Mechanical High Intensity Focused Ultrasound Research Articles

Towards in vivo immunotherapy using high intensity focused ultraosund

High-intensity focused ultrasound (HIFU) has been regarded as an appealing anti-cancer treatment for over a decade. HIFU indeed offers a combination of unique advantages: it is non-invasive and capable of delivering a precise and local thermal or mechanical dose without affecting the surrounding healthy tissue. HIFU is therefore already being applied as a cancer treatment in the clinics, although mostly as a thermal modality. On the other hand, immunotherapy has demonstrated remarkable promise for durable cancer treatment. However, its cost and complexity are prohibitive. Here, we evaluate (moderate) mechanical HIFU treatment and its capacity to induce immunogenic cell death with the aim to achieve immunotherapy treatment, directly in vivo. To that end, we treat a full lummox dish using an automatic scanning setup. Immunogenic cell death is monitored via distinct biomolecular markers (ATP secretion, HMGB1 secretion, calreticulin exposure). Cavitation activity is monitored via passive cavitation detection. Beyond the correspondence between acoustic cavitation and immunogenic cell death, our results also show a major influence of the cell type, namely, standard CT26 or B16F1O cells.

High intensity focused ultrasound atomization and erosion in healthy and tendinopathic tendons

Objective. High-intensity focused ultrasound (HIFU) can induce thermal and mechanical mechanisms in a well-defined focal volume of tissues. Histotripsy is a form of mechanical HIFU that can initiate and interact with bubble(s) to cause shock scattering and perhaps atomization within the bubble(s) to fractionate most soft tissues. Ultrasonic atomization, or the ejection of fine droplets from an acoustically-excited liquid exposed to air, has been shown to erode planar soft tissue surfaces, which has led to theories that atomization is a mechanism in histotripsy. However, healthy tendons show resistance to conventional histotripsy; pre-treatment of tendons with heat increases susceptibility to histotripsy fractionation. This study investigates ultrasonic atomization and erosion from planar healthy and tendinopathic tendon surfaces as we evaluate HIFU parameters for histotripsy in tendons. Approach. Forty-six ex vivo bovine tendon-air interfaces were pre-conditioned to surface wetting, heat baths of 20 °C (unaltered), 37 °C (body temperature), and 58 °C (collagen degradation), collagenase soaks for 1, 3, 5, and 24 h (mimicking tendinopathic tendons), and phosphate buffered saline soaks for 24 h. Ejected fragments, histology, and gross analysis determined erosion success. Tissue displacement from the HIFU radiation force was monitored with high-speed photography, and tissue relaxation was pixel-tracked and fit to a Kelvin–Voigt model to evaluate changes in viscoelastic properties. Main results. Results showed that atomization produced holes in 24 h collagenase tendons and surface pitting in 58 °C, 3 h, and 5 h collagenase tendons. Increased mound heights and viscoelastic constants in pre-heated (to 58 °C) and collagenase-soaking (3+ hours) tendinopathic models caused a decrease in elasticity and/or increase in viscosity, increasing susceptibility to erosion by HIFU atomization. Significance. Therefore, tendons with chronic tendinopathies may be more susceptible than healthy tendons to histotripsy fractionation.

Combination of ultrasound-based mechanical disruption of tumor with immune checkpoint blockade modifies tumor microenvironment and augments systemic antitumor immunity

BackgroundDespite multimodal adjuvant management with radiotherapy, chemotherapy and hormonal therapies, most surgically resected primary breast cancers relapse or metastasize. A potential solution to late and distant recurrence is to augment...

Photoacoustic computed tomography of mechanical HIFU-induced vascular injury.

