Josquin Foiret

Spatial and Temporal Control of Hyperthermia Using Real Time Ultrasonic Thermal Strain Imaging with Motion Compensation, Phantom Study

Mild hyperthermia has been successfully employed to induce reversible physiological changes that can directly treat cancer and enhance local drug delivery. In this approach, temperature monitoring is essential to avoid undesirable biological effects that result from thermal damage. For thermal therapies, Magnetic Resonance Imaging (MRI) has been employed to control real-time Focused Ultrasound (FUS) therapies. However, combined ultrasound imaging and therapy systems offer the benefits of simple, low-cost devices that can be broadly applied. To facilitate such technology, ultrasound thermometry has potential to reliably monitor temperature. Control of mild hyperthermia was previously achieved using a proportional-integral-derivative (PID) controller based on thermocouple measurements. Despite accurate temporal control of heating, this method is limited by the single position at which the temperature is measured. Ultrasound thermometry techniques based on exploiting the thermal dependence of acoustic parameters (such as longitudinal velocity) can be extended to create thermal maps and allow an accurate monitoring of temperature with good spatial resolution. However, in vivo applications of this technique have not been fully developed due to the high sensitivity to tissue motion. Here, we propose a motion compensation method based on the acquisition of multiple reference frames prior to treatment. The technique was tested in the presence of 2-D and 3-D physiological-scale motion and was found to provide effective real-time temperature monitoring. PID control of mild hyperthermia in presence of motion was then tested with ultrasound thermometry as feedback and temperature was maintained within 0.3°C of the requested value.

Development of a spherically focused phased array transducer for ultrasonic image-guided hyperthermia

A 1.5 MHz prolate spheroidal therapeutic array with 128 circular elements was designed to accommodate standard imaging arrays for ultrasonic image-guided hyperthermia. The implementation of this dual-array system integrates real-time therapeutic and imaging functions with a single ultrasound system (Vantage 256, Verasonics). To facilitate applications involving small animal imaging and therapy the array was designed to have a beam depth of field smaller than 3.5 mm and to electronically steer over distances greater than 1 cm in both the axial and lateral directions. In order to achieve the required f number of 0.69, 1-3 piezocomposite modules were mated within the transducer housing. The performance of the prototype array was experimentally evaluated with excellent agreement with numerical simulation. A focal volume (2.70 mm (axial)  ×  0.65 mm (transverse)  ×  0.35 mm (transverse)) defined by the  -6 dB focal intensity was obtained to address the dimensions needed for small animal therapy. An electronic beam steering range defined by the  -3 dB focal peak intensity (17 mm (axial)  ×  14 mm (transverse)  ×  12 mm (transverse)) and  -8 dB lateral grating lobes (24 mm (axial)  ×  18 mm (transverse)  ×  16 mm (transverse)) was achieved. The combined testing of imaging and therapeutic functions confirmed well-controlled local heating generation and imaging in a tissue mimicking phantom. This dual-array implementation offers a practical means to achieve hyperthermia and ablation in small animal models and can be incorporated within protocols for ultrasound-mediated drug delivery.

Ultrasound ablation enhances drug accumulation and survival in mammary carcinoma models

Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates noninvasive image-guided conformal thermal therapy of cancer. Yet in many scenarios, the sensitive tissues surrounding the tumor constrain the margins of ablation; therefore, augmentation of MRgFUS with chemotherapy may be required to destroy remaining tumor. Here, we used 64Cu-PET-CT, MRI, autoradiography, and fluorescence imaging to track the kinetics of long-circulating liposomes in immunocompetent mammary carcinoma-bearing FVB/n and BALB/c mice. We observed a 5-fold and 50-fold enhancement of liposome and drug concentration, respectively, within MRgFUS thermal ablation-treated tumors along with dense accumulation within the surrounding tissue rim. Ultrasound-enhanced drug accumulation was rapid and durable and greatly increased total tumor drug exposure over time. In addition, we found that the small molecule gadoteridol accumulates around and within ablated tissue. We further demonstrated that dilated vasculature, loss of vascular integrity resulting in extravasation of blood cells, stromal inflammation, and loss of cell-cell adhesion and tissue architecture all contribute to the enhanced accumulation of the liposomes and small molecule probe. The locally enhanced liposome accumulation was preserved even after a multiweek protocol of doxorubicin-loaded liposomes and partial ablation. Finally, by supplementing ablation with concurrent liposomal drug therapy, a complete and durable response was obtained using protocols for which a sub-mm rim of tumor remained after ablation.

