Gene Therapy (2008) 15, 730–738 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00 www.nature.com/gt 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 ulating MSC migration to tumors is essential to ultimately tissue damage as well as the tumor microenvironment. Many develop novel clinical strategies aimed at using MSC as of the same inflammatory mediators that are secreted by vehicles to deliver antitumor proteins or suppress MSC wounds are found in the tumor microenvironment and are migration to reduce tumor growth. For example, radiation thought to be involved in attracting MSC to these sites. Cell enhances inflammatory signaling in the tumor microenviron- migration is dependent on a multitude of signals ranging from ment and may be used to potentiate site-specific MSC growth factors to chemokines secreted by injured cells and/or migration. Alternatively, restricting the migration of the MSC respondent immune cells. MSC are likely to have chemo- to the tumor microenvironment may prevent competent tactic properties similar to other immune cells that respond to tumor-stroma formation, thereby hindering the growth of injury and sites of inflammation. Thus, the well-described the tumor. In this review, we will discuss the role of model of leukocyte migration can serve as a reasonable inflammatory signaling in attracting MSC to tumors. example to facilitate the identification of factors involved in Gene Therapy (2008) 15, 730–738; doi:10.1038/gt.2008.39; MSC migration.Understanding the factors involved in reg- published online 10 April 2008

Keywords: mesenchymal stem cell; migration; tumor microenvironment; tumor stroma; inflammatory chemoattractants

Brief description of the mesenchymal The use of bone marrow-derived MSC have been employed in support and engraftment of the transplan- stem cell tation of hematopoietic stem cells (HSCs) following Mesenchymal stem cells (MSC) are non-hematopoietic high-dose chemotherapy in an effort to replenish the adult stem cells with multilineage potential. MSC are destroyed bone marrow cell population.3 Additionally, defined by plastic adherence, differentiation potential pre-clinical studies have explored the use of MSC in the and cell surface marker expression.1 MSC, or MSC-like reduction of graft-versus-host disease, for tissue repair; cells, have been isolated from nearly every organ or including cerebral injury,4 bone fracture,5 myocardial tissue in the body, making it challenging to characterize ischemia/infarction,6 muscular dystrophy,7 as well as the MSC as a completely homogenous population. MSC tumor homing. contribute to the maintenance and regeneration of Migration of MSC to tumors is thought to be due to connective tissues and have the capacity to differentiate inflammatory signaling in a tumor resembling that of an within osteoblasts, adipocytes, chondrocytes, myocytes unresolved wound.8 The innate tropism of MSC for and cardiomyocytes. MSC express markers including tumors can be exploited for the delivery of antitumor CD29, CD44, CD51, CD73 (SH3/4), CD105 (SH2), CD166 agents to the tumor microenvironment. Gene-modified (ALCAM) and Stro-1, but the expression of specific MSC expressing interferon-b have been used to signifi- combinations of markers appear microenvironment- cantly reduce tumor burden and in some cases extend dependent, suggesting a strong influence of tissue survival in murine models of melanoma,9 lung,10 breast context on MSC phenotypes. In general, MSC appear to cancer11 and glioma.12 However, the mechanism and be a non-immunogenic population of cells; however, a factors responsible for the targeted tropism of MSC to few studies demonstrate immune-repressive functions of these wounded microenvironments remain to be fully MSC through the induction of peripheral tolerance elucidated. MSC are likely to have chemotactic proper- evident in autoimmune disorders such as multiple ties similar to other immune cells that respond to injury sclerosis.2 and sites of inflammation. Thus, the well-described model of leukocyte migration can serve as a reasonable Correspondence: Dr F Marini, Molecular Hematology and Therapy, example to facilitate the identification of factors involved Department of Stem Cell Transplantation, UT-M.D. Anderson in MSC migration. Following a discussion of alterations Cancer Center, Box081, 1515 Holcombe Boulevard, Houston, in the ‘wounded’/tumor microenvironment that are TX 77030, USA. E-mail: [email protected] shown to enhance MSC tumor-specific migration, we Received 14 February 2008; accepted 18 February 2008; published will introduce alternative, injury-induced cell migration online 10 April 2008 models based on literature reviewing leukocytes and Inflammation-driven mesenchymal stem cell migration E Spaeth et al 731 their progenitor cell line, the HSC. These alternative known to have an important role in leukocyte/macro- migration systems will provide rational for the tumor- phage infiltration into tumors.14 Based on this evidence, specific MSC migration and will lead us into an overview one can speculate that inflammation-induced chemo- of the current literature concerning MSC migration kines participate in the directed migration of stem cells, specifically. Ultimately, we will conclude with a discus- such as MSC, to tumors and inflamed microenviron- sion of the potential clinical applications of tumor- ments. Previous studies have shown the importance of directed MSC migration. 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 Inflammation-targeted homing in the in migration of MSC. In addition to the secreted tumor microenvironment chemotactic molecules secreted by the tumor and its surrounding stroma, the tumor cells themselves retain a Inflammation is a cellular response that takes place chemotactic disparity amongst its cellular components. under conditions of cellular injury and in sites of tissue When fractionated, the tumor cell membrane possesses a wounding. Over two decades ago, Dvorak and co- superior chemotaxis-induction potential compared with workers described the tumor as an unhealed wound the other cellular components such as cytosolic fractions, that produces a continuous source of inflammatory including organelles such as the nuclei, mitochondria, mediators (cytokines, chemokines and other potential lysosomes, microsomes and ribosomes (Figure 1). chemoattractant molecules). Cancer progression has been correlated with an increase in inflammatory mediator gene expression, and this is thought to occur Hypoxia contribution to MSC homing via disruption, damage and cellular turnover occurring in the tumor microenvironment. This constant produc- Many tumors exhibit hypoxia, a state of reduced oxygen tion of inflammatory mediators perpetuates the main- that often parallels and perpetuates inflammation. A tenance and progression of the tumor environment and common feature between inflammation and hypoxic becomes a target for the MSC. Tumor-generated inflam- environments is the expression of pro-angiogenic mole- matory mediators have a role in determining the cules. The hypoxia-induced transcription factor HIF-1a conditions of the tumor microenvironment, as they activates the transcription of genes including vascular regulate invasion, motility, extracellular matrix interac- endothelial growth factor (VEGF), macrophage migra- tion through autocrine effects as well as coordinating cell tion inhibitory factor, tumor necrosis factors (TNF-a), movement through paracrine signaling.13 Tumor-pro- numerous proinflammatory cytokines and the activation duced and tumor-induced inflammatory chemokines are of the transcription factor nuclear factor kB.16,17 Nuclear

