HSP90 inhibition acts synergistically with heat to induce a pro-immunogenic form of cell death in colon cancer cells.

BACKGROUND: Sub-ablative heat induces pleiotropic biological effects in cancer cells, activating programmed cell death or survival processes. These processes decide the fate of the heated cell. This study investigates these and assesses whether heat, in combination with HSP90 inhibition, augments cell death and induces a pro-immune phenotype in these cells. METHODS: HCT116 and HT29 cells were subjected to thermal doses (TID) of 60 and 120CEM43 using a PCR thermal cycler. HSP90 was inhibited with NVP-AUY922. Viability was assessed using the MTT assay. Cellular ATP and HSP70 release were assessed using ATP and Enzyme-linked Immunosorbent assays, respectively. Flow cytometry and immunoblotting were used to study the regulation of biomarkers associated with the heat shock response, the cell cycle, and immunogenic and programmed cell death. RESULTS: Exposure of HCT116 and HT29 cells to TIDs of 60 and 120CEM43 decreased their viability. In addition, treatment with 120CEM43 increased intracellular HSP70 and the percentage of HCT116/HT29 cells in the G2/M cell cycle phase, ATP release and Calreticulin/HSP70/HSP90 exposure in the plasma membrane, while downregulating CD47 compared to sham-exposed cells. When combined with NVP-AUY922, treatment of HCT116/HT29 cells with 120CEM43 resulted in a synergistic decrease of cell viability associated with the induction of apoptosis. Also, the combined treatments increased Calreticulin exposure, CD47 downregulation, and HSP70 release compared to the sham-exposed cells. CONCLUSION: Sub-ablative heating can act synergistically with the clinically relevant HSP90 inhibitor NVP-AUY922 to induce a pro-immunogenic form of cell death in colon cancer cells.

Quantitative prediction of the extent of pelvic tumour ablation by magnetic resonance-guided high intensity focused ultrasound.

BACKGROUND: Patient suitability for magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) therapy of pelvic tumors is currently assessed by visual estimation of the proportion of tumor that can be reached by the device's focus (coverage). Since it is important to assess whether enough energy reaches the tumor to achieve ablation, a methodology for estimating the proportion of the tumor that can be ablated (treatability) was developed. Predicted treatability was compared against clinically achieved thermal ablation. METHODS: MR Dixon sequence images of five patients with recurrent gynecological tumors were acquired during their treatment. Acousto-thermal simulations were performed using k-Wave for three exposure points (the deepest and shallowest reachable focal points within the tumor, identified from tumor coverage analysis, and a point halfway in-between) per patient. Interpolation between the resulting simulated ablated tissue volumes was used to estimate the maximum treatable depth and hence, tumor treatability. Predicted treatability was compared both to predicted tumor coverage and to the clinically treated tumor volume. The intended and simulated volumes and positions of ablated tissues were compared. RESULTS: Predicted treatability was less than coverage by 52% (range: 31-78%) of the tumor volume. Predicted and clinical treatability differed by 9% (range: 1-25%) of tumor volume. Ablated tissue volume and position varied with beam path length through tissue. CONCLUSION: Tumor coverage overestimated patient suitability for MRgHIFU therapy. Employing patient-specific simulations improved treatability assessment. Patient treatability assessment using simulations is feasible.

Feasibility of palliating recurrent gynecological tumors with MRGHIFU: comparison of symptom, quality-of-life, and imaging response in intra and extra-pelvic disease.

