Third-generation oncolytic herpes simplex virus inhibits the growth of liver tumors in mice

Abstract

Multimodality therapies are used to manage patients with hepatocellular carcinoma (HCC), although advanced HCC is incurable. Oncolytic virus therapy is probably the next major breakthrough in cancer treatment. The third-generation oncolytic herpes simplex virus type 1 (HSV-1) T-01 kills tumor cells without damaging the surrounding normal tissues. Here we investigated the antitumor effects of T-01 on HCC and the host's immune response to HCC cells. The cytopathic activities of T-01 were tested in 14 human and 1 murine hepatoma cell line in vitro. In various mouse xenograft models, HuH-7, KYN-2, PLC/PRF/5 and HepG2 human cells and Hepa1-6 murine cells were used to investigate the in vivo efficacy of T-01. T-01 was cytotoxic to 13 cell lines (in vitro). In mouse xenograft models of subcutaneous, orthotopic and peritoneal tumor metastasis in athymic mice (BALB/c nu/nu), the growth of tumors formed by the human HCC cell lines and hepatoblastoma cell line was inhibited by T-01 compared with that of mock-inoculated tumors. In a bilateral Hepa1-6 subcutaneous tumor model in C57BL/6 mice, the growth of tumors inoculated with T-01 was inhibited, as was the case for contralateral tumors. T-01 also significantly reduced tumor growth. T-01 infection significantly enhanced antitumor efficacy via T cell-mediated immune responses. Results demonstrate that a third-generation oncolytic HSV-1 may serve as a novel treatment for patients with HCC.

Keywords: antitumor immunity; herpes simplex virus; human hepatocellular carcinoma; oncolytic immunotherapy; oncolytic virus.

Figure 1

Cytotoxic activity of T‐01 in vitro. Cell lines were treated with T‐01 virus (MOI = 0.01 [filled circles] and 0.1 [open circles]) and then incubated for the indicated days. The number of surviving cells was counted and expressed as a percentage of mock‐infected controls at each time point. Data represent the mean ± SE (n = 3 per time point)

Figure 2

Viral replication of T‐01 in vitro. The in vitro virus yields were determined using a plaque assay 48 h after infection with T‐01 (MOI = 0.01) in Vero or hepatoma/hepatoblastoma cells (5 × 10³ pfu/well). Data represent the mean ± SE (n = 3)

Figure 3

Antitumor effect of T‐01 in mice with subcutaneous tumors. Human tumor cells (HuH‐7, KYN‐2, PLC/PRF/5 and HepG2) were implanted subcutaneously into male athymic mice. Established tumors were inoculated with mock preparation (filled circles) or T‐01 (2 × 10⁶ pfu) once on day 0 (open triangles) or twice on days 0 and 3 (filled squares). Effects on tumor growth are shown in the top row; data represent the mean ± SE (n = 10 mice per time point per group). Survival is shown in the bottom row. *P < .05 and **P < .01 vs mock treatment

Figure 4

Antitumor effect of T‐01 in mice with orthotopic tumors. Human tumor cells (HuH‐7‐Luc, KYN‐2‐Luc and HepG2‐Luc) were implanted in the liver of male athymic mice. Established tumors were inoculated with mock preparation (open circles) or T‐01 (2 × 10⁶ pfu) (filled circles) once on day 0. A, Effect of T‐01 on growth of tumors from HuH‐7‐Luc cells. Tumor growth was determined by measuring the tumor volume 21 d after implantation in the mock‐ and T‐01‐treated groups (upper graph). In the middle panel, blue circles indicate orthotopic tumors in the mock‐treated and T‐01 treated mice (representative images of 5 mice in the 2 groups). Survival of mock‐treated and T‐01‐treated mice with tumors from HuH‐7‐Luc cells (A, lower panel), KYN‐2‐Luc cells (B) and HepG2‐Luc cells (C). Data represent the mean ± SE (n = 10 mice per time point per group). *P < .05 and **P < .01 vs mock treatment

Figure 5

Cytopathic effects of T‐01 in mice with peritoneal metastatic tumors. Human hepatocellular carcinoma (HCC) cells (KYN‐2‐Luc) or murine hepatoma cells (Hepa1‐6) were implanted intraperitoneally in male athymic mice or in C57BL/6 mice, respectively. Photos in KYN‐2‐Luc cells (A, lower right, mock‐infected; upper right, T‐01‐infected mice with KYN‐2‐Luc cells). Graphs show survival of KYN‐2‐Luc tumors treated by intraperitoneal inoculation (A, left), KYN‐2‐Luc tumors treated by intravenous inoculation (B) and Hepa1‐6 tumors treated by intraperitoneal inoculation (C). Tumors were inoculated as indicated with mock preparation (open circles) or T‐01 (2 × 10⁶ pfu) (filled circles) twice on days 0 and 3 (n = 8 mice per group). *P < .05 and **P < .01 vs mock treatment

Figure 6

Cytopathic effects of T‐01 in immunocompetent mice with bilateral subcutaneous tumors. C57BL/6 mice were established harboring subcutaneous Hepa1‐6 tumors in their bilateral flanks. A, Left side tumors were treated by inoculation with mock (filled circles) or T‐01 at 2 × 10⁴ pfu (open triangles), 2 × 10⁵ pfu (filled squares), or 2 × 10⁶ pfu (open diamonds) twice (days 0 and 3) (n = 8 mice per group). The growth of the left inoculated (A) and contralateral uninoculated tumors (B) was measured. Data represent the mean ± SE. **P < .01 vs mock treatment

Figure 7

IFN‐γ and IL‐4 levels in splenocytes from Hepa1‐6 tumor‐bearing mice. C57BL/6 mice harboring established subcutaneous Hepa1‐6 tumors in the left flank were treated with mock preparation or T‐01 (2 × 10⁶ pfu) twice (days 0 and 3). IFN‐γ and IL‐4 ELISPOT assays were performed in splenocytes from treated groups. Data represent the mean ± SE (n = 3 mice per group). **P < .01 vs mock treatment

Figure 8

Immunohistochemical analyses of HSV‐1 in mice with subcutaneous tumors. Tumors were induced by subcutaneously injecting HuH‐7 or Hepa1‐6 cells (5 × 10⁶) into the left flank of athymic mice or C57BL/6 mice, respectively. Subcutaneous tumors were treated with mock preparation or T‐01 (2 × 10⁶ pfu) twice (days 0 and 3). Mice were killed 7 d after inoculation, and tissue sections were stained with HE or immunostained with anti‐HSV‐1 antibody. Bar = 50 μm (magnification, ×400)

Figure 9

Immunohistochemical analyses of CD8 and CD4 expression in mice with subcutaneous tumors. Subcutaneous Hepa1‐6 tumors were established in both flanks of C57BL/6 mice, and the left‐side tumors were inoculated with T‐01 (2 × 10⁶ pfu) twice (days 0 and 3). Mice were killed 14 d after viral inoculation. Sections prepared from Hepa1‐6 tumors inoculated with T‐01 (treated and untreated sides) or mock preparation were immunostained for anti‐CD8 (top 2 rows) or anti‐CD4 (bottom 2 rows). The graphs present the numbers of CD8 or CD4 positive cells/mm². Data represent the mean ± SE (n = 3 mice/group). Bar = 100 μm (magnification ×200). **P < .01 vs mock treatment