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0 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 VEGF & PDGF PDGF & TGFB VEGF & TGFB bFGF & Serum

1 Cytokines Tumor cells Cell membrane Mixtures 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.

Gene Therapy Inflammation-driven mesenchymal stem cell migration E Spaeth et al 732 Table 1 Cell surface markers and receptors associated with cell migration that are known to be expressed on MSC

Cell surface receptors Ligands Present on other cell types found on MSC

Growth hormone EGFR (ErbB) EGF DC, neutrophil receptors 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 CCR1 CCL3, CCL5, CCL7, CCL13, CCL14, CCL15, monocyte, T cell, DC receptors CCL16, CCL23 CCR2 CCL2, CCL7, CCL8, CCL13, CCL16 Monocyte, T cell, DC CCR3 CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, T cell, DC CCL16, CCL24, CCL26, CCL28 CCR4 CCL17, CCL22 T cell, macrophage, DC CCR5 CCL3, CCL4, CCL5, CCL8, CCL11, CCL14, Monocyte, T cell, DC, HSC CCL16 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, Neutrophil, monocyte CXCL7, CXCL8 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.

factor kB is frequently activated in response to inflam- matory cytokines via nitric oxide (NO); NO has been matory mediators18 and has been shown to induce linked to the regulation/induction of MIP-1a, MCP-1, several chemokines (RANTES (CCL5), MIP-2 (CXCL2), macrophage inflammatory protein-related protein-1 and MIP-1a (CCL3), monocyte chemoattractant protein 1 osteopontin.24,25 Hypoxia has an important role in (MCP-1) (CCL2), interleukin-8 (CXCL8)) that are im- perpetuating the inflammatory process in tumors which plicated in leukocyte migration.19–22 Hypoxia in the results in the generation of chemokines that are involved tumor microenvironment is a cyclical event, and perpe- in immune cell and likely MSC migration to tumors. tuates the inflammatory response by ensuring a constant production of angiogenic and inflammatory mediators. Briefly, hypoxic conditions result in the generation of Archetype for MSC trafficking reactive oxygen species, which can increase DNA damage in neighboring cells. Tumor cells with more The mechanism behind MSC migration is still in its virulent mutations can then proliferate and invade infancy. However, factors involved in regulating migra- neighboring cells/tissues resulting in tissue damage, tion of leukocytes have been studied extensively, and it is which increases the demand for nutrients and oxygen, likely that many of the same factors are involved in which continues to be short in supply.23 The reactive regulating MSC migration. A list of receptors expressed oxygen species induced in the microenvironment has on MSC that have been previously implicated in cell also been demonstrated to increase secretion of inflam- migration is shown in Table 1. Growth factor, cytokine/

