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RESEARC H ARTIC LE Open Access
Genomics and proteomics approaches to the
study of cancer-stroma interactions
Flávia C Rodrigues-Lisoni
1,8
, Paulo Peitl Jr
2
, Alessandra Vidotto
1
, Giovana M Polachini
1
, José V Maniglia
3
,
Juliana Carmona-Raphe
1
, Bianca R Cunha
1
, Tiago Henrique
1
, Caique F Souza
1,4
, Rodrigo AP Teixeira
2
,
Erica E Fukuyama
5
, Pedro Michaluart Jr
6
, Marcos B de Carvalho
7
, Sonia M Oliani
2
,
Head and Neck Genome Project GENCAPO
9
, Eloiza H Tajara
1,4*
Abstract
Background: The development and progression of cancer depend on its genetic characteristics as well as on the
interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and
prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential
mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated
soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and
protein expression.
Methods: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a
primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach,
quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine
factors produced by stromal and neoplastic cells.
Results: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced
apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in
response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned
medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes
(ARID4A,CALR,GNB2L1,RNF10,SQSTM1,USP9X) were validated by real time PCR.
Conclusions: A significant and common denominator in the results was the potential induction of signaling
changes associated with immune or inflammatory response in the absence of a specific protein.
Background
Solid tumors are characterized by the presence of two
major components: neoplastic cells and a specialized
nonmalignant stroma in which they are immersed and
are essential for their survival and proliferation. In carci-
nomas, a basement membrane is usually present
between these components [1,2].
The tumor stroma is distinguished by an enrichment of
microvessel density, abundance of endothelial cells and
precursors, inflammatory cells including lymphocytes,
neutrophils, macrophages, dendritic and mast cells, and a
connective tissue with fibroblasts, myofibroblasts and
histiocytes responsible for remodeling and deposition of
extracellular matrix (ECM) components - fibronectin,
collagens, elastin, and glycosaminoglycans [2-4].
Although these cells are nonmalignant, they have a
unique gene expression pattern, compared to stroma
cells in normal tissues [5,6].
Substantial evidence indicates that the development
and the progression of cancer not only depend on its
genetic characteristics but also on interactions with its
microenvironment [4,7,8]. In fact, tumor cells may alter
the surrounding stroma through direct cell contact or
via the secretion of paracrine soluble factors, inducing
cell differentiation or extracellular matrix modifications
[9]. In it turn, stromal cells may promote cancer pro-
gression and acquisition of invasiveness [10-12]. It is
* Correspondence: tajara@famerp.br
1
Department of Molecular Biology, School of Medicine (FAMERP), São José
do Rio Preto, Brazil
Rodrigues-Lisoni et al.BMC Medical Genomics 2010, 3:14
http://www.biomedcentral.com/1755-8794/3/14
© 2010 Rodrigues-Lisoni et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attri bution License (http://creativecommons.org /licenses/by/2.0), which permits unrestricted use, distribution, and
reproductio n in any medium, provided the original work is properly cited.
possible that such interactions contribute to the neo-
plastic cell phenotype and behavior as observed during
the normal development process and function of organs
and tissues [13,14]. As Albini and Sporn (2008) appro-
priately propose, the microenvironment may be more
than a partner but also an essential component of the
cancer, and both should be considered as a functional
whole [15].
In this context, inflammation and infection have
gained special attention. Well known examples connect-
ing infection-related or -unrelated chronic inflammation
and increased risk for cancer development are described
in the literature [16], and probably more than 15% of
cancers are linked to these factors [17]. TNF-alpha and
NF-B transcription factor should play a central role in
this process, modulating transcription of genes encoding
angiogenic and growth factors, inflammatory cytokines
and anti-apoptotic proteins [16]. In fact, many inflam-
matory mediators may influence cell proliferation and
tumor development, as demonstrated by our recent stu-
dies on annexin A1 [18-20].
Macrophages represent one of the main inflammatory
regulators in tumor stroma and are responsible for pro-
liferation, invasion and immunosuppressive signaling,
with the production of angiogenic and growth factors,
chemokines, cytokines and matrix metalloproteinases
[21]. The key partners of macrophages in this network
are fibroblasts, the so-called carcinoma-associated fibro-
blasts (CAFs), which significantly increase the growth of
neoplastic or normal cells [22,23] and can enhance
tumor engraftment and metastasis in animal models
[24]. Recently, Hawsawi et al. (2008) [25] observed well-
defined differences in gene expression and proteomic
profiles between activated CAFs and fibroblasts from
normal stroma, emphasizing their importance in the
cancer process.
Regardless of the fact that they are easily identified by
their morphology, specific cellular markers for fibro-
blasts remain unknown, presumably because of their
large diversity [26]. In tumor stroma, fibroblasts present
a phenotype similar to those associated with wound
healing, with a large and euchromatic nucleus and
prominent rough endoplasmic reticulum [27,28]. These
signals mediating the transition of normal to reactive
fibroblasts are still not completely defined.
Many studies have analyzed the role of fibroblasts in
cancer initiation and progression. To address this issue,
several approaches have been used, as co-culture of can-
cer cells and fibroblasts and cultures with conditioned
medium, combined or not with in vivo experiments.
Thedatahaveshownthatthesecells,similartomacro-
phages, overexpress chemokines, interleukines, growth
factors and matrix metalloproteinases, promoting
inflammatory responses and facilitating angiogenesis,
cancer-cell invasion and proliferation [29-31]. In head
and neck cancer, for example, in vitro experiments have
suggested that the presence of fibroblasts is essential for
invasive features either because cancer cells express
higher levels of matrix metalloproteinases in the pre-
sence of fibroblasts [32,33] or because cancer-associated
fibroblasts themselves synthesize these proteins [34,35].
Much of the answer to the question of tumor-stroma
interactions lies in the identity of ligands, receptors and
effectors of signaling patterns expressed by stroma and
tumor cells. Numerous growth factors, cytokines, chemo-
kines, hormones, enzymes and cells responsible for their
expression have been characterized but the cross-signal-
ing between pathways in this complex network is far
from solved [7,36]. Adding complexity to the scenario,
the chemomechanical environment of the extracellular
matrix may also act in concert with signaling pathways
and affect the cancer process [37].
