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REVIEW
Inflammation and tumor microenvironments: defining
the migratory itinerary of mesenchymal stem cells
E Spaeth, A Klopp, J Dembinski, M Andreeff and F Marini
Molecular Hematology and Therapy, Department of Stem Cell Transplantation and Cellular Therapy, UT-M.D. Anderson Cancer Center,
Houston, TX, USA
Mesenchymal stem cells (MSC) exhibit tropism for sites of
tissue damage as well as the tumor microenvironment. Many
of the same inflammatory mediators that are secreted by
wounds are found in the tumor microenvironment and are
thought to be involved in attracting MSC to these sites. Cell
migration is dependent on a multitude of signals ranging from
growth factors to chemokines secreted by injured cells and/or
respondent immune cells. MSC are likely to have chemo-
tactic properties similar to other immune cells that respond to
injury and sites of inflammation. Thus, the well-described
model of leukocyte migration can serve as a reasonable
example to facilitate the identification of factors involved in
MSC migration.Understanding the factors involved in reg-
ulating MSC migration to tumors is essential to ultimately
develop novel clinical strategies aimed at using MSC as
vehicles to deliver antitumor proteins or suppress MSC
migration to reduce tumor growth. For example, radiation
enhances inflammatory signaling in the tumor microenviron-
ment and may be used to potentiate site-specific MSC
migration. Alternatively, restricting the migration of the MSC
to the tumor microenvironment may prevent competent
tumor-stroma formation, thereby hindering the growth of
the tumor. In this review, we will discuss the role of
inflammatory signaling in attracting MSC to tumors.
Gene Therapy (2008) 15, 730–738; doi:10.1038/gt.2008.39;
published online 10 April 2008
Keywords: mesenchymal stem cell; migration; tumor microenvironment; tumor stroma; inflammatory chemoattractants
Brief description of the mesenchymal
stem cell
Mesenchymal stem cells (MSC) are non-hematopoietic
adult stem cells with multilineage potential. MSC are
defined by plastic adherence, differentiation potential
and cell surface marker expression.
1
MSC, or MSC-like
cells, have been isolated from nearly every organ or
tissue in the body, making it challenging to characterize
the MSC as a completely homogenous population. MSC
contribute to the maintenance and regeneration of
connective tissues and have the capacity to differentiate
within osteoblasts, adipocytes, chondrocytes, myocytes
and cardiomyocytes. MSC express markers including
CD29, CD44, CD51, CD73 (SH3/4), CD105 (SH2), CD166
(ALCAM) and Stro-1, but the expression of specific
combinations of markers appear microenvironment-
dependent, suggesting a strong influence of tissue
context on MSC phenotypes. In general, MSC appear to
be a non-immunogenic population of cells; however, a
few studies demonstrate immune-repressive functions of
MSC through the induction of peripheral tolerance
evident in autoimmune disorders such as multiple
sclerosis.
2
The use of bone marrow-derived MSC have been
employed in support and engraftment of the transplan-
tation of hematopoietic stem cells (HSCs) following
high-dose chemotherapy in an effort to replenish the
destroyed bone marrow cell population.
3
Additionally,
pre-clinical studies have explored the use of MSC in the
reduction of graft-versus-host disease, for tissue repair;
including cerebral injury,
4
bone fracture,
5
myocardial
ischemia/infarction,
6
muscular dystrophy,
7
as well as
tumor homing.
Migration of MSC to tumors is thought to be due to
inflammatory signaling in a tumor resembling that of an
unresolved wound.
8
The innate tropism of MSC for
tumors can be exploited for the delivery of antitumor
agents to the tumor microenvironment. Gene-modified
MSC expressing interferon-bhave been used to signifi-
cantly reduce tumor burden and in some cases extend
survival in murine models of melanoma,
9
lung,
10
breast
cancer
11
and glioma.