OBJECTIVE: To document longitudinal symptom, quality-of-life and imaging response in patients with recurrent gynecological tumors treated with magnetic resonance guided high intensity focused ultrasound (MRgHIFU), and compare changes in patients with intra- versus extra-pelvic lesions. METHODS: Eleven symptomatic patients with painful recurrent gynecological tumors were treated with MRgHIFU (Profound Sonalleve) in a prospective single center study (NCT02714621). Pain scores, analgesic intake and quality-of-life metrics, whole tumor volume, and perfused tumor volume from Gadolinium-enhanced T1W imaging documented before and up to 90 days after treatment were compared between patients with intra- and extra-pelvic tumors. RESULTS: Two of five patients with intra-pelvic and three of six patients with extra-pelvic tumors were classified as responders (>2 point reduction in NRS pain score without analgesia increase or a > 25% reduction in analgesic use). Cohort reductions in worst pain scores were not significant for either group. Emotional functioning for the whole cohort improved, although physical functioning did not. Ablative thermal temperatures were achieved in three patients with extra-pelvic tumors, but in none whose tumors were intra-pelvic. Pain response did not correlate with thermal dose. Tumor volume increased by 18% immediately post-treatment in the extra-pelvic but not in the intra-pelvic group. Ratio of perfused to whole lesion volume decreased by >20% by day 30 in extra-pelvic, but not intra-pelvic tumors although at day 30 both extra-pelvic and intra-pelvic tumors increased in volume. CONCLUSION: MRgHIFU treatments can be delivered safely to patients with recurrent gynecological tumors. Extra-pelvic tumors responded better than intra-pelvic tumors and showed immediate swelling and reduction in perfused volume by day 30.

Safety Aspects of Perinatal Ultrasound.

Ultrasound safety is of particular importance in fetal and neonatal scanning. Fetal tissues are vulnerable and often still developing, the scanning depth may be low, and potential biological effects have been insufficiently investigated. On the other hand, the clinical benefit may be considerable. The perinatal period is probably less vulnerable than the first and second trimesters of pregnancy, and ultrasound is often a safer alternative to other diagnostic imaging modalities. Here we present step-by-step procedures for obtaining clinically relevant images while maintaining ultrasound safety. We briefly discuss the current status of the field of ultrasound safety, with special attention to the safety of novel modalities, safety considerations when ultrasound is employed for research and education, and ultrasound of particularly vulnerable tissues, such as the neonatal lung. This CME is prepared by ECMUS, the safety committee of EFSUMB, with contributions from OB/GYN clinicians with a special interest in ultrasound safety.

Prediction of pelvic tumour coverage by magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU) from referral imaging.

BACKGROUND: Patient suitability for magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) ablation of pelvic tumors is initially evaluated clinically for treatment feasibility using referral images, acquired using standard supine diagnostic imaging, followed by MR screening of potential patients lying on the MRgHIFU couch in a 'best-guess' treatment position. Existing evaluation methods result in ≥40% of referred patients being screened out because of tumor non-targetability. We hypothesize that this process could be improved by development of a novel algorithm for predicting tumor coverage from referral imaging. METHODS: The algorithm was developed from volunteer images and tested with patient data. MR images were acquired for five healthy volunteers and five patients with recurrent gynaecological cancer. Subjects were MR imaged supine and in oblique-supine-decubitus MRgHIFU treatment positions. Body outline and bones were segmented for all subjects, with organs-at-risk and tumors also segmented for patients. Supine images were aligned with treatment images to simulate a treatment dataset. Target coverage (of patient tumors and volunteer intra-pelvic soft tissue), i.e. the volume reachable by the MRgHIFU focus, was quantified. Target coverage predicted from supine imaging was compared to that from treatment imaging. RESULTS: Mean (±standard deviation) absolute difference between supine-predicted and treatment-predicted coverage for 5 volunteers was 9 ± 6% (range: 2-22%) and for 4 patients, was 12 ± 7% (range: 4-21%), excluding a patient with poor acoustic coupling (coverage difference was 53%). CONCLUSION: Prediction of MRgHIFU target coverage from referral imaging appears feasible, facilitating further development of automated evaluation of patient suitability for MRgHIFU.

Heating technology for malignant tumors: a review.

The therapeutic application of heat is very effective in cancer treatment. Both hyperthermia, i.e., heating to 39-45 °C to induce sensitization to radiotherapy and chemotherapy, and thermal ablation, where temperatures beyond 50 °C destroy tumor cells directly are frequently applied in the clinic. Achievement of an effective treatment requires high quality heating equipment, precise thermal dosimetry, and adequate quality assurance. Several types of devices, antennas and heating or power delivery systems have been proposed and developed in recent decades. These vary considerably in technique, heating depth, ability to focus, and in the size of the heating focus. Clinically used heating techniques involve electromagnetic and ultrasonic heating, hyperthermic perfusion and conductive heating. Depending on clinical objectives and available technology, thermal therapies can be subdivided into three broad categories: local, locoregional, or whole body heating. Clinically used local heating techniques include interstitial hyperthermia and ablation, high intensity focused ultrasound (HIFU), scanned focused ultrasound (SFUS), electroporation, nanoparticle heating, intraluminal heating and superficial heating. Locoregional heating techniques include phased array systems, capacitive systems and isolated perfusion. Whole body techniques focus on prevention of heat loss supplemented with energy deposition in the body, e.g., by infrared radiation. This review presents an overview of clinical hyperthermia and ablation devices used for local, locoregional, and whole body therapy. Proven and experimental clinical applications of thermal ablation and hyperthermia are listed. Methods for temperature measurement and the role of treatment planning to control treatments are discussed briefly, as well as future perspectives for heating technology for the treatment of tumors.

