Crosstalk between glial and glioblastoma cells triggers the “go-or-grow” phenotype of tumor cells
Oliveira et al. Cell Communication and Signaling
Crosstalk between glial and glioblastoma cells triggers the “go-or-grow” phenotype of tumor cells
Ana Isabel Oliveira 1 2 4
Sandra I. Anjo 3 5
Joana Vieira de Castro 1 2 4
Sofia C. Serra 1 2 4
António J. Salgado 1 2 4
Bruno Manadas 0 3
Bruno M. Costa 0 1 2 4
0 Equal contributors
1 ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Campus de Gualtar, University of Minho , 4710-057 Braga , Portugal
2 Life and Health Sciences Research Institute (ICVS), School of Medicine, Campus de Gualtar, University of Minho , 4710-057 Braga , Portugal
3 CNC - Center for Neuroscience and Cell Biology, University of Coimbra , 3004-504 Coimbra , Portugal
4 ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Campus de Gualtar, University of Minho , 4710-057 Braga , Portugal
5 Faculty of Sciences and Technology, University of Coimbra , Coimbra , Portugal
Background: Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells. Methods: Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways. Results: The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation. Conclusions: Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.
Glioblastoma; Glial cells; Tumor microenvironment; Secretome; Paracrine effect
Glioblastoma (GBM), the most common of all malignant
brain and central nervous system (CNS) tumors in adults
], is characterized by a poor outcome and limited
therapeutic options. Standard GBM management involves
surgical resection followed by radiotherapy with concomitant
and adjuvant chemotherapy with temozolomide (TMZ),
but in most cases GBM rapidly relapses [
]. Hence, the
treatment remains mostly palliative, with a median overall
survival of only 15 months after diagnosis [
difficulty in effectively treating GBM patients is largely due to
the heterogeneous nature of these tumors [
] and also to
the complex network of interactions that they establish
with the tumor microenvironment (TME) [
the TME has been suggested to play vital roles in
controlling the course of GBM and influencing its treatment
In brain tumors, the microenvironment consists of a
complex network of interactions between a large variety
of glioma-associated stromal cells, including, for example,
astrocytes, endothelial cells, and infiltrating inflammatory
cells like microglia, among others [
]. The crosstalk
between these cells has been shown to favor tumor growth,
invasiveness and therapy resistance, driven by paracrine
signals and disseminated by secreted factors [
particular, the role of microglia cells and astrocytes in glioma
progression and aggressiveness has been studied (reviewed
in ), confirming the existence of paracrine interactions
between GBM cells and TME.
Astrocytes are the most abundant non-neuronal cells
in the brain (consisting of approximately 50% of the
human brain volume), and are therefore likely to establish
myriad direct contacts with tumor cells, potentially
influencing glioma pathophysiology. Indeed, the
invasiveness of glioma cancer stem cells was shown to be
increased in the presence of astrocytes or
astrocytesderived conditioned media (ACM) [
]. Similarly, the
migration capacity of human and murine glioma cell
lines is increased in the presence of ACM [
GDF-15, known to be upregulated in reactive astrocytes
], was found to increase proliferation of glioma cells
]. Taken together, these findings suggest a role for the
secretome of astrocytes in the processes of glioma
initiation and progression. Importantly, a cell secretion is a
highly dynamic and sensitive process that can change
dramatically and rapidly, such as in a disease context
]. Thus, while the secretome of naive astrocytes has
already been mapped [
], its characterization in the
context of exposure to glioma cells is still missing. In
this study, we have investigated the bidirectional
communication between GBM and glial cells. Our data
suggest that factors secreted by GBM cells are capable of
modulating the secretome of glial cells leading to the
upregulation of several proteins related to cellular
homeostasis, cell adhesion and defense response. Additionally,
we found that this modulation functions in a paracrine
fashion to regulate the “go-or-grow” phenotypic switch
of GBM cells.
Mouse glioma cell line
The murine glioma cell line GL261 was maintained in
Dulbecco’s Modified Eagle Medium (DMEM, Biochrom) with
10% fetal bovine serum (FBS, Biochrom) and 1%
penicillinstreptomycin (Gibco). Incubations were performed at 37 °C
in a humidified atmosphere containing 5% CO2.
Primary cultures of cortical glial cells
Cortical glial cells were isolated from the brain of P5-P7
C57Bl/6 wild type pups. Brain macro-dissection was
performed in ice-cold HBSS, under a conventional light
microscope. Upon dissection, cortices were cut into
smaller fragments and incubated for 30 min at 37 °C in
HBSS supplemented with 0.6% trypsin and 750 units of
DNase I (both from Sigma). The digested tissue
fragments were washed twice with DMEM supplemented
with 10% FBS and 1% penicillin-streptomycin and
mechanically dissociated through a 5 mL pipette and a P1000.