An important perspective in the study of tumor
stroma is the potential use of the gene expression pat-
tern of their cells for diagnostic or prognostic evaluation
and as a target for therapy. Supporting this idea are the
results from studies on outcome prediction and molecu-
lar marker analysis of the stroma [6,38], drugs targeting
inflammatory cells [39] and mediators of angiogenesis
[40,41].
In order to investigate potential mechanisms by which
the tumor microenvironment might contribute to cancer
phenotype, we asked whether soluble paracrine factors
produced by stromal and neoplastic cells in vitro may
influence proliferation, and gene and protein expression.
For these purposes, we exploited purified fibroblasts iso-
lated from a primary oral cancer and an epithelial can-
cer cell line linked by conditioned medium and genomic
and proteomic approaches. Both cells were treated with
the conditioned medium of each other and submitted to
analysis by rapid subtraction hybridization methodology,
two-dimensional electrophoresis and mass spectrometry.
Based on the results of the rapid subtraction hybridiza-
tion (RaSH) approach, a comparative quantitative real-
time PCR was performed to validate the expression of
several genes, focusing on those involved in tumorigen-
esis and inflammation. The results pointed to the parti-
cipation of several inflammatory mechanisms that might
have biological significance in epithelial tumors.
Methods
Primary tumor samples
For conditioned medium experiments, a primary epider-
moid (squamous cell) carcinoma of the retromolar area
was obtained from a 49-year-old male patient, prior to
radiation and/or chemotherapy. Twenty-four laryngeal
and 23 oral tongue squamous cell carcinoma (SCC)
samples from patients undergoing tumor resection were
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used for gene expression analysis. All carcinoma samples
were reviewed by senior pathologists and exhibited the
presence of at least 70% tumor cells; the corresponding
surgical margins were classified to be free of tumor cells.
The study protocol was approved by the National
Committee of Ethics in Research (CONEP 1763/05, 18/
05/2005), and informed consent was obtained from all
patients enrolled.
Epithelial cancer cell line and primary tumor cell cultures
The Hep-2 cell line, originally established from an epider-
moid carcinoma of the larynx (ATCC, Rockville, Mary-
land, USA), was seeded at a density of 1 × 10
6
cells/mL
per 75 cm
2
culture flask (Corning, NY, USA) in medium
MEM-Earle (Cultilab, Campinas, SP, Brazil), pH 7.5,
supplemented with 20% fetal calf serum (Cultilab), 1%
non-essential amino acids, 0.1% antibiotic/antimycotic
(Invitrogen Corporation, Carlsbad, CA, USA), and cul-
tured at 37°C in a humid atmosphere of 5% CO
2
.
A primary carcinoma of retromolar area sample show-
ing epithelium and adjacent connective tissues was
rinsed multiple times with 100× antibiotic and antimy-
cotic solutions (Invitrogen) and minced into 2-4 mm
fragments. Single-cell suspensions were obtained by
digestion at 37°C for 1 hour with 40 mg/mL collagenase
type I (Sigma Chemical, St Louis, USA). After centrifu-
gation, the cells were washed with PBS, resuspended in
DMEM medium supplemented with 20% fetal calf
serum (Cultilab), 2 mM glutamine (Invitrogen), 1% non-
essential amino acids (Invitrogen), and 0.1% antibiotic/
antimycotic (Invitrogen). The cells were seeded at a
density of 1 × 10
6
cells/mL per 75 cm
2
culture flasks
(Corning) and cultured at 37°C in a humid atmosphere
of 5% CO
2
. Cell medium was changed at 72 h intervals
until the cells became confluent. Since fibroblasts were
mixed with the epithelial tumor cells at the time of
initial plating, fibroblasts were selected by plating the
cells growing in medium supplemented with 20% serum
for at least 3 weeks [42-44].
Preparation of conditioned medium
Conditioned medium (CM) was prepared from Hep-2
cell or tumor stromal fibroblast cultures showing 80%
confluence. Twenty-four, 48 and 72 hours after medium
replacement, the supernatant or conditioned medium
(CM24, CM48 and CM72, respectively) from three repli-
cas was aspirated and filtered through a 0.22 μmmem-
brane (Millipore) to remove any cell debris and stored
at -80°C. Before using, the CM was diluted 1:1 in com-
plete medium. The dilution 1:1 and CM72 were chosen
to maximize the chance of detecting a cell response to
soluble factors. Optimization experiments showed that
dilutions lower than 1:1 resulted in higher numbers of
dead cells.
Hep-2 cell-conditioned medium is referred to as HCM
and fibroblast-conditioned medium is referred to as
FCM.
Growth curve
Hep-2 cells were seeded at a density of 5 × 10
4
cells in
plastic 6-well plates in two sets of quadruplicates.
Twenty-four hours later, when cells had already
adhered, Hep-2 cultures were incubated with FCMs.
One replica in each set was treated with self-conditioned
medium and one replica was treated with complete
medium.
Medium was replaced on day 4 and cell morphology
was observed every day. After 1, 3, 5 and 7 days, cells
were harvested and counted using a Neubauer hemocyt-
ometer. The same experiment was repeated twice.
Immunofluorescence analysis
The Hep-2 cell line or tumor stromal fibroblasts were
grown in culture chambers (Nunc, Naperville, IL, USA)
and, after 3 days, the chambers were carefully removed,
and the slides with adherent cells were fixed in 4% par-
aformaldehyde and 0.5% glutaraldehyde, 0.1 mol/L
sodium phosphate buffer, pH 7.4, for 2 hours at 4°C.
The slides were washed in the same buffer and incu-
bated with 0.1% albumin bovine and 3% normal serum
in PBS (PBSA) to block nonspecific binding. The cells
were immunostained with primary mouse monoclonal
antibodies (Ab) anti-vimentin (NCL-VIM-V9, Novocas-
tra, Benton Lane, Newcastle, UK) or anti-cytokeratin
(M3515, antibodies to all types of cytokeratins; AE1-
AE3; Dako, Carpinteria, CA, USA) diluted at 1:200 in
1% PBSA, followed by overnight incubation at 4°C. For
negative controls, the cells were incubated with nonim-
mune mouse serum (1:200 working dilution; Sigma-
Aldrich). After repeated washings in 1% PBS, a goat
anti-mouse IgG (Fc fragment-specific, Dako, Glostrup,
Denmark) antibody conjugatedtoFITC(1:50;British
BioCell International, Cardiff, UK) was added, followed
by 1 hour incubation at room temperature. Thus, the
cells were washed thoroughly in PBS. Analysis was con-
ducted using an Axioskop 2 light microscope (Zeiss,
GR) equipped with a digital camera. Digital images were
captured by using software AxioVision (Zeiss, GR).
