Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines.

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.

Real-Time Intravital Imaging of Acoustic Cluster Therapy-Induced Vascular Effects in the Murine Brain.

OBJECTIVE: The blood-brain barrier (BBB) is an obstacle for cerebral drug delivery. Controlled permeabilization of the barrier by external stimuli can facilitate the delivery of drugs to the brain. Acoustic Cluster Therapy (ACT®) is a promising strategy for transiently and locally increasing the permeability of the BBB to macromolecules and nanoparticles. However, the mechanism underlying the induced permeability change and subsequent enhanced accumulation of co-injected molecules requires further elucidation. METHODS: In this study, the behavior of ACT® bubbles in microcapillaries in the murine brain was observed using real-time intravital multiphoton microscopy. For this purpose, cranial windows aligned with a ring transducer centered around an objective were mounted to the skull of mice. Dextrans labeled with 2 MDa fluorescein isothiocyanate (FITC) were injected to delineate the blood vessels and to visualize extravasation. DISCUSSION: Activated ACT® bubbles were observed to alter the blood flow, inducing transient and local increases in the fluorescence intensity of 2 MDa FITC-dextran and subsequent extravasation in the form of vascular outpouchings. The observations indicate that ACT® induced a transient vascular leakage without causing substantial damage to the vessels in the brain. CONCLUSION: The study gave novel insights into the mechanism underlying ACT®-induced enhanced BBB permeability which will be important considering treatment optimization for a safe and efficient clinical translation of ACT®.

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain. Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

Ultrafast Microscopy Imaging of Acoustic Cluster Therapy Bubbles: Activation and Oscillation.

Acoustic Cluster Therapy (ACT®) is a platform for improving drug delivery and has had promising pre-clinical results. A clinical trial is ongoing. ACT® is based on microclusters of microbubbles-microdroplets that, when sonicated, form a large ACT® bubble. The aim of this study was to obtain new knowledge on the dynamic formation and oscillations of ACT® bubbles by ultrafast optical imaging in a microchannel. The high-speed recordings revealed the microbubble-microdroplet fusion, and the gas in the microbubble acted as a vaporization seed for the microdroplet. Subsequently, the bubble grew by gas diffusion from the surrounding medium and became a large ACT® bubble with a diameter of 5-50 µm. A second ultrasound exposure at lower frequency caused the ACT® bubble to oscillate. The recorded oscillations were compared with simulations using the modified Rayleigh-Plesset equation. A term accounting for the physical boundary imposed by the microchannel wall was included. The recorded oscillation amplitudes were approximately 1-2 µm, hence similar to oscillations of smaller contrast agent microbubbles. These findings, together with our previously reported promising pre-clinical therapeutic results, suggest that these oscillations covering a large part of the vessel wall because of the large bubble volume can substantially improve therapeutic outcome.

SonoVue® vs. Sonazoid™ vs. Optison™: Which Bubble Is Best for Low-Intensity Sonoporation of Pancreatic Ductal Adenocarcinoma?

The use of ultrasound and microbubbles to enhance therapeutic efficacy (sonoporation) has shown great promise in cancer therapy from in vitro to ongoing clinical studies. The fastest bench-to-bedside translation involves the use of ultrasound contrast agents (microbubbles) and clinical diagnostic scanners. Despite substantial research in this field, it is currently not known which of these microbubbles result in the greatest enhancement of therapy within the applied conditions. Three microbubble formulations-SonoVue®, Sonazoid™, and Optison™-were physiochemically and acoustically characterized. The microbubble response to the ultrasound pulses used in vivo was simulated via a Rayleigh-Plesset type equation. The three formulations were compared in vitro for permeabilization efficacy in three different pancreatic cancer cell lines, and in vivo, using an orthotopic pancreatic cancer (PDAC) murine model. The mice were treated using one of the three formulations exposed to ultrasound from a GE Logiq E9 and C1-5 ultrasound transducer. Characterisation of the microbubbles showed a rapid degradation in concentration, shape, and/or size for both SonoVue® and Optison™ within 30 min of reconstitution/opening. Sonazoid™ showed no degradation after 1 h. Attenuation measurements indicated that SonoVue® was the softest bubble followed by Sonazoid™ then Optison™. Sonazoid™ emitted nonlinear ultrasound at the lowest MIs followed by Optison™, then SonoVue®. Simulations indicated that SonoVue® would be the most effective bubble using the evaluated ultrasound conditions. This was verified in the pre-clinical PDAC model demonstrated by improved survival and largest tumor growth inhibition. In vitro results indicated that the best microbubble formulation depends on the ultrasound parameters and concentration used, with SonoVue® being best at lower intensities and Sonazoid™ at higher intensities.