A single-cell suspension in complete DMEM was
obtained and cells were plated according to the following
experiments. For conditioned media experiments,
2 × 106 cells were seeded in T75 flasks; for
immunocytochemistry, cells were plated on coverslips coated with
poly-D-lysine (Sigma-Aldrich) at a density of 25,000
cells/cm ; and for viability assay (MTT) cells were
seeded at the same density in culture wells. The culture
medium was changed every 2–3 days and glial cells were
maintained in culture for 15–18 days, at 37 °C in a
humid atmosphere (5% CO2).
Conditioned media collection and experiments
To obtain conditioned medium (CM) from GL261, cells
were plated at a density of 8600 cells/cm2 and allowed
to grow for 72 h. Following this, cells were washed 2
times with PBS and one with serum-free DMEM and
the culture medium was replaced by serum-free DMEM.
The CM was collected after 24 h and filtered through
0.22 μm pore size filters. Upon collection, CM was
snapfreeze in liquid nitrogen and kept at −80 °C until the day
For glial cells CM production, cells were allowed to
grow until they reach a monolayer (15–18 days) and then
washed twice with PBS and once with serum-free DMEM.
Flasks were randomly distributed in two groups: one
received serum-free DMEM for 72 h (unexposed) and the
other GL261 CM (exposed). In both groups, the medium
was replaced after 48 h by fresh medium (serum-free
DMEM or GL261 CM, respectively). After 3 days, glial
cells growing in serum-free DMEM (unprimed) and glial
cells exposed to GL261 CM (primed) were washed as
previous and serum-free DMEM was added to both groups.
Twenty-four hours thereafter, CM from unprimed and
primed glial cells was collected and stored as described
above for GL261 CM. Glial cells were then submitted to
viability assay and immunocytochemistry.
Cell viability assays
Cell viability of glial cells unexposed and exposed to
GL261 CM was quantified using
3-(4,5-Dimethylthiazol2-yl)-2,5-Diphenyltetrazolium Bromide assay (MTT,
Invitrogen). Cells were exposed to 0.5 mg/mL of MTT
in PBS for 2 h in a humidified atmosphere at 37 °C and
5% CO2. The formazan was then solubilized in acidified
isopropanol (0.04 M HCl in absolute isopropanol) and
the optical density was determined at 570 nm.
For GL261 glioma cells viability, 5 × 104 cells were
plated/well in a 6-well plate and incubated at 37 °C and
5% CO2 for 72 h. Following this, cells were washed and
the culture medium was replaced by CM from unprimed
or primed glial cells. After 48 h of incubation, total
viable cells were determined by trypan blue exclusion
assay (Trypan Blue Solution, 0.4%, Gibco).
Glial cells unexposed or exposed to GL261 CM were
fixed in 4% paraformaldehyde at room temperature (RT)
for 30 min and permeabilized with 0.3% Triton X-100 in
PBS for 5 min. Cells were then blocked with 10% FBS in
PBS for 1 h at RT followed by the incubation with
antiGlial Fibrillary Acidic Protein (GFAP, Dako), diluted
1:1000 in 10% FBS in PBS, for 1 h at RT. Cells were
washed with 0.5% FBS in PBS and incubated for 1 h at
RT with Alexa Fluor® 594 conjugate (Invitrogen) diluted
in 10% FBS in PBS (1:1000). Finally, cells were washed
with 0.5% FBS in PBS and the glass coverslips were
mounted in VECTASHIELD® Mounting Medium with
DAPI (Vector Laboratories). Fluorescence analysis and
image capture were performed under an Olympus
BX61 Fluorescence Microscope (Olympus).
RNA extraction, cDNA synthesis and qRT-PCR
Total RNAs from glial cells unexposed or exposed to
GL261 CM were extracted with Trizol (Invitrogen)
according to the manufacturer’s instructions. cDNA
synthesis was performed using 1 μg of total RNA with High
Capacity cDNA Reverse Transcription Kit (Applied
Biosystems). Gene-specific mRNA levels were assessed by
quantitative real-time PCR (qPCR) in a real-time
thermocycler (CFX96; Bio-Rad) using Fast SYBR Green (Qiagen)
or PowerUp SYBR (Applied Biosystems; for p21 gene)
according to the manufacturer’s instructions, by the 2-ΔΔCt
method. The list of primers used and the PCR conditions
can be found in Additional file 1: Table S1.