Gene Therapy Inflammation-driven mesenchymal stem cell migration E Spaeth et al 733 chemokine, adhesion molecules and toll-like receptors (collagen), and is able to both induce inflammation and (TLRs) are expressed on MSC, as described by Cham- serve as a receptor mediator in the homing toward sites berlain et al.,26 Ringe et al.,27 Allen et al.28 and Viola et al.29 of inflammation.36,37 Hyaluronan, which is a by-product Although the table does not encompass all receptors, it is of tissue repair, results in persistent inflammation38 and a comprehensive list of those receptors that have been in addition to being critical to cell motility during studied on other cell types, including leukocytes and development, the inhibition of hyaluronan synthesis in HSC. The T cell, macrophage and dendritic cells are all prostate tumors impairs growth and vascularization.39 considered relevant to the understanding of MSC Of importance, CD44, the receptor for hyaluronan, is homing because of their functional similarities in expressed on MSC, and a recent publication suggests targeting inflamed/injured tissues. The HSC is consid- CD44 roles in the migration of MSC toward injured ered because it is the precursor to the myeloid and kidney tissue.40 In addition to CD44, hyaluronan by itself lymphoid lineages in addition to being resident of the can interact with TLR2 and TLR4, thereby enhancing the bone marrow where we also find the MSC. inflammatory response.38 The participation and involve- ment of TLRs in MSC migration will be addressed to a Leukocytes further extent in a subsequent section. There are three main participants in leukocyte traffick- ing: adhesion molecules, integrins and chemoattrac- HSC migration tants.30 The latter is the most important when Similar to the leukocytes, the migratory machinery of considering the paracrine-mediated gradient trafficking HSC are also well characterized in regards to defined and migration without cellular contact, such as what has receptors/ligands required for migration and should be been observed for MSC migration to sites of tumors and considered in the evaluation of potential candidates for sites of injury. the elucidation of MSC migration. In fact, one of the most Cytokine and chemokine receptors have an important widely recognized receptor/ligand pairs for HSC traf- role in leukocyte and likely MSC migration. A decrease ficking is CXCR4/CXCL12 (SDF1); in addition, CXCR4/ in leukocyte migration has been observed in a mouse SDF1 appears important in dictating migration of several knockout model of the IL-8 receptors, CXCR1 and tumor cell lines to metastatic sites.41,42 However, in CXCR2.31 Similarly, neutrophil recruitment in ischemia- contrast to the key role of CXCR4/SDF1 in HSC reperfusion models was inhibited by blocking the migration, a recent publication by Ip and co-workers CXCR1 and CXCR2 receptors.32 The presence of both showed blocking of the CXCR4 receptor had no impact CXCR1 and CXCR227 on MSC suggests that they may on MSC migration, suggesting key differences have a similar function in MSC migration. Nearly every between the migration signals of these two stem cells.43 chemokine receptor has been found on the surface of This controversial receptor indicates that receptor MSC,3 while CCR2 and CCR3 are two receptors that may function differs between cell types and enhances the have a particularly important role in leukocyte and MSC importance of examining receptors found mutually trafficking. Macrophage trafficking has been shown to be expressed on MSC- and HSC-like CXCR4, CCR544 mediated by CCR2, a chemokine receptor with ligands and VEGFR.45 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, MSC trafficking—finding the tumor MCP3 and MCP4 (CCL 8, 7, 13) has been effective in reducing trafficking in leukocytes. Yet another seven The past year has revealed a surge of publications transmembrane receptor, CD97, an epidermal growth attempting to define the homing properties of MSC. Due factor receptor appears necessary for the migration of to the more comprehensive command of knowledge in neutrophils to sites of inflammation33 and has been the field of leukocyte migration, those chemokines and implicated in the promotion of angiogenesis. Increased corresponding receptors chosen for evaluation on MSC is expression of CD97 in tumor cell lines, such as colorectal based on prior data illustrated in leukocyte models. carcinoma, correlates with the increased migration However, as noted above, the migratory competence potential of leukocytes.34 Of note, MSC express CD55, of these receptors clearly varies: CXCR4/CXCL12 are the ligand to CD97, suggesting a potential CD55–CD97 crucial in bone marrow retention and homing of HSC interaction thereby influencing both MSC and leukocyte where as in MSC, it appears that CXCR4/CXCL12 do not migration. posses the same migration importance unless enforced Other than cytokines/chemokines, numerous bioac- expression of CXCR4 is employed.43,46,47 Bearing in mind tive molecules can also serve as leukocyte chemoattrac- that each of the following receptors and their respective tants. Such molecules include lipids, (leukotrienes and ligands (chemokines, cytokines, growth factors, peptides prostaglandins), peptides such as chemerin and other small biomolecules) have not been implicated as a single elements of the extracellular matrix.35 The adhesion primary mechanism for the migration of MSC; however, molecule, CD44, which is widely expressed on leuko- altogether may function in an additive manner. Thus, the cytes and parenchymal cells interacts with components coalescence of known migratory mechanisms is vital to of the extracellular matrix known to be involved in the understanding of the complete migratory disposition inflammation including hyaluronic acid, collagen, lami- of the MSC in relationship to cancer biology. nin and fibronectin. The presence of CD44 is thought to mediate and enhance localized inflammation leading to Preconditioning the MSC the increased migration of leukocytes, it also serves as a Activation of MSC with proinflammatory cytokines (that receptor for growth factors, integrins, cytokines (osteo- is, TNF-a) prior to reinfusion has been demonstrated to pontin), glycosaminoglycans (hyaluronic acid), peptides increase MSC in vivo migratory and adhesion capacity

Gene Therapy Inflammation-driven mesenchymal stem cell migration E Spaeth et al 734 1.E-05 chemoattractant for MSC migration toward breast carcinomas.52 However, prior studies show conflicting data on the migratory response to CCL2. The incon- 1.E-04 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 1.E-03 in breast-tumor explants revealing the cytokine presence not only in the tumor cells, but also surprisingly showing