Immunohistochemical analysis
Apoptosis was assayed using AnxA5 staining as
described [45]. Fixed Hep-2 cell line or tumor stromal
fibroblast in slides from culture chambers were incu-
bated with the following reagents: 2.1% sodium citrate
for 30 min at 96°C; 3% hydrogen peroxide for 15 min;
0.1% Tween 20 (Sigma-Aldrich) diluted in 0.4% PBS for
15 min; non-specific binding sites were blocked with
10% albumin bovine (BSA) diluted in TBS (20 mM Tris
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buffer in 0.9% NaCl, pH 8.2) for 30 min. The slides were
then incubated overnight with a rabbit polyclonal anti-
body anti-AnxA5 (sc8300, Santa Cruz Biotechnology,
California, USA), diluted 1:200. After repeated washings
in 1% PBSA, a goat anti-rabbit IgG (Fc fragment speci-
fic) antibody conjugated to 5 nm colloidal gold particles
(N24916, Invitrogen) was added. Silver enhancing solu-
tion (L24919, Invitrogen) was used to augment gold par-
ticle staining. At the end of the reaction, cells were
washed thoroughly in distilled water, counterstained
with haematoxylin and examined using an Axioskop2
microscope (ZEISS, GR).
RNA extraction for Rapid Subtraction Hybridization
(RaSH) and real time PCR experiments
Hep-2 cells and stromal fibroblasts were seeded at a
density of 1 × 10
6
cells/mL per 75 cm
2
culture flasks in
complete medium (controls) and in conditioned med-
ium. Hep-2 cells and fibroblasts were cultured for 5 and
3 days, respectively, and harvested by addition of TRIzol
Reagent, following treatment with DNase (Invitrogen).
Total RNA from primary tumor samples was also
extracted using TRIzol Reagent and treated with DNase.
cDNA synthesis was performed using a High Capacity
cDNA Archive kit (Applied Biosystems, Foster City, CA,
USA) as described by the manufacturer.
RaSH
RaSH technique was performed as described by Jiang et
al. (2000) [46]. Aliquots (20 μg) of total RNA from con-
trol cells (driver) or treated cells (tester) were used for
double-stranded cDNA synthesis using standard proto-
cols [47].
The cDNA was digested with MboI (Invitrogen) at
37°C for 3 h followed by phenol/chloroform extraction
and ethanol precipitation. The digested cDNAs were
mixed with the adaptors XPDN-14 5′-CTGATCACTC-
GAGA and XPDN-12 5′-GATCTCTCGAGT (Sigma
Chemical, final concentration 20 μM) in 30 μlof1×
ligation buffer (Gibco BRL), heated at 55°C for 1 min,
and cooled down to 14°C within 1 h. After adding 3 μl
of T4 DNA ligase (5 U/μl) (Gibco, BRL), ligation was
carriedoutovernightat14°C.Afterphenol/chloroform
extraction and ethanol/glycogen precipitation, the mix-
tures were diluted to 100 μl with TE buffer (10 mM
Tris/1 mM EDTA); 40 μl of the mixtures were used
for PCR amplification.
The PCR mixtures were set up using 10 μM XPDN-18
5′-CTGATCACTCGAGAGATC, 0.4 mM dNTPs, 10 ×
PCR buffer, 1.5 mM MgCl
2
and 1U Taq DNA polymer-
ase (Invitrogen). Thermocycler conditions were one
cycle at 72°C for 5 min, followed by 25 cycles of 94°C
for 1 min, 55°C for 1 min, 72°C for 1 min, ending in a
final extension at 72°C for 3 min. Ten μgofpurified
PCR product (tester) was digested with 20U XhoI (Invi-
trogen) followed by phenol/chloroform extraction and
ethanol precipitation.
One-hundred nanograms of the tester cDNA were
mixed with 5 μg of the driver cDNA in hybridization
solution (0.5 M Nacl, 50 mM Tris/HCl, SDS2% and 40%
formamide) and, after heating at 95°C, incubated at
42°C for 48 h. After extraction and precipitation, the
hybridization mixture (1 μg) was ligated with XhoI-
digested pZero plasmid and transformed into competent
bacteria. Bacterial colonies were picked and used as
DNA template for PCR. Clones were sequenced using
an automated DNA sequencer and sequence homologies
were searched using the BLAST program [48]. Gene
ontology (GO) annotation was used for the functional
classification of up- and down-regulated genes [49].
Quantitative PCR
For validation experiments, cells were seeded at a den-
sity of 1 × 10
6
cells/mL per 75 cm
2
culture flasks in two
sets of quadruplicates. Twenty-four hours later, when
cells had already adhered, Hep-2 culture replicas were
treated with FCMs and fibroblast cultures were treated
with HCMs. One replica in each set (control) was trea-
ted with self-conditioned medium. Hep-2 cells and
fibroblasts were harvested after 5 and 3 days, respec-
tively, and RNA was extracted as described above.
Nine differentially expressed genes were selected for
validation by quantitative real time PCR experiments
according to their direct or indirect involvement in
tumorigenesis. Their expression was checked in treated
samples relative to matched non-treated samples. One
of these genes (ARID4A) was also selected for quantita-
tive real time PCR validation in fresh tumor samples of
24 laryngeal SCC and in 23 oral tongue SCC relative to
matched normal samples.
The primers were manually designed with: 19-23 bp
length, 30-70% GC content and a short amplicon size
(90-110 bp). Their sequences are available upon request.
Real time PCR was performed in triplicate using a 7500
Fast Real-Time PCR System (Applied Biosystems).
Reaction mixture consisted of a 20 ul volume solution
containing 10 ul of Power SYBR Green PCR Master Mix
(Applied Biosystems), 500 nM of each primer and 100 ng
cDNA. The PCR conditions were 95°C for 10 min
followed by 40 cycles of 95° for 15 s and 60° for 1 min.