Sample preparation for proteomics analysis
Glial cells CM (unprimed and primed) spiked with the
recombinant protein malE-GFP (to be used as internal
standard) was firstly concentrated using a Vivaspin®
Turbo 15 sample concentrator (5 kDa; Sartorius) by
ultracentrifugation at 4000×g. Concentrated CM was
precipitated with Trichloroacetic acid (TCA) - Acetone
]. The washed pellets were ressuspended in 40 μL of
2× Laemmli buffer (BioRad), aided by ultrasonication
and denaturation at 95 °C [
]. Ten microlitres of each
replicate (in a total of 4 replicates per condition) were
used to create a pooled sample for protein identification.
After denaturation, samples were alkylated with
acrylamide and subjected to gel digestion using the
shortGeLC approach [
]. The entire lanes were sliced into 3
parts and each part was sliced in small pieces and
processed. Gel pieces were destained, dehydrated and
rehydrated in 75 μL of trypsin (0.01 μg/μL solution in
10 mM ammonium bicarbonate) for 15 min, on ice.
After this period, 30 μL of 10 mM ammonium
bicarbonate were added and in-gel digestion was performed
overnight (ON) at RT. After the digestion, the formed
peptides were extracted from the gel pieces and the
peptides extracted from the three fractions of each
biological replicate were combined into a single sample for
quantitative analysis. All the peptides were dried
subjected to SPE using OMIX tips with C18 stationary
phase (Agilent Technologies) as recommended by the
manufacture. Eluates were dried and ressuspended with
a solution of 2% ACN and 0.1% FA.
Protein quantification by SWATH-MS
Samples were analyzed on a Triple TOF™ 5600 System
(ABSciex®) in two phases: information-dependent
acquisition (IDA) of the pooled samples and SWATH-MS
acquisition of each individual sample. Peptides were
resolved by liquid chromatography (nanoLC Ultra 2D,
Eksigent®) on a MicroLC column ChromXP™ C18CL
(300 μm ID × 15 cm length, 3 μm particles, 120 Å pore
size, Eksigent®) at 5 μL/min with a multistep gradient:
0–2 min linear gradient from 5 to 10%, 2–45 min linear
gradient from 10% to 30% and, 45–46 min to 35% of
ACN in 0.1% FA. Peptides were eluted into the mass
spectrometer using an electrospray ionization source
(DuoSpray™ Source, ABSciex®) with a 50 μm internal
diameter (ID) stainless steel emitter (NewObjective).
Information dependent acquisition, experiments were
performed for each pooled sample and the mass
spectrometer was set to scanning full spectra (350–1250 m/
z) for 250 ms, followed by up to 100 MS/MS scans
(100–1500 m/z from a dynamic accumulation time –
minimum 30 ms for precursor above the intensity
threshold of 1000 – in order to maintain a cycle time of
3.3 s). Candidate ions with a charge state between +2
and +5 and counts above a minimum threshold of 10
counts per second were isolated for fragmentation and
one MS/MS spectra was collected before adding those
ions to the exclusion list for 25 s (mass spectrometer
operated by Analyst® TF 1.7, ABSciex®). Rolling collision
was used with a collision energy spread of 5. Peptide
identification and library generation were performed
with Protein Pilot software (v5.1, ABSciex®), using the
following parameters: i) search against a database
composed by Mus musculus from SwissProt (release at
December 2015), and malE-GFP; ii) acrylamide alkylated
cysteines as fixed modification; iii) trypsin as digestion
type. An independent False Discovery Rate (FDR)
analysis using the target-decoy approach provided with
Protein Pilot software was used to assess the quality of the
identifications and positive identifications were
considered when identified proteins and peptides reached a 5%
local FDR [
For SWATH-MS based experiments, the mass
spectrometer was operated in a looped product ion mode
] and the same chromatographic conditions used as
in the IDA run described above. The SWATH-MS setup
was designed specifically for the samples to be analyzed
(Additional file 2: Table S2), in order to adapt the
SWATH windows to the complexity of the set of
samples to be analyzed. A set of 60 windows of variable
width (containing 1 m/z for the window overlap) was
constructed covering the precursor mass range of 350–
1250 m/z. A 250 ms survey scan (350–1500 m/z) was
acquired at the beginning of each cycle for instrument
calibration and SWATH MS/MS spectra were collected
from 100 to 1500 m/z for 50 ms resulting in a cycle time
of 3.25 s from the precursors ranging from 350 to
1250 m/z. The collision energy for each window was
determined according to the calculation for a charge +2
ion centered upon the window with variable collision
energy spread (CES) according with the window.
