pFUS Treatment Research Articles

An in-vivo study of the combined therapeutic effects of pulsed non-thermal focused ultrasound and radiation for prostate cancer

Purpose The purpose of this study was to investigate the in vivo combined effects of pulsed focused ultrasound (pFUS) and radiation (RT) for prostate cancer treatment. Materials and Methods An animal prostate tumor model was developed by implanting human LNCaP tumor cells in the prostates of nude mice. Tumor-bearing mice were treated with pFUS, RT or both (pFUS + RT) and compared with a control group. Non-thermal pFUS treatment was delivered by keeping the body temperature below 42 °C as measured real-time by MR thermometry and using a pFUS protocol (1 MHz, 25 W focused ultrasound; 1 Hz pulse rate with a 10% duty cycle for 60 sec for each sonication). Each tumor was covered entirely using 4–8 sonication spots. RT treatment with a dose of 2 Gy was delivered using an external beam (6 MV photon energy with dose rate 300MU/min). Following the treatment, mice were scanned weekly with MRI for tumor volume measurement. Results The results showed that the tumor volume in the control group increased exponentially to 142 ± 6%, 205 ± 12%, 286 ± 22% and 410 ± 33% at 1, 2, 3 and 4 weeks after treatment, respectively. In contrast, the pFUS group was 29% (p < 0.05), 24% (p < 0.05), 8% and 9% smaller, the RT group was 7%, 10%, 12% and 18% smaller, and the pFUS + RT group was 32%, 39%, 41% and 44% (all with p < 0.05) smaller than the control group at 1, 2, 3, and 4 weeks post treatment, respectively. Tumors treated by pFUS showed an early response (i.e. the first 2 weeks), while the RT group showed a late response. The combined pFUS + RT treatment showed consistent response throughout the post-treatment weeks. Conclusions These results suggest that RT combined with non-thermal pFUS can significantly delay the tumor growth. The mechanism of tumor cell killing between pFUS and RT may be different. Pulsed FUS shows early tumor growth delay, while RT contributes to the late effect on tumor growth delay. The addition of pFUS to RT significantly enhanced the therapeutic effect for prostate cancer treatment.

Accelerating cutaneous healing in a rodent model of type II diabetes utilizing non-invasive focused ultrasound targeted at the spleen.

Healing of wounds is delayed in Type 2 Diabetes Mellitus (T2DM), and new treatment approaches are urgently needed. Our earlier work showed that splenic pulsed focused ultrasound (pFUS) alters inflammatory cytokines in models of acute endotoxemia and pneumonia via modulation of the cholinergic anti-inflammatory pathway (CAP) (ref below). Based on these earlier results, we hypothesized that daily splenic exposure to pFUS during wound healing would accelerate closure rate via altered systemic cytokine titers. In this study, we applied non-invasive ultrasound directed to the spleen of a rodent model [Zucker Diabetic Sprague Dawley (ZDSD) rats] of T2DM with full thickness cutaneous excisional wounds in an attempt to accelerate wound healing via normalization of T2DM-driven aberrant cytokine expression. Daily (1x/day, Monday-Friday) pFUS pulses were targeted externally to the spleen area for 3 min over the course of 15 days. Wound diameter was measured daily, and levels of cytokines were evaluated in spleen and wound bed lysates. Non-invasive splenic pFUS accelerated wound closure by up to 4.5 days vs. sham controls. The time to heal in all treated groups was comparable to that of healthy rats from previously published studies (ref below), suggesting that the pFUS treatment restored a normal wound healing phenotype to the ZDSD rats. IL-6 was lower in stimulated spleen (-2.24 ± 0.81 Log2FC, p = 0.02) while L-selectin was higher in the wound bed of stimulated rodents (2.53 ± 0.72 Log2FC, p = 0.003). In summary, splenic pFUS accelerates healing in a T2DM rat model, demonstrating the potential of the method to provide a novel, non-invasive approach for wound care in diabetes.

Abstract 2790: Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound

Abstract Various cellular and biological immunotherapies have revolutionized the treatment of human malignancies by augmenting endogenous immune responses within the tumor microenvironment (TME). Although non-ablative pulsed-FUS (pFUS) has received less attention, it has been successfully used as a noninvasive treatment of solid malignancies by stimulating a pro-inflammatory immune response within the TME. Such an approach could be useful to treat peripheral micro-metastases in normal-appearing tissues. In the present study, we interrogated the immune-modulatory changes that occur within the TME following pFUS exposure. Murine B16 melanoma and 4T1 mammary carcinoma cells were subcutaneously implanted into both mice flanks (n=10 tumors/group/time point). The natural histories cytokine, chemokine, trophic factor (CCTF), and cell adhesion molecule (CAM) expression was evaluated within the TME of untreated flank tumors over 11 days. Ultrasound-guided pFUS at 1 MHz and peak negative pressures of 6 MPa was administered to tumors after reaching ~5 mm in diameter. Temporal immune-related changes in tumors were assessed following sonication using using proteomic, transcriptomic and histological analyses. Natural history growth characteristics showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. An anti-tumor TME was detected following pFUS treatment with increased expression of various pro-inflammatory CCTF and CAM as well as reduced tumor growth rates. Transcriptomic analyses following sonication identified inverted gene expression patterns in the two tumor types. Functional analysis of B16 tumors revealed increased intracellular signaling pathways associated with immune response regulations, while 4T1 tumors demonstrated reduced expression of proliferation genes. This study provides a macroscopic overview on the temporal dynamics of the TME and underline the profound magnitude of tumoral heterogeneity between tumor types as well as through progressive stages of similar primary tumors. Nevertheless, the induced immune-modulation changes suggest that pFUS may alter the expression of pro-inflammatory CCTF, CAM, towards an anti-tumoral TME, and supports the potential use of pFUS as a neoadjuvant treatment approach. Citation Format: Gadi Cohen, Parwathy Chandran, Rebecca M. Lorsung, Lauren E. Tomlinson, Robert B. Rosenblatt, Scott R. Burks, Joseph A. Frank. Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2790.

