RH2 is a neurovirulent γ134.5 gene-deficient herpes simplex virus type 1 (HSV-1) with a lytic ability in human squamous cell carcinoma (SCC) cells; it is related to spontaneously occurring HSV-1 mutant HF10. The effect of RH2 on SCC was examined using a syngeneic C3H mouse model. After infection of mouse SCCVII cells with RH2, cell viability was decreased at first, but recovered by prolonged culture, indicating the limited replication of RH2. The antitumor ability of RH2 was examined using a bilateral SCCVII tumor model. The growth of the RH2-injected tumors was suppressed compared with that of phosphate-buffered saline-injected tumors. Moreover, the growth of contralateral tumor of RH2-treated mice was also suppressed significantly. The splenocytes of C3H mice treated with RH2 lysed more SCCVII cells than NFSaY83 cells and YAC-1 cells. The cytotoxicity of the splenocytes on SCCVII cells was significantly greater than that of splenocytes from tumor-bearing mice. Removal of CD8+ T cells from splenocytes decreased their cell killing activity remarkably. The antitumor effect of RH2 on SCCVII xenografts in nude mice was not demonstrated. These results indicate that RH2 exhibited a suppressive effect on mouse SCC, even if the replication of RH2 was limited. This is ascribed to the ability of RH2 to enhance existing tumor-specific cytotoxic T lymphocyte activity.
Oncolytic virotherapy with herpes simplex virus type 1 (HSV-1) is based on the ability of an attenuated virus to destroy infected tumor cells.1, 2, 3, 4, 5 Most cancer patients have immunity against HSV-1 and the treatment with oncolytic virus is repeated, so HSV-1 vectors are administered locally to prevent inactivation by circulating neutralizing antibodies. In this respect, this therapy’s application for head and neck cancer has an advantage in terms of accessibility.6
A number of HSV-1 vectors have been developed for solid tumors. Most of them have the main neurovirulence gene γ134.5 removed, which severely restricts their ability to replicate in the adult central nervous system and to cause latent infection.7, 8 OncoVEXGM-CSF has the γ134.5 gene deleted as well as ICP47, which otherwise blocks antigen presentation. A granulocyte macrophage colony-stimulating factor gene is also inserted to enhance the immune response to tumor antigens released after the virus replicates.3 Another type of HSV-1 vector is fusogenic viruses with improved local antitumor activity. This group includes OncSyn, OncdSyn, Synco-2D, FusOn-H2, OncoVex (GALV/CD) and HF10, a spontaneously occurring, highly attenuated virus.9, 10, 11, 12, 13 HF10, a clone of HF, forms a syncytium in a variety of cell types and has strong antitumor activity. In clinical trials, HF10 was found to be effective against breast cancer, pancreatic cancer, and head and neck cancer.14, 15, 16
Oncolytic HSV-1 can induce T-cell-mediated tumor immunity and cause regression of distant metastasis.10, 17, 18, 19 For tumor regression, CD8+ T cells and NK cells are required because depletion of these cells was shown to abolish the antitumor ability of these oncolytic viruses. Thus, in addition to their proven efficacy against a variety of tumors through a direct cytotoxic effect, these viruses can activate innate and/or adaptive tumor immunity.20, 21, 22, 23
We produced a recombinant of γ134.5 gene-deficient HSV-1 R84924 and HF,25, 26, 27 RH2,28 and determined its genome structure by DNA sequencing.29 As the fusogenic RH2 was shown to have a large part of its genome derived from HF10 and inhibited the growth of human squamous cell carcinoma (SCC) xenografts in nude mice,28 it is a novel HF10-related HSV-1 vector. However, there were differences between RH2 and HF10 in their structures, for example, the presence of UL56 gene and the absence of γ34.5 gene in RH2.29, 30 Previous studies have shown that fusogenic HSV-1 vectors have an advantage over wild-type virus in terms of antitumor activity.10, 12 In this study, we examined whether the novel oncolytic HSV-1 RH2 could suppress tumor growth and enhance tumor immunity in a syngeneic mouse model.