CpG expedites regression of local and systemic tumors when combined with activatable nanodelivery.

Ultrasonic activation of nanoparticles provides the opportunity to deliver a large fraction of the injected dose to insonified tumors and produce a complete local response. Here, we evaluate whether the local and systemic response to chemotherapy can be enhanced by combining such a therapy with locally-administered CpG as an immune adjuvant. In order to create stable, activatable particles, a complex between copper and doxorubicin (CuDox) was created within temperature-sensitive liposomes. Whereas insonation of the CuDox liposomes alone has been shown to produce a complete response in murine breast cancer after 8 treatments of 6 mg/kg delivered over 4 weeks, combining this treatment with CpG resolved local cancers within 3 treatments delivered over 7 days. Further, contralateral tumors regressed as a result of the combined treatment, and survival was extended in systemic disease. In both the treated and contralateral tumor site, the combined treatment increased leukocytes and CD4+ and CD8+ T-effector cells and reduced myeloid-derived suppressor cells (MDSCs). Taken together, the results suggest that this combinatorial treatment significantly enhances the systemic efficacy of locally-activated nanotherapy.

Super-localization of contrast agents in moving organs, first experiments in a rat kidney

Using individual microbubbles (MBs) to image vasculature with a spatial resolution below the diffraction limit has the potential to greatly improve the in vivo characterization of healthy and diseased tissue. Recent studies have demonstrated a theoretical resolution on the order of microns in stationary tissue using standard imaging arrays. However, the application of this technique to abdominal imaging brings new challenges due to the presence of physiological motion which is far larger than the achievable resolution. In this work, single MBs were localized in vivo in a rat kidney using a dedicated high frame rate (300 Hz) contrast pulse sequence (CPS) with spatial compounding (-5°, 0°, 5°). A stack of 60000 frames was acquired, providing a B-mode image to track tissue motion and a CPS image to track MB position at each time point. Acquisition was accomplished with a standard imaging array (CL15-7, ATL) driven at 6.9 MHz and a programmable ultrasound system (Verasonics). 3.4 million positions were detected and a density map of the MB positions was obtained after compensation for cardiac motion and changes in kidney position. Blood velocity was also estimated by tracking selected MBs over time.

