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STEM CELL GENETICS AND GENOMICS

Differential Gene Expression Associated with Migration of Mesenchymal Stem Cells to Conditioned Medium from Tumor Cells or Bone Marrow Cells

^aDepartment of Medicine, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey, USA;
^bDepartment of Pharmacology, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey, USA;
^cGraduate School of Biomedical Sciences, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey, USA;
^dDepartment of Pediatric Oncology, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey, USA

Key Words. In vivo migration • Gene expression • Stromal cell-derived factor-1 • Microenvironment • Chemotaxis Mesenchymal stem cells

Correspondence: Debabrata Banerjee, Ph.D., Cancer Institute of New Jersey, RWJMS/UMDNJ, 195 Little Albany Street, New Brunswick, New Jersey 08903, USA. Telephone: 732-235-6458; Fax: 732-235-8181; e-mail: banerjed{at}umdnj.edu

Received April 25, 2006; accepted for publication October 10, 2006.
First published online in STEM CELLS EXPRESS   October 19, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Distinct signals that guide migration of mesenchymal stem cells (MSCs) to specific in vivo targets remain unknown. We have used rat MSCs to investigate the molecular mechanisms involved in such migration. Rat MSCs were shown to migrate to tumor microenvironment in vivo, and an in vitro migration assay was used under defined conditions to permit further mechanistic investigations. We hypothesized that distinct molecular signals are involved in the homing of MSCs to tumor sites and bone marrow. To test this hypothesis, gene expression profiles of MSCs exposed in vitro to conditioned medium (CM) from either tumor cells or bone marrow were compared. Analysis of the microarray gene expression data revealed that 104 transcripts were upregulated in rat MSCs exposed to CM from C85 human colorectal cancer cells for 24 hours versus control medium. A subset of 12 transcripts were found to be upregulated in rat MSCs that were exposed to tumor cell CM but downregulated when MSCs were exposed to bone marrow CM and included CXCL-12 (stromal cell-derived factor-1 [SDF-1]), CXCL-2, CINC-2, endothelial cell specific molecule-1, fibroblast growth factor-7, nuclear factor-B p105, and thrombomodulin. Exposure to tumor cell CM enhanced migration of MSCs and correlated with increased SDF-1 protein production. Moreover, knockdown of SDF-1 expression in MSCs inhibited migration of these cells to CM from tumor cells, but not bone marrow cells, confirming the importance of SDF-1 expression by MSCs in this differential migration. These results suggest that increased SDF-1 production by MSCs acts in an autocrine manner and is required for migratory responses to tumor cells.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The bone marrow is known to harbor two major types of stem cells, the hematopoietic stem cell and the nonhematopoietic or mesenchymal stem cell, termed MSC. Under appropriate conditions, MSCs can give rise to cells of muscle, bone, fat, and cartilage lineage [1]. Like true stem cells, MSCs have the capacity for self-renewal and differentiation, and based on this potential, MSCs hold promise for clinical applications for regenerative medicine as well as for use as delivery vehicles [2, 3]. Several reports indicate that MSCs migrate to various in vivo locations, including sites of hematopoiesis such as the bone marrow, sites of injury, and sites of inflammation [4–6]. MSCs have also been reported to migrate to tumors when injected systemically [7, 8]. During embryonic and fetal development, MSCs circulate in the bloodstream to nidate emerging sites of hematopoiesis. They are present in large numbers in human blood for the first 12 weeks of gestation, and there is evidence for the existence of circulating MSCs, albeit in low numbers, even in the adult [9, 10].

The ability of MSCs to migrate to areas of injury and to tumors has encouraged investigation of MSCs as therapeutic tools. For example, systemically administered MSCs have been shown to improve recovery in animal models of stroke and myocardial infarction [11, 12]. MSCs have also been used for targeted delivery of therapeutic gene products to the tumor microenvironment in animal models [7, 8, 13], and the therapeutic use of MSCs is being explored for various disease conditions [14–18].

Although the migratory behavior of MSCs has now been extensively documented, distinct signals that guide migration of MSCs to specific in vivo targets are unknown. A better understanding of the molecular events that govern MSC homing may permit efficient targeted delivery of MSCs to desired sites for therapeutic purposes. We hypothesized that gene expression profiling of MSCs exposed to specific chemotactic stimuli such as conditioned medium (CM) from tumor cells or bone marrow cells would provide clues to the molecular pathways involved in MSC migration. We chose to study rat MSCs isolated from rat bone marrow because these cells have been extensively used as a model system for MSC behavior ([19] and references therein).

