Ultrasound-stimulated Microbubbles Research Articles

Ultrasound-stimulated microbubbles enhanced vascular disruption in fractionated radiotherapy-treated tumours via ASMase activation.

Recent studies have indicated that radiotherapy affects tumour vasculature as well as tumour cells. The use of ultrasound-stimulated microbubbles (USMB) can potentially enhance the effects of radiotherapy through the activation of the acid sphingomyelinase [ASMase or sphingomyelin phosphodiesterase 1 (SMPD1)]-ceramide pathway. ASMase knockout (ASMase-/-) and wild-type (WT) mice bearing fibrosarcoma (MCA/129 tumour line) were treated with 10 Gy or 20 Gy in five fractions alongside or independently of USMB treatments. The results indicated that tumour responses to fractionated radiotherapy (fXRT) were enhanced when fXRT was coupled with USMB as part of the treatment regimen. Sphingosine-1-phosphate (S1P)-treated mice and ASMase-/- mice demonstrated radioresistance against fXRT alone, whereas only ASMase-/- mice showed radioresistance against fXRT treatment alone and when combined with USMB. Results indicated that in WT and S1P-treated cohorts, the use of USMB with fXRT enhanced the tumour response compared to use of USMB or fXRT alone. Although in WT and S1P-treated cohorts, there was enhanced vascular disruption, ASMase-/- cohorts demonstrated no significant vascular disruption, indicating the importance of ASMase in facilitating vascular changes in response to fXRT and USMB treatment.

Fluid flow influences ultrasound-assisted endothelial membrane permeabilization and calcium flux.

The local fluid dynamics experienced by circulating microbubbles vary across different anatomical sites, which can influence ultrasound-mediated therapeutic delivery efficacy. This study aimed to elucidate the effect of fluid flow rate in combination with repeated short-pulse ultrasound on microbubble-mediated endothelial cell permeabilization. Here, a seeded monolayer of human umbilical (HUVEC) or brain endothelial cells (HBEC-5i) was co-perfused with a solution of microbubbles and propidium iodide (PI) at either a flow rate of 5 or 30ml/min. Using an acoustically coupled inverted microscope, cells were exposed to 1MHz ultrasound with 20-cycle bursts, 1ms PRI, and 2s duration at a peak negative pressure of 305kPa to assess the role of flow rate on ultrasound-stimulated endothelial cell permeability, as well as Ca2+ modulation. In addition, the effect of inter-pulse delays (∆t=1s) on the resulting endothelial permeability was investigated. Our results demonstrate that under an identical acoustic stimulus, fast-flowing microbubbles resulted in a statistically significant increase in cell membrane permeability, at least by 2.3-fold, for both endothelial cells. Likewise, there was a substantial difference in intracellular Ca2+ levels between the two examined flow rates. In addition, multiple short pulses rather than a single pulse ultrasound, with an equal number of bursts, significantly elevated endothelial cell permeabilization, at least by 1.4-fold, in response to ultrasound-stimulated microbubbles. This study provides insights into the design of optimal, application-dependent pulsing schemes to improve the effectiveness of ultrasound-mediated local therapeutic delivery.

Evaluating the effects of radiation and acoustically-stimulated microbubble therapy in an in vivo breast cancer model.

Ultrasound-stimulated microbubbles (USMB) cause localized vascular effects and sensitize tumors to radiation therapy (XRT). We investigated acoustic parameter optimization for combining USMB and XRT. We treated breast cancer xenograft tumors with 500 kHz pulsed ultrasound at varying pressures (570 or 740 kPa), durations (1 to 10 minutes), and microbubble concentrations (0.01 to 1% (v/v)). Radiation therapy (2 Gy) was administered immediately or after a 6-hour delay. Histological staining of tumors 24 hours after treatment detected changes in cell morphology, cell death, and microvascular density. Significant cell death resulted at 570 kPa after a 1-minute exposure with 1% (v/v) microbubbles with or without XRT. However, significant microvascular disruption required higher ultrasound pressure and exposure duration greater than 5 minutes. Introducing a 6-hour delay between treatments (USMB and XRT) showed a similar tumor effect with no further improvement in response as compared to when XRT was delivered immediately after USMB.

