Efficacy and safety of a third-generation oncolytic herpes virus G47Δ in models of human esophageal carcinoma

Abstract

Treatment options are limited for esophageal carcinoma (EC). G47Δ, a triple-mutated, conditionally replicating herpes simplex virus type 1 (HSV-1), exhibits enhanced killing of tumor cells with high safety features. Here, we studied the efficacy of G47Δ using preclinical models of human EC. In vitro, G47Δ showed efficient cytopathic effects and replication capabilities in all eight human esophageal cancer cell lines tested. In athymic mice harboring subcutaneous tumors of human EC (KYSE180, TE8, and OE19), two intratumoral injections with G47Δ significantly inhibited the tumor growth. To mimic the clinical treatment situations, we established an orthotopic EC model using luciferase-expressing TE8 cells (TE8-luc). An intratumoral injection with G47Δ markedly inhibited the growth of orthotopic TE8-luc tumors in athymic mice. Furthermore, we evaluated the safety of applying G47Δ to the esophagus in mice. A/J mice inoculated intraesophageally or administered orally with G47Δ (10⁷ plaque-forming units [pfu]) survived for more than 2 months without remarkable symptoms, whereas the majority with wild-type HSV-1 (10⁶ pfu) deteriorated within 10 days. PCR analyses showed that the G47Δ DNA was confined to the esophagus after intraesophageal inoculation and was not detected in major organs after oral administration. Our results provide a rationale for the clinical use of G47Δ for treating EC.

Keywords: G47Δ; biodistribution; esophageal cancer; esophageal squamous cell carcinoma; herpes simplex virus; intraesophageal inoculation; oncolytic virus therapy; orthotopic tumor model; preclinical safety; teserpaturev.

Graphical abstract

Figure 1

Cytopathic effects and virus yields of G47Δ in human EC cell lines (A) Cells were seeded onto six-well plates at 2 × 10⁵ cells/well or onto 96-well plates at optimal cell density. After an overnight incubation, the cells were infected with G47Δ (MOI of 0.01 or 0.1 or mock). The cell viability was determined daily either by counting surviving cells with a Coulter Counter or by the CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay. The percentage of surviving cells is expressed as a percentage of mock-infected controls. G47Δ exhibited good cytopathic effects at an MOI of 0.1 in all human esophageal cancer cell lines examined. Data are presented as the mean of triplicates ±SD. One-way ANOVA followed by Dunnett's test was used to determine statistical significance (∗∗∗p < 0.001; ns, not significant; versus mock-infected controls). (B) Cells were seeded onto six-well plates at 3 × 105 cells/well. Triplicate wells were infected with G47Δ at an MOI of 0.01. At 24 or 48 h after infection, cells were collected and progeny virus was titered on Vero cells. The dotted line shows the assumed titer of administered G47Δ per well. Vero cell line was used as a control. In all cell lines tested, G47Δ showed good replication capabilities by 48 h after infection. The results presented are the mean of triplicates ±SD.

Figure 2

Efficacy of G47Δ in subcutaneous EC tumor models Subcutaneous tumors of a human esophageal cancer cell line (KYSE180 (upper left), TE8 (upper right), or OE19 (lower left)) were generated in 6-week-old female athymic mice. Established tumors, 5–6 mm in diameter, were inoculated with G47Δ (4 × 10⁴, 2 × 10⁵, or 1 × 10⁶ pfu) or mock on days 0 and 3 (n = 9–10 per group). G47Δ treatment caused a significant inhibition of tumor growth in all subcutaneous tumor models irrespective of the dose used. Each experiment was conducted at least twice with similar results. The results presented are the mean ± SEM (n = 9–10). One-way ANOVA followed by Dunnett's test was used to determine statistical significance (∗∗p < 0.01; ∗∗∗p < 0.001).

