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TRANSLATIONAL AND CLINICAL RESEARCH: MESENCHYMAL STEM CELLS SERIES |
aDepartment of Molecular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan;
bDepartment of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan;
cLaboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Osaka, Japan;
dPharmaceutical and Medical Devices Agency, Tokyo, Japan
Key Words. Mesenchymal stem cell • Gene therapy • Multiple tumors • Lung metastases
Correspondence: Yasuo Saijo, M.D., Ph.D., Department of Molecular Medicine, Tohoku University Graduate School of Medicine, 2-1 Seiryomachi Aobaku, Sendai 980-8575, Japan. Telephone: 81-22-717-8230; Fax: 81-22-717-7882; e-mail: yasosj{at}idac.tohoku.ac.jp
Received July 23, 2006;
accepted for publication March 28, 2007.
First published online in STEM CELLS EXPRESS April 5, 2007.
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ABSTRACT |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Some types of cells migrate to and reside in the tissues after systemic injection. Therefore, genetically modified cells coupling cell therapy and gene therapy may be able to deliver certain genes to the target sites. Several types of cells, including fibroblasts [1], endothelial cells [2], dendritic cells [3], and tumor-infiltrating lymphocytes [4–6], have been used to deliver therapeutic agents into tumor tissues. However, difficulties related to in vitro cell expansion, low efficiency of gene transfer to the cells, or the need for direct injection to the tumor tissue because of poor migration to the tumor sites have limited the clinical application.
Bone marrow-derived MSCs are adherent, nonhematopoietic cells that reside within the bone marrow stroma and regulate the differentiation of hematopoietic stem cells [7]. These cells are pluripotent and have the ability to differentiate into various mesoderm-type cells, osteoblasts, adipocytes, chondrocytes, myoblasts, and endothelial precursor cells [8–10]. Because MSCs can be relatively easily expanded in vitro and retain an extensive multipotent capacity for differentiation, they are being used to develop therapies for tissue regeneration in animal models [9–11]. Recent data revealed another important biological feature of MSCs. MSCs can selectively migrate to and proliferate in solid tumors after systemic injection and become stromal cells [12]. A few studies have reported that systemic administration of genetically modified MSCs targeting to multiple tumor sites showed antitumor effects in animal tumor models [13–15].
Although adenoviral vectors efficiently infect and highly express the transgene in many types of cells, high titers of the adenoviral vector would be needed to increase the efficiency of transduction into MSCs because of the poor expression of coxsackie adenovirus receptor (CAR) in MSCs [16]. It has previously been shown that an adenoviral vector with the Arg-Gly-Asp (RGD)-4C peptide in the fiber knob (AdRGD) increased the tropism and thus improved the efficacy of transduction into MSCs by over 10 times [16, 17].
CX3CL1 fractalkine is a member of CX3CL and exists in both a membrane-bound form and a soluble form. The soluble form of CX3CL1 induces the migration of cell expressing its receptor, CX3CR1, in a manner similar to that of other soluble chemokines [18–20]. We have previously demonstrated that intratumoral injection of an adenoviral vector expressing CX3CL1 induced strong antitumor effects through the activation of both natural killer (NK) cells and T cells [21]. This treatment affected only the tumors injected with the adenoviral vector but not distant tumors. Therefore, our goal was to develop an effective gene therapy for multiple tumors by MSCs.
In this study, we infected MSCs with AdRGD expressing CX3CL1 and administered them systemically to tumor-bearing mice. Systemic administration of MSCs expressing CX3CL1 resulted in a strong inhibitory effect on lung metastases and thus prolonged the survival of the tumor-bearing mice.
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MATERIALS AND METHODS |
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tm1M
) mice that had been backcrossed to the C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). All animal experiments were approved by the institutional review board for animal experiments of Tohoku University.
Isolation and Culture of Mouse MSCs
The bone marrow of 6–10-week-old BALB/c, C57BL/6, or GFP-expressing mice was flushed out with cultured medium and expelled from a 5-ml syringe through a 25-gauge needle. The marrow suspension was then transferred to six-well plates at a concentration of 1.5 x 106 nucleated cells per cm2. The cells were cultured in low glucose Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Grand Island, NY, http://www.gibcobrl.com) with 10% FBS (Gibco-BRL). After 72 hours, nonadherent cells were removed, and fresh medium was added. When the adherent cells reach 70%–80% confluence, the cells were trypsinized (0.05% trypsin for 3 minutes), harvested, and expanded. When a homogenous cell population was obtained after 3–5 passages, these cells were used for the subsequent experiments.