A specific library of precursor masses and fragment
ions was created by combining all files from the IDA
experiments, and used for subsequent SWATH processing.
Libraries were obtained using Protein Pilot™ software
(v5.1, ABSciex®) with the same parameters as described
above. Data processing was performed using SWATH™
processing plug-in for PeakView™ (v2.0.01, ABSciex®) as
described in [
]. After retention time adjustment using
the malE-GFP peptides, up to 15 peptides, with up to
five fragments each, were chosen per protein, and
quantitation was attempted for all proteins in library file that
were identified below 5% local FDR from ProteinPilot™
searches. Peptides’ confidence threshold was determined
based on a FDR analysis using the target-decoy approach
and those that met the 1% FDR threshold in at least
three of the four biological replicates were retained, and
the peak areas of the target fragment ions of those
peptides were extracted across the experiments using an
extracted-ion chromatogram (XIC) window of 4 min
with 100 ppm XIC width.
The levels of the mouse proteins were estimated by
summing all the filtered transitions from all the filtered
peptides for a given protein (an adaptation of [
normalized to the internal standard (malE-GFP).
The MS proteomics data have been deposited to the
ProteomeXchange Consortium [
] via the Proteomics
Identifications (PRIDE) partner repository with the
dataset identifier PXD006007.
Functional clustering of the differentially secreted
proteins was performed using the Database for Annotation,
Visualization and Integrated Discovery (DAVID) and
displayed in Kyoto Encyclopedia of Genes and Genomes
(KEGG) and Gene Ontology (GO).
GL261 cell line was plated at an initial density of
1.0 × 105 cells per T25 flask in 3 mL of complete
DMEM. After 72 h, cells were washed and the medium
was replaced by CM from unprimed or primed glial
cells. Cell death was evaluated after 48 h of CM
exposure by Annexin V-FITC staining, according to the
manufacturer’s instructions (BD Biosciences), followed by
flow cytometry analyses. A total of at least 10,000 events
were acquired. Results were analyzed by FlowJo Software
GL261 cells (7.5 × 104) were seeded in 12-well plates
and incubated for 72 h. Monolayer cells were scraped
with a plastic pipette tip creating a gap in the monolayer
and then incubated with unprimed or primed glial cells
CM. Gap closure was evaluated every 24 h for a total of
48 h. The relative migration distance was calculated by
the following formula: percentage of gap closure (%) =
100 - (B*100)/A, where A is the width of cell gaps before
incubation (0 h), and B is the width of cell gaps after
Western blot analysis
GL261 cells were seeded in T25 flasks at a density of
1 × 105 cells per flask and allowed to grow for 72 h.
Cells were then washed and the medium replaced by
CM from glial cells (unprimed and primed). After 48 h,
cells were trypsinized, washed with PBS and lysed on ice
for 20 min in lysis buffer [50 mM Tris pH 7.5, 150 mM
NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 mM NaF,
10 mM NaPyrophosphate, 1% NP-40 and 1× Protease
inhibitors cocktail (Roche)]. Western blotting was
performed using standard 12% SDS-PAGE gel, loading
20 μg of protein per lane. Proteins were transferred onto
Hybond nitrocellulose membranes (GE Healthcare),
blocked with 5% non-fat milk in TBS + 0.1% Tween-20
(TBS-T) and incubated ON at 4 °C with primary
antibody. The following antibodies were used: Phospho-p44/
42 MAPK [Erk1/2, Thr202/Tyr204, (1:1000)],
PhosphoSAPK/JNK [Thr183/Tyr185, (1:2000)], Phospho-Akt
[Ser473, (1:1000)], p44/42 MAPK [Erk1/2, (1:1000)],
SAPK/JNK (1:1000), Akt (1:2000), all from Cell Signaling
Technology, and α-tubulin (1:1000, Santa Cruz
Biotechnology). HRP conjugated goat anti-mouse and goat
antirabbit (Santa Cruz Biotechnology) were used as
secondary antibodies. Subsequently ECL detection
(SuperSignal® West Femto, Thermo Scientific) was performed.
Band intensity was quantified using Image J software.
