Gene induction following wounding of wild-type versus macrophage-deficient Drosophila embryos.

Abstract

By using a microarray screen to compare gene responses after sterile laser wounding of wild-type and 'macrophageless' serpent mutant Drosophila embryos, we show the wound-induced programmes that are independent of a pathogenic response and distinguish which of the genes are macrophage dependent. The evolutionarily conserved nature of this response is highlighted by our finding that one such new inflammation-associated gene, growth arrest and DNA damage-inducible gene 45 (GADD45), is upregulated in both Drosophila and murine repair models. Comparison of unwounded wild-type and serpent mutant embryos also shows a portfolio of 'macrophage-specific' genes, which suggest analogous functions with vertebrate inflammatory cells. Besides identifying the various classes of wound- and macrophage-related genes, our data indicate that sterile injury per se, in the absence of pathogens, triggers induction of a 'pathogen response', which might prime the organism for what is likely to be an increased risk of infection.

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Gene induction following wounding of wild-type
versus macrophage-deficient Drosophila embryos
Brian Stramer1,5+, Mark Winfield2, Tanya Shaw1, Thomas H. Millard3, Sarah Woolner1,4& Paul Martin1,3
1Department of Physiology,2Department of Biological Sciences, and3Department of Biochemistry, School of Medical Sciences,
University of Bristol, Bristol, UK,4Department of Zoology, University of Wisconsin-Madison, Madison, Wisconsin, USA, and
5Royal Veterinary College, Veterinary Basic Sciences, London, UK
By using a microarray screen to compare gene responses after
sterile laser wounding of wild-type and ‘macrophageless’ serpent
mutant Drosophila embryos, we show the wound-induced
programmes that are independent of a pathogenic response and
distinguish which of the genes are macrophage dependent. The
evolutionarily conserved nature of this response is highlighted by
our finding that one such new inflammation-associated gene,
growth arrest and DNA damage-inducible gene 45 (GADD45), is
upregulated in both Drosophila and murine repair models.
Comparison of unwounded wild-type and serpent mutant
embryos also shows a portfolio of ‘macrophage-specific’ genes,
which suggest analogous functions with vertebrate inflammatory
cells. Besides identifying the various classes of wound- and
macrophage-related genes, our data indicate that sterile injury
per se, in the absence of pathogens, triggers induction of a
‘pathogen response’, which might prime the organism for what is
likely to be an increased risk of infection.
Keywords: repair; inflammation; hemocyte; antimicrobial; GADD45
EMBO reports (2008) 9, 465–471. doi:10.1038/embor.2008.34
INTRODUCTION
Similar to vertebrates, the survival of insects after tissue damage is
critically dependent on a rapid repair response to seal the injured
tissue layers and on an immune response that is necessary to
prevent microbial invasion and subsequent sepsis. In vertebrates, a
series of leukocytic lineages diapedese from nearby blood vessels
and migrate to sites of tissue damage where they kill microbes,
clear away cell and matrix debris, and release a plethora of signals
that act on local cells at the wound margin. In the Drosophila
embryo, haemocytes (Drosophila macrophages) show a similar
rapid response to wounding. This intimate association between
tissue damage and an immediate ‘inflammatory’ response has
made it difficult to distinguish which of the vast array of genes
upregulated at a wound site are triggered directly by wounding
and which are an indirect consequence of the wound-associated
inflammatory response.
Numerous studies have examined the genome-wide Drosophila
response to septic injury and these have been instrumental in our
understanding of the genetics of innate immunity, showing, for
example, the crucial role of Toll receptor signalling in the immune
response (De Gregorio et al, 2002). However, these studies do not
differentiate between the response to tissue injury and the response
to infection. Furthermore, without spatial resolution, array studies
cannot determine whether increased gene expression is due to
upregulation by local wound tissues, by activated haemocytes or by
other immunosensitive tissues such as the fat body.
By using mutant Drosophila embryos that lack haemocytes and
a laser wounding model that creates a sterile epithelial wound, we
have recently disassociated inflammation from tissue repair and
shown that in flies, as in mice that are genetically impaired in their
inflammatory response (Martin et al, 2003), wound healing does
not seem to be absolutely dependent on a cellular immune
response (Stramer et al, 2005).
