Dual-Mode Ultrasound-Guided Focused Ultrasound System Can Modulate Thermal Gene Switch-Engineered Immune Cells in Solid Tumor

Introduction: : Thermal gene switches (TGSs) enable precise control of gene expression in response to mild heating (39–43 °C), holding significant promise for therapies such as targeted gene delivery and cancer immunotherapy. However, their therapeutic potential of TGS-mediated intervention critically depends on delivering heat with high spatiotemporal precision, as TGS activity is sensitive to thermal parameters (e.g. duration, ramp rate, and duty cycle). Existing TGS activation platforms such as photothermal therapy (PTT) with infrared imaging, magnetic hyperthermia with magnetic resonance (MR) thermometry, and focused ultrasound (FUS) with MR thermometry face limitations, including poor tissue penetration, absence of real-time image guidance, or reliance on exogenous agents. To address these challenges, we present a compact ultrasound-guided focused ultrasound (USgFUS) system that seamlessly integrates anatomical and thermal imaging with localized mild hyperthermia in deep tissue. This platform enables precise modulation of TGS activation, advancing its translational potential.

Materials and

Methods: : A dual-mode USgFUS system was developed using an ultrasound (US) imaging system (Vantage 128, Verasonics Inc.) and a phased-array imaging transducer (ATL L7-4, Philips Healthcare), shared for both imaging and focused ultrasound (FUS) hyperthermia. B-mode images were generated via planewave compounding technique and used for anatomical guidance. Thermal strain imaging (TSI) was derived from B-mode images by tracking speckle displacements induced by FUS heating. For calibration, thermal strain was calculated in 4T1 breast tumors in BALB/c mice (n = 3) and linearly fitted to intratumoral thermocouple readings. This calibration was then applied in three additional tumors for in vivo USgFUS demonstration. To assess USgFUS modulation of TGS activity, bilateral subcutaneous 4T1 tumors were inoculated in BALB/c mice, and each were injected with Jurkat T cells engineered to express Firefly luciferase (Fluc) under TGS control. In one group (n=3), one tumor per mouse received continuous 20-minute FUS heating to 42°C, while the contralateral tumor remained unheated. In a second group (n=3), one tumor received continuous 20-minute FUS, and the other received two 10-minute pulsed sessions. TGS activity was quantified by luminescence using IVIS 4 hours after heating.

Results, Conclusions, and Discussions:: The USgFUS system achieved co-registered imaging and therapy using a single transducer, offering a compact design and targeting reliability. As shown in Figure 1A, representative overlaid B-mode and TSI images of a mice tumor before (top) and after 20 minutes of FUS heating (bottom) revealed a focal temperature increase of 10°C. In vivo calibration of the tissue-dependent thermal strain coefficient yielded k = 7.00 with a linear correlation (R² = 0.97) between thermal strain calculation and thermocouple measurements (Figure 1B). Using this calibration, TSI-based temperature estimation achieved a mean absolute error of 0.75°C and a peak error of 1.85°C, with a representative temperature profile shown in Figure 1C. In vivo USgFUS enabled spatially controlled TGS activation in tumor-bearing mice, resulting in a ~100-fold increase in reporter (Fluc) expression in FUS-treated tumos compared to unheated (UH) controls (Figure 1D). Moreover, pulsed FUS with 67% duty cycle led to a ~1.6-fold enhancement in Fluc expression level compared to continuous FUS with the same total heating duration (Figure 1E).
In this study, we developed a compact, non-invasive USgFUS system that integrates real-time anatomical imaging, temperature monitoring, and localized hyperthermia using a standard clinical imaging probe. By employing a single transducer for both imaging and FUS heating, the system ensures precise targeting and streamlined operation. The system demonstrated spatiotemporal modulation of TGS in preclinical models and engineered cells. TSI provided reliable temperature estimation, complemented by anatomical context from B-mode imaging. In tumor-bearing mice, USgFUS effectively induced spatially confined TGS activation. Notably, pulsed FUS enhanced TGS activity compared to continuous heating, highlighting the system's potential for precise TGS modulation. This platform overcomes key limitations of existing TGS activation methods and offers significant promise for advancing non-invasive, image-guided control of gene expression in translational applications, such as gene therapy and cancer immunotherapy.

Acknowledgements and/or References (Optional)::