Comparison of Imaging Changes and Pain Responses in Patients with Intra- or Extraosseous Bone Metastases Treated Palliatively with Magnetic Resonance-Guided High-Intensity-Focused Ultrasound.

PURPOSE: This study compared changes in imaging and in pain relief between patients with intraosseous, as opposed to extraosseous bone metastases. Both groups were treated palliatively with magnetic resonance-guided high-intensity-focused ultrasound (MRgHIFU). MATERIALS AND METHODS: A total of 21 patients were treated prospectively with MRgHIFU at 3 centers. Intraprocedural thermal changes measured using proton resonance frequency shift (PRFS) thermometry and gadolinium-enhanced T1-weighted (Gd-T1W) image appearances after treatment were compared for intra- and extraosseous metastases. Pain scores and use of analgesic therapy documented before and up to 90 days after treatment were used to classify responses and were compared between the intra- and extraosseous groups. Gd-T1W changes were compared between responders and nonresponders in each group. RESULTS: Thermal dose volumes were significantly larger in the extraosseous group (P = 0.039). Tumor diameter did not change after treatment in either group. At day 30, Gd-T1W images showed focal nonenhancement in 7 of 9 patients with intraosseous tumors; in patients with extraosseous tumors, changes were heterogeneous. Cohort reductions in worst-pain scores were seen for both groups, but differences from baseline at days 14, 30, 60, and 90 were only significant for the intraosseous group (P = 0.027, P = 0.013, P = 0.012, and P = 0.027, respectively). By day 30, 67% of patients (6 of 9) with intraosseous tumors were classified as responders, and the rate was 33% (4 of 12) for patients with extraosseous tumors. In neither group was pain response indicated by nonenhancement on Gd-T1W. CONCLUSIONS: Intraosseous tumors showed focal nonenhancement by day 30, and patients had better pain response to MRgHIFU than those with extraosseous tumors. In this small cohort, post-treatment imaging was not informative of treatment efficacy.

'Relationship between thermal dose and cell death for "rapid" ablative and "slow" hyperthermic heating'.

AIM: Thermal isoeffective dose (TID) has not been convincingly validated for application to predict biological effects from rapid thermal ablation (e.g., using >55 °C). This study compares the classical method of quantifying TID (derived from hyperthermia data) with a temperature-adjusted method based on the Arrhenius model for predicting cell survival in vitro, after either 'rapid' ablative or 'slow' hyperthermic exposures. METHODS: MTT assay viability data was obtained from two human colon cancer cell lines, (HCT116, HT29), subjected to a range of TIDs (120-720 CEM43) using a thermal cycler for hyperthermic (>2 minutes, <50 °C) treatments, or a novel pre-heated water bath based technique for ablative exposures (<10 seconds, >55 °C). TID was initially estimated using a constant RCEM>43°C=0.5, and subsequently using RCEM(T), derived from temperature dependent cell survival (injury rate) Arrhenius analysis. RESULTS: 'Slow' and 'rapid' exposures resulted in cell survival and significant regrowth (both cell lines) 10 days post-treatment for 240 CEM43 (RCEM>43°C=0.5), while 340-550 CEM43 (RCEM>43°C =0.5) delivered using 'rapid' exposures showed 12 ± 6% viability and 'slow' exposures resulted in undetectable viability. Arrhenius analysis of experimental data (activation energy ΔE = 5.78 ± 0.04 × 105 J mole-1, frequency factor A = 3.27 ± 11 × 1091 sec-1) yielded RCEM=0.42 * e0.0041*T which better-predicted cell survival than using R CEM> 43°C=0.5. CONCLUSIONS: TID calculated using an RCEM(T) informed by Arrhenius kinetic parameters provided a more consistent, heating strategy independent, predictor of cell viability, improving dosimetry of ablative thermal exposures. Cell viability was only undetectable above 305 ± 10 CEM43 using this revised measure.

