Mesenchymal stem cells: A promising targeted-delivery vehicle in cancer gene therapy

Journal of Controlled Release 147 (2010) 154–162

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Review

Mesenchymal stem cells: A promising targeted-delivery vehicle in cancer gene therapy Yu-Lan Hu a, Ying-Hua Fu b, Yasuhiko Tabata c, Jian-Qing Gao a,⁎ a b c

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, PR China Department of Pharmaceutics, School of Medicine, Jiaxing University, PR China Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Japan

a r t i c l e

i n f o

Article history: Received 29 March 2010 Accepted 12 May 2010 Available online 19 May 2010 Keywords: Targeting Mesenchymal stem cells Vehicle Gene delivery Cancer

a b s t r a c t The targeting drug delivery systems (TDDS) have attracted extensive attention of researchers in recent years. More and more drug/gene targeted delivery carriers, such as liposome, magnetic nanoparticles, ligandconjugated nanoparticles, microbubbles, etc., have been developed and under investigation for their application. However, the currently investigated drug/gene carriers have several disadvantages, which limit their future use in clinical practice. Therefore, design and development of novel drug/gene delivery vehicles has been a hot area of research. Recent studies have shown the ability of mesenchymal stem cells (MSCs) to migrate towards and engraft into the tumor sites, which make them a great hope for efficient targeteddelivery vehicles in cancer gene therapy. In this review article, we examine the promising of using mesenchymal stem cells as a targeted-delivery vehicle for cancer gene therapy, and summarize various challenges and concerns regarding these therapies. © 2010 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . The gene recombination of MSC . . . . . . . . . . . . . . . . . . 2.1. Viral vectors . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Non-viral vectors . . . . . . . . . . . . . . . . . . . . . . 2.3. Three dimensional and reverse transfection systems . . . . . . 3. MSCs as a promising targeted-delivery vehicle in cancer gene therapy . 3.1. Rationale for using MSCs as a vehicle for gene delivery. . . . . 3.2. The target of MSCs to tumor cells . . . . . . . . . . . . . . . 3.3. MSC as a tumor target vehicle for gene delivery . . . . . . . . 4. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction With the development of molecular biology, cancer gene therapy becomes a promising field in the treatment of malignant tumor. Cytokines, such as IL-2, IL-12 and IFN-β et al., can stimulate antitumor responses through the activation of T cells, which mediates ⁎ Corresponding author. 388 Yuhangtang Road, Hangzhou 310058, Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, PR China. Tel.: +86 571 88208437. E-mail address: [email protected] (J.-Q. Gao). 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.05.015

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immune response to eliminate tumors. Nevertheless, the therapeutic application of exogenously administered cytokines is limited by their short half-lives and poor accessibility to tumor sites [1]. These cytokines exhibit rapid blood clearance and poor retention time in the target, which results in the necessity for the frequent administration of such agents. Therefore, therapeutic utility of these cytokines in vivo is limited by its excessive toxicity when administered systemically at high doses and with high frequency [1]. Additionally, previous studies have largely relied on viral vectors to deliver these therapeutic genes, which are associated with safety concerns [2] and limit the clinical application of these cytokines.

Y.-L. Hu et al. / Journal of Controlled Release 147 (2010) 154–162

Targeted delivery of anticancer agents is one of promising fields in anticancer therapy. A major disadvantage of anticancer agents is their lack of selectivity for tumor tissue, which causes severe side effects and results in low therapeutic efficiency. Therefore, tumor-targeting approaches have been developed for improved efficiency and minimizing systematic toxicity by altering biodistribution profiles of anticancer agents. In recent years, the targeting drug delivery systems (TDDS) attracted extensive attention of researchers. More and more drug/gene targeted delivery carriers, such as stealth liposome [3], magnetic nanoparticles [4], ligand-conjugated nanoparticles [5], and ultrasound microbubbles [6], have been developed and under investigation for their tumor target efficiency and effectiveness for cancer treatment (Table 1) [7]. However, these drug/gene delivery vehicles are limited by their several disadvantages. For example, the magnetic nanoparticles have low drug loading capacities, nonuniform particle size distribution, and are prone to form agglomerates that may lead to an occlusion of capillaries [4]. Also, the rapid recognition and clearance of liposome themselves by the reticuloendothelial system (RES) from blood stream, limited the usefulness of liposomes as drug carriers (Table 1). Cell-based therapies are emerging as a promising therapeutic option for cancer treatment. However, the clinical application of differentiated cells is hindered by the difficulty in obtaining a large quantity of cell number, their lack of ability to expand in vitro, as well as the poor engraftment efficiency to targeted tumor sites. Mesenchymal stem cells (MSCs) have been attractive cell therapy vehicles for the delivery of agents into tumor cells because of their capability of self-renewal, relative ease of isolation and expansion in vitro, and homing capacity allowing them to migrate toward and engraft into the sites of tumor [36]. Several studies have provided evidences supporting the rationale for genetically modified MSC to deliver therapeutic cytokines directly into the tumor microenvironment to produce high concentrations of anti-tumor proteins at the tumor sites, which have been shown to inhibit tumor growth in experimental animal models. The anti-tumor effects of intravenous injections of gene-modified MSCs have been demonstrated in lung, brain, and subcutaneous tumors [33–35,37].

