A comparative study of neural and mesenchymal stem cell-based carriers for oncolytic adenovirus in a model of malignant glioma

Abstract

Glioblastoma multiforme is a primary malignancy of the central nervous system that is universally fatal due to its disseminated nature. Recent investigations have focused on the unique tumor-tropic properties of stem cells as a novel platform for targeted delivery of anticancer agents to the brain. Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) both have the potential to function as cell carriers for targeted delivery of a glioma restricted oncolytic virus to disseminated tumor due to their reported tumor tropism. In this study, we evaluated NSCs and MSCs as cellular delivery vehicles for an oncolytic adenovirus in the context of human glioma. We report the first preclinical comparison of the two cell lines and show that, while both stem cell lines are able to support therapeutic adenoviral replication intracellularly, the amount of virus released from NSCs was a log higher than the MSC (p < 0.001). Moreover, only virus loaded NSCs that were administered intracranially in an orthotopic glioma model significantly prolonged the survival of tumor bearing animals (median survival for NSCs 68.5 days vs 44 days for MSCs, p < 0.002). Loading oncolytic adenovirus into NSCs and MSCs also led to expression of both pro- and anti-inflammatory genes and decreased vector-mediated neuroinflammation. Our results indicate that, despite possessing a comparable migratory capacity, NSCs display superior therapeutic efficacy in the context of intracranial tumors. Taken together, these findings argue in favor of NSCs as an effective cell carrier for antiglioma oncolytic virotherapy.

Figure 1

Permissiveness of MSC and NSC for CRAd replication. (A) NSC and MSC were stained to detect the levels of Ad-targeted surface receptors. Numbers in top right corners of each dot plot represent the percentage of positive cells. Gates were drawn using an isotype control sample. y-Axis, SSC; x-axis, FITC-A. (B) Quantitative PCR detection of viral replication. NSC and MSC were infected with 1 IU of each vector. Seven days postinfection, the degree of viral genome amplification was assessed by measuring the number of viral E1A DNA copies/ng DNA. Bars represent mean of 3 experiments (standard error (SE). (C) CRAd-S-pk7 cytotoxicity in NSC and MSC. NSC or MSC were infected with CRAd-S-pk7 virus at the indicated iu and stained with crystal violet solution 7 days later. (D) Titration of infectious progeny assembly in NSC and MSC. Studies were conducted alongside crystal violet cytotoxicity studies. MSC were infected with CRAd-S-pk7 at the indicated MOI, and the number of infectious viral progeny was titrated 7 days later. ***, P-value <0.001; **, P-value <0.01; *, P-value <0.05.

Figure 2

Characterization of MSC and NSC migration in response to human glioma. Migration was studied by using an in vitro fluoroblock migration chamber. Conditioned media was generated by culturing each cell type shown in serum-free MEM for twenty-four hours. Migration of each cell type was assessed twenty-four hours after stem cells were plated in the top well of the migration chamber. (A) Specific migration of NSC and MSC toward human glioma. Data obtained in Supplementary Figure 1A in the Supporting Information was used to assess preferential migration (calculated as the number of cells migrating toward glioma-conditioned medium/number of cells migrating in negative control group (SF-MEM). Bar graphs represent mean fold migration ±SE. ***, P-value <0.001; **, P-value <0.01; *, P-value <0.05. (B) In vivo tumor tropic migration of MSC and NSC. Bioluminescent imaging of mice after intracranial injection of NSC-Rluc and MSC-Rluc into the left hemisphere of the mice with 7 days postimplanted U87 xenograft tumor (b, c, e, f, h, i, k, l) or mice with no tumor (a, d, g, j). Gradual disappearance of photon emission during 2 days after injection on the injection sites noted. No (for NSC; g, j) or minimal migration (for MSC; a, d) is observed in group with no tumor. (C) Evidence of loaded NSC and MSC tumor-tropic migration in vivo. Nude mice received intracranial injections of GFP-expressing U87 cells. Five days later, mice then received injections of PBS (Mock), MSC (mCherry labeled), and NSC (mCherry labeled) in the contralateral hemisphere. Seven days postimplantation of stem cells, animals were sacrificed and animals’ brains were subjected to immunohistochemical analysis; bar, 100 μm.

