ABSTRACT
Neuroblastoma accounts for 15% of childhood cancer mortality.
Amplification of the oncogene N-Myc is a well-established poor
prognostic marker for neuroblastoma. Whilst N-Myc amplification status
strongly correlates with higher tumour aggression and resistance to
treatment, the role of N-Myc in the aggressiveness of the disease is
poorly understood. Exosomes are released by many cell types including
cancer cells and are implicated as key mediators in cell-cell
communication via the transfer of molecular cargo. Hence,
characterising the exosomal protein components from N-Myc amplified and
non-amplified neuroblastoma cells will improve our understanding on
their role in the progression of neuroblastoma. In this study, a
comparative proteomic analysis of exosomes isolated from cells with
varying N-Myc amplification status was performed. Label-free
quantitative proteomic profiling revealed 968 proteins that are
differentially abundant in exosomes released by the neuroblastoma
cells. Gene ontology-based analysis highlighted the enrichment of
proteins involved in cell communication and signal transduction in
N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc
amplified SK-N-BE2 cell-derived exosomes increased the migratory
potential, colony forming abilities and conferred resistance to
doxorubicin induced apoptosis. Incubation of exosomes from N-Myc
knocked down SK-N-BE2 cells abolished the transfer of resistance to
doxorubicin induced apoptosis. These findings suggest that exosomes
could play a pivotal role in N-Myc-driven aggressive neuroblastoma and
transfer of chemoresistance between cells.
Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS
= foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS =
liquid chromatography–mass spectrometry; KD = knockdown; LTQ = linear
trap quadropole; TEM = transmission electron microscopy
KEYWORDS: Neuroblastoma, exosomes, N-Myc, intra-tumour heterogeneity,
chemoresistance
Introduction
Neuroblastoma is the most common solid tumour in children that arises
from the sympathetic nervous system and accounts for 15% of childhood
cancer mortality [[34]1]. Neuroblastoma is mostly present as a mass in
pelvis, neck, abdomen and chest [[35]2]. Neuroblastoma patients suffer
from loss of weight, fever, anaemia and irritability. Due to its highly
variable symptoms, the disease usually has metastasised to secondary
locations by the time of diagnosis in most cases. Majority of the
neuroblastoma deaths occur within two years of diagnosis due to the
aggressiveness of the cancer [[36]3]. About 20–30% of neuroblastoma
patients are presented with the amplification of the oncogene N-Myc and
is considered high-risk. Whilst N-Myc amplification status strongly
correlates with higher tumour aggression and resistance to treatment,
the role of N-Myc in the aggressiveness and progression of the disease
is poorly understood [[37]4]. As N-Myc is a transcription factor, it
can be speculated that the N-Myc can modulate the secretion of key
proteins that may play a pivotal role in tumorigenesis [[38]5].
Exosomes are small membranous extracellular vesicles that plays a key
role in intercellular communication [[39]6,[40]7]. Exosomes are
30–150 nm in size and are formed by the inward budding of the
multivesicular bodies [[41]8]. Exosomes are released by many cell types
and can transfer its molecular cargo to the target cells there by
modulating the signalling pathways in the recipient cells
[[42]9,[43]10]. The secretion of exosomes and its purported role in
intercellular communication is significantly different from the
classical protein secretory pathway. Most importantly, the lipid
bilayer of exosomes protect its cargo of proteins, RNA and DNA from
harsh environmental conditions which is not the case with the classical
secretory pathway [[44]11]. Though it is well established that exosomes
secreted by cancer cells contain a tumour specific signature [[45]12],
it is currently unknown whether exosomes from N-Myc amplified
neuroblastoma cells can transfer the aggressive phenotype including
chemoresistance between cells.
In this study, a comparative proteomic analysis of exosomes isolated
from cells with different N-Myc amplification status was performed.