Abstract 2763: Effect of murine vendor on anti-tumor immune responses to non-ablative pulsed focused ultrasound

Abstract Non-ablative image guided pulsed focused ultrasound (pFUS) has shown promise in murine tumor models as an immunomodulatory strategy for altering tumor microenvironment (TME). However, studies to date have demonstrated efficacy only in murine models procured from a single vendor. To determine whether differences in inbred mice obtained from 3 vendors would alter tumor growth and TME, we evaluated temporal expression of cytokine, chemokine and trophic factor (CCTF), and immune cell profiles following non-ablative pFUS to murine B16 melanoma flank tumors established in C57BL/6J mice. B16 cells (106 cells) were subcutaneously, bilaterally implanted into C57BL/6J mice (n=6/group/time-point) obtained from 3 vendors [Jackson Laboratories (JAX; n=36), Charles River (CR; n=36), Taconic Biosciences (Tac; n=36)]. Ultrasound guided pFUS (VIFU 2000, Alpinion) was administered at 1 MHz, at 6 MPa PNP to 5 mm tumors. Entire tumor volume was sonicated point-by-point along a 2×2 mm matrix with 20 msec pulse length, 10% duty cycle and a 5 Hz pulse repetition frequency. On days 1, 3, and 5 post-pFUS, spleens (Sp), lymph nodes (LN) and tumors were evaluated by proteomic (CCTF) and flow cytometry (FC) analyses. pFUS treatment demonstrated variable, vendor-dependent effects on tumor growth, CCTF and immune cell profiles. CR B16 volumes showed significant reduction (p&amp;lt;0.05; 2-way ANOVA) starting at day 2 through day 5, but not in JAX or Tac mice. Proteomic analyses revealed a similar trend wherein CR TME exhibited upregulation of pro-inflammatory cytokines (TNFα, IL-1β, IL-12p70) on days 1 and 3, and downregulation of anti-inflammatory cytokines (VEGF, IL-10) over 5 days. Comparatively, less robust expression of anti-tumoral cytokines were observed in Tac and JAX TME over 5 days. FC corroborated tumor growth and molecular analyses, showing significant tumor infiltration of helper and cytotoxic T cells (Th, Tcyt), M1 macrophages and dendritic cells (DC) on days 3 and 5, compared to untreated controls. Day 5 CR Sp also exhibited a pro-inflammatory profile with T cells, macrophages and DC. Tac and JAX tumor profiles over days 1, 3 and 5 were relatively unchanged by pFUS with major, anti-tumoral immunological activity restricted to draining LN on days 5 and 3 in Tac and JAX, respectively. Moreover, molecular and immune cell differences were observed in B16 tumors from control mice on days 1, 3 and 5. Collectively, B16 bearing CR mice exhibited a more pronounced anti-tumor response to pFUS, resulting in reduced tumor growth and pro-inflammatory CCTF and immune cell profiles as compared to B16 tumors in Tac and JAX mice. Despite using the same mouse strain, tumor type, and pFUS parameters, profound vendor-dependent variability was observed, suggestive of the possible role of inherent genetic disparities among C57BL/6J mice from different vendors. These results suggest that vendor choice may be critical in evaluating anti-tumor effects of pFUS. Citation Format: Parwathy Chandran, Ruby Hutchison, Lauren Tomlinson, Gadi Cohen, Scott R. Burks, Joseph A. Frank. Effect of murine vendor on anti-tumor immune responses to non-ablative pulsed focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2763.

Pulsed-Focused Ultrasound Slows B16 Melanoma and 4T1 Breast Tumor Growth through Differential Tumor Microenvironmental Changes.

Simple SummaryThis study provides a conceptual overview correlating temporal changes in the tumor immune-microenvironment (TIME) to the non-ablative pulse focused ultrasound (pFUS) in two different types of tumors. The pFUS-induced immunomodulation revealed differential anti-tumor molecular and cellular responses between the two tumor types, demonstrating tumor-type-dependent responses to identical pFUS sonication parameters. It also supports the potential use of pFUS as a possible adjuvant to ablative tumor treatment to elicit enhanced anti-tumor responses and slow tumor growth.Focused ultrasound (FUS) has shown promise as a non-invasive treatment modality for solid malignancies. FUS targeting to tumors has been shown to initiate pro-inflammatory immune responses within the tumor microenvironment. Pulsed FUS (pFUS) can alter the expression of cytokines, chemokines, trophic factors, cell adhesion molecules, and immune cell phenotypes within tissues. Here, we investigated the molecular and immune cell effects of pFUS on murine B16 melanoma and 4T1 breast cancer flank tumors. Temporal changes following sonication were evaluated by proteomics, RNA-seq, flow-cytometry, and histological analyses. Proteomic profiling revealed molecular changes occurring over 24 h post-pFUS that were consistent with a shift toward inflamed tumor microenvironment. Over 5 days post-pFUS, tumor growth rates were significantly decreased while flow cytometric analysis revealed differences in the temporal migration of immune cells. Transcriptomic analyses following sonication identified differences in gene expression patterns between the two tumor types. Histological analyses further demonstrated reduction of proliferation marker, Ki-67 in 4T1, but not in B16 tumors, and activated cleaved-caspase 3 for apoptosis remained elevated up to 3 days post-pFUS in both tumor types. This study revealed diverse biological mechanisms following pFUS treatment and supports its use as a possible adjuvant to ablative tumor treatment to elicit enhanced anti-tumor responses and slow tumor growth.

