Antitumor effects of oncolytic herpes simplex virus type 2 against colorectal cancer in vitro and in vivo

Abstract

Background: The incidence of colorectal cancer (CRC) is on the rise. Furthermore, late-stage diagnoses and limited efficacious treatment options make CRC a complex clinical challenge. Therefore, a new therapeutic regimen with a completely novel therapeutic mechanism is necessary for CRC. In the present study, the therapeutic efficacy of oncolytic herpes simplex virus type 2 (oHSV2) in CRC was assessed in vitro and in vivo. oHSV2 is an oncolytic agent derived from herpes simplex virus type 2 that encodes granulocyte-macrophage colony-stimulating factor.

Materials and methods: We investigated the cytopathic effects of oHSV2 in CRC cell lines using the MTT assay. Then, cell cycle progression and apoptosis of oHSV2 were examined by flow cytometry. We generated a model of CRC with mouse CRC cell CT26 in BALB/c mice. The antitumor effects and adaptive immune response of oHSV2 were assessed in tumor-bearing mice. The therapeutic efficacy of oHSV2 was compared with the traditional chemotherapeutic agent, 5-fluorouracil.

Results: The in vitro data showed that oHSV2 infected the CRC cell lines successfully and that the tumor cells formed a significant number of syncytiae postinfection. The oHSV2 killed cancer cells independent of the cell cycle and mainly caused tumor cells necrosis. The in vivo results showed that oHSV2 significantly inhibited tumor growth and prolonged survival of tumor-bearing mice without weight loss. With virus replication, oHSV2 not only resulted in a reduction of myeloid-derived suppressor cells and regulatory T cells in the spleen, but also increased the number of mature dendritic cells in tumor-draining lymph nodes and the effective CD4⁺T and CD8⁺T-cells in the tumor microenvironment.

Conclusion: Our study provides the first evidence that oHSV2 induces cell death in CRC in vitro and in vivo. These findings indicate that oHSV2 is an effective therapeutic cancer candidate that causes an oncolytic effect and recruits adaptive immune responses for an enhanced therapeutic impact, thus providing a potential therapeutic tool for treatment of CRC.

Keywords: colorectal cancer; gene therapy; granulocyte; herpes simplex virus type 2; immunotherapy; macrophage colony-stimulating factor; oncolytic virus.

Figure 1

Oncolytic effect of oHSV2. Notes: (A) The antitumor effects of oHSV2 in various colorectal cancer cell lines. Human murine CT26, LOVO, HT29 and HCT116 cell lines infected by oHSV2 at MOI =1.0. After the indicated time points, OV shows effective killing on tumor cells. (Images were observed with an inverted phase contrast microscope at 40× objective magnification.) (B) oHSV2 was used to infect various colorectal cancer cells at MOI =1.0; typical syncytia were observed after 48 h of treatment. (Images were observed with an inverted phase contrast microscope at 40× objective magnification.) Abbreviations: oHSV2, oncolytic herpes simplex virus type 2; MOI, multiplicity of infection; OV, oncolytic virus.

Figure 2

The cell viability of cancer cell was examined by using the MTT assay. Notes: (A) Cytotoxic effect of oHSV2 on CT26 and LOVO. Cancer cells were treated with oHSV2 at indicated MOI and time. Virus-induced cytotoxicity was assessed using the MTT assay. The oHSV2-mediated cytotoxicity was increased in a time- and dose-dependent manner. Each value represents the mean ± SD of three independent samples. (B) The CT26 and LOVO cells were treated with oHSV2 of different MOIs for 48 h. 5-FU was used as a positive control. Each value represents the mean ± SD of three independent samples. Abbreviations: oHSV2, oncolytic herpes simplex virus type 2; MOI, multiplicity of infection; 5-FU, 5-fluorouracil; SD, standard deviation.

Figure 3

In vitro comparison of oHSV2 with 5-FU in cell cycle and apoptosis. Notes: (A and B) Effects of oHSV2 infection after 48 h on cell cycle progression in CT26 and LOVO cells. Tumor cells were treated with oHSV2 or 5-FU or PBS, and then the distribution of cells in different phases of cell cycle was analyzed by flow cytometry after propidium iodide staining. The images show oHSV2 infection independent of cell cycle phase, whereas 5-FU induced S arrest with a marked increase of percentage of S cells. A: CT26, B: LOVO, *P<0.05 significantly different vs PBS. (C and D) Induction of apoptosis in oHSV2-treated cells. CT26 and LOVO cells infected oHSV2 at the indicated MOI for 48 h by flow cytometric analysis. It leads to cancer cells necrosis in a dose-dependent manner. However, there was no statistical difference in early stage of apoptosis. **P<0.01. Each value represents the mean ± SD of three independent samples. Abbreviations: oHSV2, oncolytic herpes simplex virus type 2; MOI, multiplicity of infection; SD, standard deviation; 5-FU, 5-fluorouracil; PBS, phosphate-buffered saline.