12
However, the mechanism and
factors responsible for the targeted tropism of MSC to
these wounded microenvironments remain to be fully
elucidated. MSC are likely to have chemotactic proper-
ties similar to other immune cells that respond to injury
and sites of inflammation. Thus, the well-described
model of leukocyte migration can serve as a reasonable
example to facilitate the identification of factors involved
in MSC migration. Following a discussion of alterations
in the ‘wounded’/tumor microenvironment that are
shown to enhance MSC tumor-specific migration, we
will introduce alternative, injury-induced cell migration
models based on literature reviewing leukocytes and
Received 14 February 2008; accepted 18 February 2008; published
online 10 April 2008
Correspondence: Dr F Marini, Molecular Hematology and Therapy,
Department of Stem Cell Transplantation, UT-M.D. Anderson
Cancer Center, Box081, 1515 Holcombe Boulevard, Houston,
TX 77030, USA.
E-mail: Fmarini@mdanderson.org
Gene Therapy (2008) 15, 730–738
&
2008 Nature Publishing Group All rights reserved 0969-7128/08
$
30.00
www.nature.com/gt
their progenitor cell line, the HSC. These alternative
migration systems will provide rational for the tumor-
specific MSC migration and will lead us into an overview
of the current literature concerning MSC migration
specifically. Ultimately, we will conclude with a discus-
sion of the potential clinical applications of tumor-
directed MSC migration.
Inflammation-targeted homing in the
tumor microenvironment
Inflammation is a cellular response that takes place
under conditions of cellular injury and in sites of tissue
wounding. Over two decades ago, Dvorak and co-
workers described the tumor as an unhealed wound
that produces a continuous source of inflammatory
mediators (cytokines, chemokines and other potential
chemoattractant molecules). Cancer progression has
been correlated with an increase in inflammatory
mediator gene expression, and this is thought to occur
via disruption, damage and cellular turnover occurring
in the tumor microenvironment. This constant produc-
tion of inflammatory mediators perpetuates the main-
tenance and progression of the tumor environment and
becomes a target for the MSC. Tumor-generated inflam-
matory mediators have a role in determining the
conditions of the tumor microenvironment, as they
regulate invasion, motility, extracellular matrix interac-
tion through autocrine effects as well as coordinating cell
movement through paracrine signaling.
13
Tumor-pro-
duced and tumor-induced inflammatory chemokines are
known to have an important role in leukocyte/macro-
phage infiltration into tumors.
14
Based on this evidence,
one can speculate that inflammation-induced chemo-
kines participate in the directed migration of stem cells,
such as MSC, to tumors and inflamed microenviron-
ments. Previous studies have shown the importance of
inflammation to the successful homing of systematically
infused stem cells (HSC) to cardiac tissue,
15
thus reinfor-
cing the notion of inflammation and chemokine production
in migration of MSC. In addition to the secreted
chemotactic molecules secreted by the tumor and its
surrounding stroma, the tumor cells themselves retain a
chemotactic disparity amongst its cellular components.
When fractionated, the tumor cell membrane possesses a
superior chemotaxis-induction potential compared with
the other cellular components such as cytosolic fractions,
including organelles such as the nuclei, mitochondria,
lysosomes, microsomes and ribosomes (Figure 1).
Hypoxia contribution to MSC homing
Many tumors exhibit hypoxia, a state of reduced oxygen
that often parallels and perpetuates inflammation. A
common feature between inflammation and hypoxic
environments is the expression of pro-angiogenic mole-
cules. The hypoxia-induced transcription factor HIF-1a
activates the transcription of genes including vascular
endothelial growth factor (VEGF), macrophage migra-
tion inhibitory factor, tumor necrosis factors (TNF-a),
numerous proinflammatory cytokines and the activation
of the transcription factor nuclear factor kB.