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2.2. Non-viral vectors An increasing number of non-viral vectors are being developed for the purpose of gene delivery nowadays. These vectors have several advantages such as ease of synthesis, cell/tissue targeting, low immune response, and unrestricted plasmid size [34,51]. Till now, a variety of non-viral delivery carriers, including calcium phosphate [52], liposomes [53], and niosomes [54], as well as the nanoparticles [55], have been under development [56]. More recently, a novel transfection vector, spermine–pullulan, was synthesized by cationization pullulan with the chemical introduction of spermine [57]. We compared its transfection efficiency on rat MSCs with other vectors, including peptide TAT modified polyethylenimine (PEI)-beta-cyclodextrin (Tat-cyd), PEI-cyclodextrin (Cyd), and Lipofectamine2000 (Lipofectamine2k, Invitrogen) (Fig. 1). It was proved that the transfection efficiency of spermine–pullulan was equal to that of Cyd and was higher than that of Tat-cyd and lipofectamine2000, indicating spermine–pullulan could be used as an effective non-viral gene transfection vector [58]. Although the non-viral vectors hold promise in delivering therapeutic genes to MSCs, most current studies on these vectors are still limited to the in vitro evaluation of their transfection efficiency. As viral vectors can integrate into the host genome, they could result in a stable and long-term gene expression in vivo [59]. However, the gene expression by the non-viral system is transient, so that this system is not suitable to carry out the studies involving the cells for the treatment of diseases for which gene expression is required over long periods of time, as the non-viral vectors have a shorter expression time for the transgenes and a relatively lower transfection efficiency. It was reported that non-viral gene carrier also can prepare cells stably expressed through an antibiotic selection [60]. However, this procedure showed not practical, as it takes a long time to perform and to optimize the conditions. Therefore, to tackle this issue, the controlled release of anticancer agents inside the cells or in the tissue will be promising. Hence, targeting drug delivery system (TDDS) could be practically used to modify and regulate the level and time period of gene expression [57]. In this case, MSCs could be used for the targeted delivery of tumor therapeutic genes transfected by non-viral vectors for their engraftment efficiency to tumor sites.

2. The gene recombination of MSC 2.3. Three dimensional and reverse transfection systems To develop MSCs as therapeutic agents, efficient gene transfer to the cells is a prerequisite. The strategies for gene delivery into MSCs include using viral vectors, non-viral vectors and three dimensional/ reverse transfection systems.

2.1. Viral vectors The vector of retrovirus, lentivirus, adenovirus, and adenoassociated virus has been widely used for gene transfer. Viral method using viral vectors for gene transfer to tumor tissue is proved to be a very effective approach, which allows the achievement of high transfection efficiency. In a series of studies, fiber-mutant adenovirus vectors were developed and used for cancer gene therapy and induced significant anti-tumor activity when cytokines and/or chemokines were employed as therapeutic genes [38–42]. Furthermore, viral vectors modified with PEG induced the evasion ability to against the neutralizing antibody and the accumulation of vectors in tumor tissue as well as the effective tumor-targeted gene transfer [38,43]. A variety of studies have been reported to use different viral vector systems to transduce MSCs [44–47]. However, many clinical trials in which viral vectors were used have been terminated since the application of these vectors had induced unexpected adverse effects such as toxicities, immunogenicity and oncogenicity [48–50].

