Bone Marrow Mesenchymal Stem Cells Loaded With

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original article

Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model Atique U Ahmed1, Cleo E Rolle1, Matthew A Tyler1, Yu Han1, Sadhak Sengupta1, Derek A Wainwright1, Irina V Balyasnikova1, Ilya V Ulasov1 and Maciej S Lesniak1 The Brain Tumor Center, The University of Chicago, Chicago, Illinois, USA

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Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow–derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. Received 20 May 2010; accepted 26 May 2010; advance online publication 29 June 2010. doi:10.1038/mt.2010.131

Introduction Oncolytic adenoviral vectors have emerged as a novel treatment modality for multiple malignancies. Our laboratory has focused on the development of adenoviral vectors that selectively target cancer cells while leaving normal cells unharmed. We have previ ously shown that by introducing the human survivin promoter to control oncolytic adenovirus (Ad) replication, we could enhance viral replication in tumor cells that overexpress survivin.1–3 Despite advances in adenoviral targeting to malignant cells,

difficulties remain regarding the adequate delivery and persistence of oncolytic adenoviral vectors. For instance, the host immune sys tem is thought to play a role in diminishing the efficacy of onco lytic adenoviral agents. Immunocompetent hosts mount robust immune responses against Ads, characterized predominantly by the generation of neutralizing antibodies. Moreover, strong T-cell responses are often generated resulting in the production of interferons (IFNs). As such, the immune response represents a potential hurdle in the clinical advancement of adenoviral agents. To overcome this limitation, we sought to explore the use of immunosuppressive cells to deliver an oncolytic adenoviral vector in immunocompetent hosts. Bone marrow–derived mesenchymal stem cells (MSCs) have been shown to suppress lymphocyte proliferation and cytokine production. MSCs suppress allogeneic and antigen-specific T-cell proliferative responses in vitro.4 Furthermore, Samuelson et al. described MSC-mediated suppression in mixed lymphocyte reac tions.5 Accordingly, Di Nicola et al. demonstrated suppressed T-cell proliferation in response to mitogenic stimuli when cells were cultured in the presence of MSCs.6 These findings show cased the potential use of MSCs in transplant models, and as such, MSCs have been shown to enhance engraftment by limit ing graft-versus-host disease.7,8 Despite clear evidence supporting the notion that MSCs function as immunosuppressors, the exact mechanism of their immunosuppression remains unclear. More recent studies have shown that MSCs suppress T-cell activation and IFNγ production, but MSCs are limited in their ability to sup press preactivated cytotoxic T cells.9 The immunosuppressive properties of MSCs make them an attractive vehicle for the delivery of oncolytic viruses. Previously, Sonabend et al. successfully demonstrated enhanced delivery and persistence of oncolytic adenoviral vectors using human MSCs in an immunodeficient mouse model.10 Although this study showed that MSCs were efficient vehicles for oncolytic virus delivery, it did not investigate the immunosuppressive properties of MSC. Therefore, to better understand the impact of MSC-mediated immunosuppression in immunocompetent hosts, we utilized a previously described model system. Cotton rats (CRs) are a very

Correspondence: Maciej S Lesniak, The Brain Tumor Center, The University of Chicago, 5841 South Maryland Avenue, MC 3026, Chicago, Illinois 60637, USA. E-mail: [email protected] Molecular Therapy

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MSCs Suppress the Antiadenoviral Immune Response

useful animal model for infectious disease research, based on their susceptibility to many human pathogens (for review see refs. 11,12). In particular, human Ad serotype 5 has been shown to rep licate in the lung of the CR and cause pathology resembling that seen in Ad-infected humans.12 CR is therefore, at least partially, a permissive host for Ad replication (semipermissive). Recently, Toth et al. have characterized CR as a useful animal model to study the host, the tumor, and the oncolytic Ad interaction in its full complexity.13,14 This study was designed to evaluate the antiadenoviral immune response following delivery of oncolytic adenoviral vectors using MSCs as carriers, utilizing the CR model to approximate the antiadenoviral immune response described in humans. We hypothesized that because Ad is rapidly cleared in immunocompetent hosts, the use of MSCs as a delivery vehicle would enhance adenoviral persistence by suppressing the anti viral immune response. Initially, we isolated and characterized CR MSCs and evaluated their permissivity for Ad replication. CR MSCs were successfully transduced with Ad and supported viral replication. Moreover, CR MSCs displayed immunosuppressive properties both in vitro and in vivo. Interestingly, MSC-mediated immunosuppression was not compromised by adenoviral infec tion. Collectively, our results underscore the potential benefits of using MSCs as delivery vehicles for oncolytic Ads and warrant further study in tumor models to establish the efficacy of MSCmediated immunosuppression.

