A preclinical evaluation of neural stem cell-based cell carrier for targeted antiglioma oncolytic virotherapy

Abstract

Background: Oncolytic adenoviral virotherapy (OV) is a highly promising approach for the treatment of glioblastoma multiforme (GBM). In practice, however, the approach is limited by poor viral distribution and spread throughout the tumor mass.

Methods: To enhance viral delivery, replication, and spread, we used a US Food and Drug Administration-approved neural stem cell line (NSC), HB1.F3.CD, which is currently employed in human clinical trials. HB1.F3.CD cells were loaded with an oncolytic adenovirus, CRAd-Survivin-pk7, and mice bearing various human-derived GBMs were assessed with regard to NSC migration, viral replication, and therapeutic efficacy. Survival curves were evaluated with Kaplan-Meier methods. All statistical tests were two-sided.

Results: Antiglioma activity of OV-loaded HB1.F3.CD cells was effective against clinically relevant human-derived glioma models as well as a glioma stem cell-enriched xenograft model. Median survival was prolonged by 34% to 50% compared with mice treated with OV alone (GBM43FL model median survival = 19.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.26, 95% confidence interval [CI] = 1.21 to 12.23, P = .02; GBM12 model median survival = 43.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.53, 95% CI = 1.21 to 10.38, P = .02). OV-loaded HB1.F3.CD cells were shown to effectively migrate to the contralateral hemisphere and hand off the therapeutic payload of OV to targeted glioma cells. In vivo distribution and migratory kinetics of the OV-loaded HB1.F3.CD cells were successfully monitored in real time by magnetic resonance imaging. OV-loaded NSCs retained their differentiation fate and were nontumorigenic in vivo.

Conclusions: HB1.F3.CD NSCs loaded with CRAd-Survivin-pk7 overcome major limitations of OV in vivo and warrant translation in a phase I human clinical trial for patients with GBM.

Figures

Figure 1.

Permissiveness of neural stem cell (NSC) lines for adenovirus replication and efficacy in vitro/vivo. A) A transwell chamber assay was used to measure the viral progeny released from NSCs and subsequently capable of infecting glioma cells. HB1.F3.CD and ReNcell were infected with 50 IU per cell of CRAd-S-pk7 in the upper chambers of the transwell assay in the following ratio to glioma cells: 1:2, 1:10, 1:50, and 1:100. Glioma cells were placed on the bottom chambers of the transwell assay, and cells were harvested and infectivity was measured by quantitative real-time polymerase chain reaction for the viral E1A gene. Bars represent means from three independent experiments; error bars refer to 95% confidence intervals. Student t test was used. *P < .05; **P < .01. B) Cytotoxicity by trypan blue exclusion 96 hours after coculture. Bars represent means from five independent experiments; error bars refer to 95% confidence intervals. Student t test was used. *P < .05; **P < .01. C) CRAd-S-pk7 virus-loaded NSCs inhibit xenograft growth and prolong survival of mice with orthotopic glioblastoma. We stereotactically injected 2.5×105 U87MG cells into the right hemisphere of the brains of athymic nude mice (n = 8 per group). Three days after glioma establishment, both NSC lines were infected with 50 IU per cell of CRAd-S-pk7. Separate groups of mice received an injection of either 5×105 HB1.F3.CD or ReN cells loaded with CRAd-S-pk7 in a volume of 2.5 µL per mouse 2 to 3mm away from the original tumor site. Two additional groups of mice received either 2.5×107 IU of CRAd-S-pk7 alone or phosphate-buffered saline (PBS) in an identical volume and location in the brain. P1 represents P value 1 in this table where PBS treated group served as reference and P2 represents P value 2 where CRAd-S-pk7+HB1.F3.CD serve as reference. Survival curves were obtained by the Kaplan–Meier method, and overall survival time was compared between groups using log-rank test. All statistical tests were two-sided.

Figure 2.