Here, we used microarray analysis to compare the transcriptional
profiles of unwounded and wounded wild-type and haemocyte-
null embryos. Besides identifying new haemocyte and wound-
specific genes, this approach distinguishes which of the wound-
induced genes are triggered by inflammatory signals and which
are independent of haemocytes. Furthermore, this screen shows
that in the absence of pathogenic infection, mechanical wounding
is able to induce an antibacterial response that might prime
the organism to fight what is perceived to be an increased
likelihood of infection.
RESULTS AND DISCUSSION
To investigate haemocyte-specific genes and the macrophage-
dependent and macrophage-independent gene induction after
wounding of Drosophila embryos, we used a microarray approach
to compare unwounded and wounded embryos that were either
wild-type or serpent (srp) mutant and lacked the haemocyte
Received 3 July 2007; revised 7 February 2008; accepted 7 February 2008;
published online 14 March 2008
+Corresponding author. Tel: þ44 (0) 207 121 1905; Fax: þ44 (0) 117 928 8923;
E-mail: bstramer@rvc.ac.uk
1Department of Physiology,2Department of Biological Sciences, and3Department of
Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
4Department of Zoology, University of Wisconsin-Madison, 1117 West Johnson Street,
Room 120, Madison, Wisconsin 53706, USA
5Royal Veterinary College, Veterinary Basic Sciences, Royal College Street, London
NW1 0TU, UK
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lineage (Fig 1A). Homozygous mutant embryos of the strong
srp3allele die during embryogenesis as a result of a failure in
germ band retraction, whereas srpAS/srp3embryos maintain srp
expression in all the wild-type embryonic expression zones except
for the head mesoderm from which the haemocyte primordia are
derived (Rehorn et al, 1996). This results in an embryo that seems
to be morphologically normal, with the majority of srp-dependent
tissues, including the fat body, present, but which lacks
haemocytes entirely (Fig 1A).
Many wild-type enriched genes are expressed by haemocytes
We began our microarray analysis by comparing the RNA profiles
of unwounded wild-type versus srp mutant embryos (Fig 1B).
Despite the fact that haemocytes make up only a small percentage
of the total population of cells in a Drosophila embryo, our array
comparison seemed to be an efficient filter for identifying
haemocyte-specific genes. Approximately 20% of the transcripts
we found to be expressed X1.5-fold higher in wild-type versus srp
unwounded embryos have previously been shown to be enriched
in haemocytes (supplementary Table 1 online). Furthermore,
approximately 8% of the differentially expressed genes encode
proteins with a likely role in haemocyte function or immunity in
larvae and adult flies (supplementary Table 2 online). Others with
no previous link to haemocytes or infection are shown here to be
expressed by either plasmatocytes or crystal cells, two subpopulations
of haemocytes (Fig 1C).
Of those genes not previously linked to haemocytes, several
encode proteins with likely roles in the normal functioning of
these cells during embryonic development. For example, haemo-
cytes are known to be the principal synthesizers of extracellular
matrix (ECM) and to lay down embryonic basement membranes
(Olofsson & Page, 2005); therefore, it is not surprising that one of
our haemocyte-enriched genes is an orthologue of human
procollagen-lysine dioxygenase 3 (PLOD3), which is involved in
post-translational modification of collagens for normal basement
membrane maturation (Myllyla et al, 2007). For other genes
suggested to be haemocyte enriched by our microarray analysis,
mutant studies showed reduced haemocyte numbers during
embryogenesis; for example, the cell-cycle regulator string
(cdc25; Milchanowski et al, 2004). The wild-type versus srp
WT
srp
WT
Haemocyte-
enriched
genes
Haemocyte- and
systemically
induced genes
Acy PLOD 3
Fat body
Fat body
Systemically
induced genes
Fat body
srp
WT
srp
A
B
C
Fig 1|Microarray schematic to elucidate haemocyte-specific, wound-
induced and inflammation-dependent genes. (A) To illustrate the absence
of macrophages without loss of other srp-dependent tissues, we used an
srp-gal4 driver to drive GFP in wild-type (WT) and srpAS/srp3mutants.