Materials and methods
Cells and virus
Mouse SCC cell line SCCVII, mouse fibrosarcoma cell line NFSa Y83 and mouse lymphoma cell line YAC-1 were obtained from the Riken Cell Bank (Ibaraki, Japan). SCCVII and NFSa Y83 were derived from C3H mouse tumors and YAC-1 was induced by Molony virus in A/Sn mouse.31, 32 SCCVII cells and NFSa Y83 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, 2 mM L-glutamine, 100 U ml–1 penicillin and 100 μg ml–1 streptomycin and grown in an incubator at 37 °C in a humidified atmosphere with 5% CO2. YAC-1 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum. For Vero monkey kidney cells, Eagle’s minimal essential medium containing 5% calf serum and 2 mM L-glutamine was used. HSV-1 mutant RH2 was grown in semi-confluent Vero cell monolayers. The infectivity of HSV-1 was determined by plaque formation on Vero cell monolayers covered with 0.3% methylcellulose. HSV-1 RH2 was grown in Vero cells and its infectivity was determined by plaque assay on Vero cell monolayers.26, 27
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cells grown in 96-well culture dishes were infected with HSV-1 at various multiplicities of infection (MOI), while controls were mock infected. After incubation for various intervals, 10 μl of a 5 mg ml–1 MTT solution was added to each well with 100 μl of medium. Cells were incubated for 4 h at 37 °C, and then 100 μl of 0.04N HCl in isopropanol was added. The sample was then mixed thoroughly to dissolve the dark blue crystal. After standing overnight at room temperature, the plates were read on a Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) with a reference wavelength of 630 nm and a test wavelength of 570 nm. Background absorbance at 690 nm was subtracted from the 570 nm reading. Changes from controls (room air) were calculated.
Five-week-old C3H female mice were obtained from Clea Japan (Tokyo, Japan). Bilateral tumors were produced by subcutaneous injection of 1 × 106 SCCVII cells into the back of C3H mice. Once the tumor reached approximately 5 mm in diameter, animals were divided into two groups of seven animals each. Right-side tumors received an intratumor injection of 1 × 106 plaque-forming units (PFUs) of RH2 suspended in 50 μl of phosphate-buffered saline (PBS) twice at an interval of 3 days. Animals in the control group received PBS instead of HSV-1. The experiment was started at the time HSV-1 was injected. Bidimensional tumor measurements were performed for 21 days with calipers, and tumor volume was determined using the formula for a rotational ellipsoid (L × W2 × 0.5). For immunohistochemical staining, HSV-1 was injected into the right tumors once at a dose of 1 × 106 PFU while contralateral tumors were left untreated. Athymic 5-week-old BALB/c (nu/nu) female mice were obtained from Clea Japan. Experiments were performed with the approval of the Institute of Laboratory Animals, Osaka University Graduate School of Dentistry.
Isolation of spleen cells and lactate dehydrogenase (LDH) release assay
C3H mice were treated by intratumor injection of RH2 at a dose of 1 × 106 PFU. RH2 was injected three times at 3-day intervals. Three days after the last injection, the spleen was removed and tissue homogenates were passed through meshes. Single-cell suspension was layered on Lympholyte rat (Cedarlane, Burlington, ON, Canada), centrifuged at 1500 × g for 20 min and the leukocyte layers were harvested. The leukocytes were then washed with PBS and cultured in RPMI 1640 medium. The cytotoxicity of the splenocytes was measured by LDH release assay33 using MTX-LDH (Kyokuto, Tokyo, Japan).34 Target cells were plated in 96-well plates at 2 × 103 per well and splenocytes were added at various effector/target ratios and incubated at 37 °C for 20 h. After the experiment, the culture medium was harvested and cells were dissolved in 0.1% Triton-X 100 to release intracellular LDH. LDH reagents were added to the medium. After incubation for 15 min at 37 °C, the plates were read on a Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories) at wave length of 560 nm. The values for the medium were divided by those for the medium and cells and percentages were determined as the released LDH.27
Negative selection of lymphoid cells and flow cytometry
For negative selection of CD4+T cells or CD8+ T cells in vitro,35, 36 splenocytes dispersed in 400 μl of RPMI 1640 medium were incubated according to the manufacturer’s instructions with 100 μl of CD4 (L3T4) MicroBeads mouse or CD8a (Ly-2) MicroBeads mouse (Miltenyi Biotec, Bergisch Gladbach, Germany), to which monoclonal antibody against CD4 or CD8 had been coupled, at 4 °C for 15 min. Rat anti-mouse immunoglobulin G1 Microbeads were also used as control. They were then diluted with 10 ml of RPMI 1640 medium, centrifuged at 200 × g for 10 min and the pellet was suspended in 500 μl of RPMI 1640 medium. CD4+-depleted or CD8+-depleted fraction was obtained by loading onto MACS columns (Miltenyi Biotec).