p Value that the majority of the CCL2 detected was from the 1.E-02 stromal fibroblast population.27 This finding further enforces the importance of the tumor microenvironment participation in MSC migration as well as the tumor cells 1.E-01 themselves. The cytokines, VEGFa and platelet-derived growth factor ab (PDGFab) both harness chemoattractant prop- 1.E+00 a -7 -5 -3 -1 137 5 9 erties; Ball and co-workers showed that VEGF was able Log2 (Fold Difference) to stimulate migration through the PDGF receptor, confirming the intricacies involved in the induction of Figure 2 Tumor-induced changes in mesenchymal stem cell (MSC) signaling pathways.54 In support of this, data from our inflammatory gene expression after 24 h. Expression of 84 genes involved in inflammation was compared to human MSC group demonstrate the combined potential of PDGF and co-cultured with a pancreatic tumor cell line. Individual genes VEGF acting as chemoattractants inducing MSC migra- are plotted as a function of P-value and fold difference in tion in an in vitro matrigel migration assay; this expression. The blue line represents the P ¼ 0.05 threshold. The combination of growth factors is more potent than either pink lines represent a threshold for fold difference (>2). Seven growth factor alone (Figure 1). chemokine and inflammatory cytokines, shown within the red line, Inflammatory chemokine receptor expression on MSC exhibited a significant increase in expression after non-contact co-culture conditions with the tumor cell line. See online version is influenced by microenvironmental conditions. For for colour figure. 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 through the increased expression of receptors. Existing to cellular signals may have a role in tissue-specific data suggest that the cytokines IL-1b and TNF-a activate homing.3,27,55 Additionally, the cytokines produced by adherence properties of the MSC including the upregula- the MSC themselves may not have a direct role in MSC tion of the VCAM-1-VLA-4 adhesion pathway.48 Addi- migration, but may increase adhesion molecule expres- tional receptors known to be upregulated by TNF-a sion in preparation for contact with the site of inflamma- priming include CCR3 and CCR4; these findings correlate tion/injury as a mechanism of self-conditioning. In vitro with the observation of increased in vitro migration to co-culture experiments demonstrate that tumor-secreted RANTES (a CCR3 ligand) and macrophage-derived factors influence expression of chemokines and chemo- cytokine (MDC (CCL22)—a CCR4 ligand).3 kine receptors in MSC (Figure 2). Furthermore, different Several growth factors have been shown to induce tumor cell lines produce different patterns of gene MSC migration. Insulin-like growth factor 1 (IGF-1) expression in MSC (Figure 3). Further investigation of increases the expression of chemokine receptors on the these differentially expressed factors may shed light on MSC, thereby enhancing migration. Li and co-workers mechanisms of MSC migration to tumors. demonstrated that IGF-1-induced upregulation of CXCR4 expression on MSC increased the migratory Additional receptors implicated in MSC migration capacity of the cells toward an in vitro SDF-1 gradient Toll-like receptors are a vital component of the innate through a phosphoinositide-3 kinase-dependent path- immune response. There are 11 TLR found in humans, way without altering the proliferation status of the each recognize a conserved yet broad range of molecules MSC.49 The inability for MSC migration to occur in vivo known as pathogen-associated molecular patterns. through a CXCL12/CXCR4 mechanism was mentioned Depending on the stimulation, downstream TLR previously; accordingly, IGF-1 is capable of stimulating signaling can regulate the expression of both CC and the expression of other chemokine receptors such as CXC chemokines via nuclear factor kB activation.56 Motif CCR5—the RANTES (CCL5) receptor.50 Other growth recognition varies between receptors: the dimers, TLR1/ factors including basic fibroblast growth factor and 2 and 2/6 recognize lipopeptides and peptidoglycans; VEGF are associated with angiogenesis and are secreted TLR3 recognizes double-stranded RNA; TLR4 recognizes under hypoxemic stress. MSC demonstrate an increased lipopolysaccharide; TLR5 recognizes extracellular matrix migratory propensity in the presence of basic fibroblast molecules; TLR7 and 8 recognize synthetic antiviral growth factor through a phosphoinositide-3 kinase/AKT compounds; TLR9 recognizes unmethylated CpG pathway downstream of the basic fibroblast growth DNA.57 TLR signaling results in the activation of several factor receptor on the MSC.51 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 Chemokine/cytokine secretion and the respective factor kB and AP-1, which ultimately lead to the receptors found on MSC transcription of proinflammatory chemokines and cyto- Monocyte-chemoattractant protein 1 (CCL2), a chemo- kines.57 TLR1–6 have been identified on primary human kine secreted by tumor cells, was shown to be a potent MSC by both reverse transcription-PCR and flow

Gene Therapy Inflammation-driven mesenchymal stem cell migration E Spaeth et al 735

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0.01 0.01 H 12 11 G 12 H 10 F 11 10 G 9 8 E 9 F 7 6 D 8 7 E 5 4 C Row 6 5 D Column 3 B Column 4 C Row 2 1 A 3 2 B 1 A 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.

50000 45000 Renilla luciferase 40000 4T1 tumor 35000 30000 25000

counts 20000 15000 Firefly luciferase 10000 MSC 5000 0 left right (irradiated) (unirradiated) 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.

cytometry.58 Tomchuck and co-workers reported that the stimulation of TLR3. Likewise, the inhibition of TLR3 TLR stimulation enhanced the migratory function of signaling through a neutralizing antibody decreased MSC; the most potent migratory induction occurred with MSC migration capacity by over 50%.