16,17
Nuclear
Matrigel Migration Assay
0
50
100
150
200
250
300
350
1
# of GFP Positive Cells
24 Hour
48 Hour
72 Hour
% of Migrated MSC
100
50
C
y
tokines Tumor cells Mixtures
VEGF
PDGF
bFGF
IGF
TGFB
FBS
Live 293s
Live MDA231
Live A375sm
bFGF
Live HEY
Live SKOV3
Cell Membrane - 293s
Cell Membrane - MDA23
Cell Membrane - A375sm
Cell Membrane - OVAR3
Cell Membrane - HEY
Cell Membrane - SKOV3
bFGF & IGF
bFGF & Serum
VEGF & PDGF
PDGF & TGFB
VEGF & TGFB
Cell membrane
Figure 1 Cellular membrane components and mixtures of cytokines are potent mesenchymal stem cell (MSC) attractants. In an in vitro
matrigel assay, the migratory capacity of human MSC was analyzed. MSC preferentially migrated toward selected chemoattractive regions of
the matrigel matrix. The more effective migratory inducers include the growth factors, platelet-derived growth factor and FBS when alone. In
combination, growth factors were more potent than when used as a single chemoattractant. The attractiveness of the tumor cell membrane is
evident in comparison to other chemoattractants tested. The most potent cell membrane fraction was from the SKOV3 tumor cell lines,
followed by the MDA-231 tumor cell lines.
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factor kB is frequently activated in response to inflam-
matory mediators
18
and has been shown to induce
several chemokines (RANTES (CCL5), MIP-2 (CXCL2),
MIP-1a(CCL3), monocyte chemoattractant protein 1
(MCP-1) (CCL2), interleukin-8 (CXCL8)) that are im-
plicated in leukocyte migration.
19–22
Hypoxia in the
tumor microenvironment is a cyclical event, and perpe-
tuates the inflammatory response by ensuring a constant
production of angiogenic and inflammatory mediators.
Briefly, hypoxic conditions result in the generation of
reactive oxygen species, which can increase DNA
damage in neighboring cells. Tumor cells with more
virulent mutations can then proliferate and invade
neighboring cells/tissues resulting in tissue damage,
which increases the demand for nutrients and oxygen,
which continues to be short in supply.
23
The reactive
oxygen species induced in the microenvironment has
also been demonstrated to increase secretion of inflam-
matory cytokines via nitric oxide (NO); NO has been
linked to the regulation/induction of MIP-1a, MCP-1,
macrophage inflammatory protein-related protein-1 and
osteopontin.
24,25
Hypoxia has an important role in
perpetuating the inflammatory process in tumors which
results in the generation of chemokines that are involved
in immune cell and likely MSC migration to tumors.
Archetype for MSC trafficking
The mechanism behind MSC migration is still in its
infancy. However, factors involved in regulating migra-
tion of leukocytes have been studied extensively, and it is
likely that many of the same factors are involved in
regulating MSC migration. A list of receptors expressed
on MSC that have been previously implicated in cell
migration is shown in Table 1. Growth factor, cytokine/
Table 1 Cell surface markers and receptors associated with cell migration that are known to be expressed on MSC
Cell surface receptors
found on MSC
Ligands Present on other cell types
Growth hormone
receptors
EGFR (ErbB) EGF DC, neutrophil
HGFR (c-met) HGF Leukocytes, macrophages
IGF1R IGF1 Leukocytes, HSC
PDGFR (Ra-b) PDGFa/b HSC
VEGFR1 VEGF HSC, monocytes, neutrophils
VEGFR2 VEGF HSC
FGFR2 FGF2 HSC, leukocytes
Tie-2 Ang-1 HSC, leukocytes
Chemokine/cytokine
receptors
CCR1 CCL3, CCL5, CCL7, CCL13, CCL14, CCL15,
CCL16, CCL23
monocyte, T cell, DC
CCR2 CCL2, CCL7, CCL8, CCL13, CCL16 Monocyte, T cell, DC
CCR3 CCL5, CCL7, CCL8, CCL11, CCL13, CCL15,
CCL16, CCL24, CCL26, CCL28
T cell, DC
CCR4 CCL17, CCL22 T cell, macrophage, DC
CCR5 CCL3, CCL4, CCL5, CCL8, CCL11, CCL14,
CCL16
Monocyte, T cell, DC, HSC
CCR6 CCL20 T cell, B cell, DC
CCR7 CCL19, CCL21 T cell, DC
CCR8 CCL1 monocyte, T cell, DC
CCR9 CCL25 T cell
CCR10 CCL27, CCL28 T cell
CXCR1 CXCL6, CXCL7, CXCL8 Neutrophil, monocyte
CXCR2 CXCL1, CXCL2, CXCL3, CXCL5, CXCL6,
CXCL7, CXCL8
Neutrophil, monocyte
CXCR3-A/B CXCL4, CXCL9, CXCL10, CXCL11 T cell, microvascular cells
CXCR4 CXCL12 T cell, B cell, monocyte, macrophage, DC
CXCR5 CXCL13 B cell, Th cells, HSC
CXCR6 CXCL16 CD8 T cells, NK cells, CD4 T cells
CX3CR1 CX3CL1 Macrophage
XCR1 XCL1, XCL2 T cell, NK cell
Adhesion molecules VCAM-1 (VLA-4) b1 integrin/a4 integrin Leukocytes
ICAM-1/3 LFA-1 Leukocytes, DC
ALCAM CD6 Leukocytes
Endoglin (CD105) TGFb1/3 Leukocytes, HSC
TLR1 Lipopeptides Leukocytes
Innate immune TLR2 Peptidoglycans, lipopeptides Monocytes, DC
surveillance TLR3 dsRNA DC
TLR4 LPS Monocytes, DC
TLR5 ECM molecules Monocytes
TLR6 Peptidoglycans Epithelium
The known ligands for each respective receptor are listed in the third column. The fourth column lists various cell types expressing the same
receptor.