For glial cells protein assessment, cells were washed
with PBS immediately after CM collection and stored at
−80 °C. For protein isolation, cells were lysed on ice for
20 min in lysis buffer [50 mM Tris-HCl, pH 7.4; 1% (v/
v) Igepal; 0.25% (v/v) sodium-deoxycholate; 150 mM
NaCl; 1 mM DTT; 1 mM EDTA, Complete Mini
protease inhibitor mixture and Complete Mini phosphatase
inhibitor mixture (Roche)]. Western blotting was
performed using 12.5% SDS-PAGE gel, loading 30 μg of
protein per lane. Proteins were transferred onto low
fluorescence polyvinylidene fluoride (PVDF) membranes (TBT
RTA TRANSFER KIT, Bio-Rad). The following antibodies
were used: p16, p21 and GLB1 (all from Abcam), and
Lamin B1 (Santa Cruz Biotechnology). Alkaline
phosphatase conjugated anti-rabbit and anti-goat were used as
secondary antibodies. Protein-immunoreactive bands were
developed using the “Enhanced Chemifluorescence (ECF)
detection system” (GE Healthcare) and visualized in a
Molecular Imager FX System (Bio-Rad). For determination of
the total intensity of the sample loaded, the membrane
was further stained using the ServaPurple Total Protein
Staining kit (SERVA Electrophoresis GmbH) according
with the manufacturer’s instructions. After staining, the
membrane was dried and the signal was visualized in a
Molecular Imager FX System (Bio-Rad) using the SYPRO
Red filter. The adjusted volumes (total intensities in a
given area with local background subtraction) for each
band and the total intensity of each lane were obtained
using the Image Lab software (version 5.1, Bio-Rad).
For the MS data analysis, primed/unprimed ratios were
calculated per each replicate and Grubbs test was used to
remove outliers. One-sample Student’s t-test against a
theoretical value of one was applied to the ratios using SPSS
21.0 (IBM SPSS Statistics, IBM®). For in vitro assays, single
comparisons between the different conditions studied
were done using a paired Student’s t test. Statistical
analysis was done using Graph Pad Prism version 6. Data are
presented as mean ± SEM. The level of significance in all
the statistical analysis was set at p < 0.05.
CM from GBM cells does not affect glial cell viability or cellular composition
Increasing evidence indicates that glial cells have a role
in tumor progression [
], so we investigated whether
glial cells alter their secretion pattern in response to
GBM cells. For that, we designed an experimental
approach where primary mouse glial cell cultures were
exposed to control medium or medium conditioned by a
GBM murine cell line (GL261), and collected the
medium from these glial cells (unprimed or primed,
respectively) for subsequent proteomic and functional
analyses (Fig. 1a).
We started by characterizing the glial cell cultures
exposed or unexposed to GL261 CM, by evaluating their
cell composition and viability. No significant differences
were observed in glia metabolic cell viability (Fig. 1b), nor
in cell type composition, being both conditions composed
of more than 95% of GFAP-positive cells (Fig. 1c). To rule
out the possibility that glial cells exposed to GBM CM
could be displaying a senescence-associated secretory
phenotype (SASP), that turns senescent cells into
proinflammatory cells that have the ability to promote tumor
progression (for a review [
]), we assessed the expression
levels of some SASP markers [
]. No significant
differences were detected in p21, p16, Lamin B1 and GLB1
mRNA (Additional file 3: Figure S1A) or protein levels
(Additional file 3: Figure S1B) between glial cells
unexposed or exposed to GBM CM, suggesting that glial cells
are not undergoing senescence in response to GBM CM.
Proteomic analyses of unprimed and primed glial cells’
CM identify key secreted proteins with potential functional impact in glioma
In order to characterize the secretome of unprimed and
GBM-primed glial cells, a non-targeted systematic
proteomic-based quantitative analysis was performed.
Our data showed that GBM CM is able to modulate glial
cells’ secretome (primed) to establish a different pattern
of protein secretion when compared to the CM collected
from unprimed glial cells (Fig. 2). Additionally, the
preexposure of glial cells to GBM CM (primed condition)
led to a prominent increased secretion profile of glial
cells as compared to unprimed glial cells (651 proteins
identified in primed glial cells vs 410 in unprimed cells,
of which 385 were common to the both conditions; Fig.
2a and Additional file 4: Table S3). SWATH-MS analysis
allowed the relative quantification of 534 secreted
proteins. From these, 169 proteins were found up-regulated
in primed glial cells and only 1 down-regulated (Fig. 2b).
To gain further insights into the biological functions
of the up-regulated proteins in primed glial cells, the
DAVID online annotation term enrichment tool was
used (Fig. 2c-d). GO analysis showed glial cells exposed to
GBM cells CM have an enrichment in proteins related to
cellular homeostasis, cell adhesion, inflammatory
responses, and extracellular structure organization, among
others (Fig. 2c). Further analyses using KEGG integration
revealed these cells’ secretome has significant enrichments
for lysosome and biological processes related to
complement and coagulation cascades, extracellular matrix
(ECM)-receptor interaction and glycosaminoglycan
degradation (Fig. 2d).