We have identified a set of genes, including stromal cell-derived factor-1 (SDF-1), which are differentially expressed in MSCs exposed to tumor cell CM compared with bone marrow CM. Knockdown of SDF-1 in MSCs inhibited migration to tumor cells but not to bone marrow cells. This is the first report to describe differential gene expression by MSCs during migration to specific targets. The study of molecular events associated with migration of MSCs to distinct sites such as bone marrow and tumor microenvironment will be critical in understanding the role of MSCs in physiological and pathophysiological conditions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Isolation and Culture of MSCs
Rat MSCs were isolated as previously described [19]. Briefly, rats (6-week-old male Wistar rats, weighing approximately 200 g; Taconic, Hudson, NY, http://www.taconic.com) were euthanized by CO₂ inhalation and the bilateral femora and tibias were dissected under aseptic conditions and washed in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). Bone marrow cells were extruded by flushing media through the bone, and cells were filtered through a 70-µm nylon mesh. The cells were then plated in T150- or T75-cm² flasks with -minimum essential medium (-MEM) containing 10% FBS and penicillin/streptomycin. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Nonadherent cells were removed after 24 hours, and the medium was changed every other day. Adherent cells were detached from the flasks by treatment with 0.05% trypsin and EDTA and subcultured every 4–5 days, and aliquots from passages 3 to 5 were frozen in liquid nitrogen for future use. Early-passage cells were tested for their capacity to differentiate in culture as described below. All work with animals was carried out under the auspices of a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Robert Wood Johnson Medical School (RWJMS) (protocol number NI05-015-3, approval date March 2, 2005).

In Vitro Differentiation Assays
The adherent cells were trypsinized and replated in six-well plates for differentiation assays in vitro according to the following culture conditions.

Myogenesis.   Myogenic differentiation was induced by incubating early-passage MSCs in complete growth medium containing 3 µM 5-azacytidine for 24 hours as described by Wakitani et al. [20]. Differentiated myogenic cells characterized by long myotube-like structures were identified by phase-contrast microscopy.

Osteogenesis.   This was essentially carried out as described by Neuhuber et al. [21]. MSCs were seeded in six-well plates at 3,000 cells per square centimeter. After 2 days of growth, the normal growth medium was replaced by osteogenic medium containing 100 nM dexamethasone (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 50 µM ascorbate-2-phosphate (Wako Pure Chemicals, Osaka, Japan, http://www.wako-chem.co.jp/english), and 10 mM ß-glycerol phosphate (Sigma-Aldrich). The osteogenic medium was replaced twice weekly. After 3 weeks, the medium was aspirated and cells were stained using the von Kossa method to visualize calcium phosphate deposition in the differentiated cells [1].

Adipogenesis.   MSCs were plated in six-well plates at 20,000 cells per cm² and cultured in growth medium until they reached confluence. The growth medium was discarded and adipogenic induction medium was added (Dulbecco's modified Eagle's medium [DMEM] with 4.5 mg/ml glucose, 10% FBS, regular antibiotics, 0.5 mM methylisobutylxanthine, 1 µM dexamethasone, 10 µg/ml insulin, and 100 mM indomethacin; all reagents from Sigma-Aldrich). Cells were incubated for 3 days and subsequently incubated in adipogenic maintenance medium (DMEM, FBS, and insulin as before) for 1 day. After three cycles of induction and maintenance, cells were maintained for 7 days in maintenance medium and then fixed with 4% paraformaldehyde. Cells were stained with oil red O for microscopic evaluation of accumulated neutral lipid deposits characterizing adipogenic cells [1].

Tumor Cell Lines
Human tumor cell lines used for this study included breast cancer lines MCF-7 and MDAMB231; prostate cancer lines PC3, DU145, and LnCaP; and colorectal cancer lines HCT-8 and C85. All cell lines with the exception of C85 were obtained from American Type Culture Collection (Manassas, VA, http://www.atcc.org). Cells were cultured in RPMI medium (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% FBS and penicillin-streptomycin at 37°C in 5% CO₂.

Human Colorectal Cancer Xenografts
One million C85, human colorectal cancer cells were injected into 8-week-old nude mice (Taconic) to generate subcutaneous tumors in flanks. This cell line has been described by Longo et al. [22]. Palpable tumors were obtained within 8 days following injection. Xenograft studies in nude mice were carried out according to guidelines outlined in an animal use protocol approved by the IACUC at RWJMS (protocol number NI05-019-3, approval date March 8, 2005).