Gene Therapy Inflammation-driven mesenchymal stem cell migration E Spaeth et al 736 Manipulation of MSC migration which is also upregulated under stress responses such as radiation treatment.62 The angiogenic factor, VEGF, is Augmentation of MSC migration via genetic secreted by both the tumor cells and the tumor stroma, manipulation of MSC and is a known migratory inducer of both HSC and With the mechanistic understanding behind MSC migra- MSC.63,64 In the presence of irradiated tumor cell- tion being slowly deciphered, the potential for enhancing conditioned media, MSC increase their expression of MSC-targeted therapies appears promising. The genetic VEGFa, VEGFc and PDGFb. The expression of these manipulation of MSC to overexpress target receptors growth factors may be an autocrine feedback loop should enhance their migration to site-specific locations. mechanism to enhance receptor expression, or to counter The introduction of exogenous DNA into MSC, as balance an increased apoptotic mRNA expression seen in reviewed by Damme and co-workers, enables enforced these cells. 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 Future directions receptor for CCL2 (MCP-1) on MSC may improve their migration efficiency to specific tumor cells. Such a Targeted migration of MSC to tumor sites will have a specific and directed approach will prove promising to significant impact on the field of antitumor therapy. MSC the field of gene therapy for the treatment of cancers, exhibit an intrinsic homing property enabling them to allowing the cellular targeting of specific tumors, direct migration to sites of inflammation. The exploita- inflammatory diseases and other tissue injuries including tion of this process will be a valuable asset to directed myocardial infarction and ischemic cerebral damage. therapy. Their capability to express exogenous gene The diverse collection of receptors present on MSC products, genetic stability and allogeneic properties suggest the formation of a cascade of competitive events make MSC excellent delivery vehicles for antitumor that enable a hierarchy of chemoattractants that are therapy, previously demonstrated not only in tumor responsible for a step-wise chemotaxis to the tumor models but also for other diseases such as graft-versus- microenvironment. Conflicting chemotactic signals will host, multiple sclerosis and arthritis.9,65,66 lead to a biased migration to previously encountered The induced expression of receptors critical to the chemoattractants in leukocyte-trafficking models.60 This migratory competence of MSC to tumors will allow an heterologous signaling pathway activation based on the increased number of MSC to reach the target location. variation of surface receptors expression may justify both The increase in migratory efficiency will improve the the preconditioning of MSC as discussed previously and therapeutic value of the overall system. the manipulation of surface receptor expression to enhance chemotaxis. Conclusions Alteration of MSC migration via changes in external There have been many advances in determining the environment factors involved in the migration of MSC to ‘wounded’/ As discussed previously, inflammatory mediators have inflammatory/tumor environments; however, their full been shown to increase targeted trafficking. Inflamma- potential as therapeutic-vehicle candidates can only be tion induction as an element of targeted treatment utilized when the mechanistic understanding behind enables a broader scope of secreted inflammatory their migration is elucidated. Many different receptors molecules that may influence migration. This global have been implicated in the homing of MSC: (1) the enhancement of inflammatory chemokines, cytokines, broad activation of growth factor receptors that activate along with tissue damage by-products including, lipids further chemokine receptor expression like CXC and CC (leukotriene), glycosaminoglycans (hyaluran), enzymes, receptors; (2) the activation of TLR that also target free radicals, complement and fibrinopeptides will downstream expression of CXC and CC receptors; (3) the exacerbate the migratory response seen in MSC. Mice activation of adhesion molecules and (4) integrins that with irradiated tumors, as compared with unirradiated may or may not be implicated in the direct role of tumors, show an increase in MSC migration. Klopp and paracrine cell movement. The key players implicated co-workers recently demonstrated this in an elegant in MSC migration to date include the chemokines experiment upon which bilateral hind leg tumor im- MCP-1 (CCL2),52 CXCL8,27 RANTES (CCL5);3 LL-37,58 plants were used. The tumor in the left hind leg integrinb1,43 receptors CD44,40 CCR2,3 CCR3,3 and the remained an internal control, whereas the right hind receptor tyrosine kinases for the following growth leg received local irradiation treatment after which factors, IGF-1,3 PDGF-bb,67 HGF,68 and VEGF.54 intravenous injection of MSC revealed a higher number The ability to extract from previously described cell- of MSC localizing to the irradiated tumor (Figure 4). migration models in the elucidation of MSC homing Similar experiments using HSC have shown an increased properties is an arduous task. Many common receptors migratory propensity post-irradiation treatment of glio- have been identified on MSC; however, multiple ligands ma cells. The attraction of HSC to irradiated glioma cells and co-receptors have the ability to alter the downstream was attributed to an increase in stress signaling that signaling pathway through coupling, crosstalk or in- induced hypoxia-induced transcription factor 1a tran- hibitory mechanisms thereby rendering an alternative scriptional activity dependent on a functional TGF-b mechanistic feature for a common ligand/receptor pair signaling cascade to induce CXCL12 promoter activity.61 depending on the cell-lineage. The future of targeted Low-dose irradiation promotes VEGF release that is therapy using MSC will depend on the exploitation of dependent on matrix metalloproteinase 9 expression, these previous ligand/receptor interactions for the