Although a great improvement of the transfection efficiency has been made by using non-viral vectors, in most cases, non-viral systems could not reach the high transfection efficiency and allow long-term transfection as viral vectors do. To enhance the transfection efficiency, several other transfection approaches, such as using reverse transfection method and/or three dimensional (3D) systems, were developed currently. By optimizing the transfection system, such as changing the order of adding gene complexes and cells, cell culture environment can be greatly improved, not only in the increased transfection efficiency of the gene carrier, but also in a longer gene expression time [56]. The technology and methodology of gene transfection have become more and more important to enhance the efficiency of gene therapy for several diseases. As the transfection system can greatly affect the growth status of cells, the sensitivity of the carriers to gene uptake as well as the condition of carriers in serum [57]. Up to now, reverse transfection approach, which is different from the conventional approach, has been demonstrated to be able to protect the carriers from the inhibit effects of the serum. In our studies, it was shown that using the reverse method, a high transfection efficiency of MSCs was observed even in the presence of serum. For the conventional method, in which the complex is added to the culture medium, the presence of serum reduced the transfection level to 25 ± 1.5% and 1.25 ± 0.5% using PEI and spermine–pullulan as the vector,

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Table 1 Current targeting drug delivery systems (TDDS) for cancer therapy. TDDS Nanocarriers

Disadvantages

References

1) Can carry both hydrophilic and hydrophobic agents. 2) Are composed of natural material and essentially non-toxic.

[8–10]

Polymeric nanoparticles

1) Could reduce dosage and minimizes side effects; 2) Protects drugs against degradation and enhances drug stability 3) Potential for tumor targeting and could improve cancer therapy

Microbubbles

1) Used for site-specific delivery of the bioactive agents 2) Could reduce the dosage 1) Are relatively safe after entering into the body 2) Have sustained-release properties 1) Inert and non-toxic 2) Ease of synthesis 3) Ready functionalization 4) Have photophysical properties which could trigger drug release at remote place 1) Has high selectivity and specificity for tumor site

1) Rapidly cleared from the bloodstream by the reticuloendothelial defence mechanism. 2) Instability of the carrier and burst drug release 3) Non-specific uptake by the mononuclear phagocytic system (MPS) 1) Inherent structural heterogeneity of polymers 2) Low drug loading capacities 3) Prone to form agglomerates and may lead to an occlusion of capillaries 1) Burst drug release 1) Physiochemical stability problems

[15]

1) Not biodegradable or small enough to be cleared easily 2) Potential accumulation in the body and may cause long-term toxicity.

[16,17]

1) The receptor may be expressed on normal cells and may cause side effects 2) Low ligand conjugation efficiency 3) The different expression level of antigen/receptors on different tumor cells may decrease the site-specific targeting 1) Only effective for tumor sites close to the body's surface and sites deeper within the body are difficult to target. 1) Difficulty in obtaining sufficient numbers 2) Not easy to transducing these cells with the plasmids. 3) Suffer from the immunosuppressive nature of many tumors. 1) The vectors and cytokines used are limited 2) Little was understood about the fate of MSCs in vivo

[18–21] [22–25]

Nano-emulsions Inorganic nanoparticles

Surface modified delivery system

Antibody-conjugated delivery system Ligand-conjugated delivery system

Other carriers

Magnetically targeted drug carrier

Cell-based drug carriers

Immune cells (macrophages, T cells, natural killer (NK) cells) MSCs

1) Magnetic fields are focused over the target site and allow the particles to be captured at the target sites. 1) Could enhance the tumor site target efficiency and improve the therapeutic effect. 2) Have tumoricidal activity. 1) Easily available and to expand by simple propagation procedures. 2) Have high migration potential to the tumor sites 3) Have a low immunogenicity and intrinsic mutation rate 4) Do not possess neurotoxicity or tumorigenicity

[11–13]

[14]

[26–30] [31,32]

[33–35]

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Advantages Liposome

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Fig. 1. Transfection efficiency of different non-viral vectors, including spermine– pullulan, peptide TAT modified PEI-beta-cyclodextrin (Tat-cyd), PEI-cyclodextrin (Cyd), and Lipofectamine2000 (Lipofectamine2k). For the transfection assay, known amount of MSCs was seeded into the 24-well plate and cultured for 18 h before transfection. Vector/DNA complex was prepared by mixing 1 µg of pGL3 plasmid with appropriate amounts of vector. The vector/DNA complex was added to the 24-well plate and incubated for 6 h at 37 ° C under 5% CO2 atmosphere in serum-free DMEM. Then the DMEM was replaced with the fresh DMEM with 10% serum. After 96 h, the luciferase assay was carried out according to the manufacturer’s instruction (Promega, USA). Light units (LUs) due to luciferase activity were measured with chemiluminometer (Autolumat LB953, EG&G Derthold, Germany). All the experiments were carried out in triplicate to ascertain the reproducibility.