Results Isolation of CR MSCs Our laboratory and others have previously demonstrated adenoviral transduction of human MSCs.10 In these earlier studies, MSCs were used as vehicles to deliver viral vectors, and for the most part the immune responses generated were not studied. Understanding that MSCs act as immunosuppressors, we sought to study the immune response to adenoviral-loaded MSCs in a semipermissive model system. We began our study by isolating and characterizing MSCs from the bone marrow of 6-week-old CRs. Single cell suspensions of bone marrow cells from CR femurs were plated and allowed to adhere and proliferate for 6–8 weeks, with periodic culture medium changes.15,16 After three passages in cell culture, MSCs were iso lated based on their selective adherence to plastic culture dishes. Although there are limited reagents to confirm the phenotype of the isolated CR MSCs, the fibroblast-like morphology of these cells suggests that they are indeed MSCs (Figure 1a). A representative micrograph of the MSCs after three passages in vitro shows mature MSCs with the characteristic flat, polygonal morphology.

Ostogenic and adipogenic differentiation of CR MSCs We next performed differentiation assays to ensure that our MSCs retained their pluripotency along multiple lineages. For the analy sis of ostogenic differentiation capacity, CR bone marrow–derived MSCs were cultured under osteogenic conditions (STEMPRO Osteogenic differentiation media) for 14 days, fixed, and stained for Alizarin Red S, a dye that specifically binds to calcium matrix formations. CR MSCs were able to differentiate into osteocytes, as demonstrated by staining of calcium deposits with Alzarin Red solution (Figure 1b). 2

Next, we investigated whether CR MSCs could differentiate into adipogenic lineage. Within 7 days of culture in adipogenesis differentiation media, the number of lipid vesicle containing cells had significantly increased compared to the control culture media. As shown in Figure 1d, CR MSCs were positive for oil red O stain ing, which shows adipocytes filled with lipid vesicles (see white arrows). Data presented in this experiment confirmed that MSCs isolated from CR bone marrow maintained their pluripotency and differentiated into different lineages. These MSCs were main tained as low passage cultures and further experiments were con ducted to assess their permissivity for adenoviral replication and immunosuppressive properties.

Efficient adenoviral replication and cytolysis of CR MSCs Having successfully isolated CR MSCs, we next sought to deter mine the permissivity of these cells by comparing the cytotoxic activity and replication of two Ads: wild-type human Ad type 5 (AdWT), and a conditionally replicating Ad, CRAd-S-pk7. The cytotoxic activity of each viral vector was assessed by crystal violet staining 7 days after initial infection. Both AdWT and CRAd-Spk7 transduced and replicated in CR MSCs, albeit CRAd-S-pk7 was more efficient (Figure 2a). Both AdWT and CRAd-S-pk7 showed complete replicative cytotoxicity at 1,000 infectious units (i.u.)/cell; however, CRAd-S-pk7 demonstrated a higher level of toxicity at 100 i.u. These findings suggest that CRAd-S-pk7 is more toxic to CR MSCs, and this may be related to the rate of viral replication within the infected cells.

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Figure 1 Morphological and functional characterization of cotton rat mesenchymal stem cells (CR MSCs). MSCs were isolated from the bone marrow of 6-week-old CRs by selective adherence to the plastic cell culture dishes over a 6-week period. (a) The micrographs of live passage cells were captured using an inverted microscope and Metamorph software. (b) The ability of the MSCs to differentiate into osteoclasts was assessed in vitro upon culturing the cells and staining as described in the materials and methods. The ability of the CR MSCs to differentiate into adipocyte was examined. CR bone marrow MSCs were cultured in (c) growth media (negative control) or (d) the adipogenic differentiation media for 14 days before staining for lipid droplets using oil red O (see white arrows). The images were captured using a microscope and Metamorph software. Representative photos of two independent experiments. Bar = 10 μm for a and b, 20 μm for c and d.