10 MHz catheter-based annular array for thermal strain guided intramural cardiac ablations

A yearly global population of well over 100,000 ventricular tachycardia patients could benefit from guided cardiac ablation of otherwise untreatable intramural arrhythmogenic substrates. A guided catheter-based HIFU device used epicardially with a subxiphoid approach is proposed which is placed with electroanatomical mapping. After the HIFU device is positioned, the thermal strain feedback at low intensities can confirm myocardial contact and establish a projected power titration guideline for full HIFU ablation. This PZT based spherical array design is an early adjunct prototype of a fully beam steerable CMUT array design in development at Stanford. The multifunctional catheter is designed to direct an axially steerable 10 MHz HIFU beam into the myocardium without damaging the epicardium itself and the vitally important coronary vessels. The spherical array is 7 mm in diameter with a radius of curvature of 7 mm; it is housed in a custom 3D printed tip housing which is 10 mm in total diameter and 4 mm in profile and joined to a 7 Fr (2.3 mm) catheter shaft. The prototype array is water cooled and has a built-in thermistor to monitor transducer temperature. The ablation axial steering addresses the 4 to 10 mm depth range, while 10 MHz can produce a high focus gain to produce a 23 C rise in temperature with bursts of 200 msec durations. A unique feature of this work is the use of the Verasonics Vantage 256 system as both the HIFU power source and thermal strain echo data receiver; a custom 32 channel power combiner/splitter (4 annuli x 8:1) interface was built to permit this development. Two 10 MHz 7 mm prototype spherical devices have been built and tested. Both are made from PZT-5A with no backing material and only a 100 micron EPOTEK-301 epoxy front matching/insulation layer. The first device is a 9.6 MHz single element spherical HIFU transducer which was driven with an ENI amplifier and produced heating intensities sufficient to visibly ablate (0.5 mm ablation diameter by 1.5 mm length, or 0.3 mm3) beef tissue in a room temperature bath in less than 0.5 sec. The second device is a spherical four element annular array which has been used to collect thermal strain echo data from a laboratory phantom. Continued development is underway to demonstrate both heating and thermal strain data from in-vivo animal experiments.

Spatial and temporal control of hyperthermia using real time thermal strain imaging with motion compensation

Ultrasound mild hyperthermia has been employed to induce physiological changes that can directly treat cancer and enhance local drug delivery. For mild hyperthermia, temperature monitoring is essential to avoid undesirable biological effects. In previous work by our group, control of mild hyperthermia was achieved using a proportional-integral-derivative (PID) controller based on thermocouple measurements [1]. Despite good temporal control of heating, this method was limited by the fact that temperature was measured only at the thermocouple position. Ultrasound thermometry techniques based on exploiting the thermal dependence of the longitudinal wave speed can be extended to create thermal maps and allow accurate monitoring of temperature with good spatial resolution. However, in vivo applications of this technique have not been fully developed due to the high sensitivity to tissue motion. Here, we address translational and compressional motion compensation and demonstrate effective thermometry in the presence of physiological-scale motion.

Acoustical structured illumination for super-resolution ultrasound imaging

Structured illumination microscopy is an optical method to increase the spatial resolution of wide-field fluorescence imaging beyond the diffraction limit by applying a spatially structured illumination light. Here, we extend this concept to facilitate super-resolution ultrasound imaging by manipulating the transmitted sound field to encode the high spatial frequencies into the observed image through aliasing. Post processing is applied to precisely shift the spectral components to their proper positions in k-space and effectively double the spatial resolution of the reconstructed image compared to one-way focusing. The method has broad application, including the detection of small lesions for early cancer diagnosis, improving the detection of the borders of organs and tumors, and enhancing visualization of vascular features. The method can be implemented with conventional ultrasound systems, without the need for additional components. The resulting image enhancement is demonstrated with both test objects and ex vivo rat metacarpals and phalanges.Tali Ilovitsh et al. describe a new method, acoustical structured illumination, for generating super-resolution ultrasound images. The method applies principles from structured illumination microscopy and can be adapted to existing ultrasound systems without the need for additional components.

Dynamic contrast enhanced MRI detects changes in vascular transport rate constants following treatment with thermally-sensitive liposomal doxorubicin