For flow cytometric analysis, splenocytes were incubated with either rat monoclonal antibody against CD4 (GK1.5; Abcam, Cambridge, UK) or rat monoclonal antibody (YTS169.4) against CD8 (Abcam) for 20 min on ice. After washing twice, the cells were incubated with Alexa Fluor488 goat anti-rat immunoglobulin G (Life Technologies, Grand Island, NY, USA), followed by further washing and analysis by FACS caliber TM (BD, Franklin Lakes, NJ, USA).
Tumors were removed, placed in 10% buffered formalin for fixation and embedded in paraffin wax. Sections were stained with hematoxylin and eosin. For immunohistochemical staining, endogenous peroxidase was blocked by incubation in 3% H2O2 in water for 5 min at room temperature. Sections were washed in PBS and then incubated with rabbit polyclonal antibody against HSV-1 (diluted 1:500, DAKO, Glostrup, Denmark) for 30 min at room temperature. After another wash, slides were reacted with Envision+System-HRP Labeled Polymer Anti-Rabbit (DAKO) for 30 min at room temperature. HSV-1 antigen was visualized by treating with diaminobenzidine (DAKO), counterstained with hematoxylin and mounted with Entellan (Merck, Darmstadt, Germany).
SPSS for Windows (SPSS, Chicago, IL, USA) was used for statistical analyses. Results are reported as means±s.d. Comparison of mean cytotoxicity of splenocytes was achieved using one-way analysis of variance, followed by Tukey’s honestly significant differences test. For the repeated measures part of the analyses of tumor volumes, a general linear model procedure was used to conduct a repeated measures design. When overall analyses determined significance, Tukey’s honestly significant differences test was used to examine pairwise differences. The differences between right-side tumors and left-side tumors of C3H mice were also examined using Student’s t-test. P-values of <0.05 were considered statistically significant.
Infection of RH2 in mouse SCCVI cells
HSV-1 RH2 induces a large syncytium in Vero cells and oral SCC cells.28 The cytopathic effect on mouse SCCVII cells was examined at a variety of input MOI. At an MOI of 1 or 2, the infected cells did not show apparent cell fusion, but focal cell rounding appeared in a small proportion of cells. When SCCVII cells were infected at an MOI of 2, the virus titers at 48 and 72 h after infection were 5 × 106 and 1 × 105 PFU ml–1, respectively. There was no increase in the virus amounts (Figure 1).
The effect of RH2 infection on cell viability was examined by MTT assay. When cells were infected with RH2 at MOI of 1 and 2, the cell viability was decreased to 30% and 11%, respectively. However, at 72 h after infection, they recovered to 65% and 32%, respectively (Figure 2).
Histological findings in RH-2-injected SCCVII tumors in C3H mouse
Bilateral SCCVII tumors were produced subcutaneously on the back. Right-side tumors received an injection of RH2 and contralateral tumors were left untreated. Tumors were removed 1-day later and subjected to histological examination. There was necrosis in the center of the tumors, but no syncytia were demonstrated. No dense immune cell infiltration in the tumors was observed. Tumors in the contralateral side did not show any histological changes. Immunohistochemical staining using anti-HSV-1 antibody revealed HSV-1 antigen-positive cells around the center of the tumors (Figure 3). Small number of cells showed virus antigen adjacent to the degradation of tumor on 2 days, but not 3 days after RH2 injection. In the contralateral side, HSV-1 antigen was not observed in the tumors. To examine the infiltration of immune cells further, SCCVII tumors in C3H mice were injected twice at an interval of 3 days. Nine days after the first inoculation of RH2, intratumoral accumulation of round cells was observed (Figures 4a and b). Similar round cell infiltration was observed in the contralateral tumor (Figures 4c and d).
Effect of HSV-1 infection on the growth of C3H mouse tumors
Subcutaneous right-side tumors in C3H mice were injected with RH2 twice at an interval of 3 days. Contralateral tumors were left untreated. The PBS-treated tumors grew in a manner similar to that of untreated tumors, and the tumor volume at 21 days was 1520 mm3. In contrast, the growth of RH2-injected tumors was markedly suppressed and the tumor volume was <300 mm3. When tumor volumes were compared at day 21, there was a significant (P<0.05) difference between RH2-injected tumors and PBS-injected tumors (Figure 5). Contralateral-side tumors in RH2-treated mice were suppressed to 920 mm3, whereas those in PBS-treated mice did not decrease in volume. There was a significant difference (P<0.05) in tumor volumes between RH2-treated mice and PBS-treated mice. No symptoms of neurological abnormality and skin reaction at the injected sites were observed during the experiment.