Label-free quantitative proteomic profiling revealed 968 proteins that
are differentially abundant in exosomes released by the neuroblastoma
cells with varying N-Myc amplification status. Gene ontology-based
analysis highlighted the enrichment of proteins involved in signal
transduction, cell communication and growth in N-Myc amplified
cell-derived exosomes. Incubation of N-Myc amplified cell-derived
exosomes with non-N-Myc amplified cells induced migration, colony
forming abilities and protected the cells against doxorubicin-induced
apoptosis. These findings suggest that exosomes could play a pivotal
role in N-Myc-driven aggressive neuroblastoma by transferring the
aggressive phenotype to the neighbouring cells thereby aiding in cancer
progression.
Materials and methods
SK-N-BE2 and SH-SY5Y cell culturing
SK-N-BE2 (ATCC) and SH-SY5Y (kind donation by Dr. Julie Atkins, La
Trobe University) were cultured in DMEM (GIBCO, Life Technologies)
supplemented with 10% FCS (GIBCO, Life Technologies) and 100 Unit/mL of
penicillin-streptomycin (GIBCO, Life Technologies). Cells were
incubated at 37°C in 5% CO[2].
Isolation of exosomes by ultracentrifugation
When the cells reached 70–80% confluency, the cells were washed with
PBS and supplemented with exosomes depleted FCS containing DMEM. After
24 h of incubation, the condition media was collected and subjected to
differential centrifugation (500 g for 10 min then 2,000 g for 20 min).
The supernatants were then subjected to ultracentrifugation at
100,000 g for 1 h at 4°C to pellet the vesicles. The pellets were
washed with 1 mL PBS and subjected to ultracentrifugation at 100,000 g
for 1 h at 4°C. The obtained pellets were resuspended in PBS and stored
in −80°C.
OptiPrep™ density gradient centrifugation
To isolate exosomes, an iodixanol based OptiPrep^TM density gradient
separation method was utilized as described previously [[46]12].
Briefly, an iodixanol gradient was set by diluting 60% w/v stock of
OptiPrep^TM aqueous solution (Sigma Life Sciences®) in 0.25 M
sucrose/10 mM Tris (pH 7.5) to achieve a gradient consisting of 40%,
20%, 10% and 5% w/v solutions. Next, the gradient was layered with 3 mL
fractions from 40% followed by 20% and 10% w/v iodixanol solution.
Lastly, 2.5 mL of 5% w/v iodixanol solution was added in a 12 mL
polyallomer tube (Beckman Coulter). Next the exosomes pellets were
resuspended in OptiPrep™ solution before over laying on top of the
gradient. The tubes containing the gradients were then subjected to
100,000 g ultracentrifugation for 18 h at 4°C. Each fraction (1 mL
each) was then collected and subjected to another round of
ultracentrifugation at 100,000 g for 1 h at 4°C. Pellets were then
washed with 1 mL of PBS and resuspended in 200 µL of PBS before storing
in −80°C. As a control, OptiPrep^TM gradient was run in parallel to
determine the density of each fraction using 0.25 M sucrose/10 mM Tris,
pH 7.5.
Transmission electron microscopy
Exosomes samples (0.2 mg/mL) were examined in JEM-2010 transmission
electron microscope (JEOL, 80 kV). Preparations were fixed in 400 mesh
carbon-layered copper grids for 2 mins. Surplus samples were drained by
blotting and then the samples were negatively stained twice with 10 µL
of uranyl acetate solution (2% w/v; Electron Microscopy Services).
Western blot analysis
Equal amounts of exosomal proteins and cell lysates (30 µg) were
separated using SDS-PAGE at 150V. Next, Invitrogen XCell gel transfer
stack system (Life technologies) was employed to transfer the proteins
to nitrocellulose membrane. Membranes were blocked with skim milk
before overnight probing with primary antibodies (1:1000 dilution) at
4°C overnight. The blots were then washed three times with TTBS. For
visualization of protein bands, ODYSSEY CLx (LI-COR) was used after
probing with fluorescent conjugated secondary antibodies (1:10,000
dilution) for 1 h at room temperature.
