Mesenchymal stem cells (MSCs) are a kind of adult stem cells that can be isolated easily from bone marrow, adipose tissue, umbilical cord and many other tissues. MSCs have been shown to specifically migrate to inflammatory sites, including tumors, and hold great promise as tumor-specific vectors to deliver antitumor agents. Interferon-α (IFNα) has been used in clinic to treat various types of tumors; however, because of its short half-life, significant therapeutic effects require high doses that often results in serious side effects. Here, we tested whether MSCs continuingly secreting IFNα can exert a persistent antitumor effect and eliminate the side effects associated with high clinical doses of recombinant IFNα. We found that even a small number of IFNα-secreting MSCs could potently halt B16 tumor growth in vivo. The antitumor activity of IFNα-secreting MSCs was largely abolished in immunodeficient mice, an effect largely attributed to natural killer cells and CD8+ T cells. Therefore, IFNα-secreting MSCs provide an innovative strategy for tumor therapy.
Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) were first described by Friedenstein and colleagues in the 1970s in bone marrow, according to their adherence to tissue culture plastic and differentiation into osteoblasts, chondrocytes and adipocytes.1, 2 Cells with similar characteristics as those isolated from bone marrow exist in many other tissues, including adipose tissue, umbilical cord and amniotic fluid.3 It is now believed that MSCs reside in almost all tissues and play important roles in maintaining tissue homeostasis.4 Interestingly, MSCs have been isolated from not only normal tissues, but also pathological tissues such as inflamed synovial tissues and tumors.5 MSCs have no specific surface markers, and the identification of MSCs mainly rely on the following characteristics: (1) adherence to plastic culture plates; (2) expression of CD90, CD105, CD146, Stro-1 for human MSCs and Sca-I for mouse MSCs; and (3) differentiation into osteoblasts, adipocytes and chondrocytes.6, 7 MSCs have been extensively studied for tissue regeneration and treating immune disorder diseases.8, 9, 10, 11 There are several advantages for the clinical use of MSCs: ease in isolation and culture from various tissues, rapid expansion without accumulation of mutations and no adverse immune response.12
It has been reported that exogenous MSCs specifically migrate into inflammatory sites in vivo after administration.13 As tumors are wounds that never heal, MSCs were also shown to migrate specifically into tumors in vivo.14, 15, 16 In fact, MSCs are being tested as a tumor-specific vector to deliver antitumor agents.17, 18 Interferon-α (IFNα) has been used to treat leukemia and melanoma in clinic, but its use has been hindered by its short half-life and serious side effects because of the high-dose requirement.19, 20 However, most studies on tumor-specific delivery of IFNα used human tumor cell lines in immunodeficient mice, which fail to address the potential roles played by the immune system in the process. To overcome these problems, we tested whether MSCs continuingly secreting IFNα can exert a persistent antitumor effect in syngeneic mouse melanoma model and explore the underlying mechanism of MSC-mediated antitumor effect. In our study, we employed immune-competent mice and revealed that continuous low levels of IFNα could exert strong antitumor effect via modulating the activity of immune cells such as natural killer (NK) and CD8+ cells.
Results and Discussion
MSCs can be successfully transduced to release functional IFNα
We transduced MSCs with lentivirus encoding green fluorescent protein (MSC-GFP) or GFP together with mouse IFNα (MSC-IFNα) and over 90% cells were successfully transduced, as shown by GFP expression on flow cytometry (Figure 1a). No apparent change in morphology and proliferation rate between MSC-GFP and MSC-IFNα was observed. To examine the IFN production level of MSC-IFNα, we quantified IFNα level in the supernatant of MSCs cultured at 5 × 105 cells per ml for 48 h (Figure 1b). Enzyme-linked immunosorbent assay analysis showed that there was 19 ng/ml of IFNα in the supernatant of MSC-IFNα, whereas no IFNα was detected in the supernatant of MSC-GFP cultured under the same condition. To test whether IFNα released by MSC-IFNα possesses biological function, we examined the expression of major histocompatibility complex class-I molecule H-2Kb on MSC surface by flow cytometry, as IFNα is known to increase the expression level of H-2Kb. As shown in Figure 1c, the expression of H-2Kb was low in MSC-GFP, an intrinsic property of MSCs. As expected, after treatment with recombinant IFNα, the expression of H-2Kb was dramatically increased in MSC-GFP. Interestingly, similar increased expression of H-2b was also observed on cells treated with the supernatant of MSC-IFNα. Correspondingly, H-2Kb surface expression on MSC-IFNα was also increased to similar level as that of MSC-GFP treated with IFNα. These data clearly demonstrated that MSC-IFNα produced biologically functional IFNα.