The induction of adipogenic differentiation from MSCs was performed according to the report by Arai et al. [22]. MSCs were cultured with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 1 µM dexamethasone (Dex), 10 mg/ml insulin, and 10% FBS in DMEM (adipogenic induction medium) for 1 week. Then, the medium was changed to adipogenic maintenance medium (10% FBS in DMEM containing 1 µM Dex and 10 mg/ml insulin) and cultured for an additional 4 days. The cells were fixed in 10% formalin for 10 minutes and stained for 20 minutes with fresh oil red O (Sigma-Aldrich) solution. Osteogenic differentiation was induced by culturing cells for 3 weeks in DMEM supplemented with 10% FBS, 0.2 µM ascorbic acid (Sigma-Aldrich), 10 mM ß-glycerophosphate (Sigma-Aldrich), and 0.1 µM dexamethasone (Sigma-Aldrich). The medium was changed every 3 days. Osteogenic differentiation was detected by Alizarin red S (Sigma-Aldrich) staining.
Adenoviral Vectors
A replication-deficient recombinant adenoviral vector carrying the ß-galactosidase reporter gene LacZ (AdLacZ) under the control of the cytomegalovirus promoter was constructed as previously reported [21]. A genetically modified adenoviral vector with an integrin-binding motif (Arg-Gly-Asp) in the HI loop of the fiber knob carrying fractalkine and ß-galactosidase (AdRGDFKN and AdRGDLacZ) was also constructed as previously reported [23, 24]. These recombinant adenoviral vectors were propagated using 293 cells and purified by CsCl gradient centrifugation. The total numbers of viral particles in the viral sample were measured by optical density (OD)260 (where an OD260 of 1 is equal to 1012 particles). The titers (expressed as plaque-forming units [pfu] per milliliter) of the viral stocks were quantified by a plaque-forming assay using 293 cells.
ß-Galactosidase Staining
To determine the expression of ß-galactosidase after AdLacZ or AdRGDLacZ vector infection, the MSCs were plated 24 hours before infection and incubated with the adenoviruses at different multiplicities of infection (MOIs) for 48 hours. LacZ expression by adenoviral vector (Ad)-transduced MSCs was evaluated by staining with a ß-galactosidase (ß-gal) staining kit (Invitrogen, Carlsbad, CA, http://www.invitrogen.com).
Expression of CX3CL1 Fractalkine and Its Receptor CX3CR1 on MSCs
To determine the fractalkine expression after AdRGDFKN infection (100 MOI), the total cellular RNA was extracted from the AdRGDFKN-transduced (or control-transduced) MSCs (MSCs/RGDFKN) using Isogen (Nippon Gene, Tokyo, http://www.nippongene.com) after 24 hours of infection. Total RNA (2 µg) was converted into cDNA by oligo(dT)12–18 primers and Superscript II reverse transcription (Gibco-BRL) in a final volume of 20 µl. One microliter of this cDNA was amplified with the following primers specific for either vector-derived fractalkine (FKN) or the control ß-actin transcripts: for endogenous FKN, 5'-GTCAGCACCTCGGCATGACGAAATG-3 (sense); for exogenous FKN, 5'-TGCCAAGAGTGACGTGTCCA-3 (sense) and 5'-CACTGGCACCAGGACGTATG-3' (antisense); for CX3CR1, 5'-TTCGGTCTGGTGGGAAATCTG-3 (sense) and 5'-CGTCTGGATGCGGAAGTAG-3 (antisense); for ß-actin, 5'-CTCTTTGATGTCACGCACGATTTC-3' and 5'-GTGGGCCGCTCTAGGCACCAA-3'. The amplification profile was 95°C for 5 minutes and 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds.
To assess the fractalkine expression on the cell membrane of MSCs, after 48-hour infection of AdRGDFKN, the cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 10 minutes, and stained for 30 minutes with a rat monoclonal antibody to murine fractalkine (20 µg/ml) or nonspecific control rat IgG antibodies followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Cells were then visualized after 4,6-diamidino-2-phenylindole counterstaining and examined by fluorescent microscopy. The secreted form of fractalkine in the supernatant of MSCs was measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) according to the manufacturer's instructions. The assay was performed in triplicate.
