Ultrasound Targeted Microbubble Destruction-Mediated Delivery of a Transcription Factor Decoy Inhibits STAT3 Signaling and Tumor Growth

Abstract

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers where it acts to promote tumor progression. A STAT3-specific transcription factor decoy has been developed to suppress STAT3 downstream signaling, but a delivery strategy is needed to improve clinical translation. Ultrasound-targeted microbubble destruction (UTMD) has been shown to enhance image-guided local delivery of molecular therapeutics to a target site. The objective of this study was to deliver STAT3 decoy to squamous cell carcinoma (SCC) tumors using UTMD to disrupt STAT3 signaling and inhibit tumor growth. Studies performed demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles inhibited STAT3 signaling in SCC cells in vitro. Studies performed in vivo demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles induced significant tumor growth inhibition (31-51% reduced tumor volume vs. controls, p < 0.05) in mice bearing SCC tumors. Furthermore, expression of STAT3 downstream target genes (Bcl-xL and cyclin D1) was significantly reduced (34-39%, p < 0.05) in tumors receiving UTMD treatment with STAT3 decoy-loaded microbubbles compared to controls. In addition, the quantity of radiolabeled STAT3 decoy detected in tumors eight hours after treatment was significantly higher with UTMD treatment compared to controls (70-150%, p < 0.05). This study demonstrates that UTMD can increase delivery of a transcription factor decoy to tumors in vivo and that the decoy can inhibit STAT3 signaling and tumor growth. These results suggest that UTMD treatment holds potential for clinical use to increase the concentration of a transcription factor signaling inhibitor in the tumor.

Keywords: Head and Neck Cancer; STAT3 Decoy; STAT3 Signaling Knockdown; Tumor Growth Inhibition; Ultrasound Targeted Microbubble Destruction.

Figure 1

Illustration of cyclic STAT3 decoy structure and sequence. Two hexa-ethyleneglycol linkages were added to generate the completely circularized cyclic decoy structure in order to increase stability in circulation. The mutant decoy contains a G to T substitution in the starred (*) position.

Figure 2

(A) Experimental setup for in vivo studies: STAT3 decoy loaded MBs were infused intravenously into mice as the tumor was insonified with ultrasound pulses. (B) Representative Contrast Mode ultrasound images of tumor immediately before and after therapy ultrasound pulses were delivered, indicating that MBs perfuse tumor and are destroyed by the therapy ultrasound pulses.

Figure 3

Acrylamide‐urea gel electrophoresis demonstrating resistance of circular decoy DNA bound to MBs against digestion by DNAseI. MBs loaded with decoy DNA were washed 3 times to remove unbound DNA and exposed to a range of DNAseI concentrations from 0 to 500 ng/ml (lanes 1, 3, 5, 7 and 9). As a control, free decoy was also challenged with DNAseI at these same concentrations (lanes 2, 4, 6, 8, and 10).

Figure 4

Luciferase expression by cultured SCC-VII cells expressing luciferase driven by a STAT3-responsive promoter, after treatment with STAT3 decoy relative to expression after treatment with mutant decoy for each test condition. STAT3 decoy MB + UTMD treatment reduced luciferase expression by 28% relative to mutant decoy MB + UTMD treatment (p < 0.01), demonstrating inhibition of STAT3 signaling with STAT3 decoy MB + UTMD treatment in vitro.

Figure 5

STAT3 decoy MB + UTMD treatment downregulates expression of Bcl-xL and cyclin D1 (STAT3 target genes) in tumors 24 hours after treatment. (A) Representative Western blot (N=4 per group). (B) Quantification of all Western blots (N=8 per group, Bcl-xL/Tubulin ratio: p=0.03 vs mutant decoy MB + UTMD treatment, cyclin D1/Tubulin ratio: p=0.04 vs mutant decoy MB + UTMD treatment).

Figure 6

(A) Representative 3D reconstructions of tumor volumes acquired using a high resolution ultrasound imaging system, indicating that tumor growth was blunted after treatment with STAT3 decoy MB + UTMD compared to mutant decoy MB + UTMD. (B) Tumor volume plotted as a function of time for each treatment group. Tumor growth was significantly inhibited after STAT3 decoy MB + UTMD treatment compared to control groups (ANOVA p = 0.003, N=6-8 per group). DT represents tumor doubling time. Asterisks (*) indicate ANOVA p < 0.05.

Figure 7

Amount of radiolabeled STAT3 decoy in tumors treated with UTMD after decoy + MB infusion compared to infusion of STAT3 decoy MB without ultrasound or STAT3 decoy only.