In gel digestion
Equal amount of exosomal protein samples (30 µg) were separated using
SDS-PAGE. The separated protein bands were then stained with Coomassie
Brilliant Blue stain for visualization. Using scalpel blades, the bands
were extracted from the gel lanes and were subjected to reduction
(10 mM DTT (Bio-Rad)), alkylation (25 mM iodoacetamide (Sigma)) and
tripsinization (150 ng of trypsin (Promega)) as previously described
[[47]13]. Acetonitrile (50% (w/v)) and 0.1% (v/v) trifluoroacetic acid
were used for extracting digested peptides.
LC-MS/MS
LC-MS/MS was conducted using a LTQ Orbitrap Velos (Thermo Scientific)
coupled with a nanoelectrospray interface, the nanoLC system was
equipped with an Acclaim Pepmap nano-trap column (Dionex – C18, 100 Å,
75 μm× 2 cm) and an Acclaim Pepmap RSLC analytical column (Dionex –
C18, 100 Å, 75 μm × 15 cm). For each sample run, 1 μL of the peptide
mix was loaded onto the enrichment (trap) column at an isocratic flow
of 3 μL/min of 3% (v/v) acetonitrile containing 0.1% (v/v) formic acid
for 4 min before the enrichment column is switched in-line with the
analytical column. The eluents used for the liquid chromatography were
0.1% (v/v) formic acid (solvent A) and 100% (v/v) acetonitrile 0.1%
(v/v) formic acid (solvent B). A gradient of 3% B to 8% B for 1 min, 8%
B to 35% B for 30 min, 35% B to 85% B in 5 min and was maintained at
85% B for the final 15 min. The LTQ Orbitrap Velos mass spectrometer
was operated in the data dependent mode with nano ESI spray voltage of
+1.6 kv, capillary temperature of 250°C and S-lens RF value of 60%. An
m/z change range of 300–2000 Da was accepted in the FT mode using a
resolution of 30,000 after amassing to 1.00e^6, where all spectra were
acquired in positive mode. A maximum accumulation of 500 ms was
expected, and the 20 abundant precursor ions above two charged states
were segregated at a target value of 1000. Normalised collision energy
of 30, activation Q of 0.25 and activation time of 10 ms were all set
as standard parameters. Dynamic exclusion settings of two repeat counts
over 30 sec and exclusion duration of 70 sec were applied.
Identification of proteins
The following parameters were used when generating the peak lists using
Extract-MSn as part of Bioworks 3.3.1 (Thermo Scientific). Minimum mass
300; maximum mass 5,000; intermediate scans 200; minimum group count 1;
10 peaks minimum and total ion current of 100. Next, each peak list
obtained by LC-MS/MS runs were merged into a single mascot generic
format files to enable MASCOT searches. Due to the high-resolution
survey scan (30,000), automatic charge state recognition was used. A
target decoy fashion using MASCOT, the LC-MS/MS spectra were then
searched against the human RefSeq protein database. Search parameters
used includes, fixed modification (carboamidomethylation of cysteine;
+57 Da), variable modifications (oxidation of methionine; +16 Da),
three missed tryptic cleavages, 20 ppm peptide mass tolerance and
0.6 Da fragment ion mass tolerance. Peptide identifications were deemed
significant if the ion score was greater than the identity score.
Significant protein identifications contained at least two unique
peptide identifications with a false-discovery rate was less than 1%.
Label- free spectral counting
The relative protein abundance between exosomal samples was obtained by
estimating the ratio of normalized spectral counts (Rsc) as previously
described [[48]14]. When RSc is less than 1, the negative inverse RSc
value was used
[MATH: Rsc=[(sY+c)TX−sX+
c/sX+c(TY−sY+c)] :MATH]
s = Significant MS/MS spectra for protein A
T = Total number of significant MS/MS spectra in the exosome sample
C = Correction factor set to 1.25
X and Y = Exosome samples
Nanoparticle tracking analysis
To analyse the size distribution of exosomes, NanoSight N300 (Malvern
Instruments, Malvern, UK) was used. The samples were monitored with the
use of a 640 nm laser. The exosomal sample volume was normalized to
equal number (1 × 10^6) of SK-N-BE2 and SH-SY5Y cells. The exosomal
aggregations were separated using needle and syringe and simultaneously
injected to the NanoSight sample cubicle. The frame rate used was 30
frames per second and NanoSight NTA3.2 software was used for data
analysis.