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chemokine, adhesion molecules and toll-like receptors
(TLRs) are expressed on MSC, as described by Cham-
berlain et al.,
26
Ringe et al.,
27
Allen et al.
28
and Viola et al.
29
Although the table does not encompass all receptors, it is
a comprehensive list of those receptors that have been
studied on other cell types, including leukocytes and
HSC. The T cell, macrophage and dendritic cells are all
considered relevant to the understanding of MSC
homing because of their functional similarities in
targeting inflamed/injured tissues. The HSC is consid-
ered because it is the precursor to the myeloid and
lymphoid lineages in addition to being resident of the
bone marrow where we also find the MSC.
Leukocytes
There are three main participants in leukocyte traffick-
ing: adhesion molecules, integrins and chemoattrac-
tants.
30
The latter is the most important when
considering the paracrine-mediated gradient trafficking
and migration without cellular contact, such as what has
been observed for MSC migration to sites of tumors and
sites of injury.
Cytokine and chemokine receptors have an important
role in leukocyte and likely MSC migration. A decrease
in leukocyte migration has been observed in a mouse
knockout model of the IL-8 receptors, CXCR1 and
CXCR2.
31
Similarly, neutrophil recruitment in ischemia-
reperfusion models was inhibited by blocking the
CXCR1 and CXCR2 receptors.
32
The presence of both
CXCR1 and CXCR2
27
on MSC suggests that they may
have a similar function in MSC migration. Nearly every
chemokine receptor has been found on the surface of
MSC,
3
while CCR2 and CCR3 are two receptors that may
have a particularly important role in leukocyte and MSC
trafficking. Macrophage trafficking has been shown to be
mediated by CCR2, a chemokine receptor with ligands
including MCP1, 2, 3 and 4 (MCP-1, 2, 3, 4, or CCL2, 8, 7,
13, respectively).
30
Of note, blocking CCR3, the receptor
for eotaxtin (CCL11), RANTES (CCL5), MCP2,
MCP3 and MCP4 (CCL 8, 7, 13) has been effective in
reducing trafficking in leukocytes. Yet another seven
transmembrane receptor, CD97, an epidermal growth
factor receptor appears necessary for the migration of
neutrophils to sites of inflammation
33
and has been
implicated in the promotion of angiogenesis. Increased
expression of CD97 in tumor cell lines, such as colorectal
carcinoma, correlates with the increased migration
potential of leukocytes.
34
Of note, MSC express CD55,
the ligand to CD97, suggesting a potential CD55–CD97
interaction thereby influencing both MSC and leukocyte
migration.
Other than cytokines/chemokines, numerous bioac-
tive molecules can also serve as leukocyte chemoattrac-
tants. Such molecules include lipids, (leukotrienes and
prostaglandins), peptides such as chemerin and other
elements of the extracellular matrix.