Facilitating islet transplantation using a three-step approach with mesenchymal stem cells, encapsulation, and pulsed focused ultrasound

BackgroundThe aim of this study was to examine the effect of a three-step approach that utilizes the application of adipose tissue-derived mesenchymal stem cells (AD-MSCs), encapsulation, and pulsed focused ultrasound (pFUS) to help the engraftment and function of transplanted islets.MethodsIn step 1, islets were co-cultured with AD-MSCs to form a coating of AD-MSCs on islets: here, AD-MSCs had a cytoprotective effect on islets; in step 2, islets coated with AD-MSCs were conformally encapsulated in a thin layer of alginate using a co-axial air-flow method: here, the capsule enabled AD-MSCs to be in close proximity to islets; in step 3, encapsulated islets coated with AD-MSCs were treated with pFUS: here, pFUS enhanced the secretion of insulin from islets as well as stimulated the cytoprotective effect of AD-MSCs.ResultsOur approach was shown to prevent islet death and preserve islet functionality in vitro. When 175 syngeneic encapsulated islets coated with AD-MSCs were transplanted beneath the kidney capsule of diabetic mice, and then followed every 3 days with pFUS treatment until day 12 post-transplantation, we saw a significant improvement in islet function with diabetic animals re-establishing glycemic control over the course of our study (i.e., 30 days). In addition, our approach was able to enhance islet engraftment by facilitating their revascularization and reducing inflammation.ConclusionsThis study demonstrates that our clinically translatable three-step approach is able to improve the function and viability of transplanted islets.

Abstract 3577: Pulsed focused ultrasound induces cytosolic calcium transients that increase free radical production and dsDNA breaks in tumor cells

Abstract Non-ablative pulsed focused ultrasound (pFUS) has shown promise as an immunomodulatory anti-cancer therapeutic. While largely nondestructive to tissues compared ablative ultrasound, pFUS can generate anti-tumor molecular microenvironments and enhance migration of anti-tumor immune cells into B16 and 4T1 flank tumors in immune-competent mice. pFUS also increases the number of dsDNA breaks in nuclear DNA of tumor cells by TUNEL reactions. DNA damage can elicit immune responses by causing cells to express ligands for immune cells and thus may be a critical component of generating observed immune responses in flank tumors following pFUS treatment. Here we investigate potential phyisological mechanisms underlying pFUS-associated DNA damage. B16 melanoma, 4T1 or MBA-MD-231 breast tumors, or C6 glioma cells were cultured and treated with pFUS in vitro (1 MHz, 6 MPa peak negative pressure, 10% duty cycle, 100 pulses). Cells were analyzed for DNA damage by TUNEL, reactive oxygen species (ROS) production by DCFDA, and intracellular Ca2+ by fluo-4. pFUS increased TUNEL reactivity and ROS concentrations in all cell types 2-6 hr after pFUS. Increased TUNEL reactivity was not observed when cells were incubated with the ROS scavenger Trolox (need concentration) following pFUS. pFUS generated Ca2+ transients in all cell types during pFUS treatment. Ca2+ dysregulation can increase cellular ROS production and when cytosolic Ca2+ transients were blocked by BAPTA-AM, both ROS production and TUNEL reactivity was reduced in all cell types. Further investigating mechanisms of Ca2+ transients, application of verapamil (10 uM) during pFUS to inhibit voltage-gated Ca2+ channels (VGCC) also suppressed Ca2+ transients, ROS production, and TUNEL reactivity. Lastly, the magnitudes of Ca2+ transients, ROS production, and TUNEL reactivity induced by pFUS were nonetheless different for different cell types (C6 glioma exhibited the smallest changes after pFUS, while MBA-MD-231 exhibited the largest). This suggested different sensitivities for each cell type to pFUS-induced DNA damage. As Ca2+ homeostasis can be disrupted in tumor cells, we measured resting cytosolic Ca2+ concentrations in each cell type following plasma membrane disruption with digitonin (20 uM). Resting cytosolic Ca2+ concentrations inversely correlated with increased magnitudes of ROS production or TUNEL reactivity following pFUS treatment. In conclusion, pFUS causes DNA damage in tumor cells by initiating cytosolic Ca2+ transients, in part, through VGCC that increase cellular ROS production and lead to dsDNA breaks. The effectiveness of pFUS to stimulate this physiological pathway is related to resting cytosolic Ca2+ concentrations, which different tumor cells could display varying levels of disrupted homeostasis and, thus differing magnitudes of response to pFUS. Future directions will assess potential involvement of mitochondrial dynamics in Ca2+-induced ROS production and in vivo experiments to further understand these dynamics in solid tumor models. Citation Format: Robert B. Rosenblatt, Joseph A. Frank, Scott R. Burks. Pulsed focused ultrasound induces cytosolic calcium transients that increase free radical production and dsDNA breaks in tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3577.