Systemic Injection of Carboxyfluorescein Diacetate Succinimidyl Ester-Labeled MSCs
To examine in vivo migration of MSCs to tumor microenvironment, rat MSCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) according to the manufacturer's instructions and labeling efficiency was determined by flow cytometry. One million labeled MSCs were injected via the tail vein into tumor-bearing animals (C85 tumors were grown subcutaneously, two injections of half a million cells suspended in 50 µl of RPMI without serum were given 15 minutes apart). Seven days later, tumors were resected from euthanized animals, sectioned, and examined by fluorescence microscopy to determine whether CFDA-SE-labeled MSCs could be detected in the tumor mass. Briefly, tumors were excised from euthanized animals and placed in OCT cryo embedding compound (number NC 9159334; Fisher, Hampton, NH, http://www.fishersci.com) and sections were processed for microscopy. Sections were also stained with 4',6-diamidino-2-phenylindole (DAPI). Cells were harvested from some tumors after resection and analyzed by flow cytometry. Tumors were disaggregated manually and suspended in RPMI without serum, treated with collagenase and DNAse for 1 hour at 37°C. Single-cell suspension was filtered through a strainer cap fitted on flow cytometry tubes and analyzed by flow cytometry.

Exposure of MSCs to Tumor Cell CM and Bone Marrow CM
Tumor cells were grown in RPMI+10% heat-inactivated FBS (heat inactivation was carried out at 56°C for 30 minutes) culture medium, and CM from tumor cells was harvested 12–14 hours after change of culture medium for cells in logarithmic growth phase. Bone marrow was harvested as described earlier and cultured in -MEM with 10% FBS and antibiotics. MSCs were exposed once to freshly harvested tumor cell or bone marrow CM for 24 hours or repeatedly (six times in 24 hours) to CM from tumor cells and analyzed by cDNA microarray gene expression profiling (see below).

Transwell Chamber Migration Assays
A Falcon cell culture insert system along with a companion Falcon tissue culture plate with 24 wells was used for the migration assay. The insert was removed aseptically with sterile forceps from the package and gently placed in the well with the flanges resting in the notches on the top edge of each well (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). The polyethylene terepthalate membrane pore size of 8 µm was selected to allow passage of mammalian cells. The bottom chamber either contained tumor cells or bone marrow cells or CM from tumor cells or bone marrow cells. Bone marrow was harvested as described earlier. CM was prepared from bone marrow cells (cultured for 24 hours in -MEM supplemented with 10% FBS and penicillin/streptomycin at 37°C and 5% CO₂) by centrifuging (for 5 minutes at 1,200 rpm) the supernatant collected from 2 x 10⁶ cells per milliliter (in -MEM supplemented with 10% FBS and penicillin/streptomycin) of plated bone marrow cells, and 700 µl of CM was plated in the bottom chamber of transwell migration setup. For migration toward bone marrow cells, 1.5 x 10⁵ bone marrow cells from the same aliquot was plated in the bottom chamber (in -MEM supplemented with 10% FBS and antibiotics). The top chamber contained 2 x 10⁴ MSCs. Migration assays were terminated after 16 hours (or shorter times for faster migrating cells), and MSCs that had migrated through the membrane were then stained (after removal of cells remaining on top with a wet Q-tip) using a Diff Quik staining kit obtained from Dade Behring, Inc. (Deerfield, IL, http://www.dadebehring.com) according to the manufacturer's instructions. Stained cells in 10 fields were counted manually under high-power magnification (x20). Results presented are from three independent experiments for each condition. For the migration assays toward tumor cells or CM from tumor cells, MSCs were acclimated for 72 hours in RPMI + 10% heat-inactivated FBS (the growth medium for tumor cell lines) prior to plating in the transwell chambers. For migration toward bone marrow cells and CM from bone marrow, MSCs were plated in -MEM supplemented with 10% heat-inactivated FBS and antibiotics.

Gene Expression Profiling
Cells were harvested after exposure to tumor cell CM or bone marrow or control medium and RNA was isolated using the Invitrogen RNA isolation kit (Invitrogen Corporation). Ten µg of total RNA was processed for microarray analysis after verification of quality at DNA microarray core facility of Cancer Institute of New Jersey/RWJMS. Briefly, the RNA was reverse-transcribed and hybridized to Affymetrix cDNA GeneChip Rat Genome 230 2.0 Array comprised of more than 31,000 probe sets, analyzing more than 30,000 transcripts and variants from more than 28,000 well-substantiated rat genes (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). Comparative analyses of expressed genes that were either upregulated or downregulated under various experimental conditions were carried out using proprietary software from Affymetrix and the freely available KEGG (Kyoto Encyclopedia of Genes and Genomes) and DAVID (Data base for Annotation, Integration and Discovery) tools. Three independent sets for each of the experimental conditions were analyzed to control for intrasample variation. Genes were considered increased or decreased only if the levels changed by greater than twofold with p < .0001 for increased expression and p > .9997 for decreased expression to reduce false discovery rate.