The efficacy of HB1.F3.CD neural stem cells (NSCs) as a cell carrier for CRAd-S-pk7 virus in human-derived glioma xenografts. A) To demonstrate a major difference between glioma cells maintained in culture versus in vivo, in vitro–cultured or in vivo–cultured GBM43 cells were harvested at 2 weeks and stained for CD133 and CD15 (markers of glioma stem cells) and analyzed by fluorescent-activated cell sorting (FACS). B) To test the HB1.F3.CD cell line as a cell carrier for adenovirus against the GBM43FL glioma xenograft, 5×104 cells were implanted in the right hemisphere of nude mice. Three days after implantation, mice received intratumoral therapy of either 5×105 HB1.F3.CD cells infected with 50 IU per cell of CRAd-S-pk7, CRAd-S-pk7 alone (2.5×107 IU), HB1.F3.CD cells alone, or phosphate-buffered saline (PBS). Mice were monitored for tumor volume by bioluminescence imaging at 14 days post-therapy. C) Overall survival of mice bearing GBM43 human glioma xenografts (n = 7 per group). Survival curves were obtained by the Kaplan–Meier method, and overall survival time was compared between groups using log-rank test. All statistical tests were two-sided. P1 represents P value #1 in this table where PBS treated group served as reference and P2 represents P value #2 where CRAd-S-pk7+HB1.F3.CD serves as reference. D) The same injection strategy was used for mice bearing GBM12 human glioma xenografts (n = 10 per group). NSCs significantly increased the efficacy of CRAd-S-pk7 in both GBM43 and GBM12 models, as shown by the survival increase between the CRAd-S-pk7 group and the CRAd-S-pk7–loaded HB1.F3.CD group (P = .02 for both GBM43 and GBM12 models). P1 represents P value 1 in this table where PBS treated group served as reference and P2 represents P value 2 where CRAd-S-pk7+HB1.F3.CD serve as reference.

Figure 3.

The efficacy of HB1.F3.CD neural stem cells (NSCs) as a cell carrier for CRAd-S-pk7 virus in a glioma stem cell–derived xenograft model. A) GBM43FL glioma cells were cultured and exposed to a total of 10 Gy radiotherapy (XRT) treatment (5 days × 2 Gy), temozolomide (50 uM), or cocultured with HB1.F3.CD-GFP+ cells loaded with CRAd-S-pk7 (50 IU per cell). After 72 hours of incubation, cells were collected and stained for glioma stem cell (GSC) markers CD133 and CD15 and subjected to fluoresecent-activated cell sorting (FACS) analysis. Representative FACS plots show the percentage of CD15+, CD133+, or CD15+CD133+ GBM43FL cells. B) The percentage of positive populations of GSCs in the three treatment groups. All three populations of GSCs were statistically significantly reduced in oncolytic adenoviral virotherapy (OV)–loaded NSC treatment group compared with the XRT or chemotherapy (TMZ) treatment groups (P < .001), compared using Student t test. Bars represent means from three independent experiments; error bars refer to 95% confidence intervals. C) The OV-loaded HB1.F3.CD therapy was tested in vivo. GBM43FL cells were FACS sorted, and 5×103 CD133+ cells were intracranially implanted in the brains of nude mice (n = 7 per group). Three days after tumor implantation, mice were treated either with phosphate-buffered saline (PBS), 5×105 HB1.F3.CD cells, 5×105 HB1.F3.CD cells loaded with 50 IU per cell of CRAd-S-pk7, or 2.5×107 IU of CRAd-S-pk7 intratumorally. P1 represents P value 1 in this table where PBS treated group served as reference and P2 represents P value 2 where CRAd-S-pk7+HB1.F3.CD serve as reference. Survival curves were obtained by the Kaplan–Meier method, and overall survival time was compared between groups using the log-rank test. All statistical tests were two-sided. ***P < .001; **P < .01; *P < .05.

Figure 4.

Magnetic resonance imaging of tumor-tropic migration of oncolytic adenoviral virotherapy (OV)–HB1.F3.CD neural stem cells (NSCs) in vivo. Microparticles of iron oxide (MPIO)–labeled HB1.F3.CD cells loaded with CRAd-S-pk7 (50 IU per cell) were implanted in the left hemisphere of control mice (no tumor) or mice bearing U87 xenografts in the contralateral hemisphere. A) Serial axial T1 weighted images of a mouse brain 3 days after implantation without tumor (top panel) and with tumor (bottom panel). Arrowheads point to the area of hypointense signal extending from the site of NSC injection toward the tumor graft. B) Serial coronal T1 weighted images show an alternative view of oncolytic adenovirus–loaded NSCs without tumor (top panel) or with tumor (bottom panel). C) Prussian blue staining of the corresponding animal brains confirmed the presence of iron MPIOs at the (C1) NSC implantation site, (C2) at the tumor border, and (C3) inside the tumor mass. Scale bars, (size).