MSC-IFNα exerted potent anti-tumor effect in vivo
To investigate the effect of MSC-IFNα on tumor growth in vivo, mouse B16 melanoma model was employed. In this system, all cells and mice are in the C57BL/6 background. We inoculated 1 × 106 B16 melanoma cells alone, or with either 1 × 106 MSC-GFP or MSC-IFNα intramuscularly, and tumors were removed and weighed 12 days later. Strikingly, MSC-IFNα completely halted tumor growth, whereas MSC-GFP slightly enhanced tumor growth (Figure 2a). To examine the potency of the MSC-IFNα, we injected 1 × 106 B16 melanoma cells with different numbers of MSC-IFNα. We found that even 1 × 104 MSC-IFNα (ratio of MSC-IFNα/B16=1:100) could still potently prevent tumor growth in vivo (Figure 2b). Moreover, all mice inoculated with tumor cells alone died within 30 days, whereas nearly half of mice that received tumor cells together with MSC-IFNα survived for >100 days (Figure 2c). Even when MSC-IFNα cells were injected 3 or 4 days after B16 melanoma cell inoculation, tumor growth was also effectively inhibited (Figures 2d and e). To compare the antitumor capacity of MSC-IFNα with recombinant IFNα, we treated mice with 5 μg recombinant IFNα (50 000 U) or 1 × 106 MSC-IFNα 3 days after B16 cell inoculation. Based on our in vitro assay, we roughly estimate that the 1 × 106 injected MSC-IFNα cells only produce ∼19 ng of IFNα daily. This is far below the 5 μg recombinant IFNα injected. Importantly, even with this low amount of IFNα produced (250-folds lower than the amount of recombinant IFNα injected), MSC-IFNα had much more potent antitumor effect than recombinant IFNα (Figure 2f). We also showed that repeated recombinant mouse IFNα administration also exerted potent antitumor effect in vivo (Supplementary Figure 1). These data clearly demonstrated that IFNα-secreting MSCs possess highly potent antitumor activity in vivo.
MSCs persisted in the tumor, decreased tumor cell proliferation and induced tumor cell apoptosis
To investigate the mechanisms of the potent antitumor effect of MSC-IFNα, we tracked the fate of the administered MSC-IFNα in vivo. MSC-IFNα was labeled with luciferase, whose activity was monitored in vivo with live imaging technology using Berthod NC100 imaging system (Bad Wildbad, Germany). When coinjected with B16 cells, MSC-IFNα persisted only in tumors for >2 weeks with gradual decrease (Figures 3a and b). Considering the potent antitumor effect of MSC-IFNα (still effective in the ratio MSC-IFNα/B16=1:100), we speculate that MSC-IFNα stay inside tumors and consecutively secret low but effective concentration of IFNα locally in the tumor for at least 2 weeks. This is likely superior to the short half-life and high-dose requirement of recombinant IFNα in vivo. When tumors were examined histologically, massive lymphocyte infiltration was found in the B16 plus MSC-IFNα group. Furthermore, MSC-IFNα inhibited tumor cell proliferation as shown by the decreased ratio of Ki-67-positive cells and increased tumor cell apoptosis, as shown by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay (Figure 3c).