Colony formation assay
SH-SY5Y cells (200 cells per well) were seeded in 6-well plates and
were allowed to form colonies for 14 days. The colonies were stained
using crystal violet (0.5% (w/v) crystal violet in 20% (v/v) methanol)
and the number of colonies were quantified.
Transwell migration assay
Cell culture inserts (BD, San Diego, CA) with 0.8 μm pore size were
used to perform the migration assay according to the manufacturer’s
protocol. Upper chambers were inoculated with 1 × 10^5 SH-SY5Y cells/mL
in serum-depleted culture media. FCS (10% (v/v) containing culture
media) was added to the bottom chambers and the cells were allowed to
invade for 22 h at 37°C. Non-migratory cells were gently removed before
fixing the migrated cells with 3% (v/v) paraformaldehyde. The cells
were then stained with 0.5% (w/v) crystal violet in 20% (v/v) methanol.
The stained cells were analysed by light microscopy.
Establishment of N-Myc shRNA expressing SK-N-BE2 stable cell line
The retroviral shRNA constructs for N-Myc were purchased from Origene.
SK-N-BE2 cells (5 × 10^5 cells per 2 mL of medium) were seeded prior to
the transfection and Turbofectin (Origene) was used. After 24 h of
transfection with 1 μg of shRNA plasmid, the medium was supplemented
with 2 μg/mL puromycin for selection of transfected cells. Healthy
colonies were then further transferred in to 6-well plates without
puromycin. The efficiency of the knockdown was then monitored using
Western blotting.
Wound healing assay
SH-SY5Y cells were seeded in 6-well plates and were allowed to reach
monolayer of cells of 100% confluence. The monolayer of cells was then
gently scratched with a pipette tip and the detached cells containing
media was replaced with fresh media (FCS-depleted exosomes). The cells
were then incubated in the presence and absence of exosomes varying in
concentration (5 μg/mL, 10 μg/mL and 20 μg/mL) at 37ºC in 5% CO2. The
area of wound closure was observed under the microscope at 0 h and
12 h.
FACS apoptosis assay
Equal number of SH-SY5Y cells were seeded in 24-well plates before
treating the cells with 5 μg/mL, 10 μg/mL and 20 μg/mL of exosomes
derived from SK-N-BE2, SH-SY5Y and N-Myc KD cells. For combinational
and doxorubicin only treatments, 1 µM of doxorubicin was used. Then the
cells were incubated for 48 h at 37ºC followed by staining with PI
(Sigma Aldrich) and Annexin V (Life technologies) and were subjected to
FACS apoptosis analysis.
Bioinformatics and statistical analysis
FunRich analysis tool was used to generate the Venn diagrams and
heatmaps. Statistical significance of an experiment was analysed by
student’s t-test.
Results
Isolation and characterization of exosomes from N-Myc amplified and
non-amplified neuroblastoma cells
To understand the role of exosomes in neuroblastoma progression,
SK-N-BE2 and SH-SY5Y cells were used. SK-N-BE2 cells have N-Myc
amplification and are known to be more aggressive than the non-N-Myc
amplified SH-SY5Y cells. To isolate exosomes, the conditioned media
were collected from these cells and subjected to differential
centrifugation followed by ultracentrifugation. The isolated exosomes
were separated further by iodixanol density gradient (OptiPrep™)
centrifugation. Density based separation produced 12 fractions of
increasing density, ranging from 1.04 to 1.45 g/mL. To identify exosome
enriched fractions, the samples were subjected to Western blot analysis
(fractions 4–10) and then probed with exosomal enriched proteins Alix
and TSG101 [[49]15]. As shown in [50]Figure 1(a), the exosomal markers
were enriched in fractions 7 and 8 for SK-N-BE2 cells (density of
1.13 g/mL and 1.15 g/mL respectively). Similarly, Alix and TSG101 were
enriched in fraction 7 corresponding to the density of 1.13 g/mL in
SH-SY5Y cells ([51]Figure 1(b)). In order to further confirm the
presence of SH-SY5Y and SK-N-BE2 cell-derived exosomes, the samples
were subjected to transmission electron microscopy (TEM). Morphological
analysis by TEM revealed a homogenous population of cup-shaped
membranous vesicles, with the range of 30–150 nm, confirming the
presence of exosomes ([52]Figure 1(c)).