respectively (Fig. 2). Also, the reverse method employing biodegradable or non-biodegradable three dimensional scaffolds have been reported [56,61,62]. 3. MSCs as a promising targeted-delivery vehicle in cancer gene therapy 3.1. Rationale for using MSCs as a vehicle for gene delivery In 1987, Friedenstein et al. [63] found the bone marrow single-cell can differentiate into bone, cartilage-forming, adipocytes cells under certain conditions. These cells retain the ability of forming bone and cartilage after being transplanted in diffusion chambers after 20–30 cell doublings in vitro, and were called as mesenchymal stem cells or

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bone marrow stromal cells. Mesenchymal stem cells could also be isolated from other tissues, such as adipose tissue [64] and placenta [65]. It was reported [63] that human mesenchymal stem cells had been thought to be multipotent cells, which are present in adult marrow, that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Stem cells, which have the characteristics of human mesenchymal stem cells, were isolated from marrow aspirates of volunteer donors. And it was shown [66] that these cells displayed a stable phenotype and remained as a monolayer in vitro, and could be induced to differentiate into several mesenchymal lineages, including osteoblasts, adypocytes and chondrocytes. It was also find by Pittenger et al. [66] that individual stem cells retained their multilineage potential when they were expanded to colonies. Several properties of MSCs favor the development of engineered MSCs as a vehicle to deliver therapeutic genes aimed for cancer therapy (Table 1): (1) MSCs are easily obtained using less invasive methods and easy to expand by simple propagation procedures in vitro [67]. It was reported that they can be expanded for more than 50 population doublings in culture without the loss of their phenotype and multilineage potential [65]; (2) MSCs have high migration potential to the injury or tumor sites [68]. Using a human–sheep in utero xenotransplantation model, Mackenzie et al. [69] demonstrated that human MSC have the ability to engraft, undergo site-specific differentiation into multiple cell types, and survive for more than 1 year in fetal lamb recipients. Also, MSC-derived cells appear to be present in increased numbers in wounded or regenerating tissues. Tumor microenvironments are pathologically altered tissues that resemble unresolved wounds [70–72], which favor the homing of MSCs to the tumor sites; (3) MSCs have a low immunogenicity and intrinsic mutation rate. The low immunogenicity of MSCs is associated with the lack or low expression levels of the expression of MHC class I and class II molecules as well as costimulatory molecules [73]. No immunosuppression was observed when MSCs were engrafted into the bone marrow cavities of the recipient rats for at least 13 weeks after transplantation. Therefore, the MSCs are adult stem cells with unique immunologic tolerance allowing their engraftment into a xenogeneic environment, while preserving their ability to be recruited to an injured myocardium through the blood circulation [74]; (4) MSCs do not possess neurotoxicity or tumorigenicity. Sato et al. [75] injected EGFR-MSCs to the C57BL/6 background athymic mice and no tumors were found during 100 days observation, supporting the lack of tumorigenicity of these cells; (5) It is possible to genetically modify MSCs for the purpose of expressing therapeutic proteins and secreting these proteins into the tumor microenvironment [76,77]. These features have led to the suggestion that MSCs may be useful as cellular vehicles for gene delivery to multiple tumor sites. 3.2. The target of MSCs to tumor cells

Fig. 2. The transfection efficiency of non-viral vector using different transfection methods. (A) PEI;(B) spermine–pullulan. For the reverse transfection, the reverse gene transfection system was composed of anionic gelatin, pronectin and gene complexes, all of which were coated on the surface of culture substrate before the introduction of MSCs. And the level of gene expression was compared with that of cells transfected in the medium containing the gene complexes (conventional transfection method) either in the presence or absence of serum.

