Efficacy of a third-generation oncolytic herpes simplex virus in refractory soft tissue sarcoma xenograft models

Abstract

Malignant soft tissue tumors, particularly highly malignant leiomyosarcomas, are resistant to chemotherapy and associated with a poor prognosis. T-01, a third-generation genetically modified herpes simplex virus type 1, replicates in tumor cells alone and exerts a cell-killing effect. The current study aimed to investigate the antitumor effect of T-01, which is a novel treatment for leiomyosarcoma. In vitro, six human cell lines and one mouse sarcoma cell line were assessed for T-01 cytotoxicity. In vivo, the efficacy of T-01 was examined in subcutaneously transplanted leiomyosarcoma (SK-LMS-1) cells and subcutaneously or intraperitoneally transplanted mouse sarcoma (CCRF S-180II) cells. Cytokines were assessed using ELISpot assay with splenocytes from the allogeneic models for immunological evaluation. T-01 showed cytotoxicity in all seven cell lines (p < 0.001). In the SK-LMS-1 xenotransplantation model, tumor growth was suppressed by T-01 administration (p = 0.02). In the CCRF S-180II subcutaneous tumor model, bilateral tumor growth was significantly suppressed in the T-01-treated group compared with the control group (p < 0.001). In the peritoneal dissemination model, T-01 treatment caused significant survival prolongation compared with the control (p < 0.01). In conclusion, third-generation genetically modified herpes simplex virus type 1 may be an effective novel therapy against refractory sarcomas.

Keywords: T-01; herpes simplex virus; malignant soft tissue tumor; mouse xenograft model; oncolytic virus.

Graphical abstract

Figure 1

Cytotoxic activity of T-01 in vitro Cell lines (such as SKN, RKN, SK-UT-1, SK-LMS-1, RD, RMS-YM, and CCRF S-180II) were treated with the T-01 virus (MOI = 0.01 [filled circles] or 0.1 [open circles]) and were incubated for a specific number of days. The number of surviving cells was counted and expressed as a percentage relative to that in the PBS control at each time point. Data are expressed as mean ± SE (n = 6/time point).

Figure 2

Viral replication of T-01 in vitro In vitro virus yields were determined using plaque assays 48 h after infection with T-01 (MOI = 0.01) in vero or sarcoma cells (such as SKN, RKN, SK-UT-1, SK-LMS-1, RD, RMS-YM, and CCRF S-180II) (5 × 10⁵ cells/well). The bold line indicates the initial virus concentration. Data are expressed as mean ± SE (n = 6).

Figure 3

Dose-dependent antitumor effect of T-01 using different administration protocols in mice with subcutaneous tumors (A) SK-LMS-1 and (B) RMS-YM cells were implanted subcutaneously in female athymic mice. Tumors were inoculated twice weekly (days 0 and 3) with T-01 (2.0 × 10⁵ PFU [open circle] or 2.0 × 10⁶ PFU [filled squares]) or PBS (solid circles). Data are expressed as mean ± SE (n = 10 mice/group). (C) SK-LMS-1 cell-derived tumors in female athymic mice were inoculated twice weekly (days 0 and 3) with PBS (filled circles) or T-01 (2.0 × 10⁶ PFU) at various time points: 2 (open circle), 4 (filled squares), or 8 (open squares) inoculations. Data are expressed as mean ± SE (n = 10 mice/group). ∗p < 0.05 and ∗∗p < 0.01 versus PBS treatment.

Figure 4

Immunohistochemical analysis of HSV-1 in xenograft tumors Athymic mice were subcutaneously transplanted with SK-LMS-1 (A–D) and RMS-YM (E–H) cells, and the resulting subcutaneous tumors were inoculated with T-01 (2.0 × 10⁶ PFU) or PBS twice weekly (days 0 and 3). Mice were euthanized on day 7 after inoculation, and tissue sections were stained with H&E (A, B, E, and F), anti-HSV-1 antibody (C, D), or X-gal (G, H). ICR mice with bilateral subcutaneous tumors arising from the CCRF S-180II cells (I–N) were established, and one of the bilateral subcutaneous tumors was inoculated with T-01 (2.0 × 10⁶ PFU) or PBS twice weekly (days 0 and 3). Mice were euthanized 7 days after inoculation, and histological images of tissue sections stained with H&E (I–K) or X-gal (L–N) are shown. Representative images from these experiments are presented.

Figure 5

Cytopathic effects of T-01 in immunocompetent mice with bilateral subcutaneous tumors Male ICR mice harboring subcutaneous CCRF S-180II tumors in their bilateral flanks were established. Tumors on one side were inoculated with T-01 (2.0 × 10⁵ PFU [solid square] or 2.0 × 10⁶ PFU [open circle]) or PBS (solid circle) twice weekly (days 0 and 3). Tumor volumes on the (A) inoculated and (B) uninoculated sides were evaluated. Data are presented as mean ± SE (n = 8 mice/group). ∗∗p < 0.01 versus PBS treatment.

Figure 6

IFNγ, IL-2, IL-4, and IL-10 levels in the splenocytes of CCRF S-180II tumor-bearing mice Male ICR mice with subcutaneously established CCRF S-180II tumors on the bilateral dorsum were treated with PBS or T-01 (2.0 × 10⁶ PFU) twice weekly (days 0 and 3). ELISPOT assays for (A) IFNγ, (B) IL-2, (C) IL-4, and (D) IL-10 were performed with the splenocytes of each group. Data are presented as mean ± SE (n = 5 mice/group). ∗p < 0.05 and ∗∗p < 0.01 versus PBS treatment.

Figure 7

Cytotoxic effect of T-01 in mice with peritoneal dissemination Mouse cells (CCRF S-180II) were transplanted into the peritoneal cavity of male ICR mice. The graph shows the survival rate of CCRF S-180II tumors after intraperitoneal inoculation of PBS (filled circles) or T-01 (2.0 × 10⁶ PFU) twice weekly (days 0 and 3) at various time points (1 week [open circles], 2 weeks [filled squares], or 4 weeks [open squares]). Data are presented as mean ± SE (n = 10 mice/group). ∗∗p < 0.01 versus PBS treatment.