Wild-type embryos show a labelled fat body and large numbers of
haemocytes (wild-type inset), whereas srp mutants have only a labelled
fat body. (B) RNA from unwounded wild-type and srp mutant embryos
was compared by using microarray analysis to uncover haemocyte-
enriched genes. RNA from unwounded wild-type embryos and embryos
with three wounds (indicated by asterisks) was compared to elucidate
haemocyte-induced and systemically induced wound genes, whereas RNA
from wounded and unwounded srp mutant embryos was compared to
show only inflammation-independent genes. All embryos in this figure
constitutively express GFP-moesin, which shows both the wounded
epithelium and recruited haemocytes. Note the absence of Drosophila
macrophages in srp wounds. (C) Procollagen-lysine dioxygenase 3
(PLOD3) was expressed in wild-type embryos by the fat body and
plasmatocytes, whereas in srp mutants it was expressed only by the fat
body. Aminoacylase (Acy) was expressed by the crystal cell haemocyte
subpopulation in wild-type embryos and was absent in srp mutants.
GFP, green fluorescent protein; srp, serpent.
b
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comparison also identified genes that indicate likely functional
parallels between Drosophila immune cells and vertebrate
leukocytes. Forexample,several
(supplementary Table 3 online) were shown to be enriched in
Drosophila macrophages, reflecting the high glutathione levels
of haemocytes (Tirouvanziam et al, 2004), and this parallels
the importance of glutathione for vertebrate immune function
(Droge & Breitkreutz, 2000).
glutathione-S-transferases
Identifying wound-induced genes
Next we used microarray analysis to compare the RNA profiles of
wounded versus unwounded wild-type and srp mutant embryos.
To increase the number of wound-activated cells per embryo, and
thus reduce the dilution of wound-induced genes, we generated
three large laser wounds in each embryo. Despite their large size,
each of these wounds healed as efficiently as smaller wounds
(Stramer et al, 2005). Even with three wounds, there are inevitable
effects of dilution and, as a result, this microarray comparison
yielded few significantly upregulated or downregulated genes.
However, this strategy does enrich for wound-induced genes, and
we have gone on to screen and elucidate the tissue-specific
induction of several candidates identified in this way by using
in situ hybridization. These studies showed several genes
thatwereexpressedby haemocytes
Examples of such wound-activated haemocyte genes are secreted
atthewound site.
phospholipase A2 (sPLA2; CG14507) and the antimicrobial
peptide drosomycin (Drs; Fig 2A–C). Induction of genes such as
phospholipase A2 suggests the existence of evolutionarily
conserved inflammatory signals, as the phospholipase A2 gene
family is pivotal in the production of eicosanoids during the
mammalian repair response and, in this way, regulates several
aspects of leukocyte behaviour (Ninnemann, 1988). Interestingly,
there is also evidence that eicosanoids might be involved in insect
haemocyte immune responses (Stanley-Samuelson et al, 1991).
Our screen also showed several genes induced in the wounded
epithelium or systemically in other distant tissues. A lysyl oxidase-
like gene (CG11335) was induced by epithelial cells as well as
other tissues during the repair process (Fig 2D). Lysyl oxidases are
involved in crosslinking elastin and collagen molecules and this
might be important in repairing ECM damage (Kagan & Li, 2003).
Also upregulated by the wound epidermal cells was the
Drosophila orthologue of the growth arrest and DNA damage-
inducible gene 45 (GADD45; CG11086; Fig 2E–G), which is a
known stress response gene with an ability to regulate MAP-kinase
signalling (Takekawa & Saito, 1998) and thus affect, in several
crucial ways, the repair process. It is intriguing that, in mammalian
cells, GADD45 has been shown to ‘unsilence’ genes by excisional
repair-dependent DNA hypomethylation (Barreto et al, 2007) and,
although it is generally believed that the Drosophila genome is not
regulated by methylation, it might be the case that GADD45 has
sPLA2
LoxGADD45
GADD45 enhancer trap
Wound
ventral side
No wound
dorsal side
Drs
A
DE
FG
BC
Fig 2|In situ hybridization showing the expression domains of wound-induced genes 90min after injury. Wound-activated haemocytes express
(A) phospholipase A2 (sPLA2) and (B) the antimicrobial peptide drosomycin (Drs). (C) Drs induction by haemocytes is confirmed by wounding of the
Drs-GFP reporter line. (D) Lox induction is seen in the wound epithelium and the underlying fat body. (E) In situ hybridization shows that GADD45
is induced in a broad band of epithelium spreading back from the wound edge. (F,G) A GADD45 enhancer trap line confirms GADD45 expression
extending from the wound site (F), but this inductive wave fails to spread to the unwounded side of the embryo (G). The asterisk indicates a closed
wound and the dotted lines indicate the wound site. GADD45, growth arrest and DNA damage-inducible gene 45; GFP, green fluorescent protein.
