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 wi th adenoviral vector-mediated Herpes Simplex Virus/thymidine kinase (AdHSV- tk) with ganciclovir (GCV) in situ gene therapy in the RM-1 orthotopic mous e 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 pr otocols, local cytotoxicity and activities against pre-established lung met astases were demonstrated. To test whether combined AdHSV-tk + GCV + IL-12 gene therapy would lead to enhanced therapeutic effects when compared to ei ther treatment alone, we used RM-9 mouse prostate cancer cells in both orth otopic and preestablished lung metastases models of prostate cancer. Combin ed treatment with a single injection of optimal doses of AdHSV-tk + GCV or AdmIL-12 led to significantly increased suppression of orthotopic tumor gro wth. IL-12 gene therapy alone was more effective than AdHSV-tk + GCV in sup pressing spontaneous lymph node metastases and pre-established lung metasta ses but combination gene therapy did not result in additional anti-metastat ic activities. Combination gene therapy also did not achieve significantly better animal survival compared to AdHSV-tk + GCV or AdmIL-12 alone. Analys is of localized antitumor activities demonstrated that AdHSV-tk + GCV thera py induced higher levels of necrosis compared to AdmIL-12 or combination th erapy. However, both treatments alone and combination therapy produced simi lar increases in apoptotic index. To address the possible mechanisms of loc ally co-operative cytotoxic activities, we analyzed the systemic natural ki ller (NK) response and the numbers of tumor-infiltrating immune cells using quantitative immunohistochemical analysis. AdHSV-tk + GCV therapy alone le d to detectable increases in iNOS-positive cells, CD4 + and CD8 + T-cells a nd moderately increased numbers of F4/80 (macrophage selective)-positive ce lls within treated tumors. In contrast, AdmIL-12 elicited a highly robust p attern of tumor infiltration for all four of these immune cells that was in general mimicked by combination therapy. Further analysis of the accumulat ion of transforming growth factor-beta1 (TGF-beta1) immunohistochemical sta ining demonstrated that AdHSV-tk + GCV treatment, but not AdmIL-12 treatmen t, produced cancer cell-associated increases in this cytokine relative to c ontrol Ad-beta -gal injections. Interestingly, local injection with AdHSV-t k + GCV induced significant splenocyte-derived NK cell cytolytic activities with maximal response 7 days following treatment, whereas AdmIL-12 injecti on produced significantly higher NK activity with maximal response 2 days f ollowing injection. The combined treatment produced a higher systemic NK re sponse over the 14-day treatment period. Depletion of NK cells in vivo demo nstrated that this immunocyte subpopulation was responsible for early local ly cytotoxic activities induced by AdHSV-tk + GCV but not AdmIL-12 and that NK activities were largely responsible for activities against pre-establis hed metastases generated by both gene therapy protocols.