In Vitro and In Vivo Migration Assay
To examine the biological activity of CX3CL1 secreted from MSCs/RGDFKN, a cell migration assay was carried out as described previously [21]. THP-1 cells (5 x 105 in 100 µl) were seeded in the upper wells of a transwell plate (24-well plate) with a polycarbonate membrane having a 5-µm pore size (Corning Costar, Corning, NY, http://www.corning.com/lifesciences). The lower wells were filled with 600 µl of medium containing supernatants of MSCs/RGDFKN at different doses of MOIs for 48 hours. After a 3-hour incubation at 37°C, the number of cells that migrated through the polycarbonate membrane were harvested from the lower chamber and counted under a microscope. The assay was performed in triplicate. The tropism of MSCs for tumor cells was determined using an in vitro migration assay according to previously described methods [15]. MSCs or fibroblasts (BLKCL4) were suspended at 3 x 106 cells per milliliter in 0.5% FBS containing DMEM, and 100 µl of these cell suspensions were loaded into the upper well of transwell plates (8-µm pore membranes; Corning Costar Inc.). Cell-free medium conditioned by B16F10 cells for 48 hours with 0.5% FBS was put in the lower wells. The MSCs or fibroblasts were allowed to migrate across the membrane for 3 hours at 37°C. After 3 hours, the membrane was disassembled from the chambers, and the cells that remained attached to both sides of the membrane were fixed with methanol. After staining of the cells using a Diff-Quick staining kit (International Reagents Corp., Kobe, Japan), the cells attached to the upper side of the membrane were wiped away, and the cells that migrated to the lower side of the membrane were counted. Cell numbers were expressed as the average number of migrated cells in 10 random fields. The assays were performed in triplicate.
To examine the ability of MSCs to migrate toward tumor tissues in vivo, 7 days after i.v. injection of MSCs or fibroblasts of GFP mice (5 x 105 cells per mouse) into mice with or without established B16F10 pulmonary metastases, halves of organs (lung, liver, kidney, spleen, and bone marrow) were fixed with 4% paraformaldehyde and embedded in paraffin. Halves were used for fluorescence-activated cell sorting (FACS). Sections were treated by autoclave-based antigen-retrieval technique with 10 mM citrate buffer at pH 6.0 and 120°C for 10 minutes. After blocking nonspecific staining and endogenous peroxidase, sections were incubated for 1 hour with rabbit polyclonal anti-GFP antibody (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) and then with Simple Stain Mouse MAX PO (Nichirei, Tokyo, http://www.nichirei.co.jp/english) for 60 minutes and applied to AEC reagents (Nichirei). The specimens were then incubated with hematoxylin for nuclear counterstaining.
Measurement of CX3CL1 Concentration in Lung Tissues
Seven days after MSCs/RGDFKN injection into mice with lung metastases, mice were sacrificed, and the pulmonary circulation was perfused with saline via the right ventricle. The whole lung was homogenized in 1 ml of homogenizing buffer (Hanks' balanced salt solution, pH 7.1) using a tissue homogenizer. The lung homogenates were centrifuged at 15,000 rpm for 20 minutes to sediment tissue debris, and the supernatants were subjected to the ELISA (R&D Systems, Minneapolis, MN, http://www.rndsystems.com) to the measure the fractalkine concentration.
Treatment of Lung Metastases by AdRGDFKN-Transduced MSCs
To establish experimental lung metastases, 5 x 105 tumor cells in 0.2 ml of PBS were injected into the lateral tail vein of the mice (B16F10 into C57BL/6, C26 into BALB/c) (day 0). Five days later, the mice were randomly divided into five groups. Sixteen mice were allocated to each group in all experiments. Mice in the first group received an i.v. injection of 5 x 105 MSCs/RGDFKN. Mice in the second group received an i.v. injection of 5 x 105 MSCs/RGDLacZ as a control for AdRGDFKN. Mice in the third group received an i.v injection of 5 x 105 fibroblasts/RGDFKN as a control for MSCs. Mice in the fourth group received an i.v. injection of AdRGDFKN vector (5 x 107 pfu) without any cells. Mice in the fifth group received an i.v. injection of PBS alone. Eight mice of each group were sacrificed at day 12, and the lungs were fixed in Bouin's solution. The metastatic colonies were easily identified macroscopically by demarcated black or white nodules on the lung surface. The numbers of metastatic nodules on the lung surface were counted three times per sample. The other eight mice of each group were monitored until death for the survival assay. Survival curves were drawn by the Kaplan-Meier method.