Melting curve analysis was performed for each gene to
check the specificity and identity of the RT-PCR products.
For each primer set, the efficiency of the PCR reaction
(linear equation: y = slope + intercept) was measured in
triplicate on serial dilutions of the same cDNA sample.
The PCR efficiency (E) was calculated by the formula
E= [10
(-1/slope)
] and ranged from 1.96 to 2.02 in the dif-
ferent assays.
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Three control genes (GAPDH, ACTB and TUBA6)
were used as internal standards. The relative expression
ratio (fold change) of the target genes was calculated
according to Pfaffl (2001) [50]. Statistical analysis was
performed by a two-tailed unpaired ttest using Graph-
Pad prism software.
Proteomic analysis
Hep-2 cells and stromal fibroblasts were seeded at a den-
sity of 1 × 10
6
cells/mL per 75 cm
2
culture flasks in com-
plete medium and in conditioned medium, as described
for RASH experiments. Hep-2 cells and fibroblasts were
cultured for 5 and 3 days, respectively, and harvested by
centrifugation at 3200 rpm for 5 min at 4°C. Cells were
disrupted by sonication, proteins were isolated and two-
dimensional electrophoresis (2-DE) was performed, as
described by de Marqui et al. (2006) [51]. Briefly, isoelec-
tric focusing was carried out in a IPGphor (GE Health-
care) using 13-cm immobilized pH 3-10 L gradient strips.
Vertical 12.5% SDS-PAGE was performed in a SE 600
Ruby electrophoresis unit (GE Healthcare) and proteins
were detected by Coomassie Blue staining. Differentially
expressed proteins were excised from gel, distained, dried
and in-gel tryptic-digested. Negative and positive control
digests were performed on gel slices that contained no
protein and on slices cut from a band of the molecular
weight marker, respectively.
Samples were analyzed using MALDI Q-TOF (Matrix
Assisted Laser Desorption Ionization - Quadrupole Ion
Filter - Time of Flight) Premier (Waters Corporation,
Milford, MA, USA) mass spectrometer (MS/MS). Dupli-
cate or triplicate runs of each sample were made to
ensure an accurate analysis.
For protein identification, the resulting MS/MS data
were interpreted by MASCOT software (MS/MS Ions
Search) [52] and searched against the Mass Spectrometry
Protein Sequence Database (MSDB). The UniProtKB/
Swiss-Prot [53] database was used for the functional clas-
sification of up- and down- expressed proteins.
Data Handling and Statistical Analysis
Quantification of apoptotic cells was performed with a high
magnification objective (×40) counting cells in 100 μm
2
areas and reported as mean ± SEM per group. Densito-
metric analysis for the immunofluorescence staining used
an arbitrary scale ranging from 0 to 255 units. Statistical
differences between groups were determined by analysis of
variance followed, if significant, by the Bonferroni test.
Results
Stromal fibroblasts: selection and immunofluorescence
analysis
Fetal calf serum concentration and culture time pro-
vided a simple method of selecting fibroblasts from a
primary carcinoma of retromolar area. Fibroblast cul-
tures at passage 78 still showed spindle-shaped cells,
which displayed the typical fibroblast markers, weak
cytokeratin and intense vimentin immunoreactivity
in cytoplasm, after immunofluorescence analysis (Figure
1B, E). Staining was obtained with both antibodies (cyto-
keratin and vimentin) in Hep-2 cells (Figure 1C, F). No
labeling was detected in sections incubated with the
control nonimmune mouse serum (Figure 1A, D).
Ultrastructural analysis showed that the stromal fibro-
blasts present large euchromatic nuclei, more granular
endoplasmatic reticulum, mitochondria and nucleoli
than normal fibroblasts (data not shown). Therefore, the
spontaneously immortalized cell line of fibroblasts
retained the characteristics of stromal cells and may cor-
respond to cancer-associated fibroblasts (CAF).
Conditioned medium inhibits proliferation and induces
apoptosis
Growth curves of Hep-2 cells treated with FCM showed
decreased proliferation (Figure 2). Growth inhibition
was observed as early as day 1 and was statistically sig-
nificant (P < 0.05) at day 3 and day 5.
The immunohistochemistry reaction with AnxA5 anti-
body showed the presence of gold particles on the cyto-
plasm of the Hep-2 apoptotic cells (Figure 3). The
AnxA5 immunoreactivity was found more in the apop-
totic process of Hep-2 cells incubated in FCM (56%)
than in cells without the treatment (24%). Apoptotic
cells displayed distinctive morphology, a notable
decrease in the nuclear size, irregular shape and cyto-
plasmic blebbing.
Genes identified using the RaSH approach
A total of 81 clones from the Hep-2 cell line and fibro-
blast libraries were sequenced. In the Hep-2 cell line,
forty-one genes exhibited changes in expression
levels in response to FCM treatment (33 down- and 8
up-regulated) and, in fibroblasts, 17 genes showed
down-regulation in response to HCM treatment. These
genes are involved in response to stimulus, apoptosis,
cell proliferation and differentiation, signal transduc-
tion, transcription, translation and transport (Table 1
and 2).
Real-time PCR validation of differentially expressed genes
Nine genes displaying down- (ARID4A, CALR, GNB2L1,
GPNMB, RNF10, SQSTM1, USP9X)orup-regulation
(DAP3,PRDX1) in Hep-2 cells treated with FCM were
selected and the expression data for six down-regulated
genes (ARID4A,CALR,GNB2L1,RNF10,SQSTM1,
USP9X) were confirmed by real time PCR (Figure 4A).
Most results were, therefore, consistent with the RaSH
data.
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ARID4A expression was also analyzed in 24 pairs of
tumor and matched normal tissues from laryngeal squa-
mous cell carcinomas and in 23 pairs of tumor and
matched normal tissues from oral tongue squamous cell
carcinomas. ARID4A mRNA levels were decreased (≥2-
fold) in almost half of the squamous cell carcinomas
samples (-1.04 to -6.9-fold change, 23 of 47 samples, i.e.,
49%) and were increased in some of these samples (1.51
to 6.26-fold change, 7 of 47 samples, i.e., 15%) (Figure
4B). In contrast, no differences in transcript levels were
observed between 17 of 47 samples (36%) and normal
tissue. Therefore, similarly to the Hep-2 cell line, most
primary head and neck tumors (49%) showed down-
regulation of ARID4A transcripts.
