Combination gene therapy with adenoviral vector-mediated HSV-tk plus GCV and IL-12 in an orthotopic mouse model for prostate cancer

We previously demonstrated significant therapeutic activities associated with adenoviral vector-mediated Herpes Simplex Virus/thymidine kinase (AdHSV-tk) with ganciclovir (GCV) in situ gene therapy in the RM-1 orthotopic mouse prostate cancer model and interleukin-12 (AdmIL-12) in situ gene therapy in the RM-9 orthotopic mouse prostate model for prostate cancer. In both protocols, local cytotoxicity and activities against pre-established lung metastases were demonstrated. To test whether combined AdHSV-tk + GCV + IL-12 gene therapy would lead to enhanced therapeutic effects when compared to either treatment alone, we used RM-9 mouse prostate cancer cells in both orthotopic and preestablished lung metastases models of prostate cancer. Combined treatment with a single injection of optimal doses of AdHSV-tk + GCV or AdmIL-12 led to significantly increased suppression of orthotopic tumor growth. IL-12 gene therapy alone was more effective than AdHSV-tk + GCV in suppressing spontaneous lymph node metastases and pre-established lung metastases but combination gene therapy did not result in additional anti-metastatic activities. Combination gene therapy also did not achieve significantly better animal survival compared to AdHSV-tk + GCV or AdmIL-12 alone. Analysis of localized antitumor activities demonstrated that AdHSV-tk + GCV therapy induced higher levels of necrosis compared to AdmIL-12 or combination therapy. However, both treatments alone and combination therapy produced similar increases in apoptotic index. To address the possible mechanisms of locally co-operative cytotoxic activities, we analyzed the systemic natural killer (NK) response and the numbers of tumor-infiltrating immune cells using quantitative immunohistochemical analysis. AdHSV-tk + GCV therapy alone led to detectable increases in iNOS-positive cells, CD4 + and CD8 + T-cells and moderately increased numbers of F4/80 (macrophage selective)-positive cells within treated tumors. In contrast, AdmIL-12 elicited a highly robust pattern of tumor infiltration for all four of these immune cells that was in general mimicked by combination therapy. Further analysis of the accumulation of transforming growth factor-beta1 (TGF-beta1) immunohistochemical staining demonstrated that AdHSV-tk + GCV treatment, but not AdmIL-12 treatment, produced cancer cell-associated increases in this cytokine relative to control Ad-beta -gal injections. Interestingly, local injection with AdHSV-tk + GCV induced significant splenocyte-derived NK cell cytolytic activities with maximal response 7 days following treatment, whereas AdmIL-12 injection produced significantly higher NK activity with maximal response 2 days following injection. The combined treatment produced a higher systemic NK response over the 14-day treatment period. Depletion of NK cells in vivo demonstrated that this immunocyte subpopulation was responsible for early locally cytotoxic activities induced by AdHSV-tk + GCV but not AdmIL-12 and that NK activities were largely responsible for activities against pre-established metastases generated by both gene therapy protocols.