Figure 1.
Figure 1.
[53]Open in a new tab
Characterization of exosomes isolated from N-Myc amplified and
non-amplified neuroblastoma cells.
(a) Western blot analysis of exosomal enriched proteins Alix and TSG101
in fractions obtained from OptiPrep density gradient centrifugation
(SK-N-BE2 cells). TSG101 and Alix were enriched in fractions 7 and 8
corresponding to the buoyant density of 1.13 and 1.15 g/mL. (b) Western
blot analysis representing the presence of Alix and TSG101 that are
enriched in exosomes derived from SH-SY5Y cells. Fraction 7 contained a
high abundance of Alix and TSG101. (c) TEM images of exosomes isolated
by OptiPrep density gradient centrifugation suggested the presence of
vesicles. (D) Venn diagram representing proteins present in exosomes
derived from N-Myc amplified (SK-N-BE2) and non-amplified (SH-SY5Y)
neuroblastoma cells. A total of 749 proteins are found to be common
between the exosomes isolated from the two neuroblastoma cell lines.
(e) Venn diagram depicting differentially abundant (>2-fold) proteins
in SK-N-BE2 and SH-SY5Y cell-derived exosomes. A total of 581 proteins
were enriched in exosomes derived from SK-N-BE2 cells compared to the
exosomes from SH-SY5Y cells. Similarly, a total of 385 proteins were
enriched in exosomes isolated from SH-SY5Y cells. The red arrow
represents proteins that are of high abundance in SK-N-BE2 cell-derived
exosomes compared SH-SY5Y cell-derived exosomes. Green arrow represents
proteins that are of lower abundance in SK-N-BE2 cell-derived exosomes
compared SH-SY5Y cell-derived exosomes.
Furthermore, nanoparticle tracking analysis (NTA) revealed that most of
the isolated exosomes were 50–150 nm (Supplementary Figure 1A).
Agreeing with the literature [[54]16], the more aggressive N-Myc
amplified SK-N-BE2 cells secreted significantly higher number of
exosomes compared to the less aggressive SH-SY5Y cells (Supplementary
Figure 1B). This observation was further confirmed by total exosomal
protein quantitation (Supplementary Figure 1C) and Western blotting
(Supplementary Figure 1D). Here, exosomes were normalised to equal
number of viable SK-N-BE2 and SH-SY5Y cells.
Proteomic analysis of SH-SY5Y and SK-N-BE2 cell-derived exosomes revealed the
presence of exosomal markers
To characterise the exosomes isolated from N-Myc amplified and
non-amplified neuroblastoma cells, label-free quantitative proteomics
analysis was performed. A total of 1435 proteins were identified with
two or more unique peptides with a false discovery rate (FDR) of less
than 1%. Among the identified proteins, 749 proteins were common to
both the exosomes while 441 proteins were unique in SK-N-BE2
cell-derived exosomes ([55]Figure 1(d)). When the proteomic profiles
were compared quantitatively, there were a total of 581 and 387
proteins that were differentially abundant in SK-N-BE2 and SH-SY5Y
cell-derived exosomes, respectively ([56]Figure 1(e)). Consistent with
the Western blotting, proteomic analysis confirmed the presence of
exosomal enriched proteins in both the samples ([57]Figure 2(a)). Alix
(PDCD6IP) was enriched in the exosomes isolated from both the
neuroblastoma cell lines. Whereas, TSG101, FlOT1 and VPS35 were more
enriched in exosomes derived from SK-N-BE2 cells compared to SH-SY5Y
cell-derived exosomes. Next the presence of RAB GTPases which are
enriched in exosomes were assessed in the proteomics data. RAB proteins
usually participate in membrane trafficking, biogenesis and secretion
of exosomes. As represented in [58]Figure 2(b), RAB13, RAB8A, RAB35,
RAB3C and RAB3D were highly abundant in exosomes derived from SK-N-BE2
cells. On the contrary, RAB1A, RAB5A, RAB6B, RAB5B and RAB39B were
highly abundant in SH-SY5Y cell-derived exosomes. Similarly, integrins
that are implicated in regulating metastatic organotropism were also
present in both the neuroblastoma cell-derived exosomes ([59]Figure
2(c)). The abundance of exosomal proteins and the proteomics data was
further validated by Western blotting analysis. As shown in [60]Figure
2(d), exosomal enriched proteins TSG101, Alix and CD81 were enriched in
exosomes isolated from the neuroblastoma cells. Similarly, the exosomes
contained signalling proteins such as NEDD4, β-catenin and RHOA.