35
The adhesion
molecule, CD44, which is widely expressed on leuko-
cytes and parenchymal cells interacts with components
of the extracellular matrix known to be involved in
inflammation including hyaluronic acid, collagen, lami-
nin and fibronectin. The presence of CD44 is thought to
mediate and enhance localized inflammation leading to
the increased migration of leukocytes, it also serves as a
receptor for growth factors, integrins, cytokines (osteo-
pontin), glycosaminoglycans (hyaluronic acid), peptides
(collagen), and is able to both induce inflammation and
serve as a receptor mediator in the homing toward sites
of inflammation.
36,37
Hyaluronan, which is a by-product
of tissue repair, results in persistent inflammation
38
and
in addition to being critical to cell motility during
development, the inhibition of hyaluronan synthesis in
prostate tumors impairs growth and vascularization.
39
Of importance, CD44, the receptor for hyaluronan, is
expressed on MSC, and a recent publication suggests
CD44 roles in the migration of MSC toward injured
kidney tissue.
40
In addition to CD44, hyaluronan by itself
can interact with TLR2 and TLR4, thereby enhancing the
inflammatory response.
38
The participation and involve-
ment of TLRs in MSC migration will be addressed to a
further extent in a subsequent section.
HSC migration
Similar to the leukocytes, the migratory machinery of
HSC are also well characterized in regards to defined
receptors/ligands required for migration and should be
considered in the evaluation of potential candidates for
the elucidation of MSC migration. In fact, one of the most
widely recognized receptor/ligand pairs for HSC traf-
ficking is CXCR4/CXCL12 (SDF1); in addition, CXCR4/
SDF1 appears important in dictating migration of several
tumor cell lines to metastatic sites.
41,42
However, in
contrast to the key role of CXCR4/SDF1 in HSC
migration, a recent publication by Ip and co-workers
showed blocking of the CXCR4 receptor had no impact
on MSC migration, suggesting key differences
between the migration signals of these two stem cells.
43
This controversial receptor indicates that receptor
function differs between cell types and enhances the
importance of examining receptors found mutually
expressed on MSC- and HSC-like CXCR4, CCR5
44
and VEGFR.
45
MSC trafficking—finding the tumor
The past year has revealed a surge of publications
attempting to define the homing properties of MSC. Due
to the more comprehensive command of knowledge in
the field of leukocyte migration, those chemokines and
corresponding receptors chosen for evaluation on MSC is
based on prior data illustrated in leukocyte models.
However, as noted above, the migratory competence
of these receptors clearly varies: CXCR4/CXCL12 are
crucial in bone marrow retention and homing of HSC
where as in MSC, it appears that CXCR4/CXCL12 do not
posses the same migration importance unless enforced
expression of CXCR4 is employed.
43,46,47
Bearing in mind
that each of the following receptors and their respective
ligands (chemokines, cytokines, growth factors, peptides
small biomolecules) have not been implicated as a single
primary mechanism for the migration of MSC; however,
altogether may function in an additive manner. Thus, the
coalescence of known migratory mechanisms is vital to
the understanding of the complete migratory disposition
of the MSC in relationship to cancer biology.
Preconditioning the MSC
Activation of MSC with proinflammatory cytokines (that
is, TNF-a) prior to reinfusion has been demonstrated to
increase MSC in vivo migratory and adhesion capacity
Inflammation-driven mesenchymal stem cell migration
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through the increased expression of receptors. Existing
data suggest that the cytokines IL-1band TNF-aactivate
adherence properties of the MSC including the upregula-
tion of the VCAM-1-VLA-4 adhesion pathway.
48
Addi-
tional receptors known to be upregulated by TNF-a
priming include CCR3 and CCR4; these findings correlate
with the observation of increased in vitro migration to
RANTES (a CCR3 ligand) and macrophage-derived
cytokine (MDC (CCL22)—a CCR4 ligand).
3
Several growth factors have been shown to induce
MSC migration. Insulin-like growth factor 1 (IGF-1)
increases the expression of chemokine receptors on the
MSC, thereby enhancing migration. Li and co-workers
demonstrated that IGF-1-induced upregulation of
CXCR4 expression on MSC increased the migratory
capacity of the cells toward an in vitro SDF-1 gradient
through a phosphoinositide-3 kinase-dependent path-
way without altering the proliferation status of the
MSC.