Abstract 5606: Characterization of temporal proteomic dynamics of murine breast and melanoma tumor microenvironments without and with pulsed focused ultrasound

Abstract Focused ultrasound (FUS) ablation has been successfully used for thermal ablation of solid malignancies. However, non-ablative pulsed-FUS (pFUS) has received less attention but could be less destructive anti-tumor therapy by stimulating a pro-inflammatory immune response within the tumor microenvironment (TME). Clinically, such an approach could be useful to treat peripheral disease (e.g. micro-metastases) in otherwise normal-appearing tissues. Here, we explored the alterations of cytokines, chemokines and trophic factors (CCTF) and cell adhesion molecules (CAM) in the TME of mouse flank tumor models following pFUS treatment at different time points of tumor progression. Mice were bilaterally inoculated with subcutaneous B16 melanoma or 4T1 breast tumors into the hind limbs and allowed to reach diameters of ~5 mm (n=6/group/timepoint) before performing experiments (Day 1). Separate groups of mice were subjected to pFUS (1 MHz; Peak negative pressure = 6 MPa; Duty cycle = 10%, 100 pulses per site) at Days 1, 5, or 10 and euthanized 24 hr post-pFUS for proteomic analyses. Separate groups of time-matched tumor-bearing mice that did not receive pFUS were also euthanized at the same time points both to assess relative changes due to pFUS at each time point and provide information regarding the natural history of TME progression in each tumor type. Proteomic analyses in untreated control tumors revealed an overall shift toward immunosuppressive (pro-tumor) TME over the 10 days. When comparing proteomic effects of pFUS treatment in compared to untreated time-matched controls, tumors treated on Day 1 exhibited greater upregulation of pro-inflammatory (anti-tumor) CCTF and CAM and pFUS had less pronounced effects on the TME when administered in groups of mice treated at Days 5 or 10. To further examine the acute effects of pFUS at on CCTF and CAM over 72hr. pFUS treatment in elicited unique CCTF and CAM profiles for the two tumor types, but both could be considered shifted towards anti-tumoral TME, especially within the first 24 hrs. These data suggest that pFUS can potentially modulate TME in a therapeutically beneficial way. However, they reveal two important caveats that require substantial investigation: 1) Different tumor types have unique molecular responses and imply pFUS may be more suitable for certain tumor types; 2) Natural histories encompass dynamic TME changes as part of disease progression and appropriate timing of pFUS in each tumor type will be necessary to maximizing the potential therapeutic benefits of pFUS. Citation Format: Gadi Cohen, Parwathay Chandran, Lauren E. Tomlinson, Maggie E. Sundby, Rebecca M. Lorsung, Scott R. Burks, Joseph A. Frank. Characterization of temporal proteomic dynamics of murine breast and melanoma tumor microenvironments without and with pulsed focused ultrasound [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5606.

Magnetic resonance imaging biomarkers for pulsed focused ultrasound treatment of pancreatic ductal adenocarcinoma.

BACKGROUNDThe robust fibroinflammatory stroma characteristic of pancreatic ductal adenocarcinoma (PDA) impedes effective drug delivery. Pulsed focused ultrasound (pFUS) can disrupt this stroma and has improved survival in an early clinical trial. Non-invasive methods to characterize pFUS treatment effects are desirable for advancement of this promising treatment modality in larger clinical trials.AIMTo identify promising, non-invasive pre-clinical imaging methods to characterize acute pFUS treatment effects for in vivo models of PDA.METHODSWe utilized quantitative magnetic resonance imaging methods at 14 tesla in three mouse models of PDA (subcutaneous, orthotopic and transgenic - KrasLSL-G12D/+, Trp53LSL-R172H/+, Cre or “KPC”) to assess immediate tumor response to pFUS treatment (VIFU 2000 Alpinion Medical Systems; 475 W peak electric power, 1 ms pulse duration, 1 Hz, duty cycle 0.1%) vs sham therapy, and correlated our results with histochemical data. These pFUS treatment parameters were previously shown to enhance tumor permeability to chemotherapeutics. T1 and T2 relaxation maps, high (126, 180, 234, 340, 549) vs low (7, 47, 81) b-value apparent diffusion coefficient (ADC) maps, magnetization transfer ratio (MTR) maps, and chemical exchange saturation transfer (CEST) maps for the amide proton spectrum (3.5 parts per million or “ppm”) and the glycosaminoglycan spectrum (0.5-1.5 ppm) were generated and analyzed pre-treatment, and immediately post-treatment, using ImageJ. Animals were sacrificed immediately following post-treatment imaging. The whole-tumor was selected as the region of interest for data analysis and subsequent statistical analysis. T-tests and Pearson correlation were used for statistical inference.RESULTSMean high-b value ADC measurements increased significantly with pFUS treatment for all models. Mean glycosaminoglycan CEST and T2 measurements decreased significantly post-treatment for the KPC group. Mean MTR and amide CEST values increased significantly for the KPC group. Hyaluronic acid focal intensities in the treated regions were significantly lower following pFUS treatment for all animal models. The magnetic resonance imaging changes observed acutely following pFUS therapy likely reflect: (1) Sequelae of variable degrees of microcapillary hemorrhage (T1, MTR and amide CEST); (2) Lower PDA glycosaminoglycan content and associated water content (glycosaminoglycan CEST, T2 and hyaluronic acid focal intensity); and (3) Improved tumor diffusivity (ADC) post pFUS treatment.CONCLUSIONT2, glycosaminoglycan CEST, and ADC maps may provide reliable quantitation of acute pFUS treatment effects for patients with PDA.