Formulation and characterisation of drug-loaded antibubbles for image-guided and ultrasound-triggered drug delivery.

The aim of this study was to develop high load-capacity antibubbles that can be visualized using diagnostic ultrasound and the encapsulated drug can be released and delivered using clinically translatable ultrasound. The antibubbles were developed by optimising a silica nanoparticle stabilised double emulsion template. We produced an emulsion with a mean size diameter of 4.23 ± 1.63 µm where 38.9 ± 3.1% of the droplets contained a one or more cores. Following conversion to antibubbles, the mean size decreased to 2.96 ± 1.94 µm where 99% of antibubbles were <10 µm. The antibubbles had a peak attenuation of 4.8 dB/cm at 3.0 MHz at a concentration of 200 × 103 particles/mL and showed distinct attenuation spikes at frequencies between 5.5 and 13.5 MHz. No increase in subharmonic response was observed for the antibubbles in contrast to SonoVue®. High-speed imaging revealed that antibubbles can release their cores at MIs of 0.6. In vivo imaging indicated that the antibubbles have a long half-life of 68.49 s vs. 40.02 s for SonoVue®. The antibubbles could be visualised using diagnostic ultrasound and could be disrupted at MIs of ≥0.6. The in vitro drug delivery results showed that antibubbles can significantly improve drug delivery (p < 0.0001) and deliver the drug within the antibubbles. In conclusion antibubbles are a viable concept for ultrasound guided drug delivery.

Acoustic Cluster Therapy (ACT®) enhances accumulation of polymeric micelles in the murine brain.

The restrictive nature of the blood-brain barrier (BBB) prevents efficient treatment of many brain diseases. Focused ultrasound in combination with microbubbles has shown to safely and transiently increase BBB permeability. Here, the potential of Acoustic Cluster Therapy (ACT®), a microbubble platform specifically engineered for theranostic purposes, to increase the permeability of the BBB and improve accumulation of IRDye® 800CW-PEG and core-crosslinked polymeric micelles (CCPM) in the murine brain, was studied. Contrast enhanced magnetic resonance imaging (MRI) showed increased BBB permeability in all animals after ACT®. Near infrared fluorescence (NIRF) images of excised brains 1 h post ACT® revealed an increased accumulation of the IRDye® 800CW-PEG (5.2-fold) and CCPM (3.7-fold) in ACT®-treated brains compared to control brains, which was retained up to 24 h post ACT®. Confocal laser scanning microscopy (CLSM) showed improved extravasation and penetration of CCPM into the brain parenchyma after ACT®. Histological examination of brain sections showed no treatment related tissue damage. This study demonstrated that ACT® increases the permeability of the BBB and enhances accumulation of macromolecules and clinically relevant nanoparticles to the brain, taking a principal step in enabling improved treatment of various brain diseases.

Ultrasound and Microbubbles Enhance Uptake of Doxorubicin in Murine Kidneys.

The use of ultrasound and microbubble-enhanced drug delivery, commonly referred to as sonoporation, has reached numerous clinical trials and has shown favourable results. Nevertheless, the microbubbles and acoustic path also pass through healthy tissues. To date, the majority of studies have focused on the impact to diseased tissues and rarely evaluated the impact on healthy and collateral tissue. The aim of this study was to test the effect and feasibility of low-intensity sonoporation on healthy kidneys in a mouse model. In our work here, we used a clinical diagnostic ultrasound system (GE Vivid E9) with a C1-5 ultrasound transducer combined with a software modification for 20-µs-long pulses to induce the ultrasound-guided drug delivery of doxorubicin (DOX) in mice kidneys in combination with SonoVue® and Sonazoid™ microbubbles. The acoustic output settings were within the commonly used diagnostic ranges. Sonoporation with SonoVue® resulted in a significant decrease in weight vs. DOX alone (p = 0.0004) in the first nine days, whilst all other comparisons were not significant. Ultrasound alone resulted in a 381% increase in DOX uptake vs. DOX alone (p = 0.0004), whilst SonoVue® (p = 0.0001) and Sonazoid™ (p < 0.0001) further increased the uptake nine days after treatment (419% and 493%, respectively). No long-standing damage was observed in the kidneys via histology. In future sonoporation and drug uptake studies, we therefore suggest including an "ultrasound alone" group to verify the actual contribution of the individual components of the procedure on the drug uptake and to perform collateral damage studies to ensure there is no negative impact of low-intensity sonoporation on healthy tissues.