Reverse Transcription-Polymerase Chain Reaction Validation Studies
Primers for genes of interest were designed using software available from ABI Biosystems and obtained from Dharmacon, Inc. (Lafayette, CO, http://www.dharmacon.com). Preparation of RNA and reverse transcription (RT) was carried out using commercially available kits from Invitrogen Corporation. Tubes were incubated at 50°C for 30 minutes for RT followed by a denaturation step at 94°C for 2 minutes. This was followed by 25 cycles of polymerase chain reaction (PCR) amplification at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 1 minute. The final elongation step was carried out at 72°C for 7 minutes.

SDF-1 sense primer sequence: 5'-TTTGAGAGCCATGTCGCCA-3';

SDF-1 antisense primer sequence: 5'-TGTCTGTTGTTGCTTTTCAGCC-3';

Monocyte chemoattractant protein-1 (MCP-1) sense primer sequence: 5'-TCAGCCAGATGCAGTTAATG-3';

MCP-1 antisense primer sequence: 5'-TTCTCTGTCATACTGGTCAC-3';

ß-actin sense primer sequence: 5'-ACCACCATGTACCCAGGCATT-3';

ß-actin antisense primer sequence: 5'-CCACACAGAGTACTTGCGCTCA-3';

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense primer sequence: 5'-CACCATGGAGAAGGCCGGGG-3';

GAPDH antisense primer sequence: 5'-GACGGACACATTGGGGGTAG-3'.

Immunofluorescence Staining for F Actin and Microtubule Organization Center
MSCs were placed on glass slides immediately adjacent to C85 tumor cells for 24 hours and allowed to adhere. Fresh growth medium was added gently to cover the MSCs and tumor cell spots. After incubation for an additional 24 hours, cells were processed for immunofluorescence staining for F actin and microtubule organization center (MTOC) according to standard techniques by using fluorescent-labeled reagents obtained from Invitrogen Corporation. Cells were fixed with 3.7%–4% paraformaldehyde for 10 minutes, washed three times in 1x PBS, permeabilized with 0.1% Triton for 5 minutes, washed twice with PBS (5 minutes), and blocked with complete media or blocking buffer (PBS + bovine serum albumin) for 15 minutes. Primary antibody was then added for 1 hour in the dark at room temperature and washed twice in PBS (5 minutes), and secondary antibody was added for 1 hour in the dark at room temperature. Samples were then washed twice in PBS, added to nuclear stain for 10 minutes, and washed in PBS. Coverslips were mounted and dried overnight, and ends were sealed with nail polish. Phalloidin-TRITC (tetramethylrhodamine B isothiocyanate) was used at final concentration of 50 ng/ml; -tubulin (Sigma-Aldrich) was diluted 1:2,000 dilution, and the nuclear stain (TOPRO-3; Invitrogen Corporation) was diluted 1:250 in PBS.

Quantitation of SDF-1
Levels of SDF-1 in CM were determined using the Quantikine kit (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com) based on an enzyme-linked immunosorbent assay according to manufacturer's instructions.

Small Interfering RNA Knockdown Experiments
MSCs plated in six-well plates were transfected with small interfering RNA (siRNA) designed against SDF-1 (CXCL-12), identified by the microarray experiments to be upregulated in cells exposed to tumor CM from both single and repeated exposures. A scrambled sequence served as control. All siRNAs were obtained from Ambion, Inc. (Austin, TX, http://www.ambion.com). Cells were harvested after 48 and 72 hours for migration assay and RNA and protein isolation.

Sequences of siRNAs used are as follows:

SDF-1 siRNA sequence:

Sense: GCAGUGAUUACUUCAAGGUtt;

Antisense: ACCUUGAAGUAAUCACUGCtt.