An in-vivo investigation of the therapeutic effect of pulsed focused ultrasound on tumor growth.

High-intensity focused ultrasound (HIFU) has been investigated for ablative therapy and drug enhancement for gene therapy and chemotherapy. The aim of this work is to explore the feasibility of pulsed focused ultrasound (pFUS) for cancer therapy using an in vivo animal model. A clinical HIFU system (InSightec ExAblate 2000) integrated with a 1.5 T GE MR scanner was used in this study. Suitable ultrasound parameters were investigated to perform nonthermal sonications, keeping the temperature elevation below 4 °C as measured in real time by MR thermometry. LNCaP cells (10(6)) were injected into the prostates of male mice (n = 20). When tumors reached a diameter of about 5 mm in 3D as measured on magnetic resonance imaging (MRI), the tumor-bearing mice (n = 8) were treated with pFUS (1 MHz frequency; 25 W acoustic power; 0.1 duty cycle; 60 s duration). A total of 4-6 sonications were used to cover the entire tumor volume under MR image guidance. The animals were allowed to survive for 4 weeks after the treatment. The tumor growth was monitored on high-resolution (0.2 mm) MRI weekly post treatment and was compared with that of the control group (n = 12). Significant tumor growth delay was observed in the tumor-bearing mice treated with pFUS. The mean tumor volume for the pFUS treated mice remained the same 1 week after the treatment while the mean tumor volume of the control mice grew 42% over the same time. Two weeks after the pFUS treatment, the control group had a mean tumor volume 40% greater than that of the treated group. There was a greater variation in tumor volume at 4 weeks post treatment for both treated and control mice and a slightly faster tumor growth for the pFUS treated mice. The authors' results demonstrated that pFUS may have a great potential for cancer therapy. Further experiments are warranted to understand the predominantly nonthermal cell killing mechanisms of pFUS and to derive optimal ultrasound parameters and fractionation schemes to maximize the therapeutic effect of pFUS.

TH‐C‐217BCD‐01: Best in Physics (Imaging) ‐ Evaluation of Apoptosis and Proliferation in Non‐Thermal Pulsed HIFU Treated Mouse Prostate Tumors

Purpose: The underlying mechanism of non‐thermal effects of pulsed high intensity focused ultrasound (pFUS) on normal and tumor tissues is not well understood. Our recent studies showed significant prostate cancer cell killing in vitro and significant prostate tumor growth delay in vivo after pFUS treatment. We hypothesized that these effects of non‐thermal pFUS are due to an increase in cell apoptosis and decrease in cell proliferation. Therefore, the purpose of this study was to analyze the biomarkers of apoptosis and proliferation in prostate tumors after pFUS exposures. Methods: An orthotopic prostate tumor model was established in nude mice. Prostate tumors were sonicated with MR guided pFUS (1MHz, 5W acoustic power, 5Hz frequency; 0.1duty cycle) for 60 sec for each sonication with temperature &lt;42°C (InSightec ExAblate 2000 with a 1.5T GE MR scanner). Untreated tumors were used as controls. The mice were sacrificed at predetermined times up to 7 days following therapy. Tumors were processed for light microscopic examination with H&amp;E staining and immunohistochemical staining for caspase 3 (a marker of apoptosis) and Ki67 (aproliferation marker) expression. Results: Light microscopy revealed the absence of thermal damage and acute destruction of tumor tissues exposed to pFUS. The microvessel walls in the tissues remained intact. There was no change in the extent of hemorrhage upon pFUS treatment over time. The apoptotic index, defined as a percentage of apoptotic cells per total number of cells, peaked at 24 hours after FUS treatment relative to control. There was no dramatic difference in the proliferation indices between different time points. Conclusions: Our results suggested that non‐thermal pulsed pFUS induced caspase 3‐mediated apoptosis and did not produce any thermal damage in the prostate tumor tissues. We are performing additional studies to evaluate blood vessel density and cellular DNA damage upon pFUS treatment.

TU-C-BRB-05: BEST IN PHYSICS (THERAPY) - Combined Effects of Pulsed Non-Thermal Focused Ultrasound and Radiotherapy for Prostate Cancer Treatment

