Intravenous injection of oncolytic picornavirus SVV-001 prolongs animal survival in a panel of primary tumor-based orthotopic xenograft mouse models of pediatric glioma

Abstract

Background: Seneca Valley virus (SVV-001) is a nonpathogenic oncolytic virus that can be systemically administered and can pass through the blood-brain barrier. We examined its therapeutic efficacy and the mechanism of tumor cell infection in pediatric malignant gliomas.

Methods: In vitro antitumor activities were examined in primary cultures, preformed neurospheres, and self-renewing glioma cells derived from 6 patient tumor orthotopic xenograft mouse models (1 anaplastic astrocytoma and 5 GBM). In vivo therapeutic efficacy was examined by systemic treatment of preformed xenografts in 3 permissive and 2 resistant models. The functional role of sialic acid in mediating SVV-001 infection was investigated using neuraminidase and lectins that cleave or competitively bind to linkage-specific sialic acids.

Results: SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, preformed neurospheres, and self-renewing stemlike single glioma cells derived from 4 of the 6 glioma models in vitro. A single i.v. injection of SVV-001 (5 × 10(12) viral particles/kg) led to the infection of orthotopic xenografts without harming normal mouse brain cells, resulting in significantly prolonged survival in all 3 permissive and 1 resistant mouse models (P < .05). Treatment with neuraminidase and competitive binding using lectins specific for α2,3-linked and/or α2,6-linked sialic acid significantly suppressed SVV-001 infectivity (P < .01).

Conclusion: SVV-001 possesses strong antitumor activity against pediatric malignant gliomas and utilizes α2,3-linked and α2,6-linked sialic acids as mediators of tumor cell infection. Our findings support the consideration of SVV-001 for clinical trials in children with malignant glioma.

Keywords: SVV-001; malignant glioma; oncolytic virus; orthotopic xenograft; sialic acid.

Fig. 1.

Effects of SVV-001 on primary cultured tumor cells and preformed neurospheres derived from GBM xenograft models. (A) Killing of primary cultured GBM xenograft tumor cells by SVV-001. Cell viability was estimated using CCK-8 assay by measuring the absorbance at 460 nm after the cells were exposed to SVV-001 ranging from 0 to 25 vp/cell for 72 h. **P < .01 compared with the untreated control. (B) Infection of preformed GBM neurospheres with SVV-GFP. Representative images (left panel) and flow cytometry (FCM) quantitative analysis (right panel) of viable GFP⁺ cells (right lower quadrant) as well as dead GFP⁺ cells (right upper quadrant) in neurospheres derived from IC-1406GBM 48 h post incubation with SVV-GFP, ranging from 0 to 2000 vp/cell. (C) Graphs showing the time-course analysis of SVV-GFP infection in the permissive models (ICb-1227AA, IC-1406GBM, IC-2305GBM, and IC-1621GBM) as examined through FCM analysis of GFP⁺ cells (*P < .05, **P < .01). By day 7, the GFP⁺ cells in ICb-1227AA and IC-2305GBM became undetectable. No GFP expression was detected in the resistant model IC-1128GBM.

Fig. 2.

Growth suppression of self-renewing single GBM cells by SVV-001 in vitro. Single GBM cells were plated in clonal density in quadruplicates in 96-well plates and incubated in serum-free medium containing growth factors (EGF and bFGF) that favor the growth of CSCs. Formation of neurospheres was examined under a phase-contrast microscope, and cell viability was examined with a CCK-8 assay. *P < .05, **P < .01 compared with untreated control.

Fig. 3.