Additionally, further literature mining was carried out
to identify which of the secreted proteins, with potential
paracrine effect, have been described to be associated
with, or potentially involved in, GBM pathophysiology.
Interestingly, the analysis of the relative protein levels of
the two glial cells’ secretomes (unprimed and primed)
for these specific proteins, allowed to find that proteins
previously associated with tumoral effects, such as
insulinlike growth factor-binging protein 2 (IBP-2, Fig. 3a),
metalloproteinase inhibitor 2 (TIMP-2, Fig. 3b), fibronectin
(FN, Fig. 3c), SPARC-like protein 1 (Fig. 3d) and
myeloidderived growth factor (MYDGF, Fig. 3e), among others,
were significantly up-regulated in primed glial cells
compared to unprimed. Globally, these results suggest that the
priming of glia with GBM CM shifts the secretome of glia
to favor a variety of tumorigenic processes.
Functional and molecular effects of glial cells’ secretome
in GBM cells
Our proteomics data suggests that glial cells secrete
paracrine factors that might have an impact in the
malignant phenotype of GBM cells. Since the factors
described in Fig. 3 have been implicated in biological
processes such as cell migration and invasion, cell
viability and cell death, we performed a set of functional
assays to investigate the phenotype of GL261 GBM cells
exposed to unprimed and primed glial cells secretome.
Viability assays showed that GBM cells exposed to CM
from primed glial cells present increased cell viability
than their unprimed counterparts (Fig. 4a). Accordingly,
GBM cells exposed to primed glial cells CM present
lower levels of cell death when compared to cells
exposed to unprimed glial cells CM (Fig. 4b).
A major cause of GBM patients’ recurrence and
resistance to therapy is the fact that GBM cells have a
prominent ability to migrate throughout adjacent brain tissue.
Thus, we evaluated the migration capacity of GL261
cells and the results show that they presented a
significantly decreased migration capacity when exposed to
primed glia, as compared to GBM cells exposed to
unprimed glia CM (Fig. 4c). Together, our data showed
that the priming of glial cells with GBM CM functionally
modulates their secretome towards a phenotype of
increased viability and cell-death resistance, while
decreasing the migration capacity.
To elucidate the molecular and signaling mechanisms
involved in the context-specific phenotypes observed in
GBM cells exposed to unprimed or primed glial cells
CM, we examined the activation of three major signaling
pathways typically activated in GBM, which are known
to regulate cell viability, cell death and migration
capacity of cancer cells (reviewed in [
]): ERK, JNK
and AKT. Interestingly, GL261 cells exposed to primed
glial cells CM led to increased levels of ERK, JNK and
AKT activation, as assessed by their phosphorylation
levels (Fig. 4d). This activation was consistent in all the
GL261 cells tested regardless of the batch of CM used.
Nevertheless, the ratio unprimed/primed for JNK and
AKT was different between batches leading to a
statistically significant increase only for ERK signaling pathway
(Fig. 4d, bar graphs). These molecular data at the level of
the GL261 cells fits well with the differential functional
effects observed in cell viability, death and migration.
Growing evidence of astrocytes as active participants in
neuropathological conditions, such as gliomas [
metastatic brain tumors [
], has stimulated recent
investigation into specific glial cells’ secreted proteins that
may mediate these functions (reviewed in [
]). In fact,
cells rarely work autonomously but rather act in concert
with or in response to the cellular physiology of their
neighboring cells, eliciting dynamic responses as a result
of secreted signals. In the last 10 years, the secretome of
15, 16, 36
] and human [
] astrocytes has
been mapped in different conditions. However, to the
best of our knowledge, understanding the bidirectional
communication between glial and glioma cells by
characterizing the alterations occurring in the secretome of
glial cells in the presence of glioma cells was still
missing. Additionally, understanding the phenotypic
alterations that the secretome of glial cells may have in
glioma cells and the subsequent initiation of intracellular
signaling events was also incomplete. Using primary
cultures of mouse glial cells and GL261 glioma cells, a
widely-used murine glioma model that has been shown
to recapitulate the characteristics of GBM , we found
that GBM CM led to the upregulation of several
proteins in glial cells secretome (Fig. 2). This general
upregulation of protein secretion is consistent with a state of
astrocyte reactive gliosis, a highly heterogeneous state in
which astrocytes respond to a specific injury [
fact, although there are many subtypes of reactive
astrogliosis, Zamanian and collaborators reported that
reactive gliosis consists of a rapid induction of gene
expression and identified over 1000 genes whose
expression levels were induced at least two-fold in reactive
]. Of note, glial cells secretome after
exposure to GBM cells CM was enriched in components
of the extracellular matrix, cell adhesion and proteins
involved in inflammatory response, partially mimicking
the secretion pattern of astrocytes exposed to a cytokine
cocktail for 7 days [
] or injured astrocytes [
Importantly, and validating our approach, approximately
80% of all the secreted proteins identified in our control
condition (unprimed, Additional file 4: Table S3) are
coincident with those previously identified in the CM of the
control group (astrocyte cultures) of another study [
despite the different mouse strains used (CD-1 versus
C57Bl/6 in our study). Additionally, 70% of the proteins
identified in another study using mass spectrometry-based
proteomics and computational analysis to identify
astrocytes-secreted proteins [
] were also present in our
unprimed glia secretome. Concordantly, a still remarkable
percentage (50%) of the secreted proteins identified in a
study that analyzed astrocytes CM from a very different
time-point of primary culture (8 days, versus ~20 days in
our study) can also be found in our data [
]. Of note, to
the best of our knowledge, all the studies reported so far
mapped the secretome of pure astrocyte cultures, while
we used mixed glial cultures (where the percentage of
astrocytes is ~97%), which can partially explain the
similarities, but also some of the differences found between our
control condition and the ones previously reported.