Purpose: Recent studies have shown non-thermal effects of pulsed focused ultrasound (pFUS) on tumor growth control. This study is to investigate the combined effects of non-thermal pFUS and radiotherapy (RT) for prostate cancer treatment. Methods: Animal prostate tumor model was developed by implanting LNCaP cells in mice prostates. Tumor-bearing mice were randomly assigned to 3 groups (n=8): (1) pFUS; (2) pFUS+RT; (3) control. MR-guided pFUS treatment was performed using InSightec ExAblate 2000 system with a 1.5T GE MR scanner. Non-thermal sonications were delivered by keeping the body temperature <42°C as measured real-time by MR thermometry. Prostate tumors in groups 1 and 2 were exposed to pFUS (1MHz, 25W focused ultrasound; 1Hz pulse rate with a 10% duty cycle) for 60 sec for each sonication. Each tumor was covered entirely using 4–8 sonication spots. Prostate tumors in group 2 also received 2 Gy radiation dose within 30 minutes after pFUS treatment using Siemens Artiste (6MV photon energy, 300MU/min dose rate). Following the treatment, mice were scanned weekly using MRI for tumor volume measurement. Results: Tumor volumes in the control group showed exponential increase from 121±5 mm3 on treatment day to 172±9, 247±15, 344±28, and 500±42 mm3 at 1, 2, 3, and 4 weeks post treatment, respectively. In contrast, tumor volumes in the pFUS group were 33% (p<0.05), 29% (p<0.05), 14%, and 15% smaller, and the pFUS+RT group were 31%, 31%, 26%, 33% smaller (p<0.05) at week 1, 2, 3, and 4 post treatment, respectively. Both pFUS and pFUS+RT groups showed significant tumor growth delay in the first two weeks. Conclusions: Our study showed that pFUS combined with radiotherapy can significantly delay the tumor growth. Radiation after pFUS treatment provided an additional benefit for tumor control (sponsored by Focused Ultrasound Surgery Foundation, Varian Medical Systems, DOD PC073127, DOD BC102806).

TU‐A‐BRA‐11: Targeted Drug Delivery Technique Employing Pulsed Focused Ultrasound for Treatment of Prostate

Purpose: The purpose of this study is to investigate an innovative approach to prostate cancer therapy using nanodroplet‐encapsulated drugs combined with pFUS (pulsed high intensity focused ultrasound) treatment. Pulsed FUS treatment provides a localized drug release from the carrier and enhanced intracellular uptake, which ensures temporal and spatial control of local drug delivery while lowering the systemic toxicity. Methods: LNCaP cells were implanted into the prostates of nude mice. Tumor growth was monitored using MRI. The tumor‐bearing mice were randomly divided into 5 groups (n=3). Group 1, animals were treated with an IV injection of paclitaxel (PTX)‐encapsulated nanodroplets (ND‐PTX) + pFUS. Animals in Group 2 were treated with pFUS alone. Animals in Group 3 were injected (IV) with PTX‐encapsulated nanodroplets alone, Group 4 received pFUS+empty nanodroplets and Group 5 was used as a control group. After treatment, animals were allowed to survive for 4 weeks. Tumor growth delay was monitored by MRI. The formulation of the PTX‐encapsulated nanodroplets is as follows: PTX dose 20 mg/kg, Nanoemulsion composition 0.5% PTX, 1% perfluorocarbon and 2% stabilizing block copolymer. Ultrasound treatment parameters were 1MHz, 25W acoustic power, 10% duty cycle and 60 sec for each sonication. Results: Compared with the control group, significant tumor growth delay was observed in Group 1 with p=0.004, 3 weeks after treatment. There was no significant tumor growth delay observed for Group 2 (p=0.285), Group 3 (p=0.452) and Group 4 (p=0.158). Conclusions: Our preliminary results showed a great potential for prostate cancer therapy using targeted PTX+ nanodroplets, which could be activated by pFUS. More animal studies are warranted to confirm the results. The enhancement effect of pFUS on targeted drug delivery needs to be investigated quantitatively. This work is supported by Focused Ultrasound Surgery Foundation, DOD PC073127 and DOD BC102806. Technical support from InSightec is graciously acknowledged. funding supported partially by Focused Ultrasound Surgery Foundation, DOD PC073127 and DODBC102806

Enhanced Homing Permeability and Retention of Bone Marrow Stromal Cells by Noninvasive Pulsed Focused Ultrasound

Bone marrow stromal cells (BMSCs) have shown significant promise in the treatment of disease, but their therapeutic efficacy is often limited by inefficient homing of systemically administered cells, which results in low number of cells accumulating at sites of pathology. BMSC home to areas of inflammation where local expression of integrins and chemokine gradients is present. We demonstrated that nondestructive pulsed focused ultrasound (pFUS) exposures that emphasize the mechanical effects of ultrasound-tissue interactions induced local and transient elevations of chemoattractants (i.e., cytokines, integrins, and growth factors) in the murine kidney. pFUS-induced upregulation of cytokines occurred through approximately 1 day post-treatment and returned to contralateral kidney levels by day 3. This window of significant increases in cytokine expression was accompanied by local increases of other trophic factors and integrins that have been shown to promote BMSC homing. When BMSCs were intravenously administered following pFUS treatment to a single kidney, enhanced homing, permeability, and retention of BMSC was observed in the treated kidney versus the contralateral kidney. Histological analysis revealed up to eight times more BMSC in the peritubular regions of the treated kidneys on days 1 and 3 post-treatment. Furthermore, cytokine levels in pFUS-treated kidneys following BMSC administration were found to be similar to controls, suggesting modulation of cytokine levels by BMSC. pFUS could potentially improve cell-based therapies as a noninvasive modality to target homing by establishing local chemoattractant gradients and increasing expression of integrins to enhance tropism of cells toward treated tissues.