Mechanical high-intensity focused ultrasound (HIFU) has been used for cancer treatment and drug delivery. Existing monitoring methods for mechanical HIFU therapies such as MRI and ultrasound imaging often suffer from high cost, poor spatial-temporal resolution, and/or low sensitivity to tissue's hemodynamic changes. Evaluating vascular injury during mechanical HIFU treatment, therefore, remains challenging. Photoacoustic computed tomography (PACT) is a promising tool to meet this need. Intrinsically sensitive to optical absorption, PACT provides high-resolution imaging of blood vessels using hemoglobin as the endogenous contrast. In this study, we have developed an integrated HIFU-PACT system for detecting vascular rupture in mechanical HIFU treatment. We have demonstrated singular value decomposition for enhancing hemorrhage detection. We have validated the HIFU-PACT performance on phantoms and in vivo animal tumor models. We expect that PACT-HIFU will find practical applications in oncology research using small animal models.

Combining Mechanical High-Intensity Focused Ultrasound Ablation with Chemotherapy for Augmentation of Anticancer Immune Responses.

As a noninvasive therapy, high-intensity focused ultrasound (HIFU) shows great potential in inducing anticancer immune responses. However, the overall anticancer efficacy of HIFU is still limited due to the rapid attenuation of ultrasound waves and inadequacy of ultrasound waves to spread to the whole tumor. Here, we combined HIFU with the ultrasound contrast agent/chemotherapeutic drug co-delivery nanodroplets to achieve synergistic enhancement of anticancer efficacy. Different from the widely used thermal HIFU irradiation, by which excessive heating would result in inactivation of immune stimulatory molecules, we used short acoustic pulses to trigger HIFU (mechanical HIFU, mHIFU) to improve anticancer immune responses. The nanodroplets displayed a mHIFU/glutathione (GSH)-dual responsive drug release property, and their cellular uptake efficacy and toxicity against cancer cells increased upon mHIFU irradiation. The generated immunogenic debris successfully induced the exposure of damage-associated molecular patterns on the cell surface for dendritic cells (DCs) maturation. In vivo experiments with tumor-bearing mice showed that the co-delivery nanodroplets in combination with mHIFU could effectively inhibit tumor growth by inducing immunogenic cell death, activating DCs maturation, and enhancing the effector T-cell infiltration within tumors. This work reveals that combined treatment with nanodroplets and mHIFU is a promising approach to eradicate tumors.

Immune modulation resulting from MR-guided high intensity focused ultrasound in a model of murine breast cancer

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1β and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.

High-Intensity Focused Ultrasound (HIFU) Triggers Immune Sensitization of Refractory Murine Neuroblastoma to Checkpoint Inhibitor Therapy.

Immunotherapy promises unprecedented benefits to patients with cancer. However, the majority of cancer types, including high-risk neuroblastoma, remain immunologically unresponsive. High-intensity focused ultrasound (HIFU) is a noninvasive technique that can mechanically fractionate tumors, transforming immunologically "cold" tumors into responsive "hot" tumors. We treated <2% of tumor volume in previously unresponsive, large, refractory murine neuroblastoma tumors with mechanical HIFU and assessed systemic immune response using flow cytometry, ELISA, and gene sequencing. In addition, we combined this treatment with αCTLA-4 and αPD-L1 to study its effect on the immune response and long-term survival. Combining HIFU with αCTLA-4 and αPD-L1 significantly enhances antitumor response, improving survival from 0% to 62.5%. HIFU alone causes upregulation of splenic and lymph node NK cells and circulating IL2, IFNγ, and DAMPs, whereas immune regulators like CD4+Foxp3+, IL10, and VEGF-A are significantly reduced. HIFU combined with checkpoint inhibitors induced significant increases in intratumoral CD4+, CD8α+, and CD8α+CD11c+ cells, CD11c+ in regional lymph nodes, and decrease in circulating IL10 compared with untreated group. We also report significant abscopal effect following unilateral treatment of mice with large, established bilateral tumors using HIFU and checkpoint inhibitors compared with tumors treated with HIFU or checkpoint inhibitors alone (61.1% survival, P < 0.0001). This combination treatment significantly also induces CD4+CD44+hiCD62L+low and CD8α+CD44+hiCD62L+low population and is adoptively transferable, imparting immunity, slowing subsequent de novo tumor engraftment. Mechanical fractionation of tumors using HIFU can effectively induce immune sensitization in a previously unresponsive murine neuroblastoma model and promises a novel yet efficacious immunoadjuvant modality to overcome therapeutic resistance.

