Volume 18, Issue 10, Pages 1846-1856 (October 2010) Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model  Atique U Ahmed, Cleo E Rolle, Matthew A Tyler, Yu Han, Sadhak Sengupta, Derek A Wainwright, Irina V Balyasnikova, Ilya V Ulasov, Maciej S Lesniak  Molecular Therapy  Volume 18, Issue 10, Pages 1846-1856 (October 2010) DOI: 10.1038/mt.2010.131 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2010 18, 1846-1856DOI: (10.1038/mt.2010.131) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2010 18, 1846-1856DOI: (10.1038/mt.2010.131) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 CRAd-S-pk7-loaded MSCs inhibit IFNγ production by PHA-activated 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. Molecular Therapy 2010 18, 1846-1856DOI: (10.1038/mt.2010.131) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

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, phosphate-buffered saline; PHA, phytohemagglutinin. Molecular Therapy 2010 18, 1846-1856DOI: (10.1038/mt.2010.131) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2010 18, 1846-1856DOI: (10.1038/mt.2010.131) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

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. Molecular Therapy 2010 18, 1846-1856DOI: (10.1038/mt.2010.131) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 CR MSCs enhanced intratumoral oncolytic viral persistence and systemic antiviral immune responses. (a) Increased viral persistence after intratumoral delivery of CRAd-S-pk7 virus loaded onto CR MSC. Seven days postinjection, tumors were harvested from each animal and the amount of viral DNA was measured by quantitative PCR. The increase in CRAd-S-pk7 E1A copy number in the group that received virus-loaded CR MSCs was statistically significant (overall increase, P = 0.028). (b) Tumor infection and intratumoral persistence of CRAd-S-pk7 virus. Intratumoral distribution of CRAd-S-pk7 is shown by a red stain of the infected cells expressing adenovirus hexon proteins. Microphotographs show tumors 7 days after viral injection, when several rounds of CRAd-S-pk7 replication have occurred. Tumors injected with CR MSC-loaded virus show higher percentages of hexon positive areas compared to tumors injected with naked virus. (c) CR MSCs are able to suppress the systemic antiviral immune response. Splenocytes from treated groups were stimulated for 72 hours with CRAd-S-pk7 virus ex vivo. The IFNγ levels were measured by enzyme-linked immunosorbent assay in the supernatant at 72 hours poststimulation. The splenocytes from the group of animals that received intratumoral CRAd-S-pk7-loaded CR MSCs produced 30-fold less IFNγ compared to the group that received CRAd-S-pk7 virus (P = 0.0286). CR, cotton rat; IFN, interferon; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline. Molecular Therapy 2010 18, 1846-1856DOI: (10.1038/mt.2010.131) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions