953. Therapeutic Ultrasound Mediated Anti- Angiogenic Gene Delivery Results with Prostate Tumors Inhibition
Authors: Maayan Duvshani-Eshet, Marcelle Machluf
Journal: Molecular Therapy (2006)

Gene therapy using angiogenic inhibitors is a promising approach for the therapy of prostate cancer (PCa), which depends on the angiogenesis process for its growth. Therapeutic ultrasound (TUS), which utilizes low intensities and non-continuous waves, has emerged as a method to safely deliver genes to cells and tissues. In the present study, the efficacy of TUS for gene-delivery, in combination with Optison, is studied on endothelial cells and PCa in vitro and on tumors in vivo using two genes encoding for anti- angiogenic proteins: PEX and Endostatin (Endo).In the in vitro studies, PCa and primary endothelial cells were successfully transfected with luciferase (Luc) and GFP using TUS and Optison. Similarly, cells were transfected with pPEX or pEndo and protein expression was detected by Western Blot analysis andRT-PCR during 26 days post TUS. Conditioned media taken from PCa cells 3 and 6 days post their transfection with pPEX or pEndo, using TUS inhibited the proliferation and migration of primary endothelial cells. The proliferation of PCa cells and endothelial cells was also hindered post their own TUS-transfection with pPEX.In the in vivo studies, PCa inoculated subcutaneously (s.c) in mice were successfully transfected with GFP and Luc using TUS. The expressed proteins were localized in the tumor, and were observed during 14 days post TUS. Optison increased transfection and the distribution of the expressed protein in the tumor, thus other organs were hardly transfected. Histological analyses of tumors, skin and liver indicated no inflammation. Temperature increase measured during TUS was insignificant.In order to show the efficacy of TUS to deliver gene encoding for anti angiogenic proteins, mice were injected i.t. with pPEX following treatment with TUS-Optison. Since the studies with pLuc and pGFP showed a decrease in transfection 7 days post TUS, the mice were treated either once, or treated repeatedly once a week, during 28 days. As expected, in mice treated once with pPEX and TUS- Optison, tumor volume and angiogenic index were lower than in mice treated with TUS-Optison alone, or pPEX alone or not treated. However, in mice receiving repeated treatments, tumor volume and angiogenic index were lower than in mice treated only once, suggesting that repeated treatments of TUS are needed to inhibit angiogenesis and tumor growth. This study shows the efficacy of TUS, in combination with Optison, for efficient delivery of anti-angiogenic genes to endothelial and PCa cells in-vitro and in-vivo without significant side effects. Future studies will focus on co-transfection of pPEX and pEndo that might have a synergistic effect on tumor growth inhibition. Gene therapy using angiogenic inhibitors is a promising approach for the therapy of prostate cancer (PCa), which depends on the angiogenesis process for its growth. Therapeutic ultrasound (TUS), which utilizes low intensities and non-continuous waves, has emerged as a method to safely deliver genes to cells and tissues. In the present study, the efficacy of TUS for gene-delivery, in combination with Optison, is studied on endothelial cells and PCa in vitro and on tumors in vivo using two genes encoding for anti- angiogenic proteins: PEX and Endostatin (Endo). In the in vitro studies, PCa and primary endothelial cells were successfully transfected with luciferase (Luc) and GFP using TUS and Optison. Similarly, cells were transfected with pPEX or pEndo and protein expression was detected by Western Blot analysis andRT-PCR during 26 days post TUS. Conditioned media taken from PCa cells 3 and 6 days post their transfection with pPEX or pEndo, using TUS inhibited the proliferation and migration of primary endothelial cells. The proliferation of PCa cells and endothelial cells was also hindered post their own TUS-transfection with pPEX. In the in vivo studies, PCa inoculated subcutaneously (s.c) in mice were successfully transfected with GFP and Luc using TUS. The expressed proteins were localized in the tumor, and were observed during 14 days post TUS. Optison increased transfection and the distribution of the expressed protein in the tumor, thus other organs were hardly transfected. Histological analyses of tumors, skin and liver indicated no inflammation. Temperature increase measured during TUS was insignificant. In order to show the efficacy of TUS to deliver gene encoding for anti angiogenic proteins, mice were injected i.t. with pPEX following treatment with TUS-Optison. Since the studies with pLuc and pGFP showed a decrease in transfection 7 days post TUS, the mice were treated either once, or treated repeatedly once a week, during 28 days. As expected, in mice treated once with pPEX and TUS- Optison, tumor volume and angiogenic index were lower than in mice treated with TUS-Optison alone, or pPEX alone or not treated. However, in mice receiving repeated treatments, tumor volume and angiogenic index were lower than in mice treated only once, suggesting that repeated treatments of TUS are needed to inhibit angiogenesis and tumor growth. This study shows the efficacy of TUS, in combination with Optison, for efficient delivery of anti-angiogenic genes to endothelial and PCa cells in-vitro and in-vivo without significant side effects. Future studies will focus on co-transfection of pPEX and pEndo that might have a synergistic effect on tumor growth inhibition.