Cytotoxic activity of splenocytes for murine tumor cells
C3H mice were intratumorally injected with RH2 three times at intervals of 3 days. Splenocytes were prepared and the cytotoxicity for SCCVII, NFSa Y83 mouse sarcoma cells and YAC-1 mouse lymphoma cells was examined by LDH release assay. At an effector/target ratio of 100, 90% of SCCVII cells were lysed, whereas lysis rates in NFSa Y83 cells and YAC-1 cells were 31 and 32%, respectively (Figure 6). Splenocytes from RH2-treated mice showed a significantly (P<0.05) greater effect on SCCVII than NFSa Y83 and YAC-1 cells. Splenocytes from the tumor-bearing mice lysed 62% of SCCVII, which was significantly higher than those for NFSa Y83 and YAC-1 cells, indicating the presence of specific anti-SCCVII immunity in tumor-bearing mice. In terms of the cytotoxicity to SCCVII cells, there was a significant (P<0.05) difference between RH2-injected mice and tumor-bearing mice at effector/target ratios of 50 and 100.
Effect of depletion of CD4+ or CD8+ T cells on the cytotoxicity of spleen cells
To deplete CD4+ or CD8+ T lymphocytes, the splenocytes were exposed to MicroBeads coupled with anti-CD4 or anti-CD8 antibody, and the unadsorbed fraction was obtained. Flow cytometric analysis revealed that the proportion of CD4+ T cells in the splenocytes was reduced from 25.3 to 4.0% after the treatment with MicroBeads. CD8+ T cells were decreased from 6.8 to 3.2% by the treatment with MicroBeads. The cytotoxicities of the CD4+-depleted fraction and the CD8+-depleted fraction were tested for the cytotoxicity to SCCVII cells after adjustment for the total number of splenocytes. The proportions with specific lysis for the CD4+ T-cell-depleted fraction and the CD8+ T-cell-depleted fraction were 25% and 10%, respectively (Figure 7).
Effect of HSV-1 infection on the growth of mouse tumors in nude mouse
To examine the possibility that RH2 enhances innate immunity to inhibit the growth of tumor, the effect of RH2 on SCCVII tumors was examined in nude mice in a manner similar to that described for C3H mice. In nude mice, SCCVII grew rapidly compared with the syngeneic model and the tumor volume reached 1480 mm3 at 12 days after the intratumoral injection of PBS. In tumors that received RH2, the tumor volume showed the lowest values until 9 days, but there was no difference between RH2-treated tumors and untreated tumors at 12 days after the initiation of the treatment (Figure 8).
Syncytial mutants of HSV-1 are expected to spread through cell-to-cell interaction and to cause extensive cell death. Nakamori et al.10 succeeded in the production of fusogenic virus and suggested that virus replication was a prerequisite for the effect of oncolytic HSV-1. Another fusogenic HSV-1, HF10, was derived from an in vitro-passaged laboratory strain of HSV-1 and can replicate in mouse ascites, melanoma and breast cancers.9, 14, 37 RH2 used in this study has a genome derived from both γ134.5-deficient R849 and HF, the parental strain of HF10.28 Large syncytial formation is the hallmark of RH2 in human SCC cells and xenografts in nude mouse. In this study, we found that the viral titer of RH2 in SCCVII did not increase from 48 to 72 h after infection, indicating the limited spread of infection. Although cell viability was temporarily decreased by RH2 infection, it recovered by prolonged incubation because of the growth of uninfected surrounding cells. Consistent with this finding, syncytium formation was not demonstrated in RH2-injected tumors and only a small number of HSV-1 antigen-positive tumor cells appeared. Thus, we concluded that the replication of RH2 was limited in SCCVII cells.
Nevertheless, RH2 significantly suppressed the growth of SCCVII tumor at a dose of 1 × 106 PFU. Another finding was the suppression of contralateral tumors, although the effect was less than that on the RH2-injected tumors. Takakuwa et al.9 treated disseminated peritoneal NFSa Y83 cells with HF10 and induced a specific antitumor immune response. Using a bilateral tumor model, a significant antitumor effect of HF10 on contralateral tumors was observed in mouse melanoma and colorectal cancer at a dose of 1 × 107 PFU.37, 38 Intratumor infiltration of lymphoid and polymorphonuclear cells was detected on 6 and 10 days later. Nine days after RH2 injection, we found massive intratumoral infiltration of round cells, indicating the induction of tumor immunity in response to RH2 infection. It can be stated that limited virus replication is sufficient to promote tumor immunity in this model.
