Combined gene therapy with cytosine deaminase plus uracil phosphoribosyl transferase and immunostimulatory IL12 and IL18 cytokines for treating prostate cancer in C57BL/6 mice

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We previously found local & distant bystander effects using cytosine deaminase (CD)+uracil phosphoribosyl transferase (UPRT) mediated gene directed enzyme prodrug therapy (CDUPRT-GDEPT) to treat mouse RM1 hormone-refractory prostate cancers (HRPC) in immunocompetent C57BL/6 mice. Treatment induced dose-dependent tumor infiltration by CD4⁺ T cells, macrophages and NK cells, suggesting potential for synergy with immunotherapy. We describe using immuno-stimulatory interleukins IL12, IL18 of proven anti-tumorigenic potential (enhanced when combined), plus GDEPT. Objective: To test the efficacy of combining CDUPRT-GDEPT with IL12 + IL18 cytokine treatment against mouse HRPC RM1 cells in vivo. Methods: HRPC mouse RM1 cells or those stably transformed with plasmid to express GFP/CDUPRT (RM1CDUPRT) were used to generate orthotopic RM1 or RM1CDUPRT tumors or RM1 lung pseudometastases in C57BL/6 mice. To assess the effects of the cytokine therapy, mice with intraprostatic RM1 tumors were injected with Adenoviruses expressing murine IL12 (AdmIL12) and/or murine IL18 (AdmIL18) on day 5; on day 6 RM1 cells were given intravenously (iv) to establish lung pseudometastases. Mice were euthanased on day 17, and prostate weight/volume, and lung colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry. Mouse sera were analyzed by Luminex technology to assess cytokine profiles. To assess combination of CDUPRT-GDEPT with cytokine therapy, C57BL/6 mice were implanted with RM1CDUPRT in the prostate; 5 days later, intraprostatic cytokine treatment was given followed by intraperitoneal prodrug injections for 11 days. To assess the efficacy of treatment in targeting remote deposits, mice were given RM1 cells iv on day 6.The protocol atnecropsy was as above. Survival of mice in different treatment groups was also assessed. Results: Unlike IL12 or IL18 used alone, their combination caused significant reduction in local PC growth and showed clear synergy in reducing RM1 lung colony growth to almost negligible. Serum cytokine analysis showed a significant increase in Th1 IFN γ and IL18 and a significant reduction in Th2 IL4 and IL10 levels in the IL12 + IL18 group. Combining CDUPRT-GDEPT with IL12 + IL18 led to a further reduction in growth of both local PCs and lung colonies compared with individual treatments. There was also a clear survival advantage with 28.7% of mice alive on day 33. However, serum cytokine levels were generally immunosuppressed compared with control mice; no significant trends were seen. Conclusions: Combined CDUPRT-GDEPT with mIL12+mIL18 therapy led to a significant reduction in growth of local and remote deposits of HRPC RM1 tumors in mice. Despite their immunosuppressed state, combination therapy was more effective and provided survival advantage.