Figure 3

In vitro comparison of oHSV2 with 5-FU in cell cycle and apoptosis. Notes: (A and B) Effects of oHSV2 infection after 48 h on cell cycle progression in CT26 and LOVO cells. Tumor cells were treated with oHSV2 or 5-FU or PBS, and then the distribution of cells in different phases of cell cycle was analyzed by flow cytometry after propidium iodide staining. The images show oHSV2 infection independent of cell cycle phase, whereas 5-FU induced S arrest with a marked increase of percentage of S cells. A: CT26, B: LOVO, *P<0.05 significantly different vs PBS. (C and D) Induction of apoptosis in oHSV2-treated cells. CT26 and LOVO cells infected oHSV2 at the indicated MOI for 48 h by flow cytometric analysis. It leads to cancer cells necrosis in a dose-dependent manner. However, there was no statistical difference in early stage of apoptosis. **P<0.01. Each value represents the mean ± SD of three independent samples. Abbreviations: oHSV2, oncolytic herpes simplex virus type 2; MOI, multiplicity of infection; SD, standard deviation; 5-FU, 5-fluorouracil; PBS, phosphate-buffered saline.

Figure 4

GM-CSF serum concentrations over time following intratumoral injection of oHSV2 in tumor-bearing mice. Notes: BALB/c mice (N=3 per group) were treated with oHSV2, 5-FU, and PBS. Ten days after first therapy, spleens were harvested from the mice. Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; oHSV2, oncolytic herpes simplex virus type 2; 5-FU, 5-fluorouracil; PBS, phosphate-buffered saline.

Figure 5

The oHSV2 increased the antitumor immunity in vivo. Notes: (A) shows the flow cytometry analysis of one representative sample from each treatment group. (B) The image shows the percentage of MDSC and Tregs. Statistical significance was determined using one-way analysis of variance (ANOVA) (with Tukey’s posttest) (**P<0.01). Error bars represent standard error of the mean. BALB/c mice (N=3 per group) were treated with oHSV2, 5-FU, and PBS. Ten days after first therapy, TDLN and tumor were harvested from the mice. (C) shows the flow cytometry analysis of one representative sample from each treatment group. (D) The image shows the percentage of DCs in TDLN, CD4⁺T, and CD8⁺T in tumor. Statistical significance was determined using one-way ANOVA (with Tukey’s posttest) (*P<0.05, **P<0.01). Error bars represent standard error of the mean. Abbreviations: MDSC, myeloid-derived suppressor cells; Tregs, T regulatory cells; oHSV2, oncolytic herpes simplex virus type 2; 5-FU, 5-fluorouracil; PBS, phosphate-buffered saline; TDLN, tumor-draining lymph nodes; DC, dendritic cell.

Figure 5

The oHSV2 increased the antitumor immunity in vivo. Notes: (A) shows the flow cytometry analysis of one representative sample from each treatment group. (B) The image shows the percentage of MDSC and Tregs. Statistical significance was determined using one-way analysis of variance (ANOVA) (with Tukey’s posttest) (**P<0.01). Error bars represent standard error of the mean. BALB/c mice (N=3 per group) were treated with oHSV2, 5-FU, and PBS. Ten days after first therapy, TDLN and tumor were harvested from the mice. (C) shows the flow cytometry analysis of one representative sample from each treatment group. (D) The image shows the percentage of DCs in TDLN, CD4⁺T, and CD8⁺T in tumor. Statistical significance was determined using one-way ANOVA (with Tukey’s posttest) (*P<0.05, **P<0.01). Error bars represent standard error of the mean. Abbreviations: MDSC, myeloid-derived suppressor cells; Tregs, T regulatory cells; oHSV2, oncolytic herpes simplex virus type 2; 5-FU, 5-fluorouracil; PBS, phosphate-buffered saline; TDLN, tumor-draining lymph nodes; DC, dendritic cell.

Figure 6

oHSV2 suppresses the growth of CT26 colorectal tumors in mice. Notes: (A) Kaplan–Meier survival curve comparing overall survival among the three groups. Median time to tumor progression with oHSV2 was 50 days compared with 36 days with PBS alone (P<0.01) and 51 days with 5-FU alone (P=0.61). Statistical significance was defined as P<0.05. (B) Subcutaneous CT26 xenografts were established in BALB/c mice and treated with injections of oHSV2, 5-FU, or PBS. The tumor volume of mice among the different groups was measured every 4 days following treatments. The data are represented as mean ± SD. Results showed that oHSV2 significantly suppressed growth of CT26 xenografts. *P<0.05 significantly different vs PBS, **P<0.01 significantly different vs PBS. There was no statistical significance observed between the 5-FU-alone and oHSV2-alone treatment groups. (C) The mice weight was measured every 4 days following treatments up until day 28 so as to measure side effect. The data represent the mean ± SD. **P<0.01 significantly different vs 5-FU. ^##P<0.01 significantly different vs PBS. Abbreviations: oHSV2, oncolytic herpes simplex virus type 2; PBS, phosphate-buffered saline; 5-FU, 5-fluorouracil; SD, standard deviation.