Acoustic Cluster Therapy (ACT) for improved treatment of cancer and brain diseases

Chemotherapy given alone or combined with radiotherapy or surgery is a common cancer therapy. A major problem in chemotherapy is to achieve sufficient amounts of drugs into the tumor and limit the amount of drugs to healthy tissue. Treating diseases in the brain such as cancer and Alzheimer?s disease is especially challenging as the brain tissue is protected by the blood-brain barrier, which effectively blocks drugs from entering brain tissue. Thus, scientists are developing new methods for tumor-specific delivery of drugs and to overcome the blood-brain barrier. ?Acoustic Cluster Therapy? (ACT) is a novel technology that shows promising results both in treating cancer and overcoming the blood-brain barrier in mice. The technology is based on microclusters of microbubbles and microdroplets, developed by the company EXACT-Tx in Oslo. After injecting the microclusters into the blood, ultrasound is applied towards the tumor or brain whereby the microbubbles transfer energy to the microdroplets, which undergo a liquid-to-gas phase shift. This results in large bubbles (ACT bubbles) that can transiently block a fraction of the small capillaries. Further exposure of ultrasound causes these large bubbles to oscillate and induce mechanical stress on the blood vessel wall. Such mechanical effects will open the blood vessel wall thereby allowing drugs to enter into tumor tissue or into brain tissue. Ultrasound will also improve the transport of the drugs further into the tissue. We have already shown that combining ACT with injection of free drugs and drugs encapsulated into nanoparticles, improved the treatment of tumors growing in mice. In fact, the majority of the mice were cured. We have also shown that ACT can open the blood-brain barrier safely, allowing drugs to enter brain tissue. In the current project, we want to understand the underlying mechanisms of the enhanced therapeutic response. Such knowledge will allow further optimization and successful translation into new clinical practice. The first human clinical trials started in 2019 using the ACT technology with clinically approved chemotherapeutics. To elucidate the dynamics of ACT® bubble formation and oscillations in capillaries, highspeed imaging was done using an ultrafast camera at University of Pittsburgh (10 million frames per second). The imaging revealed generation of ACT bubbles of various sizes and shapes, and the oscillation amplitudes depended on the size and shape of the bubbles. Furthermore, ACT bubbles were studied in the highly vascularized chicken chorioallantoic membrane model. Using fluorescence microscopy several events were observed such as: ACT bubbles blocking the vessel wall and changing the direction of blood flow, extravasation of macromolecules out of the capillary either in a slow diffusive process or in a fast, more jet-like process. Together these data will help optimizing ACT and obtain new knowledge on the behavior of ACT bubbles in blood vessels. Patients with non-resectable tumors in the pancreas (PDAC) have extremely poor prognosis. Five years after diagnosis only 2-5 % are still alive. Thus, novel treatments are needed. We use nanoparticles developed by the Dutch company Cristal Therapeutics and measure the uptake and distribution of these nanoparticles in PDAC. A novel 2-frequenct ultrasound transducer is used. Large variation in uptake of nanoparticles, both in the untreated control tumours and in the treated tumours were found. To understand what causes this variation, uptake of nanoparticles was correlated with structure and amount of collagen fibers and with blood vessel density. Interesting, tumours with less collagen did show a higher uptake of the nanoparticles, whereas vessels density did not seem to have any impact. We have studied whether ACT induces an immune response, i.e. increases the infiltration of macrophages and neutrophils into tumors; changes the amount of functional blood vessels and vessel diameter; reduces solid stress, tissue elasticity or interstitial fluid pressure. The results are currently not conclusive. We have previously shown that ACT can temporarily open the blood-brain barrier. Now we have demonstrated that ACT safely delivered CriPec nanoparticles into the brain tissue. The nanoparticles appeared in groups distributed throughout the brain. To understand the delivery mechanisms involved, we imaged ACT bubbles and macromolecules in brain tissue through a cranial window during ACT, using a specific designed ultrasound transducer fitting around the objective on a multiphoton microscope. Extravasation occurred at particular locations and seemed to correlate with the groups of nanoparticles observed previously on brain tissue sections. Enhanced accumulation of nanoparticles in the brain is a promising strategy for treating brain disorders such as neurodegenerative dementias and structural pathologies such as malignant brain tumours.

Outcome Establishing the cranial window model to be used to image brain tissue during ultrasound treatment. Characterize and apply a novel 2 frequency US transducer. New knowledge on the activation and oscillation of ACT bubbles using high-speed imaging. New knowledge on delivery of nanoparticles to pancreatic tumours growing in mice and impact of tumour properties such as vascular density and collagen network. Impact The finding that ACT efficiently opens the blood-brain barrier and nanoparticles enter brain tissue is of great importance for the companies EXACT Therapeutics who have included this finding in one of their patents, and for Cristal Therapeutics making the nanoparticles. This finding opening new possible applications for both companies. Our findings provide new knowledge in ultrasound-mediated drug delivery which are valuable for other research groups working in this field and aiming at developing new efficient therapies for various diseases.

A major problem in cancer therapy is that less than 1 % of drugs accumulate in solid tumors. Even less is reaching the brain tissue as the blood-brain barrier (BBB) effectively blocks drugs from entering brain tissue. Acoustic Cluster Therapy (ACT) is a novel microtechnology delivery platform based on clusters of microbubbles (MBs) and microdroplets, developed by our collaborator Phoenix Solutions. After injecting these clusters, focused ultrasound (FUS) is applied to the targeted pathology whereby the MBs transfer energy to the microdroplets, which undergo a liquid-to-gas phase shift. Growing in size, these large bubbles transiently lodge and block blood flow. Further exposure of FUS causes these large bubbles to oscillate and induce biomechanical effects on the capillary wall, enhancing extravasation of molecular or NP drugs to the diseased area. We have already demonstrated that combining ACT with co-injection of the drugs paclitaxel and Abraxane®, induce a very strong increase in the therapeutic efficacy of human prostate tumors in mice. Furthermore, we have shown that ACT can open the BBB safely, allowing macromolecules to enter brain tissue. In the present project we aim to understand the underlying mechanisms of the enhanced therapeutic response. This will allow us to optimize the concept further. The behavior of ACT bubbles during US exposure will be studied using a unique high speed camera. It is of crucial importance to understand the effect of US frequency and intensity on bio-and microdistribution of differently sized therapeutic agents in different tumor models. This will be measured using advanced fluorescently-based imaging techniques. The effects induced by ACT bubbles on the capillary wall influence the transport processes and will be measured, i.e. transcapillary pressure gradient and diffusion. Based on the results from the above-mentioned studies, several therapeutic studies will be performed. The aim is to enter clinical trials in 2018.