Interestingly, there was a shift in the molecular weights of Alix,
TSG101 and β-catenin in exosomes compared to cell lysates. Although
this could be due to a post-translational modification such as
ubiquitination, further experiments need to be performed to confirm
this phenomenon.
Figure 2.
Figure 2.
[61]Open in a new tab
Proteomic analysis of SK-N-BE2 cell-derived exosomes are enriched with
exosomal and signalling proteins.
(a) Heatmap of exosomal enriched proteins identified in exosomes
isolated from neuroblastoma cells. Alix (PDCD6IP) was highly enriched
in both the exosomal samples. (b) RAB proteins were differentially
abundant in exosomes isolated from SK-N-BE2 and SH-SY5Y cells. RAB10,
RAB14, RAB1B, RAB5C and RAB7A were detected in both the exosomal
samples in high abundance. (c) The abundance of integrins in exosomes
isolated from SK-N-BE2 and SH-SY5Y cells. (d) Western blotting based
validation of proteins found in exosomes secreted from N-Myc amplified
and non-amplified neuroblastoma cells. WCL = whole cell lysates. (e)
FunRich based enrichment analysis of signalling pathways enriched in
proteins differentially abundant in SK-N-BE2 and SH-SY5Y cell-derived
exosomes. * denotes p < 0.05, ** denotes p < 0.01 and *** denotes
p < 0.001 as determined by hypergeometric test.
Exosomes derived from SK-N-BE2 cells are enriched in proteins implicated in
signal transduction and cell communication
In order to understand the biological process that are enriched in
exosomal proteins, the proteomic data was analysed using FunRich
[[62]17,[63]18]. Proteins that were highly abundant in SK-N-BE2
cell-derived exosomes were enriched in signal transduction, cell
communication and transport (Supplementary Figure 2A). On the contrary,
proteins highly abundant in SH-SY5Y cell-derived exosomes were enriched
in protein metabolism, cell growth and/or maintenance and regulation of
nucleic acid metabolism. Consistent with this observation, pathway
enrichment analysis highlighted the significant enrichment of proteins
implicated in EGFR, IL3, mTOR, integrin and TRAIL signalling in
SK-N-BE2 cell-derived exosomes ([64]Figure 2(e)). The cellular
component analysis also revealed the significant enrichment of plasma
membrane proteins in SK-N-BE2 cell-derived exosomes (Supplementary
Figure 2B). Taken together, these data suggest that SK-N-BE2
cell-derived exosomes are enriched with proteins that could regulate
various signalling pathways in the recipient cells.