SURG-33. IMMUNO-STIMULATION THROUGH SURGICALLY ADMINISTERED MECHANICAL HIGH INTENSITY FOCUSED ULTRASOUND

Abstract INTRODUCTION While immunotherapy through anti-PD1 checkpoint inhibition has demonstrated impressive clinical efficacy against a number of tumor types, its application has been ineffective against glioblastomas. METHODS We wished to determine whether high intensity focused ultrasound (HIFU) augment the efficacy of anti-PD1 checkpoint inhibition against glioblastomas. HIFU can be surgically administered through low-duty cycle to induce subcellular fragmentation without causing temperature elevation (termed mechanical HIFU). Alternatively, HIFU delivered through high duty cycle can induce foci of temperature elevation and thermal ablation (termed thermal HIFU). We tested the impact of mechanical and thermal HIFU on the anti-glioblastoma effects of the immune checkpoint inhibitor, ipililumab. RESULTS In an in vivomurine glioblastoma model, sites targeted by mechanical HIFU exhibited a 10–100 fold increase in accumulation in tumor infiltrating lymphocytes and interferon-γ (IFN-γ) in the presence of PD-1 blockade. At least 75% of mice engrafted with glioblastomas achieved remission when treated with mechanical HIFU and PD-1 blockade. In contrast, none of the mice treated with single therapies achieved durable remission. Likelihood of remission correlated with the abundance of tumor infiltrating lymphocytes (p&lt; 0.001) and IFN- γ levels (p=0.001). The synergy observed between mechanical HIFU and PD-1 blockade was not seen when combining thermal HIFU and PD-1 blockade, suggesting thermal protein denaturation is prohibitive to immune-stimulatory effects of HIFU. CONCLUSION Mechanical HIFU, but not thermal HIFU, augments the anti-glioblastoma effects of PD-1 blockade. Our findings inform the utility of HIFU as an immune-adjuvant in glioblastoma therapy that can be surgically administered.

Immuno-Stimulatory Effects of Mechanical and Thermal High Intensity Focused Ultrasound

Abstract INTRODUCTION While immunotherapy through anti-PD1 checkpoint inhibition has demonstrated impressive clinical efficacy against a number of tumor types, its application has been ineffective against glioblastomas METHODS We wished to determine whether high intensity focused ultrasound (HIFU) augment the efficacy of anti-PD1 checkpoint inhibition against glioblastomas. HIFU can be administered through low-duty cycle to induce subcellular fragmentation without causing temperature elevation (termed mechanical HIFU). Alternatively, HIFU delivered through high-duty cycle can induce foci of temperature elevation and thermal ablation (termed thermal HIFU). We tested the impact of mechanical and thermal HIFU on the anti-glioblastoma effects of the immune checkpoint inhibitor, ipilimumab RESULTS In an in Vivo murine glioblastoma model, sites targeted by mechanical HIFU exhibited a 10 to 100 fold increase in accumulation in tumor infiltrating lymphocytes and interferon-gamma in the presence of PD-1 blockade. At least 75% of mice engrafted with glioblastomas achieved remission when treated with mechanical HIFU and PD-1 blockade. In contrast, none of the mice treated with single therapies achieved durable remission. Likelihood of remission correlated with the abundance of tumor infiltrating lymphocytes (P &lt; .001) and IFN-gamma levels (P = .001). The synergy observed between mechanical HIFU and PD-1 blockade was not seen when combining thermal HIFU and PD-1 blockade, suggesting thermal protein denaturation is prohibitive to immune-stimulatory effects of HIFU CONCLUSION Mechanical HIFU, but not thermal HIFU, augments the anti-glioblastoma effects of PD-1 blockade. Our findings inform the utility of HIFU as an immune-adjuvant in glioblastoma therapy and provide a paradigm for conceptualizing the immune effects of laser thermal ablation

Abstract 4071: A novel combination therapy of high intensity focused ultrasound and PDL1 blockades against advanced breast cancer