No differences were observed in respect to clinico-
pathological features between samples presenting up-
and down-regulation of ARID4A transcripts (Additional
file 1).
Proteomics approach
Comparison between 2-DE patterns from treated cells
and controls revealed approximately 80 spots with
significant differences in intensity. Seven proteins
(Figure 5) showing expression level changes in response
Figure 1 Immunofluorescence analysis of cytokeratin and vimentin in stromal fibroblasts and Hep-2 cell line.(AandD)Absenceof
immunoreactivity in sections incubated with control nonimmune mouse serum. Stromal fibroblasts (B and E) and Hep-2 cell line (C and F) were
positive for vimentin and cytokeratin, respectively. (G): Densitometric analysis of immunofluorescence reaction to vimentin and cytokeratin in
stromal fibroblasts and Hep-2 cell line. Scale bar, 20 μm.
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to CM treatment were identified by MALDI-Q-TOF-MS
mass spectrometry (Additional file 2). Five proteins
(alpha enolase, heterogeneous nuclear ribonucleoprotein
C C1/C2, aldolase A, tubulin beta and glyceraldehyde-3-
phosphate dehydrogenase) were down-regulated in Hep-
2 cell line treated with conditioned medium (FCM72)
and two proteins (vimentin and actin) were underex-
pressed in fibroblasts treated with Hep-2 cell line condi-
tioned medium (HCM72). These proteins are involved
in transcription, growth control, response to stimulus,
RNA processing, glycolysis, cell motion and membrane
trafficking.
Discussion
The molecular crosstalk between neoplastic and the sur-
rounding tissue induces several stromal changes, includ-
ing neoangiogenesis and immune/inflammatory reaction,
as well as new extracellular matrix formation and the
activation of fibroblast-like cells, a process known as
desmoplasia [54], [55]. Initially, the desmoplastic
response was considered a barrier against tumor inva-
sion, but there is growing evidence that desmoplasia is
an unfavorable prognostic factor. For example, Sis et al.
[56] suggested that desmoplastia is related to increased
risks of regional metastases, poorly differentiated
Figure 2 Growth curve of Hep-2 cell line. Hep-2 cells were cultured in complete medium, treated with self-conditioned medium (HCM) or
with conditioned medium from fibroblast cultures (FCM) and collected 1, 3, 5 and 7 days after medium replacement. Data are means ± s.d. of
two independent experiments in duplicates. *P< 0.05. Error bars indicate S.D.
Figure 3 Immunohistochemistry reaction with AnxA5 antibody showed the presence of gold particles on the cytoplasm of apoptotic
cells. Hep-2 cells (A) without treatment and (B) treated with conditioned medium from fibroblast culture (FCM) show AnxA5 immunoreactivity.
Apoptotic cells immunolabeling for AnxA5 can be seen in Hep-2 cells treated with FCM (arrows). Staining with haematoxylin. Scale bar, 20 μm.
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primary tumors and lymphatic and venous invasion in
colorectal carcinoma. Similar results were observed for
head and neck squamous cell carcinomas, which show a
high risk of neck recurrence in presence of a desmoplas-
tic stromal pattern [57].
In the present study, we investigated the influence of
soluble paracrine factors produced in vitro by stromal
cells derived from an oral carcinoma and by a neoplastic
epithelial cell line on proliferation and gene/protein
expression. First, we noted that conditioned medium
from stromal fibroblast cultures inhibited Hep-2 cell
line proliferation and induced apoptosis, suggesting that
factors secreted by fibroblasts include proteins that
interfere in cell growth and death of neoplastic cells. In
addition, using rapid subtraction hybridization and pro-
teomic analysis, we identified gene products generated
by stromal and neoplastic cells that may influence pro-
liferation, differentiation and apoptosis, or drive
response to stimulus.
Down-regulated genes in neoplastic cells treated with
FCM are involved in signal transduction (FAS,SQSTM1,
YWHAZ), transcription (ARID4A,CALR,MYC,PARP1,
RNF10,SQSTM1), translation (AARS,RPLP0,RPS17,
RPS23), apoptosis (CALR,FAS,TPT1,YWHAZ), cell
migration (TMSB4X,GNB2L1), cell cycle and cell prolif-
eration (DYNC1H1,GPNMB,LDOC1,MYC,PSM),
Table 1 Information on biological processes based on
Gene ontology
Biological Process Down-regulated genes
Cell communication
signal transduction FAS, SQSTM1, YWHAZ
Transcription ARID4A, CALR, MYC, PARP1,
RNF10, SQSTM1
Translation AARS, RPLP0, RPS17, RPS23
Apoptosis CALR
induction FAS
anti-apoptosis TPT1, YWHAZ
Cell migration TMSB4X
Cell cycle DYNC1H1, MYC, PSMC6
Cell proliferation
negative regulation GPNMB, LDOC1
positive regulation MYC
Developmental process
epidermis development UGCG
Response to stimulus
defense response
inflammatory response LTA4H
response to stress EIF2AK1, SQSTM1
response to oxidative stress
response to external stimulus EIF2AK1
Transport CALR, NDUFA4, SQSTM1
Metabolic process COX7C, OLA1,
protein metabolic process PARP1, SQSTM1, USP9X
protein modification process GRPEL2, HSP90AB1, PPP2R2A,
PRPF4B, USP48
lipid metabolic process LTA4H, UGCG
DNA repair PARP1
RNA processing PRPF4B, SF3B1
Cellular homeostasis CALR, MYC, RPS17
No classification GNB2L1, RCN1
Up-regulated genes
Transcription ENO1
Translation EIF1, TARS
Apoptosis RTN3
induction DAP3
Cell proliferation PRDX1
negative regulation ENO1
Developmental process
organ development PRDX1
Response to stimulus
response to stress EIF1, RTN3
Metabolic process PRDX1
protein metabolic process
protein modification process P4HB
nucleic acid metabolic process
RNA processing USP39
Top down- and up-regulated genes selected by RaSH in Hep-2 samples
treated with FCM.