Recent studies have shown the ability of MSCs to migrate to and incorporate within the connective tissue stroma of tumors [33,78]. This property of MSCs can be used to achieve targeting anti-tumor agents to tumor cells and their micro-metastases with an improvement in murine tumor models of glioma [33,79], melanoma [68], and breast [35] and colon [80] cancers. The ability of MSCs to migrate toward gliomas has been assessed both in vitro and in vivo [33,75]. In vitro Matrigel invasion assays demonstrated that the MSCs derived from human bone marrow (hMSCs) have the capacity of migration towards gliomas. Furthermore, the tropism of these hMSCs for gliomas may be mediated by specific growth factors/chemokines. It was also observed that murine MSCs transfected with epidermal growth factor receptor (EGFR) could enhance migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFRMSC may be partially dependent on EGF–EGFR, PI3-, MAP kinase, MAP

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kinases, protein kinase C, and actin polymerization [75]. Another in vivo test indicated that MSCs can localize to human gliomas either after regional intra-arterial delivery or after local intracranial delivery [33]. It was also reported that intravenous injection of MSC-IFN-beta cells into mice with established MDA 231 or A375SM pulmonary metastases led to the incorporation of MSC in the tumor architecture [35]. However, in the healthy organs examined, no engraftment of intravenously administered MSCs was observed [35], indicating MSCs themselves may not cause side effects on the health organs. As compared with the tumor-targeted nanocarrier systems, which simply involves the ligand–receptor interaction, more factors were implicated in the homing of MSCs to sites of tumor, and therefore, a higher tumor target efficiency of MSCs would be expected (Fig. 3). However, the processes and factors underling the migration of MSC to tumors sites have not been well characterized. Till now, two possible mechanisms have been proposed (Fig. 3). (1) Secretion of chemokines/cytokines from tumors tissues increases the migration of MSCs. The tumor tropism of MSCs might be mediated by several receptor– ligand combinations [81]. Cytokines, such as vascular endothelial cell growth factors, transforming growth factors (TGFs), fibroblast growth factors (FGFs), platelet-derived growth factors, monocyte chemoattractant, protein-1, and IL-8 released from the neoplasm or inflammatory tissue are possible factors that mediate the activation of MSC migration [34,82]. It is already known that these factors released from cancer cells promote the migration of endothelial cell and stromal cell progenitors from the bone marrow towards the cancer bed [83,84] or tissues surrounding the tumor, therefore enhancing the formation of tumor-stroma [85]. Similar mechanisms would be anticipated for tumor-stromal formation in glioma, and the migration of implanted MSCs. Additionally, adhesion molecules, such as b1- and b2-integrins and L-selectin, may also play a significant role in the mobilization and homing of MSCs to gliomas [86,87]. MSCs injected intratumorally are mostly distributed at the border zone between tumor and normal parenchyma. They develop a capsule-like structure, and also infiltrate into the tumor bed relatively uniformly [34]. Tissue repair is a balance between damage and repair. When the balance is broken, the injured vessel requires the recruitment of more progenitor cells which contribute to lesion formation. MSCs exhibit multipotent differentiation potential, and have been shown to give rise to different mesodermal cell lineages, including osteoblasts, chondroblasts, and adipocytes under proper experimental conditions

both in vitro and in vivo [88]. Therefore, MSCs could migrate towards the tumor site and participate in the formation of tumor stroma, which provides a new strategy for tumor therapy. Using MSCs as a tumor-targeted vehicle for the delivery of tumor therapeutic gene may decrease the side effects of these genes. For example, Studeny et al. [68] demonstrated that bone marrow-derived mesenchymal stem cells (MSCs) transducted with an adenoviral vector carrying the human β-IFN gene can produce biological agents locally at tumor sites. They also showed that MSCs with enhanced expression of IFNbeta inhibited the growth of malignant cells in vivo. Importantly, this effect required the integration of MSCs into the tumors, and therefore could not be achieved by systemically delivered IFN-beta or by IFNbeta produced by MSCs at a site distant from the tumors. These results indicated that MSCs may serve as a platform for delivering biological agents into tumors. The successful engraftment of MSC in tissues would most likely triggered by tissue damage or tumor growth, which makes MSC excellent candidates for the cell-based delivery of therapeutics to tumor sites [72]. (2) The interaction of the cytokines or chemokines with its corresponding receptors would induce the migration of MSCs towards tumor microenvironment. These receptors, such as CXCR4, CX3CRl, CXCR6, CCRI, CCR7 etc., were expressed on MSCs, and could interact with their respective ligands CXCLl2, CX3CLl, CXCLl6, CCL3 or CCLl9 [89]. Currently, CXCR4 and its receptor, stromal cell-derived factor-1 (SDF-1), are thought to be the most import pair cytokines in attracting of MSCs to migrate to the tumor, as CXCR4-SDF-1α interaction plays an important role in inflammation, tumor tropism of stem cells and the pathology of gliomas [90]. Therefore, the microenvironment of the tumor site plays an important role in the migration of MSCs [91]. Also, a better understanding of the signaling transduction pathways associated with the tropism of these MSCs to gliomas will help to elucidate the role of MSCs in tumor Table 2 MSCs used for tumor delivery of anticancer agents. Animal model Cytokines and chemokines