Abstract Background: Previous studies have reported that tumor debris and inflammation made by high-intensity focused ultrasound (HIFU) therapy induced the antitumor immune response, however, HIFU as a monotherapy is still not potent enough to eradicate tumors and treat distant metastasis. We have reported that mechanical HIFU (M-HIFU), that will mechanically destroy tumor cells/tissues through acoustic cavitation, induced stronger antitumor immune response compared to conventional thermal HIFU (T-HIFU) that will cause thermal ablation of tumors. In the present study, we established an immune based combination therapy of M-HIFU and immune checkpoint blockade to enhance systemic antitumor immune response and treated distant/metastatic tumors in murine breast cancer models. Methods and Results: HER2 oncogene-dependent murine breast cancer cell line, MM3MG-HER2, was established in our lab. In mice with bilateral implantation of MM3MG-HER2 tumors, M-HIFU monotherapy induced stronger HER2-specific cellular response and inhibited the growth of HIFU-untreated distant tumors as well as HIFU treated-tumors, more potently than T-HIFU. Flow cytometry and immunohistochemical analysis of tumor microenvironment revealed significantly stronger accumulation of activated T cell and NK cells in M-HIFU-treated tumors. On the other hand, M-HIFU induced stronger expression of programmed death-ligand 1 (PD-L1) on various immune cells in both sides of tumors than T-HIFU therapy or no treatment control. Based on these findings, we investigated the combination therapy of M-HIFU and PD-1/PD-L1 axis blockades, and found significantly enhanced tumor-specific cellular immune response compared to each monotherapy, which resulted in improved therapeutic effect against distant tumors as well as HIFU treated-tumors. Furthermore, immune cell depletion studies demonstrated that both CD8+ T cells and NK cells played an essential role for the antitumor efficacy in this combinatory therapy against both HIFU-treated tumors and untreated distant tumors. Conclusion: this study provides strong rational evidence that M-HIFU combined with PD-1/PD-L1 axis blockades could be a promising treatment strategy against advanced breast cancer with metastatic lesion. Citation Format: Shinya Abe, Takuya Osada, Kensuke Kaneko, Pei Zhong, Herbert K. Lyerly. A novel combination therapy of high intensity focused ultrasound and PDL1 blockades against advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4071.

Abstract B151: Exploring the induction of immunogenic cell death (ICD) by high-intensity focused ultrasound (HIFU)