A comprehensive model for heat-induced radio-sensitisation.

Combined radiotherapy (RT) and hyperthermia (HT) treatments may improve treatment outcome by heat induced radio-sensitisation. We propose an empirical cell survival model (AlphaR model) to describe this multimodality therapy. The model is motivated by the observation that heat induced radio-sensitisation may be explained by a reduction in the DNA damage repair capacity of heated cells. We assume that this repair is only possible up to a threshold level above which survival will decrease exponentially with dose. Experimental cell survival data from two cell lines (HCT116, Cal27) were considered along with that taken from the literature (baby hamster kidney [BHK] and Chinese hamster ovary cells [CHO]) for HT and combined RT-HT. The AlphaR model was used to study the dependence of clonogenic survival on treatment temperature, and thermal dose R2 ≥ 0.95 for all fits). For HT survival curves (0-80 CEM43 at 43.5-57 °C), the number of free fit AlphaR model parameters could be reduced to two. Both parameters increased exponentially with temperature. We derived the relative biological effectiveness (RBE) or HT treatments at different temperatures, to provide an alternative description of thermal dose, based on our AlphaR model. For combined RT-HT, our analysis is restricted to the linear quadratic arm of the model. We show that, for the range used (20-80 CEM43, 0-12 Gy), thermal dose is a valid indicator of heat induced radio-sensitisation, and that the model parameters can be described as a function thereof. Overall, the proposed model provides a flexible framework for describing cell survival curves, and may contribute to better quantification of heat induced radio-sensitisation, and thermal dose in general.

Dependence of inertial cavitation induced by high intensity focused ultrasound on transducer F-number and nonlinear waveform distortion.

Pulsed high intensity focused ultrasound was shown to enhance chemotherapeutic drug uptake in tumor tissue through inertial cavitation, which is commonly assumed to require peak rarefactional pressures to exceed a certain threshold. However, recent studies have indicated that inertial cavitation activity also correlates with the presence of shocks at the focus. The shock front amplitude and corresponding peak negative pressure (p -) in the focal waveform are primarily determined by the transducer F-number: less focused transducers produce shocks at lower p -. Here, the dependence of inertial cavitation activity on the transducer F-number was investigated in agarose gel by monitoring broadband noise emissions with a coaxial passive cavitation detector (PCD) during pulsed exposures (pulse duration 1 ms, pulse repetition frequency 1 Hz) with p- varying within 1-15 MPa. Three 1.5 MHz transducers with the same aperture, but different focal distances (F-numbers 0.77, 1.02, 1.52) were used. PCD signals were processed to extract cavitation probability, persistence, and mean noise level. At the same p -, all metrics indicated enhanced cavitation activity at higher F-numbers; specifically, cavitation probability reached 100% when shocks formed at the focus. These results provide further evidence supporting the excitation of inertial cavitation at reduced p - by waveforms with nonlinear distortion and shocks.

HIFU Tissue Ablation: Concept and Devices.

High intensity focused ultrasound (HIFU) is rapidly gaining clinical acceptance as a technique capable of providing non-invasive heating and ablation for a wide range of applications. Usually requiring only a single session, treatments are often conducted as day case procedures, with the patient either fully conscious, lightly sedated or under light general anesthesia. HIFU scores over other thermal ablation techniques because of the lack of necessity for the transcutaneous insertion of probes into the target tissue. Sources placed either outside the body (for treatment of tumors or abnormalities of the liver, kidney, breast, uterus, pancreas brain and bone), or in the rectum (for treatment of the prostate), provide rapid heating of a target tissue volume, the highly focused nature of the field leaving tissue in the ultrasound propagation path relatively unaffected. Numerous extra-corporeal, transrectal and interstitial devices have been designed to optimize application-specific treatment delivery for the wide-ranging areas of application that are now being explored with HIFU. Their principle of operation is described here, and an overview of their design principles is given.