The antitumor activity of MSC-IFNα was largely immunodependent
We then further explored the mechanisms underlying the tumor inhibitory effect of MSC-IFNα in vivo. As IFNα has been known to inhibit tumor growth by mechanisms including induction of tumor cell cycle arrest or stimulation of the immune system to combat tumors, we first studied the direct effect of recombinant IFNα on tumor growth in vitro.21 We found that recombinant IFNα only inhibited B16 melanoma cells marginally even at high concentrations (up to 100 ng/ml as compared with 19 ng/ml produced by MSC-IFNα; Figure 4a). Therefore, considering the complete tumor growth inhibition observed in vivo by MSC-IFNα, we reasoned that there should be other mechanisms involved in addition to the direct inhibition of tumor growth. To test whether the immune system played any role in the antitumor effect of MSC-IFNα, we inoculated B16 melanoma cells alone, or with either MSC-GFP or MSC-IFNα into wild-type and immunodeficient NOD-SCID (nonobese diabetic/severe combined immunodeficient) mice in parallel, and compared tumor growth in these mice. In wild-type mice, MSC-IFNα completely inhibited tumor growth (Figure 4b) as observed before, whereas in immunodeficient mice the tumor inhibition effect of MSC-IFNα was greatly abolished (Figure 4c). To more clearly analyze the role of the immune system in the antitumor effect of MSC-IFNα, we injected less MSC-IFNα together with B16 tumor cells so as to minimize the contribution of direct tumor inhibition. When low numbers of MSC-IFNα (1/100 of tumor cells) were used, tumor growth was still effectively inhibited in wild-type mice (Figure 4d); however, this effect completely disappeared in immunodeficient mice (Figure 4e). Because NK cells are well recognized for their roles in tumor surveillance in vivo, we tested whether this cell type was involved in the antitumor effect of MSC-IFNα by depleting NK cells with anti-asialo GM1 antibody.22 Interestingly, tumor growth was effectively inhibited in control mice; however, this inhibition was greatly reversed in mice treated with NK cell depletion antibody (Figure 4f). CD8+ T cells also contributed to the antitumor effect of MSC-IFNα, as shown by the diminished inhibition of tumor growth by MSC-IFNα in CD8+ T cell-deficient mice, β2m-knockout mice (Figure 4g). These data clearly showed the immune system is critical in the antitumor effect of MSC-IFNα, in addition to its direct effect on tumor cells.
Tumors are one of the most devastating diseases that develop in humans, and effective therapies are still to come. Currently available radiotherapy and chemotherapy are only partly effective in limited types of cancers with frequent severe side effects under many circumstances.23 Innovative therapies for tumors are urgently needed. One problem in tumor therapy is the difficulty of targeted drug delivery.24 MSCs have great potential for regenerative medicine and autoimmune disorders, and have been evaluated in clinical trials to treat many different kinds of diseases, including liver fibrosis, diabetes, graft-versus-host disease and Crohn’s disease.10, 25 Interestingly, MSCs have an intrinsic ability to specifically migrate into tumors, and have been suggested as a tumor-specific vector to deliver antitumor agents. In fact, MSCs have been genetically engineered to express various antitumor factors, including type I IFN, TNF-related apoptosis-inducing ligand (TRAIL), interleukin-12 and lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells, and have been shown to possess potent antitumor effect in animal models.18, 26, 27 The immune system plays a key role in combating tumor development and progression. Tumors are always accompanied by an immunosuppressive microenvironment. Enhancing antitumor immune responses holds great promise for effective cancer therapy.28, 29 In our study, IFNα was delivered into tumor via MSCs in normal mice. In such immune-competent mice, IFNα was found to exert its effect through promoting antitumor immunity. Even low number of IFNα-secreting MSCs had the ability to inhibit 100-fold more tumor cell growth in normal mice, but not in immunodeficient mice. Furthermore, both NK cells and CD8+ T cells were shown to play important roles in the antitumor effect of IFNα-secreting MSCs in vivo. IFNα could overcome the immunosuppression of MSCs. IFNα effectively reverses the immunosuppressive property of MSCs induced by IFNγ and tumor necrosis factor-α (Qing Chen, unpublished data). The long-term existence of MSCs-IFNα in tumor avoided frequent injection as seen with IFNα. The low but effective level of IFNα released by MSCs-IFNα is unlikely to cause any side effect. We strongly believe that MSCs engineered to express immune-stimulating factors hold great promise for tumor therapy in the future.
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This work was supported by grants from Scientific Innovation Project of the Chinese Academy of Science (XDA 01040107 and XDA 01040110), the Ministry of Science and Technology of China (2010CB945600 and 2011DFA30630), the National Science and Technology Project of China (31010103908 and 81273316), Shanghai Municipal Key Projects of Basic Research (12JC1409200), Shanghai Municipal Natural Science Foundation (12ZR1452600), the Knowledge Innovation Program of Shanghai Institutes for Biological Sciences and the Chinese Academy of Sciences (2012KIP202).
YS and YW conceived this project, designed experiments and analyzed data and prepared the manuscript; CX conceived this project, designed and performed experiments, analyzed data and prepared the manuscript; LL, QC, GC, YW, YH and YH performed experiments.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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