Exosomes derived from N-Myc amplified cells transferred aggressiveness to
non-N-Myc amplified recipient cells
To understand the role of exosomes in the transfer of the aggressive
phenotype to the recipient cells, colony forming ability of SH-SY5Y
cells were investigated in the presence and absence of exosomes
isolated by differential centrifugation coupled with
ultracentrifugation ([65]Figure 3(a)). Interestingly, SK-N-BE2
cell-derived exosomes treated SH-SY5Y cells had the highest number of
colonies compared to untreated and SH-SY5Y exosomes treated SH-SY5Y
cells ([66]Figure 3(a)). Next the migratory ability of the SH-SY5Y
cells were investigated by transwell migration assay. The N-Myc
non-amplified SH-SY5Y cells when incubated with SK-N-BE2 cell-derived
exosomes, showed a significant increase in migration compared to
untreated and SH-SY5Y exosomes treated cells ([67]Figure 3(b)). In
concordance with the previous results, wound healing assay also
revealed that SK-N-BE2 cell-derived exosomes significantly (compared
to: untreated – p < 0.004; SH-SY5Y exosomes – p < 0.01) increased the
migratory capability of SH-SY5Y cells and the wound closure was the
highest among all the conditions ([68]Figure 3(c)). In order to
investigate whether the loss or downregulation of the oncogene N-Myc
could regulate the function of SK-N-BE2 cell-derived exosomes, N-Myc
knockdown (N-Myc KD) cells were generated using RNA inference in
SK-N-BE2 cells (Supplementary Figure 3A). Exosomes were isolated from
N-Myc KD cells (Supplementary Figure 3B) and wound healing assay was
performed after treatment of SH-SY5Y cells with these exosomes
([69]Figure 3(c)). Agreeing with our hypothesis, there was a decrease
in wound healing ability of the SH-SY5Y cells that were treated with
N-Myc KD cells compared to the exosomes derived from the wild type
SK-N-BE2 cells. However, the migration capability of the cells treated
with exosomes isolated from the N-Myc KD cells were slightly higher
than the SH-SY5Y exosomes treated cells. Taken together, these data
suggest that exosomes derived from N-Myc amplified neuroblastoma cells
can promote recipient cell cancer progression.
Figure 3.
Figure 3.
[70]Open in a new tab
Exosomes derived from SK-N-BE2 cells induce migration in SH-SY5Y cells.
(a) Colony forming abilities of SH-SY5Y cells treated with and without
exosomes. SH-SY5Y cells (200 cells per well) were incubated with
10 µg/mL of exosomes isolated from SK-N-BE2 and SH-SY5Y cells. The
SH-SY5Y cells in the presence of SK-N-BE2 cell-derived exosomes showed
a significant increase in colony forming ability (n = 3). (b) Transwell
migration assay performed on SH-SY5Y cells after treating with exosomes
(10 µg/mL) isolated from SK-N-BE2 and SH-SY5Y cells. There was a
significant increase in the migratory ability of SH-SY5Y cells upon
SK-N-BE2 cell-derived exosomes treatment (n = 3). (c) Wound healing
assay was performed on SH-SY5Y cells (n = 3). The monolayer of cells
closed the wound faster when the cells were treated with SK-N-BE2
cell-derived exosomes (10 µg/mL). Error bars represent the standard
error of mean, * denotes p < 0.05 and ** denotes p < 0.01 as determined
by student’s t-test.
Exosomes derived from N-Myc amplified cells transferred chemoresistance to
non-N-Myc amplified recipient cells
It is well established that N-Myc amplified cells are highly aggressive
and resistant to treatment. Hence, we next investigated the role of
N-Myc amplified cell-derived exosomes in transferring chemoresistance
to the recipient cells. SH-SY5Y cells were incubated with increasing
doses of exosomes (5, 10 and 20 μg/mL) isolated by differential
centrifugation coupled with ultracentrifugation and subjected to
doxorubicin treatment ([71]Figure 4). After 48 h, SH-SY5Y cells treated
with exosomes alone did not exhibit a significant difference in
apoptosis. As expected, doxorubicin (1 μM) only treatment induced more
than 42% of apoptosis in SH-SY5Y cells. However, combinational
treatment of SH-SY5Y cell-derived exosomes and doxorubicin did not
alter the percentage of apoptosis. Interestingly, combinational
treatment of SK-N-BE2 cell-derived exosomes and doxorubicin exhibited a
significant protection from doxorubicin induced cell death in
dose-dependant manner ([72]Figure 4). To confirm the role of N-Myc in
transferring this aggressiveness, N-Myc KD cell-derived exosomes were
co-treated with doxorubicin. Agreeing with our hypothesis, the transfer
of protection against doxorubicin to SH-SY5Y cells was significantly
reduced. However, SH-SY5Y cells incubated with N-Myc KD exosomes were
marginally protected presumably due to only partial loss of N-Myc in
N-Myc KD cells. Taken together, these results suggest that the exosomes
derived from N-Myc amplified neuroblastoma cells are enriched with
molecular cargo associated with cancer progression, migration and
resistance to drugs.