Abstract Temperature‐sensitive liposomal formulations of chemotherapeutics, such as doxorubicin, can achieve locally high drug concentrations within a tumor and tumor vasculature while maintaining low systemic toxicity. Further, doxorubicin delivery by temperature‐sensitive liposomes can reliably cure local cancer in mouse models. Histological sections of treated tumors have detected red blood cell extravasation within tumors treated with temperature‐sensitive doxorubicin and ultrasound hyperthermia. We hypothesize that the local release of drug into the tumor vasculature and resulting high drug concentration can alter vascular transport rate constants along with having direct tumoricidal effects. Dynamic contrast enhanced MRI (DCE‐MRI) coupled with a pharmacokinetic model can detect and quantify changes in such vascular transport rate constants. Here, we set out to determine whether changes in rate constants resulting from intravascular drug release were detectable by MRI. We found that the accumulation of gadoteridol was enhanced in tumors treated with temperature‐sensitive liposomal doxorubicin and ultrasound hyperthermia. While the initial uptake rate of the small molecule tracer was slower (k1 = 0.0478 ± 0.011 s− 1 versus 0.116 ± 0.047 s− 1) in treated compared to untreated tumors, the tracer was retained after treatment due to a larger reduction in the rate of clearance (k2 = 0.291 ± 0.030 s− 1 versus 0.747 ± 0.24 s− 1). While DCE‐MRI assesses a combination of blood flow and permeability, ultrasound imaging of microvascular flow rate is sensitive only to changes in vascular flow rate; based on this technique, blood flow was not significantly altered 30 min after treatment. In summary, DCE‐MRI provides a means to detect changes that are associated with treatment by thermally‐activated particles and such changes can be exploited to enhance local delivery. Graphical abstract Figure. No Caption available.

Abstract LB-052: Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model

We demonstrate for the first time that blocking of the programmed death-1 (PD-1) pathway in conjunction with immunogenic cell death induced by CpG-ODN and activatable nanodelivery of doxorubicin can generate curative responses in both primary and contralateral tumors. Activatable nanotherapeutics are attractive since the toxicity of chemotherapeutics can be constrained to a small region; combining such a strategy with immunotherapy is the goal of this study. We have previously shown that administration of CpG-ODN as an adjuvant, together with local release of doxorubicin from temperature sensitive liposomes (TSL) resulted in regression of directly-treated tumors, suppressed growth of contralateral tumors and reduced chemotherapeutic-mediated toxicity in a murine breast cancer model.1 Increases in cytotoxic CD8+ T lymphocytes and a reduction in regulatory T cells and myeloid-derived suppressor cells were observed in both directly treated and contralateral tumors. This combinatorial approach was curative for directly-treated tumors and overall survival was significantly extended, however, the contralateral tumor returned in all treated mice. The following is the protocol explored for the addition of anti-PD1: immune intact FVB/n mice with bilateral invasive neu deletion syngeneic transplanted tumors were treated with a combination of anti-PD1 (aPD-1, 200 μg, i.p.) and intratumoral administration of CpG-ODN (100 μg, i.t.) on days 0, 7, 14 and 0, 3, 7, 10, 17 and 24, respectively. Doxorubicin TSL were prepared from DPPC:MPPC:DSPE-PEG2k, 86:10:4 in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-drug/mg-lipid and administrated i.v. at 6 mg doxorubicin/kg body weight on days 10, 17, 24. The formation of a complex between doxorubicin and copper was created to enhance the circulation and stability of TSL and to reduce systemic toxicity. To trigger drug release, hyperthermia was induced in the primary tumor with ultrasound (peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz) at 42°C for 5 min prior to and 20 min post drug injection with a variable duty cycle. Immediately afterwards, 100 μg of CpG-ODN 1826 was administered intratumorally to the insonified tumor. Upon treatment with this combination of locally-released doxorubicin, local administration of CpG-ODN and systemic aPD-1, 100% of treated and contralateral tumors regressed by at least 80%; further, all of the directly-treated tumors and 50% of the contralateral tumors were eliminated without recurrence. Thus, a 50% complete response rate was achieved, with tumor regression observed immediately after the incorporation of the doxorubicin treatment. By contrast, administration of CpG-ODN and systemic aPD-1 alone resulted in regression of 66% of treated and contralateral tumors. *MS and AK contributed equally to this work. 1. J Control Release (2015); 220: 253-264. Citation Format: Matthew T. Silvestrini, Azadeh Kheirolomoom, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Josquin Foiret, Samantha Tucci, Neil E. Hubbard, Alexander D. Borowsky, Katherine W. Ferrara. Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-052.