Assessment Of Liposome Mediated Suicide Gene Therapy For Breast Cancer By In Vivo Imaging

Background: The continuously increasing morbidity and high mortality rate withbreast cancer has lead to seeking of novel efficient therapeutic strategies. One of thesestrategies under study involves the so-called suicide genes. TK suicide gene has seenwidespread adoption for tumor therapy strategy in preclinical research. Cationicliposome has the ability to facilitate gene transfection both in vivo and in vitro. Dueto its obvious advantages over viral vectors, such as no safety issues and high loadingcapability, cationic liposome has been successfully used in various studies focused onin vivo gene transfer and cancer treatment. However, there is still no efficient trackingsystem to monitor the biological behavior of liposome or sensitive technology toevaluate the efficacy of its gene delivery activity. A novel emerging technology thatmight have the potential to fill this blank is in vivo imaging which, based on thetechnology of molecular imaging, can provide noninvasive visualization andcharacterization of biological processes in cellular level in living organisms.Luciferases are important tools for small animal imaging, often used to label tumorcells and track their fate in living animals, or report the expression of genes of interest.When two or more luciferase reporter genes are used for imaging simultaneously orsuccessively, multiple biological processes in a single animal can be observed. Thedual reporter gene imaging technique provides us with useful platform to assess thegene transduction efficiency of cationic liposome and evaluate treatment responsesunder liposome mediated suicide gene therapy at the same time.Objective: We employed in vivo imaging technique to serve the purpose to track andmonitor the fate of implanted mammary tumor cells in living BALB/c mice, to followthe growth, regression and response to treatment of implanted tumors, and to assessthe gene transfection efficacy as well as anti-tumor effect of liposome mediated TKsuicide gene therapy. We attempted to explore the application of firefly luciferase andrenila luciferase based dual imaging technique in research of tumor management andcationic liposome mediated gene delivery. Methods: To assess liposome-mediated gene delivery efficiency and its expression invivo using molecular imaging technique,25μg of triple fusion reporter plasmid (TF)consisting of renila luciferase (Rluc), red fluorescent protein (RFP) and herpessimplex virus truncated thymidine kinase (HSV-ttk) mixed with either liposome orPBS (1:1; v:v) was injected into the muscle of the right or left hind limb in the samemouse, respectively. Serial bioluminescence imaging of Rluc activity was performedevery week for two months. For the following animal experiments,6-8weeks oldfemale BALB/c mice were injected subcutaneously with5×105DF-labeled4T1cellsinto both the right and left shoulders. The same animals underwent longitudinalbioluminescence imaging protocols as described below. On day7,8and9after4T1cell transplantation,20l freshly prepared liposome-TF complexes was injected intotumor on the right shoulder. Meanwhile, an equal amount of TF plasmid diluted inPBS at the same volume was injected into tumor on the left shoulder. Ganciclovir(GCV) at a dose of20mg/kg/day was administered intraperitoneally once a day fromday11till end of the experiment. The tumors were isolated at the end of imagingstudies and sliced, processed for immunofluorescence staining by anti-RFP or withanti-CD31antibody, and for TUNEL assay.Results: We discovered duration of a month and a half of gene expression afterintramuscular injection of Rluc-RFP-TTK plasmid. Cationic liposome was found tobe able to significantly enhance the transfection and expression level ofRluc-RFP-TTK gene. In the experiment of suicide gene therapy on4T1-DFmammary tumor model, the renilla luciferase signal intensity was found to besignificantly increased in liposome-mediated gene therapy group and could be five tosix times higher than that of PBS-DNA group. The serial optical imaging of fireflyluciferase reporter revealed in vivo tumor regression and restrained tumor growth bylong-term treatment with liposome mediated gene therapy indicating a considerableimprovement in treatment effects by liposome facilitated transfection of therapeuticTF gene. The results of TUNEL staining demonstrated a significant difference in thepercentage of apoptotic cells between liposome-mediated gene delivery group (~20%)and control group (~0.6%). The vascularization was significantly less in4T1tumorsthat received liposome-mediated HSV-ttk gene therapy compared to control group. Conclusions: Cationic liposomal nanoparticles enhanced gene delivery into4T1tumor cells which could improve the therapeutic efficacy. We have successfullysimultaneously imaged the liposome nanoparticle-mediated gene delivery andtherapeutic activity in cancer gene therapy. The study provides proof and evidencethat the therapeutic effect of HSV-ttk/GCV system on breast cancer can beremarkably enhanced by liposome nanoparticle-mediated delivery. The functioningmechanism of HSV-ttk/GCV suicide gene system was due to HSV-ttk induced cellapoptosis and neovascularization deduction in4T1tumor cells. The noninvasiveimaging technique of luciferases can be applied to track gene delivery and expression,monitor tumor cell growth and assess treatment effects in pre-clinical researches.