Abstract Immunogenic cell death (ICD) is an intriguing concept within cancer immunotherapy. Indeed, by inducing a specific means of cellular demise, a protective immune response can be triggered against the dying cells. As such, the dying cancer cells are—in a way—turned into a vaccine, attracting immune cells to the site of cell death, and activating them to trigger an anticancer immune response. Since the early discovery of ICD, more mechanistic insights into the hallmarks of ICD have been unraveled and several state-of-the-art cancer therapies have been identified as bona fide ICD inducers (1, 2).One of the cancer therapies which could be a potential candidate to induce ICD is high-intensity focused ultrasound (HIFU) (3). HIFU is currently used as a way of debulking solid tumors in a non-invasive, image-controlled and precise way without the use of ionizing radiation. Depending on the ultrasound parameters, tumor tissue can be ablated either in a mechanical (tissue disruption) or thermal way (heating and coagulative necrosis). Interestingly, there are some indications that HIFU may also affect the immune system: experiments in mice and man have shown attraction/activation of immune cells to/in the tumor site (4, 5). Of note: these immunostimulating effects were reported to be more prominent for mechanical HIFU than thermal HIFU. However, neither the mechanisms behind these observations, nor the possible link to the induction of ICD have been investigated. Therefore, the key aim of work is to investigate the potential of HIFU to induce ICD. Using a custom-designed HIFU set-up, we were able to—in a precise, automated and controlled way—expose B16F0 melanoma cells to HIFU in vitro. The HIFU-exposed tumor cells were subsequently evaluated for the occurrence of apoptosis and ICD hallmarks. More specifically, we evaluated the exposure of calreticulin (CRT) on the cell membrane and release of high mobility group box 1 (HMGB-1) and adenosine triphosphate (ATP). Our data show that application of mechanical HIFU protocols (using short, repeated ultrasound bursts) induce a significant fraction of apoptotic cells (as measured by a loss of mitochondrial membrane potential) within 4h after treatment. Importantly, already within the first hour after HIFU exposure, high levels of the ICD hallmark and Toll-like receptor 4 agonist HMGB-1 were found in the supernatant of the HIFU-treated cells, compared to untreated controls. To determine the effect of HIFU on ATP release, a quinacrine assay was performed to stain intracellular ATP-containing vesicles. Using flow cytometry, we observed that HIFU triggered exocytosis of these vesicles, with 2- to 3-fold reductions in quinacrine fluorescence at 4h and 24h after HIFU exposure, respectively. In addition, at 24h after HIFU exposure, &amp;gt;30% of the cells exposed CRT on the outer leaflet of the cell membrane, which was a drastic increase compared to blanks (&amp;lt;1% CRT exposure). Taken together, these results indicate that 3 key hallmarks of ICD can be detected after treatment of B16F0 melanoma cells with an optimized mechanical HIFU protocol. Currently, vaccination experiments are ongoing to determine whether HIFU-treated cells induce protective immunity in mice. If we could identify mechanical HIFU as a true ICD inducer, this might have important repercussions to how HIFU ablative therapies are applied in the clinic: modified HIFU regimens that not only focus on full ablation, but also trigger ICD could be envisaged to simultaneously tackle primary tumors as well as distant metastasis. Moreover, this could provide a solid basis for the rational combination of HIFU with other immunotherapies to strengthen the evoked immune response.

In vivo photoacoustics and high frequency ultrasound imaging of mechanical high intensity focused ultrasound (HIFU) ablation.

The thermal effect of high intensity focused ultrasound (HIFU) has been clinically exploited over a decade, while the mechanical HIFU is still largely confined to laboratory investigations. This is in part due to the lack of adequate imaging techniques to better understand the in-vivo pathological and immunological effects caused by the mechanical treatment. In this work, we explore the use of high frequency ultrasound (US) and photoacoustics (PA) as a potential tool to evaluate the effect of mechanical ablation in-vivo, e.g. boiling histotripsy. Two mice bearing a neuroblastoma tumor in the right leg were ablated using an MRI-HIFU system conceived for small animals and monitored using MRI thermometry. High frequency US and PA imaging were performed before and after the HIFU treatment. Afterwards, the tumor was resected for further assessment and evaluation of the ablated region using histopathology. High frequency US imaging revealed the presence of liquefied regions in the treated area together with fragmentized tissue which appeared with different reflecting proprieties compared to the surrounding tissue. Photoacoustic imaging on the other hand revealed the presence of deoxygenated blood within the tumor after the ablation due to the destruction of blood vessel network while color Doppler imaging confirmed the blood vessel network destruction within the tumor. The treated area and the presence of red blood cells detected by photoacoustics were further confirmed by the histopathology. This feasibility study demonstrates the potential of high frequency US and PA approach for assessing in-vivo the effect of mechanical HIFU tumor ablation.

Treatment of hepatic tumors by thermal versus mechanical effects of pulsed high intensity focused ultrasound in vivo

The purpose of this study is to comparatively assess the thermal versus mechanical effects of pulsed high intensity focused ultrasound (HIFU) treatment on hepatic tumors in vivo. Forty-five rabbits with hepatic VX2 tumors were randomly separated into three groups (15 animals per group) before HIFU ablation. The total HIFU energy (in situ) of 1250 J was used for each tumor for three groups. In groups I and II, animals were treated with 1 MHz pulsed ultrasound at 1 Hz pulsed repetition frequency (PRF), 0.5 duty cycle (0.5 s on and 0.5 s off) and10 s duration for one spot sonication. For group II, in addition to HIFU treatment, microbubbles (SonoVue, Bracco, Milan, Italy) were injected via vein before sonication acting as a synergist. In group III, animals were treated with 1 MHz pulsed ultrasound at 10 Hz PRF, 0.1 duty cycle (0.1 s on and 0.9 s off) and 10 s duration for one sonication. The total treatment spots were calculated according to the tumor volume. Tumors were examined with contrast-enhanced computed tomography (CECT) immediately prior to and post HIFU treatment. Histopathologic assessment was performed 3 h after treatment. Our study showed that all animals tolerated the HIFU treatment well. Our data showed that mechanical HIFU could lead to controlled injury in rabbit hepatic tumors with different histological changes in comparison to thermal HIFU with or without microbubbles.