Repair genes in Drosophila embryos
B. Stramer et al
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some role in the epigenetic regulation of wound target genes in
migrating epidermal cells.
Drs, which is expressed by haemocytes at the wound site, was
also systemically induced in the yolk after wounding (Fig 3A–E).
An immune role for the embryonic yolk nuclei is consistent with
the finding that cecropin, another antibacterial gene, is similarly
expressed in this tissue during embryonic infection (Tingvall et al,
2001). We found that in older embryos (stage 17), the fat body,
which is the main immune responsive tissue in the adult fly, also
gained the ability to upregulate Drs after laser wounding
(Fig 3F,G) in a haemocyte-independent manner (Fig 3C,H).
However, diptericin, an antimicrobial peptide that is regulated
by a different immune response pathway (De Gregorio et al, 2002),
was not induced in the fat body after wounding in a diptericin-
green fluorescent protein (GFP) reporter line (data not shown).
Although we believe that our laser wounds were sterile and did
not seem to breach the vitelline membrane (Fig 3J), we cannot
completely exclude the possibility that some pathogens were
present on or within the vitelline membrane, thus inducing Drs
after wounding. However, Drs induction after wounding was, at
the very least, independent of any present Gram-positive or fungal
infection, as Toll mutant embryos also expressed Drs following
wounding (Fig 3I). We also found an increase in susceptibility to
induce Drs within the fat body of embryos with increasing wound
severity and number, but preincubation of embryos with a Gram-
positive bacteria before wounding failed to increase the Drs
response (Fig 3K), showing that the vitelline membrane was still a
competent barrier to infection after laser ablation. These data
support the idea that tissue damage per se might be a crucial
component in triggering a pathogen response. Braun et al (1998)
ABC
D
EFG
I
H
J
K
Unwounded
Unwounded Wound
Toll wound
Unwounded
Two large
wounds
One large
wound
One small
wound
One small
wound
+ S. aureus
(56/67) (49/74)(21/49)(41/117)
80
70
60
50
40
30
20
10
WT wound
WT wound
srp wound
srp wound
Percentage of embryos
positive for Drs
Fig 3|Systemic induction of drosomycin after sterile wounding. In situ hybridization of (A) unwounded, (B) wounded wild-type (WT) and (C) srp
mutant embryos shows that Drs is upregulated in the yolk after injury independent of haemocytes. (D,E) High-magnification view to compare Drs
expression in the yolk of an unwounded and wounded embryo. (F–I) A Drs-GFP reporter line (colour inverted) shows that from stage 17, the fat
body also upregulates Drs after laser wounding of (G) wild-type, (H) srp and (I) Toll mutants. (J) A methylene blue-stained resin section through a
90min wound with recruited haemocytes indicated by arrows (top panel). The inset corresponds to an electron microscopic view (lower panel)
illustrating how the vitelline membrane (indicated by the asterisk) remains intact beyond the epithelial wound edge (arrowhead). Scale bar, 5mm.
(K) Wound severity correlates with fat body expression of Drs. Two large wounds induced the Drs reporter in 84% of embryos, one large wound
induced 66%, one small wound 43% and one small woundþStaphylococcus aureus 35%. GFP-moesin embryos were wounded to highlight wound sizes.
Drs, drosomycin; GFP, green fluorescent protein; srp, serpent.
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showed that although infection without injury was able to induce
an immune response, the level of induction was significantly
greater when infection was accompanied by injury. A more recent
study has shown that survival rates are enhanced by previous
wounding, indicating that tissue damage might prime the host’s
defence to future infections (Apidianakis et al, 2005).