Silencer Negative Control siRNA (catalog number 4611; Ambion, Inc.) was used as a control for nonspecific gene silencing. Transfection of siRNAs was carried out using Lipofectamine 2000 transfection reagent according to the manufacturer's instructions (Invitrogen Corporation).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
In Vitro Differentiation Potential of Rat MSCs and Migration to Tumor Microenvironment In Vivo
To determine the differentiation potential of MSCs, cells were assayed for myogenesis, osteogenesis, and adipogenesis under in vitro conditions according to published procedures [1]. Myogenic differentiation characterized by long fused myotubes, von Kossa-positive staining for cells of osteogenic lineage, and positive staining with oil red O for cells of adipocyte lineage was observed under the appropriate conditions (Fig. 1Aa–Ad). These results confirmed that the cells were indeed multipotential MSCs.

View larger version (49K): [in this window] [in a new window]   Figure 1. Differentiation potential of mesenchymal stem cells (MSCs). (A): MSCs can differentiate along myogenic, osteogenic, and adipogenic lineages. To confirm the differentiation potential of the rat MSCs, these cells were assayed for myogenesis, osteogenesis, and adipogenesis under in vitro conditions according to published procedures. (a) shows control rat MSCs in culture which can be induced to differentiate to cells of myogenic lineage with 5-azacytidine (b), osteogenic lineage as seen by von Kossa-positive staining for calcium deposits (c), and positive staining with oil red O for cells of adipocyte lineage (d). This result confirms that the cells are capable of differentiating along various lineages. (B): Rat MSCs, indicated as S, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) in (b), take up residence in tumor microenvironment (indicated as T) when injected to tumor-bearing animals, suggesting that MSCs migrate to and are retained at the tumor sites. Fluorescence intensity of CFDA-SE-labeled MSCs decreases as they proliferate in the tumor microenvironment. Four',6-diamidino-2-phenylindole (DAPI) staining for cell nuclei (a) reveals the outline of the dense tumor mass surrounded by the migrated MSCs. Fluorescence microscopy on tissue sections stained with DAPI (blue fluorescence for nuclei) and CFDA-SE (green fluorescence) clearly shows presence of MSCs surrounding the tumor cells.

Transwell Chamber Migration Assay
MSCs plated on the upper chamber migrated toward tumor as well as bone marrow cells or CM from tumor or bone marrow cells placed in the lower chamber. Figure 2A shows results of typical migration assays after 16 hours. Tumor cells or bone marrow cells (panel b: C85 colorectal cells; panel c: DU145 prostate cancer cells; panel d: bone marrow cells) were placed in the lower chamber and the membrane was stained for the presence of MSCs after 16 hours of migration as compared with control containing complete growth medium without cells (panel a). Figure 2B shows results of a migration assay for MSCs challenged to migrate to various cell types and CM. Also shown is the migration of MSCs toward bone marrow cells and CM from bone marrow. It is of interest that MSCs migrated toward cells derived from metastatic tumors to a greater extent than toward cells derived from primary tumors. We then asked whether the migration potential of MSCs could be enhanced ex vivo by repeated exposure to CM of tumor cells. Repeated exposure of MSCs to CM from tumor cells increased the migration potential significantly as compared with naïve MSCs (by approximately fourfold; Fig. 2C) as well as to MSCs exposed once to CM from C85 cells (by approximately twofold). Increased migration of MSCs exposed repeatedly to control medium (RPMI plus 10% FBS and antibiotics) was also observed. This is possibly due to increased activation of MSCs by growth factors present in FBS; however, we were still able to observe a significant increase in migration of MSCs after repeated exposure to CM from tumor cells as compared with repeated exposure to control growth medium.

View larger version (45K): [in this window] [in a new window]   Figure 2. Migration of mesenchymal stem cells (MSCs) toward tumor cells. (A): Transwell chamber in vitro migration assay. Figure from a typical migration assay. MSCs migrate toward C85 human colorectal cancer cells placed in the bottom chamber (b), human prostate cancer cells DU145 (c), and rat bone marrow cells (d). A significantly greater number of MSCs after 16 hours of migration (b–d) are viewed under x20 field as compared with those in control chamber containing complete growth medium without cells (a). (B): Migration of MSCs to various tumor types and conditioned medium (CM) from the respective tumor cells and to bone marrow and CM from bone marrow. The bars represent the following. 1: Control medium; 2: MCF-7; 3: MDA MB 231; 4: HCT8; 5: C85; 6: LnCaP; 7: DU145; 8: PC3; 9: control medium for rat bone marrow; 10: rat bone marrow. The dark bars represent migration to cells, and the lighter bars represent migration to CM from the same cells. p < .05 for all migration values to tumor cells and tumor CM as compared with control using the unpaired t test. (C): Migration of MSCs is enhanced after repeated exposure to CM from tumor cells. Number of migrating cells is shown on the y-axis. 1, 3, and 5: Control unexposed MSCs, exposed once for 16 hours to RPMI+10% fetal bovine serum (FBS), exposed six times to RPMI+10% FBS respectively, placed in upper chamber and migrating to control RPMI+10% FBS placed in the bottom chamber. 2, 4, and 6: MSCs previously unexposed to CM from tumor cells, exposed once for 16 hours to CM from tumor cells and exposed six times to CM from tumor cells, respectively, placed in the upper chamber and migrating to CM placed in the bottom chamber. Values are from three independent experiments for each condition. Note that, although number of migrating MSCs increases after single and repeated exposure to RPMI+10% FBS control medium, it is significantly less than number of migrating MSCs after comparable exposure to CM from tumor cells.