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Figure 2 Cotton rat mesenchymal stem cells (CR MSCs) are permissive for AdWT and CRAd-S-pk7 transduction and replication. (a) Monolayers of CR MSCs were infected with the indicated multiplicity of infection (MOI) for 1 hour, then the cells were washed and virus-free culture medium was added. On day 7 postinfection, the monolayers were stained with crystal violet to assess the cytotoxic effect of AdWT and CRAd-S-pk7. (b) The replication of AdWT and CRAd-S-pk7 in CR MSCs was measured by quantitative PCR. CR MSCs were infected with the indicated MOI and on day 7, the cells were harvested and DNA was isolated. The data represented as the mean ± SD of triplicates. *P ≤ 0.05.

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In order to determine whether the toxicity observed for AdWT and CRAd-S-pk7 was due to differences in Ad replication, we next assessed the kinetics of viral replication by conducting qPCR on days 1, 3, 7, and 10 postinfection using multiplicities of infection of 10 or 100 i.u. The detection of viral DNA correspond ing to the viral E1A gene was used as a measure of viral replica tion. On day 1, in CR MSCs infected with 10 i.u., we detected 6.3 × 107 E1A copies/ng of DNA, compared to AdWT, which was similar to the mock-infected samples (Figure 2b). The level of CRAd-S-pk7 replication peaked on day 7 (3.4 × 108 E1A copies/ ng of DNA). Replication of AdWT was not observed until day 3, when we detected 1.3 × 103 E1A copies/ng of DNA. The peak in AdWT replication also occurred on day 7, albeit at a much lower level than CRAd-S-pk7. When CR MSCs were infected with a multiplicity of infection of 100 i.u./cell, E1A copy num bers peaked on day 1 for CRAd-S-pk7-infected cells (3.4 × 109 E1A copies/ng of DNA). In the case of AdWT, replication on day 1 was low, such that only 3.3 × 102 E1A copies/ng of DNA was detected. The peak replication of AdWT was observed on day 7 at 3.4 × 107 E1A copies/ng DNA. CRAd-S-pk7 had a significantly higher level of replication than AdWT (P ≤ 0.05). However, the ability of CRAd-S-pk7 virus to replicate in the human MSCs is more efficient than CR MSCs (Supplementary Figure S1). The level of E1A copies in both human and CR MSCs peaked at day 3 postinfection with 2.5-fold more E1A copies recovered in human MSCs. Of note, human MSCs also supported higher yields of viral progeny release at all time-points. The differences between these two systems in supporting human adenoviral replication are probably due to the semipermissive nature of the CR model. Collectively, our crystal violet and qPCR data confirm that CR MSCs are indeed permissive for viral replication. Therefore, we decided to infect CR MSCs with 100 i.u./cell for all future experi ments. These data suggest the feasibility of using CR MSC loaded

Figure 3 CRAd-S-pk7-loaded MSCs inhibit IFNγ production by PHAactivated splenocytes. Naive CR splenocytes were cultured in the presence of CR MSCs or CRAd-S-pk7-loaded CR MSCs and activated with PHA. The supernatants were harvested at 24 hours (a) or 48 hours (b) and assayed by ELISA for IFNγ production. A strong decrease in IFNγ production was noticed in the CR MSC–treated group and infection with CRAd-S-pk7 did not abrogate the suppression. Data shown are the mean ± SD, and are representative of 3 independent experiments. CR, cotton rat; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; MSC, mesenchymal stem cell; PHA, phytohemagglutinin.

with CRAd-S-pk7 as carrier-vector combination for future in vivo studies.

CR MSCs suppress T-cell activation, proliferation, and IFNγ production Having shown that CR MSCs are permissive for viral replication, the next step in our study was to determine whether CR MSCs retain the characteristics described for MSCs from other species. In particular, a key property of MSCs is their ability to suppress lym phocyte proliferation and cytokine production. To investigate the 3



response by downregulating both antigen-driven proliferation and cytokine production.

immunosuppressive properties of CR MSCs in vitro, we assessed their ability to suppress the activation of phytohemagglutinin (PHA)-stimulated CR splenocytes. Upon PHA-activation, IFNγ is produced by splenocytes. Therefore, to gauge T-cell activation, we measured the amount of IFNγ in the supernatant of PHAstimulated cultures. Early after activation (24 hours), splenocytes cocultured with CR MSCs produced 6.9-fold less IFNγ than sple nocytes alone (Figure 3a), suggesting that activation was delayed in the presence of CR MSCs. To determine whether CRAd-S-pk7loaded CR MSCs retained the ability to suppress IFNγ production by activated splenocytes, we activated splenocytes with PHA for 48 hours in the presence of mock-infected CR MSCs and CRAdS-pk7-infected CR MSCs. Both groups demonstrated significantly suppressed IFNγ production by PHA-stimulated splenocytes (Figure 3b). Collectively, our in vitro data suggest that oncolytic Ad-loaded CR MSCs retain the ability to suppress the immune Media