Figure 5.

In vivo differentiation of CRAd-S-pk7–loaded HB1.F3.CD cells. To follow the differentiation status of oncolytic adenovirus–loaded HB1.F3.CD cells implanted into the brains of mice, HB1.F3.CD-GFP+ loaded with CRAd-S-pk7 (50 IU per cell) were implanted into the contralateral hemisphere of mice bearing U87 glioma xenografts. Mice were killed 24 and 72 hours after neural stem cell (NSC) implantation, and brains were prepared for immunohistochemical analysis. A) Hematoxylin and eosin staining of the migratory path of NSCs from the injection site represented by the (*) to the tumor site (A1). Magnified views of the migration path: injection site (A2), center of migration path (A3), end of migration site or tumor (A4). B) Hematoxylin and eosin staining was confirmed by 4'6-diamidino-2-phenylindole (DAPI). (B1) and green fluorescent protein (GFP) (B2) staining. HB1.F3.CD-GFP+ cells (B3) were also positive for human nestin along the migratory path. (B3-2) corresponds to the slightly elongated shape seen in (A4) along the migratory path where nestin staining is spread out (arrowhead) as opposed to (B3-1), a bunched (arrowhead) shape that corresponds to (A2) or NSC implantation site. C) Mice were also sacrificed at 72 hours after NSC implantation. (C2) shows the borders of the tumor (represented by dotted line) and HB1.F3.CD-GFP+ positive cells inside human CD44+ tumor foci (C1). (C3) HB1.F3.CD-GFP+ cells that colocalize with CD44+ cells also stained positive for nestin. C4) Merge. Sizing of scale bars: Figure 5 A1 (200 μm); Figure 5 A2-A4 (50 μm); Figure 5 B1-B2 (200 μm); Figure 5 C1 (100 μm).

Figure 6.

Neural stem cells (NSCs) hand off and expand oncolytic adenoviral virotherapy (OV) therapeutic payload at distant tumor foci in vivo. OV-loaded HB1.F3.CD cells were implanted in the contralateral hemisphere of mice bearing U87 xenograft tumors. Mice were killed 72 hours after NSC implantation, and brains were preserved and prepared for immunohistochemical analysis. A, E, I) stained with 4’6-diamidino-2-phenylindole (DAPI) only. Scale bars: A–D = 200 μm, E–H=100 μm I–L = 50 μm. B, F, J) Early stages of viral replication represented by the positive staining for E1A (gray arrowhead) inside the tumor border (dotted line). C, G, K) Intermediate stages of viral replication denoted by the costaining of E1A and hexon. D, H, L) (white arrow) Hexon positive staining represents the late phases of viral infection (white arrowhead). Sizing of scale bars: Figure 6 A-D (200 μm); Figure 6 E-H (100 μm); Figure 6 I-L (50 μm).

Figure 7.

Contralateral delivery of oncolytic adenoviral virotherapy (OV)–loaded HB1.F3.CD cells show therapeutic efficacy in a mouse model of glioma. To examine distance delivery, either 5×103 U87 or GBM43FL cells were implanted into the right hemisphere of nude mice (n = 7 per group). After 3 days, mice were treated with an injection of either phosphate-buffered saline (PBS), 5×105 HB1.F3.CD cells, 5×105 HB1.F3.CD cells loaded with 50 IU per cell of CRAd-S-pk7, or 2.5×107 IU of CRAd-S-pk7 into the contralateral left hemisphere of the brain. A) Overall survival of U87MG bearing mice. B) Overall survival of GBM43FL-bearing mice. CRAd-S-pk7–loaded HB1.F3.CD extended the efficacy of OV alone in both U87MG and GBM43FL models (P < .001, P = .03, respectively). P1 represents P value 1 in this table where PBS treated group served as reference and P2 represents P value 2 where CRAd-S-pk7+HB1.F3.CD serve as reference. Survival curves were obtained by the Kaplan–Meier method, and overall survival time was compared between groups using the log-rank test. All statistical tests were two-sided. *P < .05, **P < .01, ***P < .001