (figure
1A), while tumor cells of each histological type showed a
strong immunopositivity: figure 1B depicts a case of
low-grade astrocytoma, while panels C-F show four cases of
glioblastoma. Only a few samples of oligodendrogliomas showed a
weak expression, although this was seen in more than 50% of cells.
When the CX3CR1 score was compared in categories of tumor
severity defined as low-grade, low-grade recurrent, high-grade and
high-grade recurrent, no statistically significant difference was
found (p = 0.72). Similar results were obtained when low-grade
tumors were stratified into oligodendroglioma (n = 32) and
astrocytoma (n = 7) (p = 0.42). Overall, the marked expression of
CX3CR1 was similar across low- and high-grade tumors based on
the histopathological diagnosis (median value: 9.0; p = 0.30).
Although no statistically significant difference was found, weak
CX3CR1 scores were observed only in low-grade
oligodendroglioma. Multivariate models, including age and gender as
covariates, did not substantially changed the results.
To confirm receptor expression, CX3CR1 mRNA was studied in
selected tumor tissues. Figure 3 shows the levels
of mRNA CX3CR1 in four cases of oligodendroglioma, two
astrocytoma and three glioblastoma.
Ongoing efforts are aimed to identify biological and genetic
alterations in brain tumors that may provide additional prognostic
information, as well as guidance for making decisions about optimal
therapy. We therefore considered other pathological variables
reported to occur in malignant gliomas. Epigenetic silencing of the
MGMT gene by promoter methylation has been associated with longer
survival in glioblastoma patients receiving both radiotherapy and
chemotherapy. Methylation of the MGMT promoter was detected in
77.6% of patients tested (45/62), and was less frequent in
high-grade tumors (66.7% versus 85.3%; p = 0.09 Fisher’s exact
test).
Another biological variable currently analyzed in malignant
gliomas is the loss of heterozygosity (LOH), at chromosomes 1p and
19q because of its correlation with histology and chemotherapy
response, especially in oligodendrogliomas. [38, 39] LOH at either
1p or 19q was present in 53.3% of patients tested (32/60), and
again was less frequent in high-grade than in low-grade tumors
(34.8% versus 64.9%); p = 0.03 Fisher’s exact test.
When all the variables were tested in a multivariate logistic
regression model, the presence of chromosomal deletion at 1p or 19q
was associated with a lower risk of high-grade tumor (OR: 0.2; 95%
CI: 0.1-0.8; p = 0.02; table 1), while
there was no apparent influence of either MGMT gene methylation or
CX3CR1 expression (table 1). The
statistical model was able to explain 27.6% of the total variance
of the dependent variable.
Table 1 Multivariate logistic regression analysis.
Dependent variable: risk of having a WHO-type III tumor.
Predictors: age; gender; CX3CR1 score; chromosomal 1p and 19q
deletion; MGMT gene promoter methylation. OR: Odds ratio; 95% CI:
95% confidence intervals; P: p value according to the logistic
regression analysis
|
Variables
|
OR
|
95% CI
|
P
|
|
Age
|
1.04
|
0.9-1.2
|
0.29
|
|
Gender: Female (Ref.)
|
1.0
|
|
|
|
Male
|
0.70
|
0.2-2.5
|
0.58
|
|
CX3CR1 score
|
0.97
|
0.6-1.5
|
0.9
|
|
Chromosomal deletion 1p and/or 19q
|
0.20
|
0.1-0.8
|
0.02
|
|
MGMT gene promoter methylation
|
0.38
|
0.1-1.7
|
0.21
|
Discussion
We show in this study that human gliomas have mRNA and
immunopositivity for the chemokine receptor CX3CR1. Expression was
evident even in low-grade tumors and was highest in glioblastomas.
Expression of CX3CR1 by cancer cells has been poorly
investigated: prostate tumors express the receptor, which is
involved in metastasis to bone marrow [40, 41]. We have reported
that human pancreatic tumors upregulate CX3CR1, while the normal
pancreatic tissue is negative [34]. Recently, the involvement of
the CX3CR1 receptor in the transmigration of neuroblastoma
cells through bone-marrow endothelial cells has been reported [42].
Glial tumors have been investigated for the expression of several
chemokine receptors, mainly CXCR4 [14, 15], but poorly studied for
CX3CR1. Using a murine model of glioma obtained by intracranial
injection of 3-methylcholantrene, Liu et al. showed a
positive, in situ hybridization for CX3CR1 that corresponded
however, to the localization of CD11b-positive microglia [43]. In
human gliobastoma, Rodero et al. report a diffuse expression
of CX3CR1, but mainly concentrate on the functional defects of
polymorphic CX3CR1 receptor associated with infiltrating
immune cells [44].
Increased expression of both CX3CR1 and its ligand occurs
in different neuro-inflammatory conditions (e.g. infections, toxic
insults and nerve injuries). Higher production and shedding from
the membrane of CX3CL1 results in a higher density of CX3CR1+
inflammatory microglia recruited in the brain [30, 32, 45-47]. In
experimental conditions, cytokines such as TNF and TGFβ, were
responsible for the upregulation of both ligand and receptor in
neurons and microglia, respectively [48]. These mediators are
frequently present in tumors, including gliomas [9, 49-51], and may
well be involved in the modulation of the CX3CL1/CX3CR1 axis
in neoplastic conditions.
The biological significance of the up-regulation of
CX3CR1 by glioma cells remains unclear. The constitutive
expression, in the brain, of CX3CL1 and CX3CR1 has been
the subject of intense investigation. Experimental evidence
indicates a role for CX3CL1 in promoting neuronal survival in
glutamate-mediated excitoxicity [32]. Enhanced neuron loss occurs
in CX3CR1- deficient mice after systemic lipopolysaccharide
injection, in toxin-induced Parkinsonism, and in the
SOD1G93A transgenic mouse model of motor neuron disease
[22]. In addition, CX3CL1 regulates microglia functions,
inducing mobilization of intracellular Ca2+, chemotaxis,
inhibition of Fas-mediated apoptosis and of LPS-induced activation
[28, 29, 31].
As CX3CL1 is a membrane-bound chemokine, the
ligand/receptor axis can function as an adhesion molecule.
CX3CR1 positive tumor cells may have enhanced adhesion to
neurons expressing the ligand. In pancreatic cancer, we
demonstrated that tumor samples with high receptor scores have
higher percentage of local nerve terminations infiltrated by cancer
cells [34]. Tumor perineural tropism and dissemination along
cerebral fibre tracts also occur in malignant glioblastoma
[1-3].
In conclusion, the results reported here show that expression of
the chemokine receptor CX3CR1 is a frequent event in human
gliomas, irrespective of histology and grading. The molecular basis
underlying CX3CR1 up-regulation and its functional biological
significance remain to be determined.
Acknowledgments
This work was supported by the Associazione Italiana Ricerca sul
Cancro (AIRC) Italy, grants from the Ministry of Health and
Istituto Superiore Sanità Italy (Project Oncology 2006, Alleanza
Contro il Cancro), and the European Commission FP6 Framework
(Project Innochem).
Disclosure. None of the authors has any conflict of
interest to disclose.
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