Results

References

IL-2

Rat glioma model

[34]

IL-12

Mouse xenograft model of renal cell carcinoma (RCC)

IFN-β

Mouse prostate cancer lung metastasis model Mice metastatic lung Tumors (MDAMB231) Mice metastatic lung Tumors (LLC)

Gene-modification of MSCs by infection with an adenoviral vector encoding human interleukin-2 (IL-2) augmented the anti-tumor effect and prolonged the survival of tumorbearing rats. systemic administration of MSC/ IL-12 reduced the growth of 7860 RCC and significantly prolonged survival of tumorbearing mouse. A significant reduction in tumor volume in lungs following IFN-β expressing MSC therapy. TRAIL-expressing MSCs were able to reduce tumor growth and clear the metastatic disease. Intratracheal administration of MSCs expressing CX3CL1 suppressed the growth of multiple lung metastases and prolonged survival in mice. The active homing of MSCs into primary pancreatic tumor stroma and activation of the CCL5 promoter was verified using eGFP- and RFP-reporter genes. In the presence of ganciclovir, HSV-Tk transfected MSCs led to a significant reduction of primary pancreatic tumor growth and incidence of metastases.

TRAIL

CX3CL1

CCL5

Fig. 3. Schematic of nanocarrier systems and MSCs for site-targeted drug/gene delivery (modified from [5,92]). FR: Folate receptor; EGFR: Epidermal growth factor receptor.

Mice primary pancreatic tumor

[101]

[99]

[98]

[80]

[102]

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growth and may permit more efficient targeted delivery of MSCs to desired sites for therapeutic purposes [92]. 3.3. MSC as a tumor target vehicle for gene delivery The evidence that tumor microenvironment favors the homing of exogenous MSCs have support the rationale for developing engineered MSCs as a tool to track tumor sites, and deliver anticancer agents within these area. Several reports have proven the efficiency of MSCs as cell carrier for in vivo delivery of various clinically relevant anticancer agents following engraftment within tumor sites (Table 2). The observation that unmodified MSCs could have an anti-tumorigenic activity further support the use of MSCs in cancer immunotherapy [93]. Besides, MSCs are proved to be effective in delivering oncolytic adenovirus for the treatment of cancer with low systemic toxicity [94,95], suggesting MSCs are promising cell vehicle for cancer therapy. Several research groups have reported promising results following the injection of genetically engineered mesenchymal stem cells in animal models bearing different tumors [35,68,96,97]. For example, Nakamura et al. [34] used gene-modified MSCs as a new tool for gene therapy of malignant brain neoplasms. Gene-modification of MSCs by infection with an adenoviral vector encoding human interleukin-2 (IL-2) clearly enhanced the anti-tumor effect and further prolonged the survival of tumor-bearing rats, indicating that gene therapy employing MSCs as a targeting vehicle is promising as a new therapeutic approach for brain tumor. Studeny et al. [35] injected the MSCs transduced with an adenoviral expression vector carrying the human IFN-β gene into mice with MDA231 or A375SM pulmonary metastases, resulting in suppression of pulmonary metastases and prolonged survival in mouse. This suggests MSC may be an effective platform for the targeted delivery of therapeutic proteins to cancer sites. Loebinger et al. [98] also demonstrated that MSCs could accumulated in tumor tissues (Fig. 4) and the tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)-expressing MSCs were able to inhibit tumor growth, suggesting that the TRAIL-expressing MSCs has a wide potential therapeutic application, which includes the treatment of both primary tumors and their metastases. Also, MSCs were transduced by the rAAV-IFN-b or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-