View larger version (32K): [in this window] [in a new window]   Figure 3. Cytoskeletal changes in mesenchymal stem cells (MSCs) associated with migration. (A): Exposure of MSCs to conditioned medium (CM) from tumor cells alters F actin filament organization. Organization of F actin filaments along the length of MSCs placed adjacent to tumor cells for 24 hours as compared with unorganized F actin filaments in control MSCs as demonstrated by immunofluorescence. (a) shows control MSCs, whereas (b–d) show different activated MSCs. (B): Microtubule organization center (MTOC) organizes in direction of migration of MSCs. Organization of MTOC, shown for two different cells in (a) and (b), along the direction of migration of MSCs to tumor cells as detected by immunofluorescence. Details of experimental conditions can be found in Materials and Methods.

(a) mRNA levels of 104 genes were upregulated in MSCs exposed to CM from C85 cells for 24 hours versus control medium, of which superoxide dismutase 2, CXCL-12 (SDF-1), CXCL-2, CCL-2 (MCP-1), CINC-2, CXCL-2/ gro ß, CCL-3 (MIP-1), and MMP-9 showed the largest increase.

(b) In MSCs exposed six times in 24 hours to CM from C85 cells versus control, mRNA levels of 480 genes were upregulated. These included cytokines, their receptors, and cDNAs encoding participants in signal transduction such as JAK2, which are particularly important for cytoskeleton reorganization during locomotion. Moreover, a subset of seven genes (including expressed sequence tags) was common among the genes whose expression was increased between MSCs exposed repeatedly and MSCs exposed once to CM from C85 cells and included SDF-1, bone morphogenetic protein-6 (BMP-6), and glutathione S-transferase 1. Overlap of transcripts upregulated after single and repeated exposure to CM from tumor cells is shown in Table 1 and as a Venn diagram in Figure 4A.

View larger version (28K): [in this window] [in a new window]   Figure 4. Changes in gene expression following exposure of mesenchymal stem cells (MSCs) to tumor CM. (A): Venn diagram showing overlap of altered transcripts from single and repeated exposure to tumor cell CM. (B): Venn diagram showing overlap of altered transcripts from MSCs exposed to tumor cell CM and bone marrow CM. (C): Validation by reverse transcription-polymerase chain reaction (RT-PCR) of increased expression of SDF-1 and MCP-1 in MSCs after exposure to CM from tumor cells. Lanes 1–3 represent RT-PCR product for amplifications specific for SDF-1, MCP-1, and ß-actin from three independent samples of MSCs exposed to CM from tumor cells for 16-hour experiments, whereas lanes 4–6 represent three independent control MSC samples exposed to RPMI+10% FBS for 16 hours. (D): Increased expression of SDF-1 is observed in cells exposed to tumor CM but not in cells exposed to bone marrow CM. Lanes marked M represent size markers. Lanes 1 and 2 represent cells exposed to tumor CM, whereas lanes 3 and 4 represent cells exposed to control medium. Lanes 5 and 6 represent cells exposed to bone marrow CM, whereas lanes 7 and 8 represent cells exposed to control medium. Abbreviations: CM, conditioned medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCP-1, monocyte chemoattractant protein-1; SDF-1, stromal cell-derived factor-1.

Knockdown of SDF-1 in MSCs Resulted in Inhibition of Migration to CM from C85 Cells but Not to Bone Marrow CM
Results from the gene expression profile study indicated that SDF-1 was one of the major genes whose expression level was increased significantly when MSCs were exposed to CM from tumor cells. Knockdown of SDF-1 in MSCs using siRNA was performed to determine whether migration to CM from C85 cells was impaired under these conditions.

