Abstract 585: Treatment of squamous cell carcinoma with ultrasound and microbubble mediated gene therapy

Abstract

Abstract Background: Ultrasound contrast agents are gas-filled microbubbles (MB) that can be induced to vibrate or cavitate in an ultrasound field. When the MB are loaded with genes and systemically injected, ultrasound-targeted MB destruction (UTMD) has been shown to facilitate focused delivery and transduction of genes to the target site. An experimental model of mouse squamous cell carcinoma was used to test the hypothesis that delivery of a suicide gene via UTMD would slow tumor growth. Methods: Lipid based perfluorobutane gas-filled MBs (2 μm diameter) were synthesized incorporating a cationic phospholipid to enable loading with DNA (100 ug per 109 MB). DNA loading was confirmed using electrophoresis and DNAseI challenge. Primary cultured mouse squamous carcinoma cells were subcutaneously injected into mice. 3-5 days after cell injection, mice were intravenously infused over 30 min with MB (5 × 108) loaded with plasmids containing reporter genes GFP (n = 3) or luciferase (n = 6). During MB infusion, ultrasound (1.3MHz) was intermittently delivered using a clinical scanner at high acoustic power, with simultaneous ultrasound imaging used to confirm MB destruction within the tumor. To assess the therapeutic potential of the system, tumors were allowed to grow to 100 µl, and MB (5 × 108) loaded with plasmid containing either the suicide gene thymidine kinase (TK) (n = 6) or GFP control (n = 6) was given to separate groups of mice along with ultrasound treatment. Gancyclovir was administered 3 days after UTMD and tumors were serially measured until they were 2ml in volume, after which animals were euthanized and tissue harvested for histology. Results: UTMD-mediated delivery of reporter genes resulted in tumor expression of luciferase (1568 RLU/min/mg) and GFP (<1%) that exceeded expression in control tumors treated with microbubbles + plasmid only (p = 0.02). GFP expression was seen in both peri-vascular areas as well as individual tumor cells, both in small percentages. The doubling rate of TK-treated tumors (2.91 days) was lower than that of GFP treated tumors (3.44 days, p = 0.02) despite equal ultrasound and gancyclovir treatment regimes. Relative to GFP-treated tumors, TK-treated tumors had greater apoptosis (p = 0.04) and increased microvascular density (p = 0.03). Conclusions: Our data demonstrate the principle that the acoustic behavior of MB can be exploited to therapeutic end. Single dose UTMD-mediated delivery of suicide gene slows the growth of squamous cell carcinoma in this experimental model. Tumor growth suppression could be through an anti-angiogenic mechanism, as reporter gene expression was largely peri-vascular in location. Increased microvasculature seen in TK-treated tumors might be a result of compensatory angiogenesis, although this requires further study. UTMD is a promising non-viral method for targeting gene therapy which may be useful in the treatment of a spectrum of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 585.