Introduction

Mesenchymal stem cells (MSCs) are non-hematopoietic progenitor cells that can be easily isolated from a variety of tissues including bone marrow, adipose tissue, and the placenta/umbilical cord. They can be identified, expanded, genetically modified, and have the ability to differentiate into a variety of cell lineages, including adipocytes, osteocytes, chondrocytes, muscle cells, and stromal cells in vitro [1]. Recent studies have demonstrated that MSCs are capable of supporting hematopoiesis and can modulate immune responses. Interestingly, MSCs exhibit a homing behavior toward damaged tissue, inflammatory sites, and tumor sites (Fig. 1). Another feature of MSCs is their low immunogenicity because of a lack of expression of co-stimulatory molecules [2]. Therefore, MSCs do not activate the host immune response and escape immunological rejection when injected into HLA-non-identical recipients. These characteristics permit infusion of MSCs into recipients without HLA matching. The tumor site-homing activity of MSCs facilitates their use as cellular vehicles for the delivery of anti-cancer agents specifically to tumors [3]. This targeted therapy can reduce the systemic side effects of anti-cancer agents by facilitating their effective concentration at local tumor sites without elevating systemic concentrations. However, how and why MSCs accumulate at tumors remains poorly understood. Therefore, this review focuses on the application of MSCs for the targeted delivery of anti-cancer agents to tumors, and on the molecular mechanisms of their accumulation in these tumors.

Fig. 1
figure 1

Concept of regenerative medicine and cell therapy with MSCs. In bone marrow, there are different types of tissue stem cells including hematopoietic stem cells and MSCs. MSCs account for a small population of cells in bone marrow, and have the capacity to differentiate into a variety of mesenchymal lineages. Interestingly, MSCs have the ability to accumulate at the site of (1) damaged tissue; (2) inflammatory tissue; and (3) tumors. Therefore, MSCs can be utilized for: (1) regenerative therapy; (2) treatment of GVHD; (3) cancer gene therapy (targeted delivery)

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Applications of genetically engineered MSCs for cancer therapy

An early application of MSCs to targeted cancer therapy was directed delivery of interferon beta (IFN-β). MSCs were transduced with the IFN-β gene, followed by their infusion into mice carrying melanoma xenografts. MSC treatment resulted in reduced tumor growth and prolonged survival of tumor-bearing mice [3]. Based on this observation, genetically modified MSCs from various tissues have since been evaluated for their therapeutic efficacy and their ability to act as cellular vehicles [3–23].

Interleukins (ILs) that regulate inflammatory and immune responses are often used as therapeutic agents. IL-12-expressing MSCs (MSC-IL-12) have been used to improve tumor immunological surveillance by activating cytotoxic lymphocytes and natural killer (NK) cells. Administration of MSC-IL-12 was shown to suppress metastasis and increase tumor cell apoptosis in mice bearing melanoma, lung cancer and hepatoma. Other immunomodulatory cytokines have also been examined for anti-tumor activities, including the T cell attractant CX3CL1. For example, MSC-CX3CL1 inhibited lung metastasis of melanoma and prolonged survival of tumor-bearing mice.

Suicide cancer gene therapy has been considered in the treatment of invasive tumors such as malignant glioma. A number of prodrug activation schemes that convert non-toxic prodrugs into toxic anti-metabolites are available for the selective killing of tumor cells. Cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK), which confer sensitivity to 5-Fluorocytosine (5-FC) and ganciclovir (GCV), respectively, are being evaluated in clinical trials. HSV-TK-expressing MSCs were injected in the vicinity of tumors, reducing tumor volume through bystander-mediated tumor cell killing following administration of GCV. Our group has developed genetically modified MSCs that produce retroviral vectors encoding HSV-TK, with the aim of augmenting the therapeutic efficacy of suicide cancer gene therapy (Fig. 2). MSCs isolated from the bone marrow of Sprague–Dawley rats were transfected with plasmid DNA expressing HSV-TK alone (non-VP-MSCs) or whole retroviral vector components (LTR-HSV-TK with Gag-pol and VSV-G; VP-MSCs) by nucleofection. To evaluate therapeutic efficacy, tumor-bearing nude mice were treated with non-VP-MSCs or VP-MSCs combined with GCV, and the size of subcutaneous tumors was periodically measured. Here, tumor growth was more efficiently suppressed by injection of VP-MSCs compared with non-VP-MSCs. This efficacy was dependent on therapeutic gene amplification through production of retrovirus progeny from MSCs and transduction of tumor cells in situ.