Selecting the optimal parameters for sonoporation of pancreatic cancer in a pre-clinical model.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in the modern world, in part due to poor delivery of chemotherapeutics. Sonoporation can be used to enhance the efficacy of standard of care therapies for PDAC. Using xenograft models of PDAC we investigate sonoporation using four ifferent ultrasound contrast agents (UCAs) and two ultrasound regimens to identify the ideal parameters to increase therapeutic efficacy. MIA-PaCa2 xenografts in over 175 immunodeficient mice were treated with gemcitabine and paclitaxel and subjected to low or high power ultrasound (60 and 200 mW/cm2 respectively) in conjunction with one of four different UCAs. The UCAs investigated were Definity®, SonoVue®, Optison™ or Sonazoid™. Tumor volumes, vascularity, hemoglobin, and oxygenation were measured and compared to controls. High power treatment in conjunction with Sonazoid sonoporation led to significantly smaller tumors when started early (tumors ~50mm3; p = .0105), while no UCAs significantly increased efficacy in the low power cohort. This trend was also found in larger tumors (~250mm3) where all four UCA agents significantly increased therapeutic efficacy in the high power group (p < .01), while only Definity and SonoVue increased efficacy in the low power cohort (p < .03). Overall, the higher power ultrasound treatment modality was more consistently effective at decreasing tumor volume and increasing vascularity characteristics. In conclusion, Sonazoid was the most consistently effective UCA at decreasing tumor volume and increasing vascularity. Thus, we are pursuing a larger phase II clinical trial to validate the increased efficacy of sonoporation in conjunction with chemotherapy in PDAC patients.

Sonoporation for Augmenting Chemotherapy of Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer is the third most common cancer diagnosed in the United States, with more than 53,000 new cases in 2017. It is the fourth leading cause of cancer-related death in both men and women. Nonetheless, there has been no significant improvement in survival for pancreatic ductal adenocarcinoma (PDAC) patients over the past 30+ years. For this reason, there is a considerable and urgent clinical need to develop innovative strategies for effective drug delivery and treatment monitoring, resulting in improved outcomes for patients with PDAC.This chapter describes the development of contrast-enhanced ultrasound image-guided drug delivery (CEUS-IGDD or sonoporation) to be that method and to translate it from the lab to the clinic. The initial clinical focus has been on a Phase I clinical trial for enhancing the effectiveness of standard chemotherapeutics for treatment of inoperable PDAC, which demonstrated a median survival increase from 8.9 months to 17.6 months in ten subjects augmented with sonoporation compared to 63 historical controls (p = 0.011). Recent efforts to optimize this platform and move forward to a larger Phase II clinical trial will be described.

Ultrasound- and Microbubble-Assisted Gemcitabine Delivery to Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death worldwide. Poor drug delivery to tumours is thought to limit chemotherapeutic treatment efficacy. Sonoporation combines ultrasound (US) and microbubbles to increase the permeability of cell membranes. We assessed gemcitabine uptake combined with sonoporation in vitro in three PDAC cell lines (BxPC-3, MIA PaCa-2 and PANC-1). Cells were cultured in hypoxic bioreactors, while gemcitabine incubation ± sonoporation was conducted in cells with operational or inhibited nucleoside membrane transporters. Intracellular active metabolite (dFdCTP), extracellular gemcitabine, and inactive metabolite (dFdU) concentrations were measured with liquid chromatography tandem mass spectrometry. Sonoporation with increasing US intensities resulted in decreasing extracellular gemcitabine concentrations in all three cell lines with inhibited membrane transporters. In cells with inhibited membrane transporters, without sonoporation, dFdCTP concentrations were reduced down to 10% of baseline. Sonoporation partially restored gemcitabine uptake in these cells, as indicated by a moderate increase in dFdCTP concentrations (up to 37% of baseline) in MIA PaCa-2 and PANC-1. In BxPC-3, gemcitabine was effectively inactivated to dFdU, which might represent a protective mechanism against dFdCTP accumulation in these cells. Intracellular dFdCTP concentrations did not change significantly following sonoporation in any of the cell lines with operational membrane transporters, indicating that the gemcitabine activation pathway may have been saturated with the drug. Sonoporation allowed a moderate increase in gemcitabine transmembrane uptake in all three cell lines, but pre-existing nucleoside transporters were the major determinants of gemcitabine uptake and retention.