Gene Therapy Inflammation-driven mesenchymal stem cell migration E Spaeth et al 737 enhancement of the existing intrinsic migratory/homing cells correlates with severity of inflammation in murine propensity. myocarditis. Pflug Arch Eur J Physiol 2006; 452: 268–275. 16 Winner M, Koong AC, Rendon BE, Zundel W, Mitchell RA. Amplification of tumor hypoxic responses by macrophage migration inhibitory factor-dependent hypoxia-inducible factor Acknowledgements stabilization. Cancer Res 2007; 67: 186–193. This work was supported in part by grants from the 17 Ye J, Gao Z, Yin J, He Q. Hypoxia is a potential risk factor for National Cancer Institute (CA-1094551 and CA-116199 chronic inflammation and adiponectin reduction in adipose for FCM, CA-55164, CA-16672, and CA-49639 for MA) tissue of ob/ob and dietary obese mice. Am J Physiol Endocrinol and by the Paul and Mary Haas Chair in Genetics (MA). Metab 2007; 293: E1118–E1128. ES, AK and FCM are supported in part by grants from 18 Pold M, Zhu LX, Sharma S, Burdick MD, Lin Y, Lee PP et al. the Susan G Komen Breast Cancer Foundation. Cyclooxygenase-2-dependent expression of angiogenic CXC chemokines ENA-78/CXC ligand (CXCL) 5 and interleukin-8/ CXCL8 in human non-small cell lung cancer. Cancer Res 2004; 64: 1853–1860. References 19 Ubogu EE, Callahan MK, Tucky BH, Ransohoff RM. Determi- nants of CCL5-driven mononuclear cell migration across the 1 Prockop DJ. Marrow stromal cells as stem cells for nonhemato- blood-brain barrier. Implications for therapeutically modulating poietic tissues. Science 1997; 276: 71–74. neuroinflammation. J Neuroimmunol 2006; 179: 132–144. 2 Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, 20 Gregory JL, Morand EF, McKeown SJ, Ralph JA, Hall P, Yang YH Gerdoni E et al. Mesenchymal stem cells ameliorate experi- et al. Macrophage migration inhibitory factor induces macro- mental autoimmune encephalomyelitis inducing T-cell anergy. phage recruitment via CC chemokine ligand 2. J Immunol 2006; Blood 2005; 106: 1755–1761. 177: 8072–8079. 3 Lopez Ponte A, Marais E, Gallay N, Langonne´ A, Delorme B, 21 Montecucco F, Bianchi G, Bertolotto M, Viviani G, Dallegri F, He´rault O et al. The in vitro migration capacity of human bone Ottonello L. Insulin primes human neutrophils for CCL3- marrow mesenchymal stem cells: comparison of chemokine and induced migration: crucial role for JNK 1/2. Ann N Y Acad Sci growth factor chemotactic activities. Stem Cells 2007; 25: 1737–1745. 2006; 1090: 399–407. 4 Li Y, Chen J, Wang L, Zhang L, Lu M, Chopp M. Intracerebral 22 Lehman N, Di Fulvio M, McCray N, Campos I, Tabatabaian F, transplantation of bone marrow stromal cells in a 1-methyl-4- Gomez-Cambronero J. Phagocyte cell migration is mediated by phenyl-1,2,3,6-tetrahydropyridine mouse model of parkinson’s phospholipases PLD1 and PLD2. Blood 2006; 108: 3564–3572. disease. Neurosci Lett 2001; 316: 67–70. 23 Laconi E. The evolving concept of tumor microenvironments. 5 Niedzwiedzki T, Dabrowski Z, Miszta H, Pawlikowski M. Bone BioEssays 2007; 29: 738–744. healing after bone marrow stromal cell transplantation to the 24 Guo HT, Cai CQ, Schroeder RA, Kuo PC. Nitric oxide is Biomaterials 14 bone defect. 1993; : 115–121. necessary for CC-class chemokine expression in endotoxin- 6 Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Miyatake H stimulated ANA-1 murine macrophages. Immunol Lett 2002; 80: et al. Nonhematopoietic mesenchymal stem cells can be 21–26. mobilized and differentiate into cardiomyocytes after myocar- 25 Takahashi F, Takahashi K, Maeda K, Tominaga S, Fukuchi Y. dial infarction. Blood 2004; 104: 3581–3587. Osteopontin is induced by nitric oxide in RAW 264.7 cells. 7 Dezawa M, Ishikawa H, Itokazu Y, Yoshihara T, Hoshino M, IUBMB Life 2000; 49: 217–221. Takeda S et al. Bone marrow stromal cells generate muscle cells 26 Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: and repair muscle degeneration. Science 2005; 309: 314–317. mesenchymal stem cells: their phenotype, differentiation capa- 8 Dvorak HF. Tumors: wounds that do not heal. Similarities city, immunological features, and potential for homing. Stem between tumor stroma generation and wound healing. N Engl J Cells 2007; 25: 2739–2749. Med 1986; 315: 1650–1659. 27 Ringe J, Strassburg S, Neumann K, Endres M, Notter M, 9 Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira- Burmester G-R. Towards in situ tissue repair: human mesench- Hansen M, Bekele BN et al. Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for ymal stem cells express chemokine receptors CXCR1, CXCR2 anticancer agents. J Natl Cancer Inst 2004; 96: 1593–1603. and CCR2, and migrate upon stimulation with CXCL8 but not 10 Kanehira M, Xin H, Hoshino K, Maemondo M, Mizuguchi H, CCL2. J Cell Biochem 2007; 101: 135–146. Hayakawa T et al. Targeted delivery of NK4 to multiple lung 28 Allen SJ, Crown SE, Handel TM. Chemokine: Receptor structure, tumors by bone marrow-derived mesenchymal stem cells. interactions, and antagonism. Annu Rev Immunol 2007; 25: Cancer Gene Ther 2007; 14: 894–903. 787–820. 11 Hall B, Dembinski J, Sasser AK, Studeny M, Andreeff M, 29 Viola A, Luster AD. Chemokines and their receptors: drug Marini F. Mesenchymal stem cells in cancer: tumor-associated targets in immunity and inflammation. Annu Rev Pharmacol fibroblasts and cell-based delivery vehicles. Int J Hematol 2007; 86: Toxicol 2008; 48: 171–197. 8–16. 30 Luster AD, Alon R, von Andrian UH. Immune cell migration in 12 Nakamizo A, Marini F, Amano T, Khan A, Studeny M, Gumin J inflammation: present and future therapeutic targets. Nat et al. Human bone marrow-derived mesenchymal stem cells in Immunol 2005; 6: 1182–1190. the treatment of gliomas. Cancer Res 2005; 65: 3307–3318. 31 Becker MD, O’Rourke LM, Blackman WS, Planck SR, Rosen- 13 Balkwill F. Cancer and the chemokine network. Nat Rev Cancer baum JT. Reduced leukocyte migration, but normal rolling and 2004; 4: 540–550. arrest, in interleukin-8 receptor homologue knockout mice. 14 Zernecke A, Weber KSC, Erwig LP, Kluth DC, Schro¨ppel B, Rees Invest Ophthalmol Vis Sci 2000; 41: 1812–1817. AJ et al. Combinatorial model of chemokine involvement in 32 Sekido N, Mukaida N, Harada A, Nakanishi I, Watanabe Y, glomerular monocyte recruitment: role of CXC chemokine Matsushima K. Prevention of lung reperfusion injury in rabbits receptor 2 in infiltration during nephrotoxic nephritis. J Immunol by a monoclonal antibody against interleukin-8. Nature 1993; 2001; 166: 5755–5762. 365: 654–657. 15 Malek S, Kaplan E, Wang JF, Ke Q, Rana JS, Chen Y et al. 33 Leemans JC, te Velde AA, Florquin S, Bennink RJ, de Bruin K, van Successful implantation of intravenously administered stem Lier RA et al. The epidermal growth factor-seven transmembrane