49
The inability for MSC migration to occur in vivo
through a CXCL12/CXCR4 mechanism was mentioned
previously; accordingly, IGF-1 is capable of stimulating
the expression of other chemokine receptors such as
CCR5—the RANTES (CCL5) receptor.
50
Other growth
factors including basic fibroblast growth factor and
VEGF are associated with angiogenesis and are secreted
under hypoxemic stress. MSC demonstrate an increased
migratory propensity in the presence of basic fibroblast
growth factor through a phosphoinositide-3 kinase/AKT
pathway downstream of the basic fibroblast growth
factor receptor on the MSC.
51
Chemokine/cytokine secretion and the respective
receptors found on MSC
Monocyte-chemoattractant protein 1 (CCL2), a chemo-
kine secreted by tumor cells, was shown to be a potent
chemoattractant for MSC migration toward breast
carcinomas.
52
However, prior studies show conflicting
data on the migratory response to CCL2. The incon-
sistent results may be attributed to differences between
primary cells and passaged cell lines.
27,53
Additionally,
Dwyer and co-workers examined the expression of CCL2
in breast-tumor explants revealing the cytokine presence
not only in the tumor cells, but also surprisingly showing
that the majority of the CCL2 detected was from the
stromal fibroblast population.
27
This finding further
enforces the importance of the tumor microenvironment
participation in MSC migration as well as the tumor cells
themselves.
The cytokines, VEGFaand platelet-derived growth
factor ab (PDGFab) both harness chemoattractant prop-
erties; Ball and co-workers showed that VEGFawas able
to stimulate migration through the PDGF receptor,
confirming the intricacies involved in the induction of
signaling pathways.
54
In support of this, data from our
group demonstrate the combined potential of PDGF and
VEGF acting as chemoattractants inducing MSC migra-
tion in an in vitro matrigel migration assay; this
combination of growth factors is more potent than either
growth factor alone (Figure 1).
Inflammatory chemokine receptor expression on MSC
is influenced by microenvironmental conditions. For
example, the CC-, but not CXC-, chemokine receptors
have been shown to be upregulated by TNF-a.
3
This
upregulation of certain chemokine receptors in response
to cellular signals may have a role in tissue-specific
homing.
3,27,55
Additionally, the cytokines produced by
the MSC themselves may not have a direct role in MSC
migration, but may increase adhesion molecule expres-
sion in preparation for contact with the site of inflamma-
tion/injury as a mechanism of self-conditioning. In vitro
co-culture experiments demonstrate that tumor-secreted
factors influence expression of chemokines and chemo-
kine receptors in MSC (Figure 2). Furthermore, different
tumor cell lines produce different patterns of gene
expression in MSC (Figure 3). Further investigation of
these differentially expressed factors may shed light on
mechanisms of MSC migration to tumors.
Additional receptors implicated in MSC migration
Toll-like receptors are a vital component of the innate
immune response. There are 11 TLR found in humans,
each recognize a conserved yet broad range of molecules
known as pathogen-associated molecular patterns.
Depending on the stimulation, downstream TLR
signaling can regulate the expression of both CC and
CXC chemokines via nuclear factor kB activation.
56
Motif
recognition varies between receptors: the dimers, TLR1/
2 and 2/6 recognize lipopeptides and peptidoglycans;
TLR3 recognizes double-stranded RNA; TLR4 recognizes
lipopolysaccharide; TLR5 recognizes extracellular matrix
molecules; TLR7 and 8 recognize synthetic antiviral
compounds; TLR9 recognizes unmethylated CpG
DNA.
57
TLR signaling results in the activation of several
pathways, MAPK, MyD88, c-Jun N-terminal kinase and
inhibitor of kB kinase leading to the activation of the
downstream activation of transcription factors nuclear
factor kB and AP-1, which ultimately lead to the
transcription of proinflammatory chemokines and cyto-
kines.