Figure 3

Efficacy of G47Δ in an orthotopic EC model (A) Experimental design. Female athymic mice were inoculated with TE8-luc cells (1 × 10⁶ cells) into the esophageal wall (day −18). After confirmation of the orthotopic tumors with IVIS 3 days after the implantation (on day −15), the mice were randomly divided into two groups (n = 10 per group), and were treated with an intratumoral inoculation with G47Δ (1.0 × 10⁶ pfu) or mock on day 0. The photon counts of the tumors were calculated with IVIS every 3–4 days. On day 34, all surviving mice were euthanized and the tumor weight was compared between the groups. (B) The average of the logarithmic photon flux was plotted on a chart. G47Δ treatment significantly decreased the total bioluminescence from tumors (p < 0.01 on day 13). Each experiment was conducted at least twice with similar results. The results presented are the mean ± SEM (n = 10). The Welch's t test was used to determine statistical significance (∗∗p < 0.01). (C) The changes of IVIS images with time of both treatment groups are shown.

Figure 4

Histology and immunohistochemistry of orthotopic EC tumors treated with G47Δ Orthotopic EC tumors in athymic mice were treated with an intratumoral inoculation with G47Δ (1 × 10⁶ pfu) or mock or G47Δ. Mice were euthanized 34 days after the treatments, orthotopic tumors excised, and the paraffin-embedded tissue sections stained with HE or immunostained with an anti-HSV-1 antibody. The red dotted circles indicate the extent of local tumor invasion (T, tumor; L, lumen; E, esophageal wall). Representative HE staining images of orthotopic EC tumors inoculated with mock (A) and G47Δ (B). (A) In orthotopic EC treated with mock, tumor cells invade extensively across the layers of the esophageal wall, narrowing the lumen of the esophagus. (B) In orthotopic EC treated with G47Δ, the tumor size remains small and the original structure of the esophageal wall is preserved. (C) The same tumor from (B) immunostained for HSV-1. HSV-1 positivity, presumably indicating replicating G47Δ, localizes within the tumor. Original magnification, ×30 (A) and ×40 (B, C), as indicated. Scale bars: 1 mm (A) and 500 μm (B, C).

Figure 5

Safety evaluation of G47Δ applied to the esophagus of A/J mice A/J mice were intraesophageally inoculated or orally administered with mock, G47Δ (1 × 10⁷ pfu) or wild-type HSV-1 strain F (1 × 10⁶ pfu) (n = 5 per group). Cages were then blinded, and each mouse was monitored twice a week for clinical manifestations and body weight for 2 months. (A) Clinical manifestation scores were estimated based on three parameters: appearance, activity, and response. Each parameter has a response on a five-point (0–4 points) ranging from normal (4) to death (0). Score 0 means the death of the mouse. (B) Time course changes in clinical manifestations (top) and body weight ratio based on the body weight on day 0 (bottom) in mice inoculated intraesophageally with mock (left), G47Δ (middle), or strain F (right). (C) Time course changes in clinical manifestations (top) and body weight ratio (bottom) in mice administered orally with mock (left), G47Δ (middle), or strain F (right). In both experiments (B and C), all mice treated with G47Δ survived without remarkable manifestations, but many of the mice treated with strain F deteriorated rapidly and became moribund.

Figure 6

Biodistribution of G47Δ applied to the esophagus of A/J mice A/J mice were intraesophageally inoculated or orally administered with G47Δ (1 × 10⁷ pfu) or wild-type HSV-1 strain F (1 × 10⁷ pfu) (n = 12 per group). Three mice per group were sacrificed on days 1, 3, 5, and 7, and the amount of G47Δ DNA in major organs (esophagus, adrenal glands, spinal cord, and brain) was detected by qPCR. Each dot represents the viral DNA copy number of the specimen (n = 3). (A) In mice receiving a direct inoculation with G47Δ into the subserosa of the esophagus (top), high levels of G47Δ DNA were detected from the esophagus throughout the time course. G47Δ DNA was also detected in the adrenal glands early after inoculation, but not detected by day 7. In mice receiving a direct inoculation with strain F into the subserosa of the esophagus (bottom), the copy numbers of strain F DNA remained high throughout the time course in the esophagus and increased over time in the adrenal glands, spinal cord, and brain. (B) In mice receiving an oral administration with G47Δ, G47Δ DNA was not detected in major organs throughout the time course, except for one in the esophagus on day 1 (top). In mice receiving an oral administration with strain F, strain F DNA was detected at high levels throughout the time course in the esophagus and markedly increased over time in the spinal cord and brain (bottom).