Histological Analyses of Infiltrating Immune Cells into Tumors
Three days after i.v. injection of MSCs/RGDFKN, frozen sections of the lung were incubated with optimal dilutions of the primary antibodies, including anti-mouse CD4 (RM4–5; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), CD8 (KT15; Serotec Ltd., Oxford, U.K., http://www.serotec.com), rabbit anti-asialo GM1 (Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english), or isotype matched IgG for 30 minutes. For CD4 and CD8, the sections were then incubated with biotin-labeled second antibody for 30 minutes, followed by streptavidin-horseradish peroxidase. For NK, the sections were incubated with Simple Stain Mouse MAX PO (Nichirei) and then applied to Simple Stain AEC reagents (Nichirei). The specimens were then incubated with hematoxylin for nuclear counterstaining.
For flow cytometry, lung tissues were minced and then incubated for 90 minutes at 37°C in 3% FBS containing medium (3 ml per lung) supplemented with collagenase I (0.7 mg/ml; Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com) and DNase I (30 µg/ml; Roche Diagnostics). After incubation, a single-cell suspension was collected by removing large aggregates and debris by passage through a 70-µm Falcon cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and resuspended at 5 x 106 cells per milliliter for antibody staining (FITC-conjugated monoclonal rat anti-mouse IgGs against CD4, CD8, and NK1.1, respectively [BD Pharmingen]). The cells were then exposed to propidium iodide (1 µg/ml in PBS) to identify dead cells. These cells were analyzed on an EPICS XL cytometer with EXPO32 ADC software (Beckman Coulter, Miami, FL, http://www.beckmancoulter.com). Each group had more than three mice.
Treatment of Lung Metastases in CD8–/– and NK-Depleted Mice
CD8–/– mice received an i.v. injection of 5 x 105 MSCs/RGDFKN or PBS 5 days after the injection of 5 x 105 B16F10 cells into the lateral tail vein. For NK cell depletion, C57BL/6 mice were treated with asialo GM1 antiserum (200 µg per mouse) (Wako Chemical) or the same dose of control IgG by intraperitoneal injection five times on days –3, –2, –1, 4, and 9. Using anti-NK1.1 antibody, it was determined that the NK1.1+ population was less than 1% [21]. These NK-depleted mice and control IgG-injected mice were transplanted with B16F10 on day 0 and treated with an i.v. injection of 5 x 105 MSCs/RGDFKN or PBS on day 5. Each group had eight mice.
Statistical Analysis
The results were expressed as mean ± SE or as mean ± SD. Statistical comparisons were made using the two-tailed Student's t test, and a value of p < .05 was accepted as indicating significance. For the survival data, the log-rank test was used to assess differences among the five treatment groups, and a p value of less than .05 was considered statistically significant.
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RESULTS |
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vß integrin-targeting adenoviral vector were more efficient than those by using conventional unmodified adenoviral vectors.
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As the next step, we determined whether MSCs/RGDFKN expressed CX3CL1 in the lung tumor tissues by ELISA. Treatment of MSCs/RGDFKN increased the CX3CL1 contents in the lung with metastases, but not in the normal lung (Fig. 3C).
Effects of Systemic Administration of MSCs/RGDFKN on the C26 Lung Metastases
We next examined the in vivo effects of MSCs/RGDFKN in the C26 lung metastasis model. MSCs/RGDFKN (5 x 105) were injected intravenously once on day 5 after the injection of C26 cells. Control mice received PBS, MSCs/RGDLacZ as a control for AdRGDFKN, BALB 3T3/RGDFKN as a control for MSCs, or 5 x 107 pfu AdRGDFKN, a dose equivalent to that for infection of MSCs ex vivo. The mice treated with MSCs/RGDFKN developed fewer and smaller metastatic nodules on the lung surface than the mice of the other treatment groups (Fig. 4A). Microscopic observation of the lung confirmed that only mice treated with MSC/RGDFKN developed fewer and smaller nodules than the other treatment groups (Fig. 4B). When the metastatic nodules on the lung surface were counted macroscopically, treatment with MSCs/RGDFKN was shown to have significantly reduced the number of lung metastases compared with any other controls (p < .01). Although mice treated with AdRGDFKN alone showed reduced numbers of metastatic nodules, the inhibitory effect was minimal (36%) compared with PBS and was not statistically significant (p = .83). Mice treated with either MSCs/RGDLacZ or BALB 3T3/RGDFKN did not demonstrate any reduction of lung metastases (Fig. 4C).