‘Inflammation’-dependent and -independent wound genes
Microarray comparison of wounded srp and wild-type embryos
also allowed us to screen which of the wound-induced genes was
dependent on an inflammatory response. For example, our array
analysis suggested that Lox was equivalently upregulated after
wounding of both wild-type and srp embryos, and our in situ
hybridization data confirmed similar expression levels in both
genotypes after wounding (Fig 4A). However, it seemed that
Drosophila GADD45 was much more robustly induced by
wounding of wild-type embryos, and in situ hybridization showed
that this gene was strongly inflammation dependent (Fig 4B). Our
data suggest that Drosophila macrophages might secrete signals
that are necessary for full GADD45 induction in the wounded
epithelium. There is precedent for a paracrine signalling role for
haemocytes during an immune response; after septic injury, an
unpaired (Upd)-like cytokine is secreted by haemocytes and is
necessary for Jak/Stat signalling in the fat body (Agaisse et al,
2003). Although GADD45 is not a known Jak/Stat target, it is
responsive to Toll signalling (De Gregorio et al, 2002). However,
Toll mutants showed a similar epithelial wound induction of
GADD45 (Fig 4C,D), and expression of an activated form of the
Toll ligand, spatzle (spz), in the epithelium of Drosophila embryos
failed to induce GADD45 expression (Fig 4E,F), suggesting that
GADD45 induction following wounding is Toll independent.
Our data suggest that GADD45 is an ‘inflammation-associated’
wound response gene in insects. To determine whether this
response is conserved in mammals, we re-analysed microarray
data from an analogous experiment in mice comparing the gene
profiles of wounds in the presence and absence of an inflammatory
response (Cooper et al, 2005). These data showed that a murine
homologueof DrosophilaGADD45
102292_at) was upregulated rapidly after wounding and that
this response was much reduced in PU.1 null mice in which
inflammatory cells were missing (Fig 4G). The expression of
GADD45 protein after injury was examined by western blotting
and showed rapid and transient induction by 1 day after wounding
(Fig 4H). Furthermore, immunostaining showed that, as in
Drosophila, murine GADD45 was induced in the wound
epithelium (Fig 4I). This finding provided further evidence for an
evolutionarily conserved repair response in flies and vertebrates,
and highlights how useful Drosophila might be in elucidating new
mechanisms regulating various aspects of vertebrate tissue repair.
(AffymetrixMGU74A,
METHODS
Fly stocks. Mutant embryos for microarray analysis and for
in situ hybridization were generated by using a heteroallelic
combination of alleles srp3and srpAS(Rehorn et al, 1996); wild-type
embryos were rucuca strain (stock no. 576, Bloomington Stock
Center). To visualize Drs (Drs-GFP) and diptericin expression, GFP
reporter lines were wounded (Ferrandon et al, 1998; Tzou et al,
2000). To visualize epithelial wounds and haemocyte recruitment,
a fly line expressing moesin fused to GFP was used (Kiehart et al,
2000). To examine Drs and GADD45 expression in srp and Toll
mutants, lines containing Drs-GFP; srpAS/TTG (GFP balancer), Tlr4/
TTG and Drs-GFP; Tlr4/TTG were created and mutant embryos
were selected by their absence of fluorescence. For the GADD45
enhancer trap (stock no. 103594, Kyoto Stock Center, Kyoto,
Japan), UAS-red stinger (stock no. 8546, Bloomington Stock
Center, Bloomington, IN, USA) was recombined with this line
before wounding. To express the spz ligand in the epithelium, an
active form of spz (Ligoxygakis et al, 2002) was driven in the
epithelium with the Engal4 driver.
Laser wounding and microarray analysis. Embryos were col-
lected at stage 15 and wounded by using laser ablation (Wood
et al, 2002). Embryos were collected after 90min for in situ
hybridization and microarray analysis, and total RNA was
extracted from collections of 400 embryos for each time point
by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and the
RNeasy Cleanup Kit (Qiagen, Crawley, UK). Complementary
DNA was labelled by using amino-allyl reverse transcription
labelling. The samples were hybridized to an in-house cDNA
array (Fred Hutchinson Cancer Center, Seattle, WA, USA)
containing 12,144 probes derived from the following 3 collec-
tions: Dgcr1, Dgcr2 and the Northwest Drosophila Microarray
Consortium.