Gene Therapy Inflammation-driven mesenchymal stem cell migration E Spaeth et al 738 (EGF-TM7) receptor CD97 is required for neutrophil migration in human mesenchymal stem cells. Stem Cells 2006; 24: and host defense. JImmunol2004; 172: 1125–1131. 1750–1758. 34 Galle J, Sittig D, Hanisch I, Wobus M, Wandel E, Loeffler M et al. 52 Dwyer RM, Potter-Beirne SM, Harrington KA, Lowery AJ, Individual cell-based models of tumor-environment interac- Hennessy E, Murphy JM et al. Monocyte chemotactic protein-1 tions: multiple effects of CD97 on tumor invasion. Am J Pathol secreted by primary breast tumors stimulates migration of 2006; 169: 1802–1811. mesenchymal stem cells. Clin Cancer Res 2007; 13: 5020–5027. 35 Zabel BA, Zuniga L, Ohyama T, Allen SJ, Cichy J, Handel TM et 53 Wang L, Li Y, Chen J, Gautam SC, Zhang Z, Lu M et al. Ischemic al. Chemoattractants, extracellular proteases, and the integrated cerebral tissue and MCP-1 enhance rat bone marrow stromal cell host defense response. Exp Hematol 2006; 34: 1021–1032. migration in interface culture. Exp Hematol 2002; 30: 831–836. 36 Pisterna GV, Siragusa M. CD44 presence in inflamed pulp tissue. 54 Ball SG, Shuttleworth CA, Kielty CM. Vascular endothelial J Endodontics 2007; 33: 1203–1207. growth factor can signal through platelet-derived growth factor 37 Chaulet H, Desgranges C, Renault MA, Dupuch F, Ezan G, receptors. J Cell Biol 2007; 177: 489–500. Peiretti F et al. Extracellular nucleotides induce arterial 55 Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM, smooth muscle cell migration via osteopontin. Circ Res 2001; Silberstein LE. Human bone marrow stromal cells express a 89: 772–778. distinct set of biologically functional chemokine receptors. Stem 38 Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and Cells 2006; 24: 1030–1041. repair. Annu Rev Cell Dev Biol 2007; 23: 435–461. 56 Boyd JH, Divangahi M, Yahiaoui L, Gvozdic D, Qureshi S, Petrof 39 Simpson MA, Wilson CM, McCarthy JB. Inhibition of prostate BJ. Toll-like receptors differentially regulate CC and CXC tumor cell hyaluronan synthesis impairs subcutaneous growth chemokines in skeletal muscle via NF-kappaB and calcineurin. and vascularization in immunocompromised mice. Am J Pathol Infect Immun 2006; 74: 6829–6838. 2002; 161: 849–857. 57 West AP, Koblansky AA, Ghosh S. Recognition and signaling by 40 Herrera MB, Bussolati B, Bruno S, Morando L, Mauriello- toll-like receptors. Annu Rev Cell Dev Biol 2006; 22: 409–437. Romanazzi G, Sanavio F et al. Exogenous mesenchymal stem 58 Tomchuck SL, Zwezdaryk KJ, Coffelt SB, Waterman RS, Danka cells localize to the kidney by means of CD44 following acute ES, Scandurro AB. Toll-like receptors on human mesenchymal tubular injury. Kidney Int 2007; 72: 430–441. stem cells drive their migration and immunomodulating 41 Wright DE, Bowman EP, Wagers AJ, Butcher EC, Weissman IL. responses. Stem Cells 2007; 26: 99–107. Hematopoietic stem cells are uniquely selective in their 59 Hamada H, Kobune M, Nakamura K, Kawano Y, Kato K, migratory response to chemokines. J Exp Med 2002; 195: Honmou O et al. Mesenchymal stem cells (MSC) as therapeutic 1145–1154. cytoreagents for gene therapy. Cancer Sci 2005; 96: 149–156. 