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Effects of Systemic Administration of MSCs/RGDFKN on the B16F10 Lung Metastases
We also examined the in vivo effects of MSCs/RGDFKN in the B16F10 lung metastasis model. Again, mice treated with MSCs/RGDFKN developed fewer and smaller metastatic nodules on the lung surface than the mice of the other treatment groups (Fig. 5A, 5B). The number of metastatic nodules in mice treated with MSCs/RGDFKN was markedly reduced by 84% compared with the PBS control (p < .005) (Fig. 5C). Although treatment with MSCs/RGDLacZ reduced metastatic nodules by 43%, this reduction was not statistically significant (p = .3). The mice treated with either 5 x 107 pfu AdRGDFKN or BLKCL4/RGDFKN did not demonstrate any reduction in lung metastases (Fig. 5C).
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NK Cells and CD8+ T Cells Were Involved in the MSCs/RGDFKN-Mediated Antitumor Effects
To investigate the mechanisms of the antitumor effects by MSCs/RGDFKN, histological analyses in the tumors were performed on day 3 after MSCs/RGDFKN treatment. Metastatic lung tumors treated by MSCs/RGDFKN showed infiltrations of CD8+ lymphocytes, CD4 lymphocytes, and NK cells compared with controls (Fig. 6A). Quantification of these leukocytes by FACS revealed significant infiltration of CD8+ lymphocytes and NK cells (p < .05) (Fig. 6B), whereas no statistically significant increase of CD4+ T lymphocytes was observed.
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DISCUSSION |
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In our study, MSCs but not fibroblasts migrated to the culture supernatant of B16F10 cells in vitro. MSCs injected intravenously migrated to the tumor tissues of the lung, but there were only a few in other, normal tissues, as we demonstrated using GFP-positive MSCs, as reported previously [14]. However, we could not demonstrate colony formation of MSCs in the tumor tissues. No colony formation of MSCs might be attributed to the difficulty of expanding mouse MSCs or the short period after MSC injection. The concentration of CX3CL1 in the lung with metastases was significantly increased compared with controls. In addition, the fact that systemic administration of fibroblasts expressing CX3CL1 did not show any inhibitory effects on lung metastasis in C26 or B16F10 suggests that MSCs indeed stayed in the tumor tissues rather than just passing into the vascular circulation of the lung.
Hung et al. recently provided direct evidence for microscopic tumor targeting by exogenously administered human MSCs [12]. They used positron emission tomography (PET) imaging with [18F]-9-(4-fluoro-3-hydroxymethylbutyl)-guanine to monitor the genetically modified herpes simplex virus type 1 thymidine kinase and enhanced green fluorescent protein expressing human MSCs. In vivo PET imaging revealed that human MSCs could target microscopic tumors, proliferate, differentiate, and contribute to the formation of tumor stroma. Although the precise molecular mechanisms by which MSCs migrate to tumor tissues are still unknown, the mechanisms of migration of MSCs to injured organs are beginning to be understood. A subpopulation of MSCs expresses a restricted set of chemokine receptors and shows chemotactic migration in response to the chemokine in vitro [25]. In an in vivo model, this chemotactic migration was principally mediated by CX3CL1 and CXCL12 [26]. Since injured or inflammatory tissues upregulate CXCL12 and CX3CL1, the migration of MSCs to the injured tissues is regulated by the interaction between CXCR4/CXCL12 and CX3CR1/CX3CL1. Moreover, a recent study demonstrated that MSCs express functional c-met and exhibit chemotactic migration toward a hepatocyte growth factor (HGF) gradient and that the combination of HGF and CXCL12 promoted stronger migration of MSCs in vitro [27]. Because solid tumor tissues express and secrete growth factors, cytokines, and chemokines (e.g., CXCL12 and HGF) similar to injured tissues, MSCs are likely to migrate to tumor tissues through these factors.
