A Novel Luciferase Based Reporter System Can Monitor Activation of the Her2/neu Pathway In Vivo

Abstract

Purpose/Objective(s)To develop a split-luciferase based reporter system that allows us to monitor activation of the Her2/neu pathway in vivo in a quantitative and highly sensitive manner.Material/MethodsWe have engineered fusion proteins of the Her2 receptor to the N-terminal fragment of luciferase as well as of its downstream binding partner Shc to the C-terminal fragment of luciferase. Upon activation and binding of the Her2 receptor to Shc, the luciferase function becomes partly reconstituted and in the presence of its substrate luciferin, light is emitted which can be detected by our bioluminescence imaging chamber (Xenogen IVIS).ResultsWe have transduced the MDA 231 breast cancer cell line with our reporter system and tested it for activation with various stimuli in vitro as well as in vivo. Treatment with EGF, which is known to activate the Her2 pathway, albeit not specifically, caused a 2 fold increase of the background signal of non-treated cells. To test our reporter in vivo we injected 2∗102 MDA231 Her2-Nluc Shc-CLuc cells in the hind legs of athymic nude mice to form xenograft tumors. When tumors reached a size of 8mm, they were subjected to irradiation with 5Gy and mice imaged for 9 consecutive days. A strong induction in the irradiated leg (3-fold) was observed starting 5h after irradiation and peaking at 24h after irradiation.ConclusionsWe believe that our reporter system is a powerful tool to study the biology of the Her2-neu pathway. In particular our system is a very sensitive method to investigate newly developed drugs that interfere with the Her2/neu pathway in vivo. Purpose/Objective(s)To develop a split-luciferase based reporter system that allows us to monitor activation of the Her2/neu pathway in vivo in a quantitative and highly sensitive manner. To develop a split-luciferase based reporter system that allows us to monitor activation of the Her2/neu pathway in vivo in a quantitative and highly sensitive manner. Material/MethodsWe have engineered fusion proteins of the Her2 receptor to the N-terminal fragment of luciferase as well as of its downstream binding partner Shc to the C-terminal fragment of luciferase. Upon activation and binding of the Her2 receptor to Shc, the luciferase function becomes partly reconstituted and in the presence of its substrate luciferin, light is emitted which can be detected by our bioluminescence imaging chamber (Xenogen IVIS). We have engineered fusion proteins of the Her2 receptor to the N-terminal fragment of luciferase as well as of its downstream binding partner Shc to the C-terminal fragment of luciferase. Upon activation and binding of the Her2 receptor to Shc, the luciferase function becomes partly reconstituted and in the presence of its substrate luciferin, light is emitted which can be detected by our bioluminescence imaging chamber (Xenogen IVIS). ResultsWe have transduced the MDA 231 breast cancer cell line with our reporter system and tested it for activation with various stimuli in vitro as well as in vivo. Treatment with EGF, which is known to activate the Her2 pathway, albeit not specifically, caused a 2 fold increase of the background signal of non-treated cells. To test our reporter in vivo we injected 2∗102 MDA231 Her2-Nluc Shc-CLuc cells in the hind legs of athymic nude mice to form xenograft tumors. When tumors reached a size of 8mm, they were subjected to irradiation with 5Gy and mice imaged for 9 consecutive days. A strong induction in the irradiated leg (3-fold) was observed starting 5h after irradiation and peaking at 24h after irradiation. We have transduced the MDA 231 breast cancer cell line with our reporter system and tested it for activation with various stimuli in vitro as well as in vivo. Treatment with EGF, which is known to activate the Her2 pathway, albeit not specifically, caused a 2 fold increase of the background signal of non-treated cells. To test our reporter in vivo we injected 2∗102 MDA231 Her2-Nluc Shc-CLuc cells in the hind legs of athymic nude mice to form xenograft tumors. When tumors reached a size of 8mm, they were subjected to irradiation with 5Gy and mice imaged for 9 consecutive days. A strong induction in the irradiated leg (3-fold) was observed starting 5h after irradiation and peaking at 24h after irradiation. ConclusionsWe believe that our reporter system is a powerful tool to study the biology of the Her2-neu pathway. In particular our system is a very sensitive method to investigate newly developed drugs that interfere with the Her2/neu pathway in vivo. We believe that our reporter system is a powerful tool to study the biology of the Her2-neu pathway. In particular our system is a very sensitive method to investigate newly developed drugs that interfere with the Her2/neu pathway in vivo.