Quantitative study of focused ultrasound enhanced doxorubicin delivery to prostate tumor in vivo with MRI guidance

The purpose of this study was to investigate the potential of MR-guided pulsed focused ultrasound (pFUS) for the enhancement of drug uptake in prostate tumors in vivo using doxorubicin (Dox). An antitumor drug Dox, an orthotopic animal prostate tumor model using human prostate cancer, LNCaP cell line, and a clinical FUS treatment system (InSightec ExAblate 2000) with a 1.5T GE MR scanner were used in this study. First, experiments on a tissue mimic phantom to determine the optimal acoustic power and exposure durations with a 10% duty cycle and a 1 Hz pulse rate were performed. The temperature variation was monitored using real-time MR thermometry. Second, tumor-bearing animals were treated with pFUS. There were three groups (n = 8/group): group 1 received pFUS + Dox (10 mg/kg i.v. injection immediately after pFUS exposure), group 2 received Dox only (10 mg/kg i.v. injection), and group 3 was a control. Animals were euthanized 2 h after the pFUS treatment. The Dox concentration in the treated tumors was measured by quantifying fluorescent tracers using a fluorometer. Third, the histological changes of tumors with and without pFUS treatments were evaluated. Finally, experiments were performed to study the spatial drug distribution in tumors after the pFUS treatment, in which two animals received pFUS + Dox, two animals received Dox only, and one animal was used as control. Two hours following the treatment, animals were euthanized and processed. The Dox distribution was determined using a fluorescence microscope. Parametric measurements using a tissue phantom showed that the temperature increased with an increasing acoustic power (from 10 to 50 W) or sonication duration (from 10 to 60 s) with a given acoustic frequency of 1 MHz, duty cycle 10%, and pulse rate 1 Hz. A set of ultrasound parameters was identified with which the temperature elevation was less than 5 °C, which was used for nonthermal pFUS sonication. Increased Dox concentration (14.9 ± 2.5 μg/g) was measured in the pFUS-treated group compared to the Dox-only group (9.5 ± 1.6 μg/g), indicating an approximate 60% increase with p = 0.05. The results were consistent with the increased spatial drug distributions by fluorescence imaging. Histological analysis showed increased extravasation in pFUS-treated prostate tumors suggesting increased drug delivery with pFUS. The results showed that pFUS-enhanced drug uptake in prostate tumors was significant. This increased uptake may be due to increased extravasation by pFUS. Optimal pFUS parameters may exist to maximize the drug uptake, and this study using Dox demonstrated a quantitative method for such systematic parametric studies. In addition, this study may provide useful data for the potential application of pFUS-mediated Dox delivery for prostate tumor therapy.

MR-guided pulsed high intensity focused ultrasound enhancement of docetaxel combined with radiotherapy for prostate cancer treatment

The purpose of this study is to evaluate the efficacy of the enhancement of docetaxel by pulsed focused ultrasound (pFUS) in combination with radiotherapy (RT) for treatment of prostate cancer in vivo. LNCaP cells were grown in the prostates of male nude mice. When the tumors reached a designated volume by MRI, tumor bearing mice were randomly divided into seven groups (n = 5): (1) pFUS alone; (2) RT alone; (3) docetaxel alone; (4) docetaxel + pFUS; (5) docetaxel + RT; (6) docetaxel + pFUS + RT, and (7) control. MR-guided pFUS treatment was performed using a focused ultrasound treatment system (InSightec ExAblate 2000) with a 1.5T GE MR scanner. Animals were treated once with pFUS, docetaxel, RT or their combinations. Docetaxel was given by i.v. injection at 5 mg kg−1 before pFUS. RT was given 2 Gy after pFUS. Animals were euthanized 4 weeks after treatment. Tumor volumes were measured on MRI at 1 and 4 weeks post-treatment. Results showed that triple combination therapies of docetaxel, pFUS and RT provided the most significant tumor growth inhibition among all groups, which may have potential for the treatment of prostate cancer due to an improved therapeutic ratio.

WE-G-220-07: Treatment Assessment for Pulsed Focused Ultrasound Therapy of Prostate Tumor Using a Clinical 1.5T MRI

Purpose: Recent in vivo experiments have showed non‐thermal effects of pulsed focused ultrasound (pFUS) on tumor growth delay. This study investigates the application of a clinical 1.5T MR scanner for treatment assessment of prostate tumors after pFUS treatment, which provides a non‐ invasive and accurate way to measure tumor volume for treatment efficacy evaluation, and also an alternative way for small animaltumor model study that generally requires high‐magnetic‐field MRI facility (e.g., the Bruker NMR system). Methods: A mouse tumor model was developed by implanting LNCaP tumor cells in nude mice prostates. When tumor volume reached ∼130 mm3 on MRI, mice were randomly assigned into two groups (n=8): (1) pFUS treatment; (2) control. MRI‐guided pFUS treatment was performed using InSightec ExAblate 2000 with a 1.5T GE MR scanner. Non‐thermal pFUS was delivered for 60 sec per sonication (1MHz; 25W acoustic power; 10% duty cycle, on/off=0.1/0.9 sec). The body temperature was kept 40% reduction in tumor volume was shown in mice treated with pFUS compared to that for the control mice Conclusion: Clinical 1.5T MRI is able to assess the tumor volume change after pFUS treatment for a small animal model (tumor size: 3–10mm). It may provide an alternative way for treatment assessment for small animal studies. This work is supported by Focused Ultrasound Surgery Foundation, Varian Medical Systems, DOD PC073127 and DOD BC102806.