Table 2 Information on biological processes based on
Gene Ontology
Biological Process Down-regulated genes
Cell communication
signal transduction S100A6, FN1
Transcription FOSL1
Translation RPL37A, RPL7, RPL19, RPL27A, RPLP0
Apoptosis CTSB
anti-apoptosis TPT1
Cell adhesion FN1
Cell proliferation
positive regulation S100A6, FOSL1
Developmental process
organ development
epidermis development COL1A1
Response to stimulus
defense response FOSL1
response to stress FN1
Transport ERGIC3, STX4
Metabolic process
protein metabolic process CTSB
RNA processing PRPF3
No classification CIZ1, POLE4
Top down-regulated genes selected by RaSH in CAF samples treated with
HCM.
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epidermis development (UGCG), response to stimulus
(EIF2AK1,LTA4H,SQSTM1), transport (CALR,
NDUFA4,SQSTM1) and different metabolic processes
(USP9X). Up-regulated genes are also involved in tran-
scription and translation (ENO1,EIF1,TARS), apoptosis
(DAP3,RTN3), cell proliferation (PRDX1,ENO1), organ
development (PRDX1), response to stress (EIF1,RTN3)
and metabolic processes (PRDX1,P4HB,USP39).
In fibroblasts treated with HCM, the biological pro-
cesses of down-regulated genes include signal transduc-
tion (S100A6,FN1), transcription and translation
(FOSL1,RPL37A,RPL7,RPL19,RPL27A,RPLP0), apop-
tosis (CTSB,TPT1), cell proliferation (S100A6,FOSL1),
epidermis development (COL1A1), response to stimulus
(FN1,FOSL1), transport (ERGIC3,STX4)andprotein
and RNA metabolism (CTSB,PRPF3).
Figure 4 Real-time PCR gene expression in a conditioned medium-treated neoplastic cell line and in primary tumors. (A) Expression of
ARID4A, CALR, DAP3, GNB2L1, PRDX1, RNF10, SQSTM1 and USP9X genes in Hep-2 cells treated with conditioned medium from fibroblast cultures.
(B). ARID4A gene expression in 47 laryngeal and oral tongue carcinomas. Relative quantitation of target gene expression for each sample was
calculated according to Pfaffl [50]; GAPDH was used as the internal reference and control sample as the calibrator. Values were Log2 transformed
(y-axis) so that all values below -1 indicate down-regulation in gene expression while values above 1 represent up-regulation in tumor samples
compared to normal samples.
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Figure 5 Enlarged 2-DE gels of proteins from conditioned medium-treated Hep-2 cells and stromal fibroblasts.Fiveproteins(arrows),
tubulin beta (A-B), alpha enolase (C-D), aldolase A (E-F), glyceraldehyde-3-phosphate dehydrogenase (G-H) and heterogeneous nuclear
ribonucleoprotein C (I-J) were down-regulated in Hep-2 cell line treated with fibroblast conditioned medium (A, C, E, G and I) and two proteins
(K-L), vimentin (arrow on left) and actin (arrow on right), were underexpressed in fibroblasts treated with Hep-2 cell line conditioned medium (K).
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Two genes exhibited similar patterns in both cells
(RPLP0,TPT1), which may indicate that the transcript
levels are affected by soluble paracrine factors produced
by either fibroblasts or neoplastic cells or by other in
vitro conditions. Therefore, they may not be specific to
interactions between stroma and tumor.
After literature analysis, nine genes (ARID4A,CALR,
GNB2L1,GPNMB,RNF10,SQSTM1,USP9X,PRDX1
and DAP3) showing potential involvement in signaling
cascades related to tumorigenesis and/or stromal/tumor
cell interactions were selected for validation by real-time
RT-PCR using treated and non-treated cell lines. For six
genes (ARID4A,CALR,GNB2L1,RNF10,SQSTM1,
USP9X), the results were consistent with the RASH
data. In almost half of the primary tumors analyzed,
ARID4A transcripts also showed down-regulation,
although no correlation with clinicopathological features
was detected. These findings in primary tumors should
reflect the complex network of a multi-cellular tissue, a
situation contrasting with that of a neoplastic cell line
cultured in medium conditioned by fibroblasts.
The product of ARID4A - AT rich interactive domain
4A (RBP1-like) - also known as RBP1 or RBBP1 gene,
interacts with the tumor suppressor retinoblastoma
(pRB) and histone-modifying complexes, repressing pro-
moters of specific genes [58]. Röhl et al. [59] detected
several genes, including ARID4A, overexpressed in astro-
cytes treated with medium conditioned by activated
microglia, which protected them against stress condi-
tions. Recently, Wu et al. [60] showed that Arid4a-defi-
cient mice exhibit down-regulation of several homeobox
genes and of the forkhead box gene Foxp3, which codes
a transcription factor involved in the development and
function of regulatory T cells [61]. These mice also
show bone marrow failure with myelofibrosis and higher
frequencies of hematologic malignancies, providing evi-
dence that ARID4A functions as a tumor suppressor
gene and its absence is permissive for the proliferation
of connective tissue elements. The study of Perez et al.
[62] added data on the role of this gene in cancer.
These authors detected increased mRNA levels of
ARID4A and RB1 in normal human epidermal keratino-
cytes treated with arsenic and benzo [a]pyrene in vitro.
Since these chemicals alter proliferation and inhibit dif-
ferentiation of keratinocytes [63-65], the findings may
indicate that up-regulation of ARID4A is negatively
related to epithelial differentiation. Therefore, the poten-
tial modulation of this gene by paracrine factors pro-
duced by stromal fibroblasts may represent an attempt
to promote differentiation of neoplastic epithelial cells
and, at the same time, their proliferation.
Calreticulin (coded by CALR or CRT gene) is a cal-
cium-binding protein of the endoplasmic reticulum with
intracellular and extracellular functions related to
cellular adhesion, migration, and phagocytosis [66]. Cal-
reticulin can be observed on the surface of stressed cells
and, when bound to the plasma membrane of apoptotic
cells, drives the phagocytosis by macrophages and den-
dritic cells [67]. In absence of this protein, the cells are
not efficiently removed by phagocytes [68]. Recently,
Nanney et al. [69] showed that calreticulin stimulates
both migration and proliferation of keratinocytes and
fibroblasts and apparently attracts monocytes and
macrophages, suggesting its involvement in inflamma-
tory response. Otherwise, fibroblasts underexpressing
CARL exhibit weak adhesion and spreading [70].