Fig. 2
figure 2

Left panel non-vector-producing MSCs (non-VP-MSCs). Although non-VP-MSCs have the ability to home to tumor sites, local expression of therapeutic molecules is dependent on the continued presence of the MSCs. Right panel vector-producing MSCs (VP-MSCs). The retrovirus progeny produced by MSCs can transduce tumor cells in situ, extending the expression of therapeutic molecules from tumor cells, even when the MSCs have died off

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The efficacy of other therapeutic genes including IFN-α, cytosine deaminase (CD), NK4, and TRAIL has also been examined in various tumor models as described in Table 1. Furthermore, oncolytic virotherapy is emerging as a promising strategy for tumor treatment. MSCs can be employed as carriers to deliver oncolytic viruses to tumor sites. Experimentally, injection of genetically modified MSCs has resulted in tumor growth inhibition, metastasis suppression, and prolonged survival.

Table 1 Studies of cell and gene therapy for cancer that utilize genetically modified MSCs
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Interactions between tumors and MSCs

As detailed above, the use of MSCs to deliver anti-cancer agents is an attractive novel cancer therapeutic strategy. Endothelial cells (ECs), pericytes, and stromal cells are all known to support tumor growth and contribute to the tumor microenvironment by producing various growth factors including vascular endothelial growth factor (VEGF)-A, IL-8, transforming growth factor (TGF)-β, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) [24–29]. MSCs can also act in such a supportive fashion. It is speculated that when MSCs accumulate at tumor sites, they differentiate into pericytes or cancer-associated fibroblasts (CAFs) and are involved in facilitating tumor growth by providing structural support to the tumor microenvironment [3, 30, 31]. Interestingly, breast cancer cells stimulate MSCs and MSCs in turn support cancer cell invasion and metastasis by secreting CCL5 [32]. Conversely, MSCs can also induce apoptosis in tumor cells by blocking phosphorylation of AKT or preventing cell cycle progression [33]. Moreover, MSCs can produce DKK-1 to attenuate the potential for growth and malignancy of tumor cells [34]. As MSCs have dual and opposing effects with respect to tumor growth, modification with anti-tumor genes is required for MSC-based cancer-targeted therapy. Furthermore, it is also necessary to promote more efficient MSC accumulation at tumor sites.

Molecular mechanisms of MSC accumulation at tumors

If it becomes possible to increase the accumulation efficiency of MSCs at tumor sites, MSCs can effectively target not only primary tumors but also metastatic lesions. It is thought that MSCs are mobilized to damaged tissues, such as in injury or inflammation, by the release of inflammatory cytokines. Tumors possess a microenvironment consisting of a large number of inflammatory cells [35]. This microenvironment promotes the recruitment of MSCs via various soluble factors secreted by both tumor and inflammatory cells, including EGF, VEGF-A, PDGF, IL-8, IL-6, fibroblast growth factor, stromal cell-derived factor, granulocyte colony-stimulating factor, granulocyte–macrophage colony-stimulating factor, monocyte chemoattractant protein-1 (MCP-1), hepatocyte growth factor, TGF-β1, and urokinase-type plasminogen activator [36–42]. However, our own work found that while systemically injected MSCs accumulated at tumor sites, subcutaneously injected MSCs did not. We also compared the migration capacity of MSCs and fibroblasts (FBs) toward growth factors and chemokines in vitro, and found that FBs were more strongly attracted to these factors than MSCs [43]. These results suggest that the mechanism of MSC accumulation cannot be explained solely by cytokine-mediated migration.