Besides the categories above mentioned, the proteomic
analysis performed in the present work also revealed
many components of the astrocyte secretome that may
not only regulate the composition of the extracellular
matrix, but also serve as paracrine signaling hubs.
Interestingly, when we tested the influence of unprimed and
primed glial cells CM in GL261 glioma cells, we found
that CM from primed cells promotes GBM cells viability
and prevents apoptosis, contrasting with CM derived
from unprimed glial cells that favors cell migration (Fig.
4). These results suggest that glial cells modulated by
GBM CM, secrete factors that regulate the “go-or-grow”
phenotypic switch of GBM cells, a phenomenon
previously described for brain tumors, where proliferation
and migration are mutually exclusive behaviors [
Interestingly, this singularity seems to be regulated by
external stimuli such as ECM components and soluble
motility factors , suggesting that GBM cells enter a
less invasive state when treated with CM from primed
glial cells, mimicking GBM disease, where tumors
exponentially grow in the beginning and then invade in a
latter phase (i.e. cells in the tumor bulk, surrounded by
activated glial cells, tend to “grow”, while cells that
escape from the tumor core have a higher invasive
phenotype, the “go”, by being in contact with non-activated
glial cells). These effects were accompanied by the
activation of ERK, AKT and JNK intracellular signaling
pathways (Fig. 4d), which is in accordance with the
functions of the proteins identified in this study and
described to have a role as paracrine agents (Fig. 3).
Indeed, IBP-2 was previously described as an inducer of
proliferation in glioma cells via integrin β1/ERK
], and MYDGF is involved in cells survival as a
paracrine-acting protein and also in inhibition of cell
apoptosis in a PI3K/AKT-dependent signaling pathway
]. Additionally, we also found FN, a component of
ECM that actively participates in cell proliferation
(reviewed in ), to be upregulated in primed glia CM.
Interestingly, FN is also described as being able to
activate ERK, p38 and AKT signaling pathways [
]. On the
other hand, the upregulation of SPARC-like protein 1,
reported to inhibit pancreatic cancer cell invasion [
and TIMP-2, a tissue inhibitor of metalloproteinases that
inhibits endothelial cell migration [
] and reduces
migration and invasion of breast cancer cells [
], fits well
with the decreased migration capacity we found in GBM
cells exposed to primed CM. Together, all these proteins
have the potential to influence cancer cells phenotype
acting in a synergistic way.
By identifying an interplay between glial cells and
GBM cells, this work also opens novel opportunities for
the clinical management of GBM patients. Although we
do not describe the precise and complete mechanisms
by which glial cells affect GBM cell viability, cell death
and migration, we characterize the secretome of glial
cells after exposure to GBM CM, and describe the
intracellular pathways activated in GBM cells during this
crosstalk. This opens a wide range of opportunities
where the secretory profile of GBM cells can be
therapeutically targeted to prevent this interplay with glial
cells that benefits tumor progression, and thus improve
the clinical management of these tumors. Further studies
are warranted to identify the best targetable candidates.
Additionally, the combination of these inhibitors
together with specific pharmacological inhibitors of the
identified intracellular signaling pathways (e.g. AKT
inhibitors, revised in [
]) could be of interest in a
precision medicine rationale.