Accordingly, Kypreou et al. [71] detected a correlation
between calreticulin up-regulation and progression of
fibrosis and also that TGF-beta, a contributing factor in
fibrotic processes, up-regulated calreticulin in cultured
human epithelial cells. In light of the data, we speculate
that the low levels of this protein observed in treated
Hep-2 cells inhibit proliferation, or represent a protec-
tive response of neoplastic cells to phagocytosis and
antitumor immune process.
Guanine nucleotide binding protein (G protein), beta
polypeptide 2-like 1 or Rack1 (coded by GNB2L1 gene)
is a cytosolic protein homologous to the beta subunit of
G proteins, and contains seven WD repeats, which act
as sites for protein-protein interactions. Binding partners
of GNB2L1 include protein kinase C, Src family kinases,
components of the ERK pathway, cytokine and inter-
feron receptors, beta integrins and many others. Many
of these interactions are consistent with the participa-
tion of Rack1 in cell adhesion, movement and growth
[72-75].
Sequestosome 1 or ubiquitin-binding protein p62
(coded by SQSTM1 or p60orp62 gene) is a 62-kDa
protein that binds to the Src homology 2 (SH2) domain
of p56
lck
kinase in a phosphotyrosine-independent man-
ner [76]. It has been suggested that p62 is a signaling
adaptor which links different signal transduction path-
ways related to cell proliferation, differentiation and
death, including NF-B pathway [77-82]. SQSTM1
abnormal expression has been observed in hepatocellu-
lar, prostate and breast cancers [83-85] and is associated
with poor outcomes in breast cancer [86].
Anothergenedown-regulatedbyfibroblast-condi-
tioned medium is USP9X (ubiquitin specific peptidase 9,
X-linked), also known as DFFRX,FAF or FAM.This
gene is a member of the peptidase C19 family and
encodes a protein similar to ubiquitin-specific proteases
(USPs). These proteases regulate the production and
recycling of ubiquitin and are critically involved in the
control of cell growth, differentiation, and apoptosis
[87]. Alteration of USPs may play an important role in
the pathogenesis of cancer [88] and may exert distinct
growth regulatory activities by acting as oncoproteins or
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tumor suppressor proteins. Overexpression of certain
USPs correlates with progression towards a more malig-
nant phenotype in carcinoma of lung, kidney, breast and
prostate [89,90].
RNF10 (ring finger protein 10) is the least known gene
selected for validation. The product contains a ring fin-
ger motif, which is involved in protein-protein interac-
tions and has been described in proteins implicated in
many cellular processes such as signal transduction,
transcriptional regulation, ubiquination, and apoptosis
[91,92].
With respect to proteomic analysis, few differences
(mostly quantitative) between treated and non-treated
cells were detected. Among the proteins differentially
expressed, alpha-enolase, heterogeneous nuclear ribonu-
cleoprotein C C1/C2, aldolase A, tubulin beta and gly-
ceraldehyde-3-phosphate dehydrogenase were down-
regulated in neoplastic cells treated with FCM and
vimentin and actin were down-regulated in fibroblasts
treated with HCM. These proteins, produced by neo-
plastic cells or fibroblasts, may affect tumorigenesis. For
example, the glycolytic enzyme alpha-enolase and its
enzymatically inactive isoform MBP-1 (c-myc promoter
binding protein 1) are negative regulators for MYC
expression [93,94]. MYC is one of the most frequently
de-regulated oncogenes in cancer [95] and, in the
absence of both enzymes, may become activated and
accelerate tumor growth. Contrary to RaSH results,
alpha enolase protein was observed underexpressed by
proteomic analysis in treated Hep-2 cells, which may
indicate a nonspecific finding or a post-transcriptional/
posttranslational regulation of the RNA/enzyme.
Conclusions
Fibroblasts, as other cells in tumor microenvironments,
need to maintain close communication with cancer
cells, promoting proliferation, recruitment of inflamma-
tory cells and acquisition of invasive characteristics.
Similarly, cancer cells may influence stromal cells to
generate a favorable and supportive environment, which
would supply them with nutrients and factors necessary
for developing the tumor and spreading of metastasis. In
the present study, we observed both positive and nega-
tive effects exerted by fibroblasts on Hep-2 cells, favor-
ing or not the former. A significant and common
denominator in the results was the potential induction
of signaling changes associated with immune or inflam-
matory response in the absence of a specific protein. In
fact, ARID4A down-regulation is related to low levels of
the transcript factor Foxp3 [60], which in turn is linked
to immune responsiveness by targeting NF-Band
CREB pathways [96]. The final effect is the inhibition of
the inflammatory response and the cost is a permissive
sign for fibroblast proliferation [60]. Down-regulation of
CARL also blocks the inflammatory response but has
negative effects on stroma growth [69]. In presence of
low levels of Rack1, again a deficient or altered inflam-
matory response may occur since Rack1 underexpres-
sion has already been related to the deregulation of
cytokine production [97]. Similar results have been
observed in p62-deficient mice, which exhibit abnormal
control of NF-B activation and reduced inflammation
in experimental conditions [98]. The opposite effect is
expected for osteoactivin underexpression because this
protein has been observed as a negative regulator of
macrophage inflammatory responses [99].
The complexity of the tumor microenvironment is
immense and much information is still necessary for
better understanding how the relationship between
stroma and carcinoma cells can be used for diagnostic
and prognostic evaluation and a target for therapy.
Additional file 1: Clinicopathological features of 24 patients with
larynx SCC and of 23 patients with tongue SCC.
Additional file 2: Underexpressed proteins in Hep-2 cells and
fibroblasts treated with conditioned medium from fibroblasts (FCM)
and Hep-2 (HCM), respectively.
Acknowledgements
We acknowledge the financial support from Fundação de Amparo à
Pesquisa do Estado de São Paulo/FAPESP (Grants 04/12054-9 and 06/60162-
0), Rede Proteoma do Estado de São Paulo (Grant FAPESP 04/14846-0/FINEP
01.07.0290.00), The Ludwig Institute for Cancer Research, and the researcher
fellowships from FAPESP (FCR-L) and Conselho Nacional de Pesquisas/CNPq
(EHT, SMO).