Low-Intensity Sonoporation-Induced Intracellular Signalling of Pancreatic Cancer Cells, Fibroblasts and Endothelial Cells.

The use of ultrasound (US) and microbubbles (MB), usually referred to as sonoporation, has great potential to increase the efficacy of chemotherapy. However, the molecular mechanisms that mediate sonoporation response are not well-known, and recent research suggests that cell stress induced by US + MBs may contribute to the treatment benefit. Furthermore, there is a growing understanding that the effects of US + MBs are beyond only the cancer cells and involves the tumour vasculature and microenvironment. We treated pancreatic cancer cells (MIA PaCa-2) and stromal cells, fibroblasts (BJ) and human umbilical vein endothelial cells (HUVECs), with US ± MB, and investigated the extent of uptake of cell impermeable dye (calcein, by flow cytometry), viability (cell count, Annexin/PI and WST-1 assays) and activation of a number of key proteins in important intracellular signalling pathways immediately and 2 h after sonoporation (phospho flow cytometry). Different cell types responded differently to US ± MBs in all these aspects. In general, sonoporation induces immediate, transient activation of MAP-kinases (p38, ERK1/2), and an increase in phosphorylation of ribosomal protein S6 together with dephosphorylation of 4E-BP1. The sonoporation stress-response resembles cellular responses to electroporation and pore-forming toxins in membrane repair and restoring cellular homeostasis, and may be exploited therapeutically. The stromal cells were more sensitive to sonoporation than tumoural cells, and further efforts in optimising sonoporation-enhanced therapy should be targeted at the microenvironment.

Theranostic Attributes of Acoustic Cluster Therapy and Its Use for Enhancing the Effectiveness of Liposomal Doxorubicin Treatment of Human Triple Negative Breast Cancer in Mice.

INTRODUCTION: Acoustic cluster therapy (ACT) comprises co-administration of a formulation containing microbubble/microdroplet clusters (PS101), together with a regular medicinal drug (e.g., a chemotherapeutic) and local ultrasound (US) insonation of the targeted pathological tissue (e.g., the tumor). PS101 is confined to the vascular compartment and, when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase-shift to produce bubbles with a median diameter of 22 µm when unconstrained by the capillary wall. In vivo these bubbles transiently lodge in the tumor's microvasculature. Low frequency ultrasound (300 kHz) at a low mechanical index (MI = 0.15) is then applied to drive oscillations of the deposited ACT bubbles to induce a range of biomechanical effects that locally enhance extravasation, distribution, and uptake of the co-administered drug, significantly increasing its therapeutic efficacy. METHODS: In this study we investigated the therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple negative breast cancer using orthotopic human tumor xenografts (MDA-MB-231-H.luc) in athymic mice (ICR-NCr-Foxn1nu). Doxil® (6 mg/kg, i.v.) was administered at days 0 and 21, each time immediately followed by three sequential ACT (20 ml/kg PS101) treatment procedures (n = 7-10). B-mode and nonlinear ultrasound images acquired during the activation phase were correlated to the therapeutic efficacy. RESULTS: Results show that combination with ACT induces a strong increase in the therapeutic efficacy of Doxil®, with 63% of animals in complete, stable remission at end of study, vs. 10% for Doxil® alone (p < 0.02). A significant positive correlation (p < 0.004) was found between B-mode contrast enhancement during ACT activation and therapy response. These observations indicate that ACT may also be used as a theranostic agent and that ultrasound contrast enhancement during or before ACT treatment may be employed as a biomarker of therapeutic response during clinical use.

Therapeutic Dose Response of Acoustic Cluster Therapy in Combination With Irinotecan for the Treatment of Human Colon Cancer in Mice.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40-2.00 ml PS101/kg body weight (n = 8-15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated.