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Suppression of the antiadenoviral immune response by CR MSCs ex vivo To investigate whether CR MSCs are able to suppress a specific antiviral immune response, naive CRs were challenged intraperi toneally (i.p.) with the CRAd-S-pk7 virus (5 × 106 i.u./animal). Two weeks postchallenge, splenocytes from individual rats (n = 5) were harvested and stimulated with CRAd-S-pk7, CRAd-Spk7-loaded CR MSC, or CRAd-S-pk7-loaded CR hepatocytes ex vivo. Normally, T cells form clusters of activated cells in response to a polyclonal stimulator such as PHA. When we cocultured CR MSCs with the PHA-stimulated CR splenocytes, the cluster formation was significantly inhibited compared to PHA alone or the control cell, CR hepatocytes (Figure 4a). In the presence

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Figure 4 CRAd-loaded MSCs suppress splenocyte activation and proliferation by CRAd-S-pk7 ex vivo. To generate CRAd-S-pk7-specific immunity, cotton rats (CRs) were injected with 5 × 106 i.u. of CRAd-S-pk7 virus intraperitoneally. Two weeks postchallenge, splenocytes from the immunized animals were cultured in the presence of indicated stimuli. (a) CR splenocytes were activated for 72 hours with PHA or CRAd-S-pk7 in the presence or absence of MSCs. CR MSC inhibited PHA and CRAd-S-pk7 induced splenocyte clusters. CR hepatocytes were used as the control cells for this experiment. The micrographs of live cells were captured using an inverted microscope and Metamorph software. (b) CR MSCs inhibit IFNγ production by the splenocytes activated with CRAd-S-pk7. IFNγ production was evaluated at 72 hours by enzyme-linked immunosorbent assay in response to CRAd-S-pk7, CRAd-S-pk7-loaded CR MSC, or CR hepatocytes. In the presence of CR MSC, splenocytes produced 7.35-fold less IFNγ as compared to CRAd-S-pk7 alone (P < 0.05). The results represent the mean ± SD of four animals. These results are representative of two independent experiments. (c) CFSE-labeled splenocytes were activated for 72 hours alone or in coculture with MSC or CRAd-S-pk7-loaded MSC. Data are representative of three independent experiments. CFSE, carboxyfluorescein succinimidyl ester; CR, cotton rat; IFN, interferon; MSC, mesenchymal stem cell; PBS, phosphatebuffered saline; PHA, phytohemagglutinin.

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CR MSCs deliver Ad systemically and enhance persistence in vivo The kinetics of viral replication and dissemination were measured by quantifying viral genomic E1A copy number relative to total DNA in CR tissues on days 2, 7, and 28 after i.p. injection using qPCR (Figure 5). CRs were randomized into four groups before receiving i.p. injections of phosphate-buffered saline (PBS; con trol), MSC, CRAd-S-pk7, or CRAd-S-pk7-loaded MSC. In the blood, the level of viral DNA in the CRAd-S-pk7 group peaked on day 2, whereas the peak viral DNA level in the CRAd-S-pk7loaded MSC group occurred on day 7. At 2 days postinjection, Molecular Therapy