Fu et al.23 reported that intratumoral injection of HSV-2 into highly resistant tumors resulted in marked infiltration of neutrophils in tumor tissues and the increase of interferon-γ production, suggesting that an innate immune response mainly represented by neutrophils may be part of the virus-mediated antitumor mechanism. Miller and Fraser18 found that the effect of oncolytic HSV for metastatic melanoma was abolished in syngeneic models lacking NK and T-cell subsets. Thus, the innate immune response is implicated as a critical factor in tumor immunity as well as viral infection.39, 40Among the innate immune cell types, NK cells stand out as a key cellular factor along with their antiviral properties. To examine the cytotoxic T lymphocyte (CTL) activity, splenocytes are usually cultured in the presence of tumor antigen and cytokines, and subjected to cytotoxicity test. In this study, however, we wanted to examine the role of innate immunity as well as adaptive tumor immunity, so we used splenocytes of RH2-treated mice without further cultivation. Esaki et al.38 reported that splenocytes from HF10-treated mice contained CD4+ T cells, CD8+ T cells, B cells, NK cells, macrophages and myeloid-derived suppressor cells. Thus, these immune cells were expected to be included in the spleens of the RH2-treated mice. Indeed, splenocytes from mice treated with PBS only killed 62% of SCCVII tumors cells, indicating an on-going host response in the SCCVII tumors. Using the splenocytes, we found similar cytotoxicity for SCCVII, NF83Y cells and YAC-1 cells sensitive to NK cells, showing the presence of innate immune cells. We also found that RH2 did not inhibit the growth of mouse SCCVII tumor in nude mice. This is consistent with the findings by Thomas and Fraser,41 indicating the inability of HSV-1 1716 to reduce the growth of 4T1 mouse mammary tumors in SCID mice. It is unlikely that innate immunity has a major role in the RH2-mediated regression of SCCVII tumors. Innate immune activity may be elevated at the site of virus inoculation.
Injection of HSV into tumors induced tumor-specific CTL activity in a major histocompatibility complex class I-restricted manner.42 Li et al.43 indicated the induction of a strong T-cell response against primary and metastatic mouse mammary tumor by an HSV-2-based oncolytic virus, FusOn-H2. The overall levels of representative cytokines, namely, interferon-γ, tumor necrosis factor-α and interleukin-2, secreted by splenocytes from mice treated with FusOn-H2 were three- to fivefold higher than those from mice treated with nonfusogenic Baco-1. An increase of CD4+ T cells and CD8+ T cells was observed in the spleen of HF10-treated mice.38 In a study with fusogenic HSV-1 Synco-2D, tumor-specific CTL was undetectable in tumor-bearing mice, but was induced after intratumoral injection of Synco-2D into mouse mammary tumors. Depletion of CD4+ T cells or CD8+ T cells by intraperitoneal administration of mouse antibodies abolished the therapeutic effects of Synco-2D on both local tumor and lung metastasis.10 In this study, we found that the depletion of CD8+ T cells from splenocytes resulted in marked reduction of CTL activity on SCCVII cells, although the population of CD8+ T cells in the spleen was much smaller than that of CD4+ T cells. On the other hand, specific cytotoxicity to SCCVII was demonstrated in the splenocytes of untreated tumor-bearing mice. Thus, it can be stated that RH2 enhances the existing CTL activity of the splenocytes.
For the activation of cytotoxic T cells, tumor cells must be destroyed by HSV-1 and recognized by dendritic cells. As efficient propagation and spread distant from the injection site are blocked because of a fibrillar collagen barrier,44 a virus vector with a potent cytopathic effect and its repeated intratumoral injection would be required. In a recent clinical study on head and neck cancer, OncoVEXGM-CSF was injected into metastatic lymph node lesions several times.5 Advanced-stage patients have multiple lymph node metastases in the neck, so a large amount of virus would be required for them if the viral activity were low. Fusogenic HSV-1 RH2 exhibited a suppressive effect at doses of 1 × 106 PFU in C3H mice, whereas Synco-2D and HF10 were used at a dose of 2 × 107 and 1 × 107 PFU, respectively.10, 38 RH2 may be a favorable candidate for the treatment of multiple lesions of head and neck cancer.
In conclusion, the novel fusogenic HSV-1 vectors can infect mouse SCC cells, but do not spread to neighboring cells in vitro and in vivo. In this study, RH2 exhibited antitumor ability in both injected tumors and contralateral tumors. This can be ascribed to the ability to enhance the tumor-specific activity of CD8+ T cells.
This investigation was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
About this article
Cancer Gene Therapy (2016)