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C2 prostate cancer cells in a therapy model. Results indicated a significant reduction of tumor volume in lungs following IFN-β expressing MSC therapy. Immunohistochemical examination of the lung tissue demonstrated an increase in tumor cell apoptosis, and a decrease in both tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-b therapy correlating the anti-tumor effect. Systemic level of IFN-b was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-b therapy for prostate cancer lung metastasis [99]. Currently, most studies on gene-modified MSCs for the treatment of tumors are using viral vectors for gene transfer [100]. However, they may be associated with safe problems. Recently, we employed non-viral vector polyethylenimine (PEI) to transfer IL-12 into MSCs for the treatment of lung metastases in C57BL/6 mice, and found that MSC-IL12 injected mice showed significantly less lung metastases numbers as compared with the direct injection of IL-12 and vector (data not shown).Our study supports the effectiveness of non-viral vectors in transferring the therapeutic gene to MSCs. 4. Future perspectives Targeted delivery of anticancer drugs/genes to tumor cells/tissues can improve the therapeutic index of drugs by minimizing their toxic effects. Currently, a variety of delivery systems have been employed for developing TDDS for anticancer agents to enhance their therapeutic values [103]. For instance, nanocarrier drug delivery systems were designed to reach target cells and tissues or respond to stimuli in a well-controlled manner to induce desired physiological responses [7]. Also, cell- or tissue-specific receptor/antigens can provide a useful target for targeted delivery of anticancer drugs (Fig. 3). Clinical developments of anticancer agents utilizing TDDS are now only at their initial stages by pharmaceutical companies. And the application of MSCs to targeted gene delivery has been the subject of current experimental studies and may bring new hope to cancer patient. After transfecting MSCs with tumor therapeutic gene, the MSCs would deliver the gene towards tumor sites, which could be widely used in tumor therapy. Besides, recent evidence also suggests that MSCs selectively migrate to areas of injury/inflammation, where they are involved in the tissue repair. This effect makes MSCs as attractive

Fig. 4. DiI-labeled MSCFLTs (red) injected intravenously at day 10 and shown to localize to lung metastases on fluorescent microscopy with DAPI nuclear counterstain with H&E contiguous sections from day 30 harvested lungs (magnification, ×10 µm; bar, 20 µm and magnification, ×4; bar, 60 µm). Reproduced by the kind permission of the American Associate for Cancer Research (AACR) from [98].

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candidates of widely used delivery vehicles for site-specific therapy. In the future, these strategies could be extended by using various genes to achieve a general cell-based gene therapy platform for the treatment of different disease. MSCs have attracted considerable attention as novel tools for the delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into these cells would create a number of therapeutic opportunities [104]. However, the potential concerns in using MSC as delivery vehicles are we understand little about the fate of this cell population in vivo [105,106] and the possibility that MSC themselves might enhance or initiate tumor growth [107–109]. Moreover, although more and more studies using gene-engineered MSCs for the treatment of tumor disease and they are considered as a powerful therapeutic weapon for the targeting of advanced malignancies [100], till now, the cytokines and the vectors used are still limited. The use of MSCs for cancer gene therapy usually provides some degree of tumor selectivity, however, strategies to improve tumor homing might greatly increase their applicability as tumor-targeted cell delivery vehicles [31]. Cell homing ability may be enhanced by using specific culture agents or medium treatments to alter the expression profile of cell surface receptors [110,111]. It might be possible to manipulate cell targeting or homing properties by preprogramming the MSCs to alter their properties during the gene delivery process to make them the most effective in targeting the tumor site. This approach represents another option of investigation to enhance the efficiency of cellular vehicle strategies. Still, much work remains to be done before the clinical application of MSC-based therapy. The development of optimally targeted cellular vectors will require both the advances in the molecular and cell biology and the improvement in the methods for gene transduction to cells. A lot of efforts still need to be made to develop delivering systems capable of effectively transporting various therapeutic cytokines. Together, the further development of MSC-based therapy will benefit from a better understanding of biology, tissue stem cell engineering, pharmaceutics, as well as gene therapy.

[9] [10] [11] [12]

[13]

[14] [15]

[16] [17] [18]

[19]

[20]

[21]

[22] [23] [24]

[25] [26] [27]

Acknowledgement This work was financially supported by National Natural Science Foundation of China (30873173, 30973648), Zhejiang Provincial Natural Science Foundation of China (R2090176) and China–Japan Scientific Cooperation Program (81011140077) supported by both NSFC, China and JSPS, Japan. We would like to thank Dr. Guping Tang (Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University) for providing Cyd and TAT-cyd and thank Ms. Cai-Xia He for technical assistance.

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