Our findings add new insights to the body of knowledge
highlighting the relevance of the TME in gliomas and its
implication for tumor progression. We found that
proteins secreted by GBM cells are able to modulate glial
cells that respond by secreting several proteins related to
cell adhesion, extracellular matrix and inflammatory
response. Moreover, we show how this modulation acts in
a paracrine fashion to regulate GBM cells viability,
apoptosis, and migration capacity, by regulating ERK, AKT
and JNK signaling pathways (Fig. 5). In the future,
validation of our major findings with additional GBM
cell lines of both murine and human origin would be
important to clarify if this phenomenon is universal, and
how the remarkable heterogeneity typical of GBM
tumors can also be explained by different interactions with
the TME. Nevertheless, to study this pathway with
human-derived glioma cell lines, human glial cells are
required to avoid false negative/positive interactions due
to species-specific differences in protein composition/
structure. By characterizing the dynamic process of glial
cells secretion in the presence/absence of tumor cells,
and showing that these findings play an important role
in GBM progression, this study also contributes to the
rational development of novel combinatory antitumor
strategies to treat malignant gliomas.
Additional file 1: Table S1. Sequence of primers used for quantitative
RT-PCR analyses. (DOCX 19 kb)
Additional file 2: Table S2. SWATH-MS variable windows used in the
acquisition of the samples used for pull-down analysis. For each window
is indicated the m/z range, the window width in Dalton (Da) and the
CES. (DOCX 19 kb)
Additional file 3: Figure S1. Glial cells exposed to GBM CM do not
change the expression of senescence-associated secretory phenotype
markers. a. mRNA expression levels of p16, GLB1, Lamin B1 and p21
assessed by qPCR showing that there are no significant differences in the
transcriptional levels of these genes between glial cells unexposed and
exposed to GBM CM. b. Western Blot immunostaining for anti-p16,
antiGLB1, anti-Lamin B1 and anti-p21 in glial cells (left). Graph shows the
relative quantification based on the total intensity of the sample loaded
(right). No significant differences are found between unexposed and
exposed glial cells. Abbreviations: U, unexposed; E, exposed. Results
are representative of two independent experiments (data points
represent mean + SEM). Statistical differences were calculated by paired
Student’s t-test. (TIFF 2081 kb)
Additional file 4: Table S3. Proteins identified in pooled samples of
unprimed and primed CM. (DOCX 56 kb)
ACM: Astrocytes-derived conditioned media; CES: Collision energy spread;
CM: Conditioned media; CNS: Central nervous system; DAVID: Database for
Annotation, Visualization and Integrated Discovery; DMEM: Dulbecco’s
Modified Eagle Medium; ECM: Extracellular matrix; FBS: Fetal bovine serum;
FDR: False discovery rate; FN: Fibronectin; GBM: Glioblastoma; GFAP: Glial
Fibrillary Acidic Protein; GO: Gene Ontology; IBP-2: Insulin-like growth
factorbinding protein 2; ID: Internal diameter; IDA: Information-dependent
acquisition; IF: Immunofluorescence; KEGG: Kyoto Encyclopedia of Genes and
Genomes; MS: Mass spectrometry; MYDGF: Myeloid-derived growth factor;
ON: Overnight; PRIDE: Proteomics Identifications; RT: Room temperature;
SASP: Senescence-Associated Secretory Phenotype; TCA: Trichloroacetic acid;
TIMP-2: Metalloproteinase inhibitor 2; TME: Tumor microenvironment;
TMZ: Temozolomide; XIC: Extracted-ion chromatogram
(B.M.C.), Fundação Calouste Gulbenkian (B.M.C.) and Inter-University Doctoral
Programme in Ageing and Chronic Disease (PhDOC; to A.I.O.). Project
cofinanced by Programa Operacional Regional do Norte (ON.2—O Novo Norte),
Quadro de Referência Estratégico Nacional (QREN), Fundo Europeu de
Desenvolvimento Regional (FEDER), Programa Operacional Factores de
Competitividade (COMPETE), and by The National Mass Spectrometry
Network (RNEM) under the contract REDE/1506/REM/2005.
Availability of data and materials
The datasets generated during the current study are publicly available in
Proteomics Identifications (PRIDE) repository with the accession number
A.I.O. designed the study, produced and analyzed data, and wrote the
manuscript. S.I.A. carried out the proteomic analysis. J.V.C. produced and
analyzed expression data. S.C.S. and A.J.S. helped in the establishment of the
experimental set up, namely glial cell cultures and conditioned media
collection and processing. B.M. and B.M.C. designed the study, analyzed and
discussed data and helped writing the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
All experiments with mice were approved by institutional and national
ethical committees (Direção Geral de Alimentação e Veterinária, Portugal)
and are in accordance with European Union Directive 2010/63/EU.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published
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