RT-PCR analysis showed that SDF-1 RNA levels were reduced at 48 and 72 hours after transfection using 50 nM siRNA to SDF-1 (Fig. 5A). Reduced expression of SDF-1 (using 50 nM siRNA) impaired migration of MSCs to tumor CM but not bone marrow CM (Fig. 5B). One possible explanation for this result is that SDF-1 or another chemoattractant secreted by bone marrow cells can replace SDF-1 produced by MSCs. Because tumor cells do not secrete SDF-1, knocking down SDF-1 in MSCs prevents them from becoming activated. This result also suggests that chemokine/cytokines present in CM from tumor cells may act primarily by increasing SDF-1 production in MSCs, which then can function in an autocrine manner to elicit a chemotactic response. To test this possibility, we measured SDF-1 levels in supernatants from MSCs either stimulated or unstimulated by exposure to tumor cell CM.

View larger version (18K): [in this window] [in a new window]   Figure 5. Role of stromal cell-derived factor-1 (SDF-1) in migration of mesenchymal stem cells (MSCs) to tumor conditioned medium (CM). (A): Knockdown of SDF-1 expression using small interfering RNA (siRNA). Reverse transcription-polymerase chain reaction analysis shows knockdown of SDF-1 levels after 48 and 72 hours using either 25 or 50 nM siRNA for SDF-1 as compared with control scrambled siRNA (lanes 6 and 7). Lane 1: 100-base-pair (bp) ladder, size markers. Lane 2: SDF-1 siRNA, 48 hours, 25 nM. Lane 3: SDF-1 siRNA, 72 hours, 25 nM. Lane 4: SDF-1 siRNA, 48 hours, 50 nM. Lane 5: SDF-1 siRNA, 72 hours, 50 nM. Lane 6: scrambled siRNA, 48 hours, 50 nM. Lane 7: scrambled siRNA, 72 hours, 50 nM. (B): Knockdown of SDF-1 inhibits migration of MSCs to CM from tumor cells. SDF-1 knockdown using 50 nM siRNA inhibits migration of MSCs (bars 1 and 3) to CM from tumor cells (bar 2) but not to CM from bone marrow cells (bar 4). 1 and 3: naïve MSCs. 2 and 4: MSCs after knockdown of SDF-1 migrate to CM from C85 cells (bars 1 and 2) and to CM from BM cells (bars 3 and 4). *, p < .0008 (two-tailed) for bar 2 compared to bar 1 using the unpaired t test. (C): Exposure of MSCs to CM from tumor cells increases SDF-1 production. C85 tumor cell CM (bar 1) and RPMI medium (bar 2) have barely detectable levels of SDF-1. Exposure of MSCs to RPMI+10% FBS for 16 hours (bar 3) and to tumor cell CM for 16 hours (bar 4) leads to a significant increase in SDF-1 levels in secreted medium of MSCs in agreement with the cDNA microarray results. SDF-1 level between single exposure to RPMI+10% FBS and single exposure to CM from tumor cells is increased significantly (p < .005, unpaired t test).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
MSCs can differentiate to cells of the osteogenic, adipogenic, chondrogenic, and myogenic lineages under appropriate conditions, and in addition, MSCs can migrate to sites of injury, inflammation, and tumors. These properties of MSCs make them attractive candidates for use in regenerative medicine as well as for delivery vehicles for site-specific therapy. For example, genetically modified MSCs expressing interferon ß have been shown to impede tumor growth in animal models, indicating that this approach is feasible [7, 8, 13]. However, the molecular events underlying migration of MSCs to tumor sites are not well defined. An understanding of the signaling pathways associated with migration of MSCs will help to define the role of MSCs in tumor growth.

In this study, we demonstrate that in addition to morphological changes, MSCs undergo specific alterations in gene expression patterns in response to different stimuli. A small set of genes was found to be upregulated in response to tumor CM but not bone marrow CM. These differences in gene expression indicate that specific MSC genes respond to a particular microenvironment. Expression of 104 genes was increased by exposure to tumor cell CM and included those involved in chemotaxis, invasion, oxidoreduction, cell signaling, and cytoskeleton reorganization. Interestingly, repeated exposure to tumor CM enhanced migration of MSCs and was accompanied by an increased expression of 473 genes versus control. Expressions of seven genes were found to be increased in MSCs after a single as well as repeated exposure to CM from C85 tumor cells, and these included SDF-1, BMP-6, and glutathione S-transferase. SDF-1 mRNA level was upregulated in MSCs by tumor CM, had a more profound increase in expression in MSCs after repeated exposure to tumor CM, and was not increased by exposure to bone marrow CM.