42 Sutton A, Friand V, Brule-Donneger S, Chaigneau T, Ziol M, 60 Foxman EF, Kunkel EJ, Butcher EC. Integrating conflicting Sainte-Catherine O et al. Stromal cell-derived factor-1/ chemotactic signals: the role of memory in leukocyte navigation. chemokine (C-X-C motif) ligand 12 stimulates human hepatoma J Cell Biol 1999; 147: 577–587. cell growth, migration, and invasion. Mol Cancer Res 2007; 5: 61 Tabatabai G, Frank B, Mohle R, Weller M, Wick W. Irradiation 21–33. and hypoxia promote homing of haematopoietic progenitor 43 Ip JE, Wu Y, Huang J, Zhang L, Pratt RE, Dzau VJ. Mesenchymal cells towards gliomas by TGF-beta-dependent HIF-1alpha- stem cells use integrin beta1 not CXC chemokine receptor 4 for mediated induction of CXCL12. Brain 2006; 129 (Pt 9): myocardial migration and engraftment. Mol Biol Cell 2007; 18: 2426–2435. 2873–2882. 62 Heissig B, Rafii S, Akiyama H, Ohki Y, Sato Y, Rafael T et al. 44 Schwabe RF, Bataller R, Brenner DA. Human hepatic stellate Low-dose irradiation promotes tissue revascularization cells express CCR5 and RANTES to induce proliferation through VEGF release from mast cells and MMP-9- and migration. Am J Physiol Gastrointest Liver Physiol 2003; 285: 202 G949–G958. mediated progenitor cell mobilization. J Exp Med 2005; : 45 Novo E, Cannito S, Zamara E, Valfre` di Bonzo L, Caligiuri A, 739–750. Cravanzola C et al. Proangiogenic cytokines as hypoxia- 63 Rabbany SY, Heissig B, Hattori K, Rafii S. Molecular pathways dependent factors stimulating migration of human hepatic regulating mobilization of marrow-derived stem cells for tissue stellate cells. Am J Pathol 2007; 170: 1942–1953. revascularization. Trends Mol Med 2003; 9: 109–117. 46 Le´vesque JP, Hendy J, Takamatsu Y, Simmons PJ, Bendall LJ. 64 Schichor C, Birnbaum T, Etminan N, Schnell O, Grau S, Miebach Disruption of the CXCR4/CXCL12 chemotactic interaction S et al. Vascular endothelial growth factor A contributes to during hematopoietic stem cell mobilization induced by gcsf glioma-induced migration of human marrow stromal cells or cyclophosphamide. J Clin Invest 2003; 111: 187–196. (hMSC). Exp Neurol 2006; 199: 301–310. 47 Shi M, Li J, Liao L, Chen B, Li B, Chen L et al. Regulation of 65 Kim SK, Kim SU, Park IH, Bang JH, Aboody KS, Wang KC et al. CXCR4 expression in human mesenchymal stem cells by Human neural stem cells target experimental intracranial cytokine treatment: Role in homing efficiency in NOD/SCID medulloblastoma and deliver a therapeutic gene leading to mice. Haematologica 2007; 92: 897–904. tumor regression. Clin Cancer Res 2006; 12: 5550–5556. 48 Segers VFM, VanRiet I, Andries LJ, Lemmens K, Demolder MJ, 66 Evans CH, Robbins PD, Ghivizzani SC, Wasko MC, Tomaino De Becker AJ et al. Mesenchymal stem cell adhesion to cardiac MM, Kang R et al. Gene transfer to human joints: progress microvascular endothelium: activators and mechanisms. Am J toward a gene therapy of arthritis. Proc Natl Acad Sci USA 2005; Physiol Heart Circ Physiol 2006; 290: H1370–H1377. 102: 8698–8703. 49 Li Y, Yu X, Lin S, Li X, Zhang S, Song YH. Insulin-like growth 67 Fiedler J, Ro¨derer G, Gu¨nther KP, Brenner RE. BMP-2, BMP-4, factor 1 enhances the migratory capacity of mesenchymal stem and PDGF-bb stimulate chemotactic migration of primary cells. Biochem Biophys Res Commun 2007; 356: 780–784. human mesenchymal progenitor cells. J Cell Biochem 2002; 87: 50 Mira E, Lacalle RA, Gonza´lez MA, Go´mez-Mouto´n C, Abad JL, 305–312. Bernad A et al. A role for chemokine receptor transactivation in 68 Forte G, Minieri M, Cossa P, Antenucci D, Sala M, Gnocchi V growth factor signaling. EMBO Rep 2001; 2: 151–156. et al. Hepatocyte growth factor effects on mesenchymal stem 51 Schmidt A, Ladage D, Schinkothe T, Klausmann U, Ulrichs C, cells: proliferation, migration, and differentiation. Stem Cells Klinz FJ et al. Basic fibroblast growth factor controls migration 2006; 24: 23–33.

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