Intracellular Signaling in Key Pathways Is Induced by Treatment with Ultrasound and Microbubbles in a Leukemia Cell Line, but Not in Healthy Peripheral Blood Mononuclear Cells.

Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm2) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (-0.03), CREB (+0.0), STAT3 (-0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells.

Sonoporation with Acoustic Cluster Therapy (ACT®) induces transient tumour volume reduction in a subcutaneous xenograft model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers with survival averaging only 3months if untreated following diagnosis. A major limitation in effectively treating PDAC using conventional and targeted chemotherapeutic agents, is inadequate drug delivery to the target location, predominantly due to a poorly vascularised, desmoplastic tumour microenvironment. Ultrasound in combination with ultrasound contrast agents, i.e., microbubbles, that flow through the vasculature and capillaries can be used to disrupt such mechanical barriers, potentially allowing for a greater therapeutic efficacy. This phenomenon is commonly referred to as sonoporation. In an attempt to improve the efficacy of sonoporation, novel microbubble formulations are being developed to address the limitation of commercially produced clinical diagnostic ultrasound contrast agents. In our work here we evaluate the ability of a novel formulation; namely Acoustic Cluster Therapy (ACT®) to improve the therapeutic efficacy of the chemotherapeutic agent paclitaxel, longitudinally in a xenograft model of PDAC. Results indicated that ACT® bubbles alone demonstrated no observable toxic effects, whilst ACT® in combination with paclitaxel can transiently reduce tumour volumes significantly, three days posttreatment (p=0.0347-0.0458). Quantitative 3D ultrasound validated the calliper measurements. Power Doppler ultrasound imaging indicated that ACT® in combination with paclitaxel was able to transiently sustain peak vasculature percentages as observed in the initial stages of tumour development. Nevertheless, there was no significant difference in tumour vasculature percentage at the end of treatment. The high vascular percentage correlated to the transient decrease and overall inhibition of the tumour volumes. In conclusion, ACT® improves the therapeutic efficacy of paclitaxel in a PDAC xenograft model allowing for transient tumour volume reduction and sustained tumour vasculature percentage.

Glass-windowed ultrasound transducers.

In research and industrial processes, it is increasingly common practice to combine multiple measurement modalities. Nevertheless, experimental tools that allow the co-linear combination of optical and ultrasonic transmission have rarely been reported. The aim of this study was to develop and characterise a water-matched ultrasound transducer architecture using standard components, with a central optical window larger than 10 mm in diameter allowing for optical transmission. The window can be used to place illumination or imaging apparatus such as light guides, miniature cameras, or microscope objectives, simplifying experimental setups. Four design variations of a basic architecture were fabricated and characterised with the objective to assess whether the variations influence the acoustic output. The basic architecture consisted of a piezoelectric ring and a glass disc, with an aluminium casing. The designs differed in piezoelectric element dimensions: inner diameter, ID=10 mm, outer diameter, OD=25 mm, thickness, TH=4 mm or ID=20 mm, OD=40 mm, TH=5 mm; glass disc dimensions OD=20-50 mm, TH=2-4 mm; and details of assembly. The transducers' frequency responses were characterised using electrical impedance spectroscopy and pulse-echo measurements, the acoustic propagation pattern using acoustic pressure field scans, the acoustic power output using radiation force balance measurements, and the acoustic pressure using a needle hydrophone. Depending on the design and piezoelectric element dimensions, the resonance frequency was in the range 350-630 kHz, the -6 dB bandwidth was in the range 87-97%, acoustic output power exceeded 1 W, and acoustic pressure exceeded 1 MPa peak-to-peak. 3D stress simulations were performed to predict the isostatic pressure required to induce material failure and 4D acoustic simulations. The pressure simulations indicated that specific design variations could sustain isostatic pressures up to 4.8 MPa.The acoustic simulations were able to predict the behaviour of the fabricated devices. A total of 480 simulations, varying material dimensions (piezoelectric ring ID, glass disc diameter, glass thickness) and drive frequency indicated that the emitted acoustic profile varies nonlinearly with these parameters.

A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer.

