Suicide gene therapy and cytokine gene therapy are promising approaches for cancer gene therapy (21
). Various strategies using HSVtk
suicide gene therapy combined with local delivery of cytokine genes have been explored in preclinical models to target systemic neoplastic disease. Gene therapies using various cytokine genes combined with HSVtk
suicide gene have been shown to increase anti-tumor effects leading to prolongation of survival, partial protection against a subsequent tumor challenge, and infiltration of immune cells into the tumor (24
This study was undertaken to determine if cytokine gene therapy using IFN-γ and GM-CSF in combination with HSVtk suicide gene therapy could increase anti-tumor effects at local and distant tumor sites. For this purpose, we cloned retroviral vector expressing HSVtk and tricistronic retroviral vector to co-express IFN-γ and GM-CSF. This tricistronic vector expressed both cytokines as well as single gene expression vectors ().
Rodent tumor cells have been engineered to secrete cytokines locally in an attempt to elicit immune responses against the tumors (28
). Secretion of IL-2, IL-4, IFN-γ or GM-CSF by a variety of such tumor cells was shown to reduce the tumorigenicity of these cells in syngeneic animals (17
). In this context, the genes for GM-CSF and IFN-γ have been studied most extensively to date. GM-CSF has potential as an important anti-neoplastic agent (16
). Armstrong et al. (9
) reported that GM-CSF expression in the weakly immunogenic murine B16-derived malignant melanoma cell line HFH18 reduced tumorigenicity and induced protective immunity in animals. In addition, they showed that GM-CSF could induce an anti-tumor effect by recruiting dendritic antigen-presenting cells. IFN-γ has also been reported to induce a potent anti-tumor immune response, largely because of the induction of MHC class I antigens. In this study, the FACS results show that the MHC class I antigen was increased in the IFN-γ-transduced cell lines. This result is compatible with the finding of Webber and Rosenberg (15
In our study, HSVtk suicide gene therapy alone showed a significant local bystander effect, in which an 80% volume reduction was produced after inoculation of 50% CT26+50% CT26/TK
at the left flank compared with the control group. This means that each HSVtk
expressing cell could inhibit approximately 0.6 unmodified neighboring cells in vivo. This result is compatible with the finding of Lee et al. (18
) that two HSVtk
cells could kill one neighbouring unmodified cell in vivo. But, one HSVtk
expressing cell in vitro could kill two adjacent cells. Suicide gene treatment alone also showed a 25% tumor volume reduction at distant tumor sites, although the p
-value was 0.052. The mechanism for the distant bystander effect of HSVtk
is not fully understood. Although the transfer of toxic metabolites via gap junction has a role, it is not possible to explain the distant anti-tumor effect. It is known that HSVtk
has a distant bystander effect via an immunologic mechanism, in which the release of tumor antigens by dying cells may induce systemic anti-tumor immunity (31
Cytokine gene therapy alone also showed a local anti-tumor effect, in which a 56% volume reduction was produced, but this result was smaller than suicide gene treatment alone (56% vs. 81%). This means that cytokine gene therapy can evoke the local anti-tumor effect but it is weaker than suicidal gene therapy. The distant anti-tumor effect was not shown in our study design, which was to sacrifice animals at day 18. However, this finding is compatible with the report of Gansbacher et al. (33
) that IFN-γ producing CMS-5 cells on the left flank did not inhibit the growth of unmodified CMS-5 cells on the opposite flank. This also explains why the effect of IFN-γ production by the tumor cells initially appeared to stimulate a local rather than a systemic immune response but ultimately led to a potent long-term protective immunity. Considering the short-term duration of this study, an additional study with a longer treatment period of cytokine gene therapy is needed to show a distant anti-tumor effect.
Finally, combined gene therapy showed significantly decreased local and distant tumor volumes, in which an 81% volume reduction at the locally inoculated tumor and a 43% volume reduction at distantly inoculated tumor were obtained. In addition, combined gene therapy showed earlier tumor reduction from day 15 than did suicide treatment alone. This means that combined gene therapy has an earlier and stronger anti-tumor effect compared with each treatment alone.
In summary, our study shows that cytokine gene therapy using IFN-γ and GM-CSF genes in combination with HSVtk suicide gene therapy could be an effective treatment modality for cancer gene therapy.