Figure 4.
Figure 4.
[73]Open in a new tab
Exosomes derived from SK-N-BE2 cells conferred chemoresistance to
SH-SY5Y cells.
FACS apoptosis assay was performed on SH-SY5Y cells after treatment
with SK-N-BE2, SH-SY5Y and N-Myc KD cell-derived exosomes (5, 10 and 20
μg/mL), doxorubicin (1 µM) and combinational treatment of exosomes and
doxorubicin. As shown in the graph, SH-SY-5Y cells obtained resistance
to doxorubicin in the presence of SK-N-BE2 cell-derived exosomes
(n = 3). Error bars represent the standard error of mean, * denotes
p < 0.05, ** as determined by student’s t-test.
Discussion
It has been established that intercellular communication plays a
pivotal role in regulating the morphology and the phenotype of the
recipient cells. Cells exchange signals through both direct
interactions and via secreted molecules and exosomes. Often, these
exosomes reflect the pathophysiological status of the donor cells.
Hence, exosomes are known to transport cargo with oncogenic potential
that can aid cancer progression. In this study, we show that the
proteomic cargo of exosomes varies based on the aggressive nature of
the host cell. Whilst, it has been shown that exosomes secreted from
breast cancer cells can mediate the transfer of chemoresistance to the
recipient sensitive cells via the transfer of miRNA [[74]19–[75]21], it
has been unclear whether neuroblastoma cells can also transfer the
aggressive phenotype between cells via exosomes.
When the proteomic data obtained from the exosomes derived from the two
neuroblastoma cell lines were subjected to gene enrichment analysis, it
was evident that the samples were enriched in exosomal and RAB
proteins. The presence of high levels of proteins implicated in cell
communication and signal transduction in SK-N-BE2 cell-derived exosomes
suggested that the exosomes play a critical role in cell-cell
commutation and transfer of cargo. Moreover, exosomes secreted by N-Myc
amplified SK-N-BE2 cells also contained proteins that are implicated in
various signalling pathways such as ErbB1, mTOR and integrin cell
surface interaction signalling pathways.
ErbB1 has been implicated in cancer cell invasion [[76]22] while
integrin family of proteins are known to promote metastasis and drug
resistance in a variety of cancer [[77]23–[78]25]. Moreover, mTOR
signalling pathway is involved in angiogenesis, cancer cell mobility
and metastasis [[79]26–[80]31]. Consistent with the enrichment of these
proteins, upon incubation of SK-N-BE2 cell-derived exosomes with
SH-SY5Y cells, the migration, colony forming ability and resistance to
chemotherapeutic drug were significantly increased. Overall, this study
confirms that the exosomes derived from N-Myc amplified and
non-amplified neuroblastoma cells contain a different protein signature
and the exosomes can alter the pathophysiological status of the
recipient cells.
It is well established that intra-tumour heterogeneity results due to
the presence of different subpopulations of cancer cells that have
unique genotypes within a single tumour [[81]32–[82]34]. In
neuroblastoma, it is possible that the clonal subpopulations with
different mutational loads exist within a single tumour mass. Exosomes
could be one way by which these different clonal subpopulations
potentially interact with each other and with the surrounding normal
cells. Hence, the possibility that exosomes could transfer the
aggressive and chemoresistant phenotype between cells need to be
studied in the context of intra-tumour heterogeneity. Hence, the
advantages N-Myc amplified neuroblastoma clonal subpopulations may
provide to the entire tumour need to be examined further to identify
new therapeutic avenues to treat neuroblastoma.
Funding Statement
SM is supported by Australian Research Council (DP130100535),
(DP170102312), Australian Research Council FT (FT180100333) and
Ramaciotti Establishment Grant. The funders had no role in study
design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Acknowledgments