Ultrasound-guided therapeutic focused ultrasound: current status and future directions.

This paper reviews ultrasound imaging methods for the guidance of therapeutic focused ultrasound (USgFUS), with emphasis on real-time preclinical methods. Guidance is interpreted in the broadest sense to include pretreatment planning, siting of the FUS focus, real-time monitoring of FUS-tissue interactions, and real-time control of exposure and damage assessment. The paper begins with an overview and brief historical background of the early methods used for monitoring FUS-tissue interactions. Current imaging methods are described, and discussed in terms of sensitivity and specificity of the localisation of the FUS effects in both therapeutic and sub-therapeutic modes. Thermal and non-thermal effects are considered. These include cavitation-enhanced heating, tissue water boiling and cavitation. Where appropriate, USgFUS methods are compared with similar methods implemented using other guidance modalities, e.g. magnetic resonance imaging. Conclusions are drawn regarding the clinical potential of the various guidance methods, and the feasibility and current status of real-time implementation.

A study of thermal dose-induced autophagy, apoptosis and necroptosis in colon cancer cells.

PURPOSE: The pleiotropic effects of heat on cancer cells have been well documented. The biological effects seen depend on the temperature applied, and the heating duration. In this study we investigate the cytotoxic effects of heat on colon cancer cells and determine how different cell death processes such as autophagy, apoptosis and necroptosis play a role in cell response. MATERIALS AND METHODS: The thermal dose concept was used to provide a parameter that will allow comparison of different thermal treatments. Two human colon cancer cell lines, HCT116 and HT29, were subjected to ablative temperatures using a polymerase chain reaction thermal cycler. Temperature was recorded using thermocouples. Cell viability was assessed using the MTT assay. Induction of apoptosis was estimated using an enzyme-linked immunosorbent assay that detects cleaved cytoplasmic nucleosomes. Protein regulation was determined using immunoblotting. The percentage of cells undergoing apoptosis and autophagy was determined with annexin V/propidium iodide staining and a cationic amphiphilic tracer using fluorescence-activated cell sorting analysis. RESULTS: Exposure of colon cancer cells to ablative thermal doses results in decreased cell viability. The cytotoxic effect of heat is associated with induction of apoptosis and autophagy, the amount depending on both the thermal dose applied and on the time elapsed after treatment. Autophagy induction is mainly seen in live cells. RIPK3 protein levels are increased after exposure of cells to heat. A necroptosis inhibitor does not affect cell viability. CONCLUSIONS: Autophagy, apoptosis and necroptosis are associated with the response of these cancer cell lines to supra-normal temperatures.

Towards a dosimetric framework for therapeutic ultrasound.

There is a need for a coherent set of exposure and dose quantities to describe ultrasound fields in media other than water (including tissue and tissue-simulating materials). This paper proposes an outline dosimetry scheme, with quantities for free field exposure, in situ exposure, dose (both instantaneous and cumulative) and effect, to act as a structure for organising a more complete set of definitions. It also presents findings from a survey of the views of the therapeutic ultrasound community which generally supports the principle of using modified free field quantities to describe the in situ field, and the prioritising of dose quantities which are related to heating and thermal mechanisms. Although there is no one-to-one relationship between any known ultrasound dose quantity and a specific biological effect, this can also be said of radiotherapy and other modalities where weighting factors have been developed to calculate the degree of equivalence between different tissues and radiation types. This same separation is recommended for ultrasound, provided that an appropriate set of recognised 'engineering' quantities can be established for exposure and dose quantities.

Quality assurance for clinical high intensity focused ultrasound fields.

As the use of HIFU in the clinic becomes more widespread there is an ever increasing need to standardise quality assurance protocols, an important step in facilitating the wider acceptance of HIFU as a therapeutic modality. This article reviews pertinent aspects of HIFU treatment delivery, encompassing the closely related aspects of quality assurance and calibration. Particular attention is given to the description and characterisation of relevant acoustic field parameters and the measurement of acoustic power. Where appropriate, recommendations are made.

International consensus on use of focused ultrasound for painful bone metastases: Current status and future directions.