In vivo infection of SVV-001 in the orthotopic xenograft tumors of pediatric GBM. Mice bearing relatively large intracerebral xenografts (8–10 wk post tumor cell transplantation) received a single tail vein injection of SVV-001 (5 × 10¹²vp/kg), after which whole brains were removed at predetermined time points (24 h to 7 days), followed by IHC staining using antibodies specific to the capsid proteins of SVV-001. (A) Representative images showing the time-course increase of positively infected xenograft cells (circled in red) in the permissive model IC-1406GBM (1406, a–d), as contrast to the isolated positive reactions (arrow) in the resistant model IC-1128GBM (1128, e–h). (B) IHC staining of SVV-001 capsid protein in 2 additional permissive models, ICb-1227AA (1227, i–k) and IC-1621GBM (1621, l–m), 96 h post virus injection. The “border” between tumor mass and normal mouse brain in ICb-1277AA is marked by dotted lines (i and j). SVV-001–infected tumor cells both in the core area (i, l–m) and in the invasive front (i–k), including the single invasive cells (j and k, arrow); while sparing the normal mouse granular neurons that are in close proximity with the tumor cells in ICb-1227AA (j, green arrowhead). No SVV-001 positivity was detected in the left ventricle (*) and normal cerebral tissue (n) despite strong positive reaction in tumor cells (l–m) in the same mouse brain of IC-1621GBM. Red arrows in j and k also mark the direction of tumor cell migration. Magnification: ×40 (a–h, j, k, m), ×20 (i, l, n).

Fig. 4.

Systemic treatment of preformed GBM xenograft tumors with SVV-001 significantly prolongs survival times. (A) Graphs showing the log-rank analysis of animal survival times. Treatment of preestablished orthotopic xenografts (n = 10 per group) was performed through a single tail vein injection of SVV-001 (5 × 10¹²vp/kg) after the engrafted tumor cells (1 × 10⁵/mouse) were allowed to grow for 2 and 4 wk, respectively. Mice in the control group (n = 10) received the injection of PBS that was used to resuspend SVV-001. (B) All pairwise multiple comparison procedures were performed with the Holm–Sidak method to isolate the group or groups that differ from the others. (C) Representative images showing the elimination of ICb-1227AA xenografts by SVV-001. In the untreated animals (#3060 and #3061), the growth of xenograft tumors (arrows) almost completely destroyed the mouse cerebellum and caused severe hydrocephalus, as evidenced by enlarged bilateral ventricles (*). In the 2 mice that were treated with SVV-001 4 wk (#3079) and 2 wk (#3068) post tumor injection, respectively, no remnant of human tumor cells were detected with IHC staining using human-specific monoclonal antibodies against mitochondria, although disturbances of granular layer caused by surgical procedure were visible (arrow) compared with normal brain. (D) Western hybridization showing the decreased content of α2,6-linked (top panel) α2,3-linked (middle panel) sialic acids in the resistant model IC-1128GBM (1128) compared with the 5 permissiveness models (++ to +) toward SVV-001 in vivo and/or in vitro. Biotinylated lectins SNL (for α2,6-linkage) and MAL (for α2,3-linkage) were applied to proteins extracted from the xenograft tumors. Arrows indicate the bands that were missing or significantly reduced in the resistant model (IC-1128GBM).

Fig. 5.

Comparison of microvessel density between the small and the medium-sized xenograft tumors in IC-1227AA. (A) Representative images showing the overall sizes of xenograft tumors 2 wk (a, b) and 4 wk (d, e) post tumor cell injection. Microvessel density was estimated by counting the intratumoral blood vessels containing <8 red blood cells under high power (×40) magnifications (c, d, arrows). (B) Graph showing the significantly lower density of blood vessels in the 2-wk tumors compared with the 4-wk tumors (P < .001).

Fig. 6.

Infection of SVV-GFP in pediatric gliomas is mediated through the binding of sialic acid. (A) Representative images showing the dose-dependent suppression of SVV-GFP infection by neuraminidases, which cleave α2,3-linked and α2,6-linked terminal sialic acid residues and α2,8-linked internal sialic acid residues, in neurospheres derived from IC-1406GBM. (B) Quantitative analysis of the changes of cell viability (top panel) and the infectivity of SVV-GFP (lower panel) in pediatric GBM by treating 2 preformed neurospheres in quadruplicates with neuraminidase. Results in the treated cells were normalized to those of the untreated control. *P < .05, **P < .01. (C) Inhibition of SVV-GFP infection by linkage-specific lectins. SNL binds preferentially to α2,6-linked sialic acid, MAL to α2,3-linked sialic acid, and TRI to both α2,6-linked and α2,3-linked acids. Significant suppression was caused by all 3 lectins in the 2 models compared with untreated control (**P < .01).