Figure 3

In vitro analysis of stem cell loading capacity and glioma toxicity. As in Figure 2, this study employed an in vitro Matrigel migration chamber. U87MG cells were cultured for 48 h in SF-MEM supplemented with b-FGF and EGF to generate spheroid colonies, and to serve as an attractant for stem cell migration. After this 48 h culturing period, stem cells were infected with different iu of CRAd-S-pk7 and subsequently plated in the top wells of the migration chamber. (A) Images were taken nine days after infected stem cells were plated in the top well of the migration chamber (original objective, 4×). (B) Inhibition of glioma spheroid formation by stem cell-mediated release of infectious CRAd-S-pk7. The ability of NSC and MSC to release CRAd-S-pk7 and mediate a toxic effect in glioma cells was quantified by counting the number of glioma spheroids (indicated with arrowhead in Figure 3A) formed in the bottom of the migration chamber. Quantification and analysis of spheroid formation (as observed in 3A) mediated by each stem cell when loaded with different iu of CRAd-S-pk7. There were four wells per experimental condition, and three random field views were captured per well. Bar graphs represent mean spheroid number per field view ±SE. (C, D) The replicative capacity of CRAd-S-pk7 virus in stem cells was measured by the titer assay. Media and the cell pellets were separated from the infected well. The total viral progeny in the cell pellets (cell associated) (C) and the progeny released by the infected stem cells over time were measured by the titer assay. Data is presented as the mean infectious units/mL/well ±SE. ***, P-value <0.001; **, P-value <0.01; *, P-value <0.05.

Figure 4

Effects of oncolytic virus loading in the stem cells’ tumor-tropic migratory properties. (A) Tumor-tropic migration of CRAd-S-pk7 loaded stem cell in response to U87 cells 24 h after coculture in the Transwell plate. All conditions were done in quadruplicate and repeated in two separate experiments. CRAd-S-pk7 significantly enhanced the in vitro tumor-tropic migration of NSCs. *P < 0.05, **P < 0.0005 significant compare to uninfected control and uninfected MSC. (B) Evidence of loaded NSC and MSC tumor-tropic migration in vivo. Nude mice received intracranial injections of GFP-expressing U87MG cells. Five days later, mice received injections of PBS (Mock), MSC (nonloaded), NSC (nonloaded), CRAd-S-pk7, CRAd-S-pk7-loaded MSC (MSC + V), or CRAd-S-pk7-loaded NSC (NSC + V) in the contralateral hemisphere. Injections were performed using the same burr hole used to inject U87MG cells. Images were captured using an inverted fluorescent microscope. The presence of stem cells was detected by the expression of the red fluorescent protein, mCherry; bar 100 μm. (C) Analysis of expression of chemoattractant receptors MET, CXCR4 and VGEFR2 expression postloading with 50 IU of CRAd-S-pk7 virus per stem cells by FACS analysis 48 h postinfection.

Figure 5

Immunological consequences of oncolytic adenovirus loading into stem cells. (A) Analysis of cytokine expression postinfection with 10 IU of CRAd-S-pk7 virus per stem cells by qRT-PCR. Levels of expression were expressed relative to uninfected control for each stem cell line at 0 h; *P < 0.05, **P < 0.005, ***P < 0.0005 significant. (B) Analysis of IL-10 cytokine expression in stem cells infected with CRAd-S-pk7 (10 IU/cell). Supernatant from the infected stem cell culture was harvested at 6, 24, 48 and 72 h postinfection, and IL-10 expression was measured by ELISA; ***p <0.0005. (C) Analysis of TGF-β positive stem cells by FACS at 48 and 72 h postinfection with CRAd-S-pk7 (10 IU/cell). (D) Histological features of brains from mice injected with CRAd-S-pk7 virus alone (2.5 × 10⁷ iu/injection) or virus loaded into stem cells (5 × 10⁵ cells loaded with 50 IU of CRAd-S-pk7 per NSCs) harvested at day 14 postinjection. (1–3) Image of H&E staining assessing neuronal toxicity postinjection. Representative image of sections of the same animal brain as H&E sections stained with antiglial fibrillary acidic protein (GFAP) (4–9) and anti-mouse MHC class II antibody (10–15); bar, 50 μm.

Figure 6

Oncolytic virus loaded NSCs inhibit xenograft growth and prolong survival of mice with orthotopic glioblastoma. (A) Three animals from different groups in the experiment described below (6B) were sacrificed 30 days post therapy and their brains were subject to H&E analysis to assess the tumor burden. H&E staining of representative animals’ brains were shown (original objective, 40×). (B) Both stem cell lines were incubated with the oncolytic adenovirus (50 IU/cells) for 2 h in the room temperature, washed 3 times with PBS, and resuspended in PBS (5 × 10⁵ stem cells in 2.5 μL/mouse) before intratumoral injection. 5 × 10⁵ U87 cells were injected stereotactically into the right hemisphere of the brain of 9 to 12 week old nu/nu mice followed by intratumoral (IT) injection of CRAd-S-pk7 loaded into stem cells 5 days post tumor implantation. The median survival for animals injected with PBS was 41.4 days, with MSC loaded CRAd-S-pk7 it was 44 days (log-rank, P = 0.075), and with NSC loaded CRAd-S-pk7 it was 68.5 days, n =7 (P < 0.002 vs MSC). Data are presented from two independent experiments.