Focused ultrasound surgery (FUS), in particular magnetic resonance guided FUS (MRgFUS), is an emerging non-invasive thermal treatment modality in oncology that has recently proven to be effective for the palliation of metastatic bone pain. A consensus panel of internationally recognised experts in focused ultrasound critically reviewed all available data and developed consensus statements to increase awareness, accelerate the development, acceptance and adoption of FUS as a treatment for painful bone metastases and provide guidance towards broader application in oncology. In this review, evidence-based consensus statements are provided for (1) current treatment goals, (2) current indications, (3) technical considerations, (4) future directions including research priorities, and (5) economic and logistical considerations.

Holographic Focused Ultrasound Hyperthermia System for Uniform Simultaneous Thermal Exposure of Multiple Tumor Spheroids.

Hyperthermia is currently used to treat cancer due to its ability to radio- and chemo-sensitize and to stimulate the immune response. While ultrasound is non-ionizing and can induce hyperthermia deep within the body non-invasively, achieving uniform and volumetric hyperthermia is challenging. This work presents a novel focused ultrasound hyperthermia system based on 3D-printed acoustic holograms combined with a high-intensity focused ultrasound (HIFU) transducer to produce a uniform iso-thermal dose in multiple targets. The system is designed with the aim of treating several 3D cell aggregates contained in an International Electrotechnical Commission (IEC) tissue-mimicking phantom with multiple wells, each holding a single tumor spheroid, with real-time temperature and thermal dose monitoring. System performance was validated using acoustic and thermal methods, ultimately yielding thermal doses in three wells that differed by less than 4%. The system was tested in vitro for delivery of thermal doses of 0-120 cumulative equivalent minutes at 43 °C (CEM43) to spheroids of U87-MG glioma cells. The effects of ultrasound-induced heating on the growth of these spheroids were compared with heating using a polymerase chain reaction (PCR) thermocycler. Results showed that exposing U87-MG spheroids to an ultrasound-induced thermal dose of 120 CEM43 shrank them by 15% and decreased their growth and metabolic activity more than seen in those exposed to a thermocycler-induced heating. This low-cost approach of modifying a HIFU transducer to deliver ultrasound hyperthermia opens new avenues for accurately controlling thermal dose delivery to complex therapeutic targets using tailored acoustic holograms. Spheroid data show that thermal and non-thermal mechanisms are implicated in the response of cancer cells to non-ablative ultrasound heating.

Recommendations for Reporting Therapeutic Ultrasound Treatment Parameters.

These recommendations are intended to provide guidance and to encourage best practice in reporting therapeutic ultrasound treatment parameters. Detailed uniform reporting will allow testing of therapy ultrasound systems and protocols, cross-comparison of studies between different teams using different systems and validation of therapeutic bio-effects. These recommendations have been divided into two sets, one for clinical and one for preclinical studies, each with stratified reporting categories, to account for the disparities in expertise and access to equipment between sites. The recommendations are intended to be useful for clinicians and researchers, for ethical and funding review boards and for the editors and reviewers of scientific journals.

Latest Advances in the Use of Therapeutic Focused Ultrasound in the Treatment of Pancreatic Cancer.

Traditional oncological interventions have failed to improve survival for pancreatic cancer patients significantly. Novel treatment modalities able to release cancer-specific antigens, render immunologically "cold" pancreatic tumours "hot" and disrupt or reprogram the pancreatic tumour microenvironment are thus urgently needed. Therapeutic focused ultrasound exerts thermal and mechanical effects on tissue, killing cancer cells and inducing an anti-cancer immune response. The most important advances in therapeutic focused ultrasound use for initiation and augmentation of the cancer immunity cycle against pancreatic cancer are described. We provide a comprehensive review of the use of therapeutic focused ultrasound for the treatment of pancreatic cancer patients and describe recent studies that have shown an ultrasound-induced anti-cancer immune response in several tumour models. Published studies that have investigated the immunological effects of therapeutic focused ultrasound in pancreatic cancer are described. This article shows that therapeutic focused ultrasound has been deemed to be a safe technique for treating pancreatic cancer patients, providing pain relief and improving survival rates in pancreatic cancer patients. Promotion of an immune response in the clinic and sensitisation of tumours to the effects of immunotherapy in preclinical models of pancreatic cancer is shown, making it a promising candidate for use in the clinic.