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of CRAd‑S‑pk7 (1 × 105 i.u.), splenocytes from the immunized animals also formed cell clusters, but to a lesser extent (Figure 4a). Thus, virus-mediated formations of cell clusters were abolished when the splenocytes were stimulated with the CRAd-S-pk7loaded CR MSCs (Figure 4a). This was an MSC-specific phenom enon, because CRAd-S-pk7 loaded into CR hepatocytes induced cell clusters (Figure 4a). Next, we evaluated the splenocyte activation by measur ing IFNγ production in response to different stimuli ex vivo. The splenocytes from immunized animals produced eight times more IFNγ compared to the naive animals in response to ex vivo stimulation with CRAd-S-pk7 virus, which confirmed that a virus-specific immune response was generated after in vivo prim ing with the CRAd-S-pk7 virus (Supplementary Figure S2). When the splenocytes from the immunized rats were cocultured with virus-loaded CR MSCs, IFNγ production was inhibited 9.4fold (P < 0.05 compared to CRAd-S-pk7 alone; Figure 4b). This inhibition was specific to CR MSCs, because we did not observe any decrease in IFNγ production after coculturing with CR hepa tocytes loaded with the same amount of CRAd-S-pk7 (Figure 4b). One possibility may be that CRAd-S-pk7-loaded CR MSCs were able to hide the virus from the splenocytes, so the splenocytes may have encountered a lower dose of virus compared to when stimulated with the CRAd-S-pk7 virus alone. To investigate this possibility, we measured the number of Ad infected splenocytes from these cultures by fluorescence-activated cell sorting. The percentage of positive splenocytes for the adenoviral hexon pro tein after 72 hour coculture with the CRAd-S-pk7 virus alone or CRAd-S-pk7-loaded CR MSC was very similar (Supplementary Figure S3). Also the viability of the CR splenocytes was not altered from these two systems at the time when the superna tant was harvested (72 hours after ex vivo stimulation) for IFNγ enzyme-linked immunosorbent assay (ELISA) (Supplementary Figure S3). Thus, our findings indicate that CR MSCs can specifi cally suppress antiviral immune response ex vivo. The clusters of activation observed in CR splenocytes/CR MSC cocultures were smaller than splenocytes alone, suggesting that CR MSCs were either delaying the activation or proliferation of these splenocytes. To specifically measure proliferation, we assessed the dilution of carboxyfluorescein succinimidyl ester (CFSE) in sple nocytes (Figure 4c). At 72 hours postactivation with CRAd-S-pk7, splenocytes alone had diluted CFSE to a greater extent than cells cocultured with viral-loaded CR MSCs. These data highlight the immunosuppressive functionality of CR MSCs by showing their ability to inhibit antigen-stimulated proliferation.


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Figure 5 CR MSCs enhanced the dissemination and persistence of CRAd-S-pk7 in vivo. On the indicated days, blood, spleen, lung, liver, and bone marrow were collected from the CR. Total DNA was isolated, quantified, and subjected to real-time quantitative PCR to determine the amount of viral DNA (E1A) present. Data shown are the mean ± SEM. CR, cotton rat; MSC, mesenchymal stem cell.

the greatest amount of viral DNA was detected in the blood of the CRAd-S-pk7 group (P ≤ 0.05). By day 7 postinjection, similar levels of viral DNA were detected in the blood of CRAd-S-pk7 and CRAd-S-pk7-loaded MSC groups. In the blood, the level of viral DNA was reduced in the CRAd-S-pk7 and CRAd-S-pk7-loaded MSC groups to that of the control and MSC groups by day 28. The kinetics and level of viral DNA was similar in the spleen of the CRAd-S-pk7 and CRAd-S-pk7-loaded MSC groups. In the spleen, the peak viral DNA level occurred on day 7 postinjection. Significantly higher amounts of viral DNA were detected in the lungs of the CRAd-S-pk7-loaded MSC group compared to the CRAd-S-pk7 group at 2 days postinjection (P ≤ 0.05). In the lungs of the CRAd-S-pk7-loaded MSC group, the level of viral DNA declined rapidly and by day 7 postinjection the level was similar to that of the CRAd-S-pk7 group. By day 28 postinjec tion, viral DNA was not detected in the lungs of any of the groups. Although the kinetics of viral replication were similar in the liver of the CRAd-S-pk7 and CRAd-S-pk7-loaded MSC groups, the level of viral DNA detected on day 7 postinjection was greater in the CRAd-S-pk7-loaded MSC group. In both groups, viral DNA levels peaked on day 7 postinjection and then declined to control and MSC levels by day 28 postinjection. The level of viral DNA detected in the bone marrow of CRAdS-pk7-loaded MSC group was similar to that of the control and MSC groups, indicating that MSCs did not preferentially traffic 5



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Figure 6 CRAd-S-pk7-loaded MSCs suppressed the in vivo and in vitro immune activation. (a) Sera from each animal was collected on the indicated days and assayed by enzyme-linked immunosorbent assay (ELISA) to quantify the amount of IFNγ produced in response to CRAd-S-pk7 infection. (b) On the indicated days, spleens were harvested and splenocytes were activated in vitro with phytohemagglutinin. After 4 hours, the supernatants were collected and assayed by ELISA to determine the production of IFN-γ. Data shown are the mean ± SD. IFN, interferon; MSC, mesenchymal stem cell.

to and deliver virus to the bone marrow. Furthermore,