The tumors generated in the above study possessed tumor stroma with many blood vessels, and MSCs in particular accumulated at the boundaries between tumors and tumor stroma. Furthermore, MSC accumulation at the tumor sites was observed only when cells were injected via the left ventricular cavity. Therefore, we focused on MSC-EC adhesion to elucidate the mechanisms involved. Interestingly, stimulation of MSCs with TNF-α enhanced the adhesion of MSCs to ECs in vitro. This adhesion was partially inhibited by antibodies that block vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4). It is well known that TNF-α induces VCAM-1 expression via the NF-κB signaling pathway. Parthenolide (PTL) has anti-inflammatory activity and suppresses NF-κB activity by inhibiting IκBα phosphorylation after TNF-α stimulation, and PTL strongly inhibited TNF-α-induced VCAM-1 expression on MSCs. In vivo imaging using luciferase-expressing MSCs revealed that the bioluminescent signal gradually increased at tumor sites in mice injected with untreated MSCs. In contrast, we observed very weak signals at tumor sites in mice injected with PTL-treated MSCs. These results suggest that NF-κB activity regulates MSC accumulation at tumors by inducing VCAM-1 expression and subsequent cellular interaction with tumor vessel ECs (Fig. 3).

Fig. 3
figure 3

Proposed mechanism of MSC accumulation at tumors. Growth factors and chemokines recruit MSCs to the tumor microenvironment. Inflammatory cytokines, including TNF-α, stimulate MSCs and VCAM-1 expression is induced. Activated MSCs attach to the tumor vasculature, penetrate, and accumulate at tumor sites

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Considerations for the use of genetically modified MSCs in cancer therapy

Although we focused on the function of TNF-α in the above study, other inflammatory cytokines including IL-1β and IFN-γ have the ability to induce VCAM-1 expression on target cells, and may also be involved in MSC accumulation. TNF-α is a major inflammatory cytokine that has important roles in diverse cellular events including cell survival, proliferation, differentiation, and death. Numerous reports have demonstrated elevated TNF-α levels in the serum of cancer patients, and TNF-α correlates closely with tumor progression and metastasis [44, 45]. For example, TNF-α readily induces IL-6 and MCP-1 secretion by CAFs and normal FBs and has an indirect influence on the generation of a prometastatic microenvironment. Furthermore, TNF-α is released in cardiac infarction [46] and graft-versus-host disease [47, 48]; MSCs accumulate at the site of cardiac infarction [49, 50]. These results indicate that pro-inflammatory cytokines also promote homing of MSCs in the heart and that these cytokines have a positive effect on cardiac regeneration. Therefore, MSC-based tissue-targeting strategies could be adapted for various inflammatory diseases, and activation with TNF-α may be one of the critically important steps for MSC accumulation.

For MSC-based cancer-targeted gene therapies, it is thought that therapeutic efficacy is directly coupled with the efficiency of MSC accumulation at tumor sites. Results from our laboratory suggest that the combined use of NF-κB inhibitors, including bortezomib, or TNF-α blocking agents, such as infliximab, reduces the therapeutic efficacy of genetically modified MSCs because of inhibition of MSC accumulation at the tumor. In contrast, tumor-specific TNF-α-inducing agents would be useful in enhancing therapeutic efficacy, thus further investigation is required for identifying such agents to establish more effective therapeutic strategies.

Conclusion

The application of anti-cancer gene-expressing MSCs for targeted cancer therapy is a novel and promising strategy. Here, we propose that suicide cancer gene therapy may be improved using vector-producing MSCs. This strategy is likely to generate vectors in situ, leading to the killing of solid tumors. This could be achieved using MSCs to initiate virus production near tumor cells in situ. These viruses are then transduced into tumor cells, which themselves produce virus progeny, thereby amplifying the transgene expression at tumor sites. While the therapeutic benefit and safety of this approach requires further examination, it holds great potential for the eradication of tumors.

MSC accumulation at tumor sites is related to migratory capacity toward growth factors and chemokines and also MSC-EC adhesion following activation by TNF-α. Furthermore, NF-κB activity regulates MSC accumulation at tumor sites through the induction of VCAM-1 expression and the resultant interaction with tumor blood vessel ECs. Although MSCs are useful as cellular vehicles for cancer-targeted gene therapy, previous studies have shown that increased MSC accumulation is required to enhance therapeutic efficacy. Thus, mechanisms of enhancing MSC accumulation should be developed, and the TNF-α-NF-κB-VCAM-1 axis may represent a solution to this problem.