Development of a high-field MR-guided HIFU setup for thermal and mechanical ablation methods in small animals.

BackgroundThermal and mechanical high intensity focused ultrasound (HIFU) ablation techniques are in development for non-invasive treatment of cancer. However, knowledge of in vivo histopathologic and immunologic reactions after HIFU ablation is still limited. This study aims to create a setup for evaluation of different HIFU ablation methods in mouse tumors using high-field magnetic resonance (MR) guidance. An optimized MR-guided-HIFU setup could be used to increase knowledge of the different pathologic and immunologic reactions to different HIFU ablation methods.MethodsThree different HIFU treatment strategies were applied in mouse melanomas (B16): a thermal (continuous wave), a mechanical (5 ms pulsed wave), and an intermediate setting (20 ms pulsed wave) for HIFU ablation, all under MR guidance using a 7 tesla animal MR system. Histopathologic evaluation was performed 3 days after treatment.ResultsThe focus of the ultrasound transducer could accurately be positioned within the tumor under MR image guidance, without substantial damage to the surrounding tissue and skin. All mice retained complete use of the treated leg after treatment. Temperatures of >60, <50, and <44 °C were reached during thermal, intermediate, and mechanical HIFU ablation, respectively. Thermal-treated tumors showed large regions of coagulative necrosis. Tumors of both the mechanical and intermediate groups showed fractionated tissue with islands of necrosis and some pseudocysts with hemorrhage.ConclusionA stable small animal MR-guided HIFU setup was designed and evaluated for follow-up MR imaging and histopathologic responses of the treated tumors. This will facilitate further studies with a larger number of mice for detailed evaluation of the pathologic and immunologic response to different HIFU strategies.

M-HIFU Inhibits Tumor Growth, Suppresses STAT3 Activity and Enhances Tumor Specific Immunity in a Transplant Tumor Model of Prostate Cancer

ObjectiveIn this study, we explored the use of mechanical high intensity focused ultrasound (M-HIFU) as a neo-adjuvant therapy prior to surgical resection of the primary tumor. We also investigated the role of signal transducer and activator of transcription 3 (STAT3) in M-HIFU elicited anti-tumor immune response using a transplant tumor model of prostate cancer.MethodsRM-9, a mouse prostate cancer cell line with constitutively activated STAT3, was inoculated subcutaneously in C57BL/6J mice. The tumor-bearing mice (with a maximum tumor diameter of 5∼6 mm) were treated by M-HIFU or sham exposure two days before surgical resection of the primary tumor. Following recovery, if no tumor recurrence was observed in 30 days, tumor rechallenge was performed. The growth of the rechallenged tumor, survival rate and anti-tumor immune response of the animal were evaluated.ResultsNo tumor recurrence and distant metastasis were observed in both treatment groups employing M-HIFU + surgery and surgery alone. However, compared to surgery alone, M-HIFU combined with surgery were found to significantly inhibit the growth of rechallenged tumors, down-regulate intra-tumoral STAT3 activities, increase cytotoxic T cells in spleens and tumor draining lymph nodes (TDLNs), and improve the host survival. Furthermore, M-HIFU combined with surgery was found to significantly decrease the level of immunosuppression with concomitantly increased number and activities of dendritic cells, compared to surgery alone.ConclusionOur results demonstrate that M-HIFU can inhibit STAT3 activities, and when combined synergistically with surgery, may provide a novel and promising strategy for the treatment of prostate cancers.