WE‐E‐220‐05: On‐Thermal Pulsed Focused Ultrasound for Cancer Therapy

Purpose: MR guided focused ultrasound (MRgFUS) has been investigated for ablative therapy and drug enhancement for gene therapy and chemotherapy in our institution. The aim of this work is to explore the feasibility of pulsed focused ultrasound (pFUS) for non‐thermal cancer therapy using an in vivo animal model. Methods: An InSightec ExAblate 2000 with a 1.5T GE MR scanner was used in this study. Suitable ultrasound parameters were investigated to perform non‐thermal sonications, keeping the temperature below 42&amp;#61616;C as measured in real time by MR thermometry. LNCaP cells (106) were injected into the prostates of male mice (n=20). When tumors reached the volume of 100±50mm3 on MRI, the tumor‐bearing mice (n=8) were treated with pFUS (US frequency 1MHz; 25W acoustic power; pulse frequency 1Hz; 0.1 duty cycle) for 60sec. A total of 4–6 sonications were used to cover the entire tumor volume under MR image guidance. The animals were allowed to survive for 4 weeks after the treatment. The tumor growth was monitored on MRI and compared with the control group (n=12). Results: Significant tumor growth delay was observed in the tumor‐bearing mice treated with pFUS. The mean tumor volume for the pFUS treated mice remained the same 1 week after the pFUS treatment while the mean tumor volume of the control mice grew 42% over the same time. Two weeks after the pFUS treatment the cont Conclusions: Our results demonstrated that non‐thermal pFUS has a great potential for cancer therapy. Further experiments are needed to derive optimal ultrasound parameters and fractionation schemes to maximize the therapeutic effect. This work is supported by Focused Ultrasound Surgery Foundation, Varian Medical Systems, DOD PC073127 and DOD BC102806. Technical support from InSightec is acknowledged.

WE‐E‐220‐06: Histological Changes in the Mouse Prostate Tumors Treated with Non‐Thermal Pulsed Focused Ultrasound

Purpose: To date, detailed studies assessing histological changes in prostate tumors upon treatment with non‐thermal pulsed focused ultrasound (pFUS) are scarce in the literature. The purpose of our study was to evaluate the effect of non‐thermal pFUS on the morphology of mouse prostate tumor tissues by light microscopy. Methods: The orthotopic implantation of 106 LNCaP cancer cells in nude male mice resulted in prostate tumor growth that was monitored by MRI. When tumors reached approximately 100mm3 in volume, the animals were treated with non‐thermal pFUS using the following parameters: 1MHz; 25W acoustic power; 5Hz frequency and 0.1 duty cycle for 60sec with temperature kept below 42 degree C (InSightec ExAblate 2000 with a 1.5T GE MR scanner). On average 6–8 sonications were used to cover the tumor volume. Animals were sacrificed immediately after pFUS treatment and tumors were fixed in formalin, embedded in paraffin and 5 microns thick tissue sections were stained with H&amp;E. Tumors from untreated animals were used as controls. Results: Morphological analysis of prostate tumor tissues revealed poorly differentiated cancers. The main histological findings in both pFUS‐treated and untreated tumors were coagulation necrosis and hemorrhage. The extravasation of blood cells, hemorrhage, was predominant in the periphery, while necrosis was present in the central zones of the tumor, in both pFUS‐treated and control, untreated prostate tumors. However, the main distinction between the treated and untreated tumors was the increased hemorrhage in the treated group. This finding is consistent with our previous studies. Conclusions: Our results indicated that non‐thermal pFUS increased extravasation of blood cells in prostate tumors compared to controls. We are performing more detailed histological and immunohistological analyses at different time points post pFUS treatment to look for markers of apoptosis in prostate tumors.

WE‐G‐220‐04: Enhanced Uptake of Doxorubicin in Prostate Tumor Using Pulsed Focused Ultrasound — An in Vivo Study

Purpose: To investigate the effect of pulsed focused ultrasound (pFUS) on the uptake of doxorubicin (DOX) in prostate tumor in vivo. Methods: A mouse prostate tumor model was used in this study by implanting and growing LNCaP tumor cells in male nude mice prostate. An InSightec ExAblate 2000 system with a 1.5T GE MR scanner was used for MRI‐ guided pFUS treatment. Non‐thermal sonications were delivered by keeping the local body temperature below 42°C as measured in real‐time by MR thermometry. Tumor‐bearing mice were exposed to pFUS (1MHz; 25W acoustic power; 10% duty cycle with 0.1 sec on and 0.9 sec off) for 60 sec in each sonication. A total of 4–8 sonication spots, depending on the tumor size, were used to cover the whole tumor. Immediately after pFUS exposure, DOX (10mg/kg) was injected via tail vein. Mice were euthanized after two hours and the DOX concentration in tumor (microgram of DOX per gram of tumor weight) was measured using the fluorescence technique. Mice were randomly assigned to two groups (n=8 for each group). One group received DOX injection only (group: DOX). The other received both pFUS exposure and DOX injection (group: FUS+DOX). Results: The DOX concentration in the FUS+DOX group was 14.9+/−2.5 ug/g compared to 9.5+/−1.6 ug/g of the DOX group (p=0.051). There was an about 60% increase of the DOX uptake in the prostate tumor exposed to pFUS. Conclusion: Results show that pFUS exposures significantly enhanced the uptake of DOX in prostate tumors. This result is consistent with our previous study of enhanced [3H]‐docetaxel uptake in prostate tumors. Further studies are being conducted to investigate the optimal pFUS parameters to maximize the uptake enhancement and its clinical benefits for cancer therapy (Study supported by a grant from Focused Ultrasound Surgery Foundation and partially by DOD PC073127).