For the unwounded wild type versus srp comparison, the
experiment was conducted twice, yielding two independent
replicate measurements. In addition, wounded versus unwounded
wild-type embryosandwounded
comparisons were made, and used to screen for inflammation-
dependent and inflammation-independent transcripts. Analysis
was performed by using the GeneSpring GX programme. Briefly,
per spot and per chip intensity-dependent (Lowess) normalization
was performed. Transcripts with unreliable scores were removed
from the analysis on the basis of the cross-gene error model by
using replicate scores. Genes were screened on the basis of t-test
P-values and fold change. All raw microarray data are available in
an MIAME-compliant format under accession number GSE10225
of the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/).
To examine Drs and diptericin expression by using GFP
reporter lines, stage 17 embryos were wounded and examined 4h
later for fat body expression. To examine GADD45 induction with
the enhancer trap line, stage 15 embryos were wounded and
imaged after 12h. For the Staphylococcus aureus experiment,
embryos were preincubated in 1?107CFU of S. aureus (reference
strain ATCC 25923) before wounding.
In situ hybridization and electron microscopy. Immunohisto-
chemical whole-mount in situ hybridization was carried out using
standard methods (Lehmann & Tautz, 1994) with digoxigenin-
substituted RNA probes generated by transcription of expressed-
sequence tag clones from the Berkeley Drosophila Genome Project:
LP04931 (Lox),RH27007(CG14507),
LP03851 (Drs), LD37702 (PLOD3), RE63537 (aminoacylase).
Embryos for transmission electron microscopy were frozen under
high pressure, and then fixed and stained during the freeze
substitution step with osmium tetroxide and uranyl acetate before
embedding in resin. Thick sections were stained with methylene blue.
Murine histology, western blotting and immunostaining. Male,
8-week-old ICR mice were used according to UK Home Office
regulations. Mice were anaesthetized by halothane inhalation and
excisional wounds were made on the shaved back on either side
versusunwounded srp
RE38345 (GADD45),
Repair genes in Drosophila embryos
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No wound
No wound
Toll +/–
Toll –/–
Lox
WT wound
GADD45
WT wound
Spz
250
WT
PU.1
GADD45α
GAPDH
WoundNormal
200
150
100
0
Un
1 day
3 days
5 days
7 days
10 days
14 days
0.53
Hours after wounding
12 24
GADD45 mRNA levels
50
0
Wound
srp wound
srp wound
A
B
CDEF
G
I
H
Fig 4|Inflammation-dependent and inflammation-independent induced genes. (A) In situ hybridization studies show that Lox is induced by wounding,
independent of a haemocyte response. (B) By contrast, GADD45 is robustly induced at wounds only in wild-type (WT) but not in srp mutant embryos.
(C) Toll heterozygotes and (D) Toll mutants were wounded to show similar levels of GADD45 induction. (E) Ectopic expression of an activated form
of spz in the epithelium failed to induce GADD45 expression, (F) unless wounded (arrows). (G) Temporal GADD45a messenger RNA expression
following wounding of wild-type (solid line) and PU.1 mutant (dashed line) murine skin. (H) Western blot analysis shows transient GADD45a protein
levels following excisional wounding of murine skin. (I) Immunostaining of GADD45a in 3-day murine wounds shows induction at the epithelial
wound margin (arrowhead). The insets show a high-magnification view of increased GADD45a expression in wounded compared with normal
epithelium. GADD45, growth arrest and DNA damage-inducible gene 45; spz, spatzle; srp, serpent; Un, unwounded.
Repair genes in Drosophila embryos
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of the dorsal midline with a 4-mm biopsy punch. Tissue was fixed
in 10% formalin for embedding in paraffin. Wax sections (6mm)
were immunostained by using a GADD45a antibody, as described
previously (Yamasawa et al, 2002). Protein samples were
separated on Tris-glycine gels (Invitrogen) and blotted according
to standard protocols by using GADD45a (Santa Cruz, Santa Cruz,
CA, USA) and GAPDH (Abcam, Cambridge, UK) antibodies.
Supplementary information is available at EMBO reports online
(http://www.emboreports.org).
ACKNOWLEDGEMENTS
We thank P. Verkade, D. Carter and G. Tilly for help with transmission
electron microscopy, B. Lemaitre for fly stocks, A. Hawrani for the
bacteria and W. Wood for helpful discussions. We thank The Royal
Society, The Medical Research Council, The Wellcome Trust and The
Fred Hutchinson Cancer Center for funding this study.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
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