The GENCAPO (Head and Neck Genome) Project authors are the following:
Cury PM
7
, de Carvalho MB
8
, Dias-Neto E
3,14
, Figueiredo DLA
9
, Fukuyama EE
5
,
Góis-Filho JF
5
, Leopoldino AM
15
, Mamede RCM
9
, Michaluart-Junior P
6
,
Moyses RA
6
, Nóbrega FG
4
, Nóbrega MP
4
, Nunes FD
13
, Ojopi EPB
3
, Serafini
LN
10
, Severino P
1
, Silva AMA
8
, Silva Jr WA
11
, Silveira NJF
16
, Souza SCOM
13
,
Tajara EH
2
, Wünsch-Filho V
12
, Amar A
8
, Bandeira CM
4
, Braconi MA
4
, Brandão
LG
6
, Brandão RM
11
, Canto AL
4
, Cerione M
5
, Cicco R
5
, Chagas MJ
4
, Chedid H
8
,
Costa A
12
, Cunha BR
2
, Curioni OA
8
, Fortes CS
12
, Franzi SA
8
, Frizzera APZ
7
,
Gazito D
8
, Guimarães PEM
6
, Kaneto CM
11
, López RVM
12
, Macarenco R
4
,
Magalhães MR
8
, Meneses C
4
, Mercante AMC
8
, Pinheiro DG
11
, Polachini GM
2
,
Rapoport A
8
, Rodini CO
13
, Rodrigues-Lisoni FC
2
, Rodrigues RV
2
, Rossi L
8
,
Santos ARD
11
, Santos M
8
, Settani F
5
, Silva FAM
15
, Silva IT
11
,SouzaTB
8
,
Stabenow E
6
, Takamori JT
8
, Valentim PJ
5
, Vidotto A
2
, Xavier FCA
13
,
Yamagushi F
5
, Cominato ML
5
, Correa PMS
4
, Mendes GS
5
, Paiva R
5
, Ramos O
6
,
Silva C
6
, Silva MJ
5
, Tarlá MVC
11
(also presented in http://ctc.fmrp.usp.br/
clinicalgenomics/cp/group.asp).
Affiliations:
1
Instituto de Ensino e Pesquisa Albert Einstein, São Paulo;
2
Departamento de Biologia Molecular, Faculdade de Medicina de São José
do Rio Preto;
3
Departamento e Instituto de Psiquiatria, Faculdade de
Medicina, Universidade de São Paulo (USP), São Paulo;
4
Departamento de
Biociências e Diagnóstico Bucal, Faculdade de Odontologia, Universidade
Estadual Paulista, São José dos Campos, São Paulo,
5
Serviço de Cirurgia de
Cabeça e Pescoço, Instituto do Câncer Arnaldo Vieira de Carvalho, São Paulo;
6
Departamento de Cirurgia de Cabeça e Pescoço, Faculdade de Medicina,
USP, São Paulo;
7
Departamento de Patologia, Faculdade de Medicina de São
José do Rio Preto;
8
Hospital Heliópolis, São Paulo;
9
Serviço de Cirurgia de
Cabeça e Pescoço, Faculdade de Medicina de Ribeirão Preto, USP;
10
Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto,
USP;
11
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto,
USP;
12
Departamento de Epidemiologia, Faculdade de Saúde Pública, USP,
São Paulo;
13
Departamento de Estomatologia, Faculdade de Odontologia da
Rodrigues-Lisoni et al.BMC Medical Genomics 2010, 3:14
http://www.biomedcentral.com/1755-8794/3/14
Page 12 of 15
USP, São Paulo;
14
Centro de Pesquisas do Hospital AC Camargo. São Paulo,
15
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas,
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP;
16
Departamento de Ciências Exatas, Universidade Federal de Alfenas, UNIFAL,
MG; Brazil.
Author details
1
Department of Molecular Biology, School of Medicine (FAMERP), São José
do Rio Preto, Brazil.
2
Department of Biology, Instituto de Biociências, Letras e
Ciências Exatas (IBILCE), São Paulo State University (UNESP), São José do Rio
Preto, Brazil.
3
Department of Otorhinolaryngology and Head and Neck
Surgery, School of Medicine (FAMERP), São José do Rio Preto, Brazil.
4
Department of Genetics and Evolutionary Biology, Institute of Biosciences,
University of São Paulo (USP), São Paulo, Brazil.
5
Department of Head and
Neck Surgery, Arnaldo Vieira de Carvalho Hospital, São Paulo, Brazil.
6
Division
of Head and Neck Surgery, Department of Surgery, School of Medicine
(USP), São Paulo, Brazil.
7
Department of Head and Neck Surgery, Heliópolis
Hospital, São Paulo, Brazil.
8
Department of Biology and Zootechny, Faculty of
Engineering of Ilha Solteria (UNESP), Ilha Solteira, Brazil.
9
Author list and
addresses presented in the Acknowledgements.
Authors’contributions
FCR-L participated in the design of the study and analysis of the data,
carried out cell culture, RaSH experiments and drafted the manuscript. PPJr
helped with RaSH experiments. AV and GMP carried out proteomics analysis.
JVM was responsible for sample collection and processing. JC-R carried out
cloning and sequencing of the samples. BRC carried out cell culture
experiments. TH helped with manuscript preparation. CFS performed the
real time PCR experiments. RAPT and SMO carried out immunofluorescence
and immunohistochemical analysis. EEF and PMJr carried out clinical data
analysis for sample selection. MBdC carried out clinical data analysis for
sample selection and drafted the manuscript. GENCAPO team members
were responsible for sample collection and initial on-site sample processing,
provided the pathological analysis of the cases, obtained the informed
consent and discussed the findings. EHT participated in the study design
and coordination, carried out the analysis and interpretation of the data and
drafted the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 26 June 2009 Accepted: 4 May 2010 Published: 4 May 2010
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Cite this article as: Rodrigues-Lisoni et al.: Genomics and proteomics
approaches to the study of cancer-stroma interactions. BMC Medical
Genomics 2010 3:14.
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