BACKGROUND: The primary aim of our study was to evaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary aim was to evaluate a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance status, and prolonging survival. METHODS: Ten patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30min. Subsequently, patients were treated using a commercial clinical ultrasound scanner for 31.5min. SonoVue® was injected intravenously (0.5ml followed by 5ml saline every 3.5min) during the ultrasound treatment with the aim of inducing sonoporation, thus enhancing therapeutic efficacy. RESULTS: The combined therapeutic regimen did not induce any additional toxicity or increased frequency of side effects when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n=10) tolerated an increased number of gemcitabine cycles compared with historical controls (n=63 patients; average of 8.3±6.0cycles, versus 13.8±5.6cycles, p=0.008, unpaired t-test). In five patients, the maximum tumour diameter was decreased from the first to last treatment. The median survival in our patients (n=10) was also increased from 8.9months to 17.6months (p=0.011). CONCLUSIONS: It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available equipment with no additional toxicities. This combined treatment may improve the clinical efficacy of gemcitabine, prolong the quality of life, and extend survival in patients with pancreatic ductal adenocarcinoma.

Open-source, high-throughput ultrasound treatment chamber.

Studying the effects of ultrasound on biological cells requires extensive knowledge of both the physical ultrasound and cellular biology. Translating knowledge between these fields can be complicated and time consuming. With the vast range of ultrasonic equipment available, nearly every research group uses different or unique devices. Hence, recreating the experimental conditions and results may be expensive or difficult. For this reason, we have developed devices to combat the common problems seen in state-of-the-art biomedical ultrasound research. In this paper, we present the design, fabrication, and characterisation of an open-source device that is easy to manufacture, allows for parallel sample sonication, and is highly reproducible, with complete acoustic calibration. This device is designed to act as a template for sample sonication experiments. We demonstrate the fabrication technique for devices designed to sonicate 24-well plates and OptiCell™ using three-dimensional (3D) printing and low-cost consumables. We increased the pressure output by electrical impedance matching of the transducers using transmission line transformers, resulting in an increase by a factor of 3.15. The devices cost approximately €220 in consumables, with a major portion attributed to the 3D printing, and can be fabricated in approximately 8 working hours. Our results show that, if our protocol is followed, the mean acoustic output between devices has a variance of <1%. We openly provide the 3D files and operation software allowing any laboratory to fabricate and use these devices at minimal cost and without substantial prior know-how.

Sonoporation-enhanced chemotherapy significantly reduces primary tumour burden in an orthotopic pancreatic cancer xenograft.

PURPOSE: Adenocarcinoma of the pancreas remains one of the most lethal human cancers. The high mortality rates associated with this form of cancer are subsequent to late-stage clinical presentation and diagnosis, when surgery is rarely possible and of modest chemotherapeutic impact. Survival rates following diagnosis with advanced pancreatic cancer are very low; typical mortality rates of 50% are expected within 3 months of diagnosis. However, adjuvant chemotherapy improves the prognosis of patients even after palliative surgery, and successful newer neoadjuvant chemotherapeutical modalities have recently been reported. For patients whose tumours appear unresectable, chemotherapy remains the only option. During the past two decades, the nucleoside analogue gemcitabine has become the first-line chemotherapy for pancreatic adenocarcinoma. In this study, we aim to increase the delivery of gemcitabine to pancreatic tumours by exploring the effect of sonoporation for localised drug delivery of gemcitabine in an orthotopic xenograft mouse model of pancreatic cancer. EXPERIMENTAL DESIGN: An orthotopic xenograft mouse model of luciferase expressing MIA PaCa-2 cells was developed, exhibiting disease development similar to human pancreatic adenocarcinoma. Subsequently, two groups of mice were treated with gemcitabine alone and gemcitabine combined with sonoporation; saline-treated mice were used as a control group. A custom-made focused ultrasound transducer using clinically safe acoustic conditions in combination with SonoVue® ultrasound contrast agent was used to induce sonoporation in the localised region of the primary tumour only. Whole-body disease development was measured using bioluminescence imaging, and primary tumour development was measured using 3D ultrasound. RESULTS: Following just two treatments combining sonoporation and gemcitabine, primary tumour volumes were significantly lower than control groups. Additional therapy dramatically inhibited primary tumour growth throughout the course of the disease, with median survival increases of up to 10% demonstrated in comparison to the control groups. CONCLUSION: Combined sonoporation and gemcitabine therapy significantly impedes primary tumour development in an orthotopic xenograft model of human pancreatic cancer, suggesting additional clinical benefits for patients treated with gemcitabine in combination with sonoporation.