MSCs have been proposed for cell therapy and cell-based gene therapy because MSCs migrate to the site of tissue injury and differentiate into several types of cells. Although several vectors have been applied for gene transfer to MSCs, the low efficiency of infection remains a challenge [28]. Adenoviral vectors have been widely used for gene transfer because of their efficient gene transfer and high expression of transgenes. However, conventional adenoviral vectors cannot efficiently transduce genes to MSCs because MSCs poorly express CAR [29]. We have previously demonstrated that a modified adenoviral vector could efficiently transduce to MSCs. We generated a fiber-modified adenoviral vector that showed high expression of CX3CL1 on the cell surface, as well as the secreted form of CX3CL1 at 50 MOI [24]. In contrast, in other reports, MOIs of over 1,000 were required to express sufficient amounts of biological products from MSCs [14].
The successful treatments of multiple tumors by engineered MSCs have been reported by Studeny et al. [13, 14]. They treated multiple lung metastases of human tumors in SCID mice by i.v. injection of human MSCs expressing interferon-ß and demonstrated the inhibition of tumor growth in the lung. They extended this therapeutic strategy to the treatment of intracranial human gliomas in nude mice by injection of human MSCs expressing interferon-ß through the carotid artery [15]. Although our study also demonstrated successful treatment of multiple lung metastases by systemic administration of MSCs, we failed to treat metastases to other organs, such as subcutaneous tumors. This discrepancy may be attributed to the difference between human MSCs and mouse MSCs or the low number of migrated MSCs to induce sufficient antitumor effects [30]. Repeated injection of engineered MSCs can be a way to enhance the antitumor effects in other organs. These limitations in the effectiveness should be solved before clinical application.
Neural stem cells also display extensive tropism to gliomas after intravascular administration [31]. Glioblastoma in the rat brain was successfully treated by neural progenitor cells expressing interleukin-4 [32] or the prodrug activating enzyme cytosine deaminase [33]. Although the neural stem cells were obtained from the cortex of rat brain in these experiments, neural stem cell-like cells can be obtained from bone marrow. Genetically modified neural stem cell-like cells migrate to the tumor site and inhibit the growth of U87-MG glioblastoma [34]. Thus, neural progenitor cells derived from bone marrow could be useful for glioblastoma.
Several genes, including immunostimulatory genes and a suicide gene, have been applied for MSC-based cancer gene therapy [13, 32, 33]. We chose CX3CL1 gene for MSC gene therapy for multiple lung tumors because CX3CL1 induces both innate and adaptive immunity [21]. In this study, dominant infiltration of CD8+ lymphocytes and NK cells was observed in the tumors treated by MSCs/RGDFKN. Depletion of NK cells or CD8+ lymphocytes resulted in the complete disappearance of the antitumor effect by MSCs/RGDFKN, suggesting that the effects of MSCs/RGDFKN depend on both innate and adaptive immunity. Although we did not demonstrate in vitro cytotoxic effects by lymphocytes after MSCs/RGDFKN treatment, it is likely that CX3CL1 induced cytotoxic T lymphocytes as we showed previously [21]. All mice eventually died after MSCs/RGDFKN treatment due to incomplete eradication of pulmonary metastases. This incomplete eradication may be due to an insufficient number of migrated MSCs and/or evasion of tumor cells from antitumor immune response induced by CX3CL1.
We isolated and propagated mouse MSCs from bone marrow by their adherence to plastic and confirmed that these MSCs were capable of differentiating to adipocytes and osteoblasts. In several reports, CD45-positive cells were depleted from bone marrow cells before starting the culture for MSCs [35], because cultured adherent cells from bone marrow contain heterogeneous cell populations [36]. Although we did not deplete CD45+ cells for isolating MSCs, we assume that the majority of these adherent cells would be MSCs because these cells could differentiate to multiple mesenchymal lineages.
In conclusion, we have demonstrated that mouse MSCs could be efficiently transduced by an adenoviral vector with the RGD motif. Systemic administration of MSCs could target multiple lung tumors and induce antitumor effects after tail vein injection. MSCs can serve as cellular vehicles to deliver biological agents, which exhibit antitumor effects against multiple lung tumors.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
, MSCs/RGDFKN; 
, MSCs/RGDLacZ; 
, 3T3/RGDFKN; 
, AdRGDFKN vector; 
, PBS. Survival curves were drawn by the Kaplan-Meier method (n = 8 in each treatment group). Abbreviations: AdRGDFKN, adenoviral vector carrying fractalkine; PBS, phosphate-buffered saline; RGDFKN, AdRGD expressing fractalkine; RGDLacZ, AdRGD expressing ß-galactosidase reporter gene LacZ.