57
TLR1–6 have been identified on primary human
MSC by both reverse transcription-PCR and flow
1.E-05
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
Lo
g
2
(
Fold Difference
)
p Value
-7 -5 -3 -1 1 5 937
Figure 2 Tumor-induced changes in mesenchymal stem cell (MSC)
inflammatory gene expression after 24 h. Expression of 84 genes
involved in inflammation was compared to human MSC
co-cultured with a pancreatic tumor cell line. Individual genes
are plotted as a function of P-value and fold difference in
expression. The blue line represents the P¼0.05 threshold. The
pink lines represent a threshold for fold difference (>2). Seven
chemokine and inflammatory cytokines, shown within the red line,
exhibited a significant increase in expression after non-contact
co-culture conditions with the tumor cell line. See online version
for colour figure.
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E Spaeth et al
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cytometry.
58
Tomchuck and co-workers reported that
TLR stimulation enhanced the migratory function of
MSC; the most potent migratory induction occurred with
the stimulation of TLR3. Likewise, the inhibition of TLR3
signaling through a neutralizing antibody decreased
MSC migration capacity by over 50%.
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Figure 3 Tumor-specific induction of mesenchymal stem cell (MSC) gene expression. Represented by graphs are the gene expression profiles
from real-time reverse transcription-PCR analysis on 84 inflammatory cytokines and chemokine genes expressed in human MSC. hMSC were
co-cultured with a tumor cell line for 24 h before RNA was extracted for the expression analysis comparison between the MSCs alone and the
MSC in the presence of the tumor paracrine factors. (a) Pediatric sarcoma: RD54; (b) pancreatic cancer: PANC1; (c) breast cancer: MDA-231;
(d) ovarian cancer: SKOV-3.
Renilla luciferase
4T1 tumor
Firefly luciferase
MSC
0
5000
10000
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20000
25000
30000
35000
40000
45000
50000
left
(irradiated)
right
(unirradiated)
counts
Figure 4 Targeted tropism of mesenchymal stem cell (MSC) using focal irradiation. (a) Ten days after 4T1 tumors were implanted into the
bilateral hindlimbs of Balb/C mice, tumors were imaged with renilla luciferase (upper panel). The right hindlimb was irradiated with 2 Gy
(approximate irradiated volume represented by white triangle). MSC were injected i.v. the following day and imaged with firefly luciferase
48 h later (lower panel). (b) Quantitation of MSC in tumors.
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Manipulation of MSC migration
Augmentation of MSC migration via genetic
manipulation of MSC
With the mechanistic understanding behind MSC migra-
tion being slowly deciphered, the potential for enhancing
MSC-targeted therapies appears promising. The genetic
manipulation of MSC to overexpress target receptors
should enhance their migration to site-specific locations.
The introduction of exogenous DNA into MSC, as
reviewed by Damme and co-workers, enables enforced
expression/secretion of a desired therapeutic factor into
the targeted environment.
59
One could imagine the
potential in the overexpression of TLR3 and CCR2, the
receptor for CCL2 (MCP-1) on MSC may improve their
migration efficiency to specific tumor cells. Such a
specific and directed approach will prove promising to
the field of gene therapy for the treatment of cancers,
allowing the cellular targeting of specific tumors,
inflammatory diseases and other tissue injuries including
myocardial infarction and ischemic cerebral damage.
The diverse collection of receptors present on MSC
suggest the formation of a cascade of competitive events
that enable a hierarchy of chemoattractants that are
responsible for a step-wise chemotaxis to the tumor
microenvironment. Conflicting chemotactic signals will
lead to a biased migration to previously encountered
chemoattractants in leukocyte-trafficking models.
60
This
heterologous signaling pathway activation based on the
variation of surface receptors expression may justify both
the preconditioning of MSC as discussed previously and
the manipulation of surface receptor expression to
enhance chemotaxis.
Alteration of MSC migration via changes in external
environment
As discussed previously, inflammatory mediators have
been shown to increase targeted trafficking. Inflamma-
tion induction as an element of targeted treatment
enables a broader scope of secreted inflammatory
molecules that may influence migration. This global
enhancement of inflammatory chemokines, cytokines,
along with tissue damage by-products including, lipids
(leukotriene), glycosaminoglycans (hyaluran), enzymes,
free radicals, complement and fibrinopeptides will
exacerbate the migratory response seen in MSC. Mice
with irradiated tumors, as compared with unirradiated
tumors, show an increase in MSC migration. Klopp and
co-workers recently demonstrated this in an elegant
experiment upon which bilateral hind leg tumor im-
plants were used. The tumor in the left hind leg
remained an internal control, whereas the right hind
leg received local irradiation treatment after which
intravenous injection of MSC revealed a higher number
of MSC localizing to the irradiated tumor (Figure 4).
