Holographic Focused Ultrasound Hyperthermia System for Uniform Simultaneous Thermal Exposure of Multiple Tumor Spheroids

SIMPLE SUMMARY: Acoustic holograms were investigated as a method of delivering simultaneously controlled ultrasound hyperthermia to multiple tumor spheroids. Findings show that holographic lenses designed to deliver a uniform thermal dose rather than a uniform acoustic field can tune the natural focus of a transducer, allowing the exposure to ultrasound-mediated hyperthermia of several tumor spheroids simultaneously, and improving experimental throughput for future studies. Preliminary in-vitro findings suggest that hyperthermia delivered by ultrasound and polymerase chain reaction heating have different outcomes, with ultrasound being more lethal for the same thermal dose. ABSTRACT: Hyperthermia is currently used to treat cancer due to its ability to radio- and chemo-sensitize and to stimulate the immune response. While ultrasound is non-ionizing and can induce hyperthermia deep within the body non-invasively, achieving uniform and volumetric hyperthermia is challenging. This work presents a novel focused ultrasound hyperthermia system based on 3D-printed acoustic holograms combined with a high-intensity focused ultrasound (HIFU) transducer to produce a uniform iso-thermal dose in multiple targets. The system is designed with the aim of treating several 3D cell aggregates contained in an International Electrotechnical Commission (IEC) tissue-mimicking phantom with multiple wells, each holding a single tumor spheroid, with real-time temperature and thermal dose monitoring. System performance was validated using acoustic and thermal methods, ultimately yielding thermal doses in three wells that differed by less than 4%. The system was tested in vitro for delivery of thermal doses of 0–120 cumulative equivalent minutes at 43 °C (CEM(43)) to spheroids of U87-MG glioma cells. The effects of ultrasound-induced heating on the growth of these spheroids were compared with heating using a polymerase chain reaction (PCR) thermocycler. Results showed that exposing U87-MG spheroids to an ultrasound-induced thermal dose of 120 CEM(43) shrank them by 15% and decreased their growth and metabolic activity more than seen in those exposed to a thermocycler-induced heating. This low-cost approach of modifying a HIFU transducer to deliver ultrasound hyperthermia opens new avenues for accurately controlling thermal dose delivery to complex therapeutic targets using tailored acoustic holograms. Spheroid data show that thermal and non-thermal mechanisms are implicated in the response of cancer cells to non-ablative ultrasound heating.