In agreement with the gene expression profiling data, SDF-1 protein levels were also increased in MSCs upon exposure to CM from tumor cells. Conversely, knockdown of SDF-1 in MSCs reduced migration toward tumor CM but not bone marrow CM. SDF-1 is produced in the bone marrow. However, tumor CM contains barely detectable levels of SDF-1. Therefore, upregulation of SDF-1 production by MSCs is required for migration toward tumor cells but not bone marrow. SDF-1, induced by factors present in CM from tumor cells, then functions in an autocrine manner to support chemotaxis of MSCs to tumor sites.

An important role for SDF-1 in survival and proliferation of MSCs has been described previously [23]. Although SDF-1 is produced by a number of cell types in the bone marrow [24, 25], tumor cells do not produce SDF-1 in significant quantities. The major source of SDF-1 in the MCF-7 tumor xenograft model is fibroblasts [24]. Our data expand these previous studies by demonstrating that production of SDF-1 by the MSCs themselves has an important effect on MSC behavior under certain conditions. This suggests that in some microenvironments, SDF-1 signaling through its receptor, CXCR4, may act in an autocrine manner to promote chemotaxis and may also impact other MSC functions. A recent report demonstrating that migration of bone marrow- and cord blood-derived MSCs in vitro is regulated by SDF-1 and HGF-1 and by the CXCR4 c-met axes, respectively, lends further support to our contention [26].

It should be appreciated that the in vitro migration assay recapitulates only a small part of the process of MSC migration to tumors or bone marrow. In vivo, it has been suggested that a small number of MSCs are circulating and migrate specifically to sites of injury, inflammation, or tumor growth [27]. The transwell chamber assay reflects only chemotaxis of MSCs. It is known that there are few MSCs in the postnatal circulation ([27] and references therein). However, MSCs populate sites of injury, inflammation, and tumor growth. It is likely that MSCs, being pluripotent cells, proliferate "on site" and produce prosurvival signaling molecules in an environment that is otherwise not conducive to their survival. SDF-1 and other chemokines produced on site by MSCs may participate in all three functions (that is, survival, proliferation, and migration toward the tumor microenvironment). In addition to SDF-1, MSCs exposed to CM produce a number of additional chemokines, including CXCL-2 and CCL2, both of which have previously been demonstrated to protect cardiomyocytes from apoptosis during myocardial ischemia [28].

Based on our preliminary data, we suggest a model, shown in Figure 6, for recruitment of MSCs to tumor microenvironment and activation. We are currently attempting to identify soluble factors produced by tumor cells as well as the downstream signal transduction pathways after interaction of constituents of tumor cell CM with chemokine/cytokine receptors on MSCs to understand how these factors act on MSCs. It will also be important to understand whether increased SDF-1 production is a direct or indirect consequence of such ligand receptor interaction.

View larger version (17K): [in this window] [in a new window]   Figure 6. Model showing putative molecular mechanisms underlying activation of MSCs. 1: Secretion of chemokines/cytokines from tumor microenvironment recruits MSCs to tumor sites. 2: Tumor microenvironment-derived cytokines interact with receptors on MSC surface and activate signal transduction, leading to increased SDF-1. 3: Increased SDF-1 acts in an autocrine manner to increase survival, proliferation, and migration of MSCs in the tumor microenvironment. Abbreviations: MSC, mesenchymal stem cell; SDF-1, stromal cell-derived factor-1.

Additionally, it will be important to determine the role of chemokine/cytokine factors in differential localization of MSCs to target sites such as tumors, bone marrow, and wound and injury sites. Our data show that there is differential gene regulation in MSCs exposed to different microenvironments and that these changes influence an important MSC function such as chemotaxis. Elucidating the full repertoire of SDF-1/CXCR-based signaling in MSCs as well as identifying other pathways critical to microenvironment-specific MSC biology will be important in understanding the role of MSCs in clinically important processes such as tumor growth and metastasis, bone marrow engraftment, and wound healing.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
We thank Dr. Tulin Budak-Alpdogan and Dr. Daniel Medina for help and guidance with CFDA-SE labeling and flow cytometry studies. We are indebted to Dr. Joseph R. Bertino and Dr. Barton A. Kamen for critical reading of the manuscript. This study was supported by National Institutes of Health CA-86438-05, a Collaborative Research grant from the Cancer Institute of New Jersey, and research grants from The New Jersey Commission on Cancer Research (05-2406-CCR-EO), The New Jersey Commission on Science and Technology (HESC-06-04-00), The Beez Foundation, and The Foundation of UMDNJ.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 

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