Similar experiments using HSC have shown an increased
migratory propensity post-irradiation treatment of glio-
ma cells. The attraction of HSC to irradiated glioma cells
was attributed to an increase in stress signaling that
induced hypoxia-induced transcription factor 1atran-
scriptional activity dependent on a functional TGF-b
signaling cascade to induce CXCL12 promoter activity.
61
Low-dose irradiation promotes VEGF release that is
dependent on matrix metalloproteinase 9 expression,
which is also upregulated under stress responses such as
radiation treatment.
62
The angiogenic factor, VEGF, is
secreted by both the tumor cells and the tumor stroma,
and is a known migratory inducer of both HSC and
MSC.
63,64
In the presence of irradiated tumor cell-
conditioned media, MSC increase their expression of
VEGFa, VEGFc and PDGFb. The expression of these
growth factors may be an autocrine feedback loop
mechanism to enhance receptor expression, or to counter
balance an increased apoptotic mRNA expression seen in
these cells.
Future directions
Targeted migration of MSC to tumor sites will have a
significant impact on the field of antitumor therapy. MSC
exhibit an intrinsic homing property enabling them to
direct migration to sites of inflammation. The exploita-
tion of this process will be a valuable asset to directed
therapy. Their capability to express exogenous gene
products, genetic stability and allogeneic properties
make MSC excellent delivery vehicles for antitumor
therapy, previously demonstrated not only in tumor
models but also for other diseases such as graft-versus-
host, multiple sclerosis and arthritis.
9,65,66
The induced expression of receptors critical to the
migratory competence of MSC to tumors will allow an
increased number of MSC to reach the target location.
The increase in migratory efficiency will improve the
therapeutic value of the overall system.
Conclusions
There have been many advances in determining the
factors involved in the migration of MSC to ‘wounded’/
inflammatory/tumor environments; however, their full
potential as therapeutic-vehicle candidates can only be
utilized when the mechanistic understanding behind
their migration is elucidated. Many different receptors
have been implicated in the homing of MSC: (1) the
broad activation of growth factor receptors that activate
further chemokine receptor expression like CXC and CC
receptors; (2) the activation of TLR that also target
downstream expression of CXC and CC receptors; (3) the
activation of adhesion molecules and (4) integrins that
may or may not be implicated in the direct role of
paracrine cell movement. The key players implicated
in MSC migration to date include the chemokines
MCP-1 (CCL2),
52
CXCL8,
27
RANTES (CCL5);
3
LL-37,
58
integrinb1,
43
receptors CD44,
40
CCR2,
3
CCR3,
3
and the
receptor tyrosine kinases for the following growth
factors, IGF-1,
3
PDGF-bb,
67
HGF,
68
and VEGF.
54
The ability to extract from previously described cell-
migration models in the elucidation of MSC homing
properties is an arduous task. Many common receptors
have been identified on MSC; however, multiple ligands
and co-receptors have the ability to alter the downstream
signaling pathway through coupling, crosstalk or in-
hibitory mechanisms thereby rendering an alternative
mechanistic feature for a common ligand/receptor pair
depending on the cell-lineage. The future of targeted
therapy using MSC will depend on the exploitation of
these previous ligand/receptor interactions for the
Inflammation-driven mesenchymal stem cell migration
E Spaeth et al
736
Gene Therapy
enhancement of the existing intrinsic migratory/homing
propensity.
Acknowledgements
This work was supported in part by grants from the
National Cancer Institute (CA-1094551 and CA-116199
for FCM, CA-55164, CA-16672, and CA-49639 for MA)
and by the Paul and Mary Haas Chair in Genetics (MA).
ES, AK and FCM are supported in part by grants from
the Susan G Komen Breast Cancer Foundation.
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