MR-guided ultrasound-stimulated microbubble therapy enhances radiation-induced tumor response

High intensity focused ultrasound (HIFU) systems have been approved for therapeutic ultrasound delivery to cause tissue ablation or induced hyperthermia. Microbubble agents have also been used in combination with sonication exposures. These require temperature feedback and monitoring to prevent unstable cavitation and prevent excess tissue heating. Previous work has utilized lower power and pressure to oscillate microbubbles and transfer energy to endothelial cells in the absence of thermally induced damage that can radiosensitize tumors. This work investigated whether reduced acoustic power and pressure on a commercial available MR-integrated HIFU system could result in enhanced radiation-induced tumor response after exposure to ultrasound-stimulated microbubbles (USMB) therapy. A commercially available MR-integrated HIFU system was used with a hyperthermia system calibration provided by the manufacturer. The ultrasound transducer was calibrated to reach a peak negative pressure of − 750 kPa. Thirty male New Zealand white rabbits bearing human derived PC3 tumors were grouped to receive no treatment, 14 min of USMB, 8 Gy of radiation in a separate irradiation cabinet, or combined treatments. In vivo temperature changes were collected using MR thermometry at the tumor center and far-field muscle region. Tissues specimens were collected 24 h post radiation therapy. Tumor cell death was measured and compared to untreated controls through hematoxylin and eosin staining and immunohistochemical analysis. The desired peak negative pressure of − 750 kPa used for previous USMB occurred at approximately an input power of 5 W. Temperature changes were limited to under 4 °C in ten of twelve rabbits monitored. The median temperature in the far-field muscle region of the leg was 2.50 °C for groups receiving USMB alone or in combination with radiation. Finally, statistically significant tumor cell death was demonstrated using immunohistochemical analysis in the combined therapy group compared to untreated controls. A commercial MR-guided therapy HIFU system was able to effectively treat PC3 tumors in a rabbit model using USMB therapy in combination with radiation exposures. Future work could find the use of reduced power and pressure levels in a commercial MR-guided therapy system to mechanically stimulate microbubbles and damage endothelial cells without requiring high thermal doses to elicit an antitumor response.

Focused Ultrasound and Ultrasound Stimulated Microbubbles in Radiotherapy Enhancement for Cancer Treatment.

Radiation therapy (RT) has been the standard of care for treating a multitude of cancer types. However, ionizing radiation has adverse short and long-term side effects which have resulted in treatment complications for decades. Thus, advances in enhancing the effects of RT have been the primary focus of research in radiation oncology. To avoid the usage of high radiation doses, treatment modalities such as high-intensity focused ultrasound can be implemented to reduce the radiation doses required to destroy cancer cells. In the past few years, the use of focused ultrasound (FUS) has demonstrated immense success in a number of applications as it capitalizes on spatial specificity. It allows ultrasound energy to be delivered to a targeted focal area without harming the surrounding tissue. FUS combined with RT has specifically demonstrated experimental evidence in its application resulting in enhanced cell death and tumor cure. Ultrasound-stimulated microbubbles have recently proved to be a novel way of enhancing RT as a radioenhancing agent on its own, or as a delivery vector for radiosensitizing agents such as oxygen. In this mini-review article, we discuss the bio-effects of FUS and RT in various preclinical models and highlight the applicability of this combined therapy in clinical settings.

Quantitative Ultrasound for Evaluation of Tumour Response to Ultrasound-Microbubbles and Hyperthermia.

Objectives: Prior study has demonstrated the implementation of quantitative ultrasound (QUS) for determining the therapy response in breast tumour patients. Several QUS parameters quantified from the tumour region showed a significant correlation with the patient's clinical and pathological response. In this study, we aim to identify if there exists such a link between QUS parameters and changes in tumour morphology due to combined ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) using the breast xenograft model (MDA-MB-231). Method: Tumours grown in the hind leg of severe combined immuno-deficient mice were treated with permutations of USMB and HT. Ultrasound radiofrequency data were collected using a 25 MHz array transducer, from breast tumour-bearing mice prior and post-24-hour treatment. Result: Our result demonstrated an increase in the QUS parameters the mid-band fit and spectral 0-MHz intercept with an increase in HT duration combined with USMB which was found to be reflective of tissue structural changes and cell death detected using haematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labelling stain. A significant decrease in QUS spectral parameters was observed at an HT duration of 60 minutes, which is possibly due to loss of nuclei by the majority of cells as confirmed using histology analysis. Morphological alterations within the tumour might have contributed to the decrease in backscatter parameters. Conclusion: The work here uses the QUS technique to assess the efficacy of cancer therapy and demonstrates that the changes in ultrasound backscatters mirrored changes in tissue morphology.

Intravital imaging and cavitation monitoring of antivascular ultrasound in tumor microvasculature.

Rationale: Focused ultrasound-stimulated microbubbles have been shown to be capable of inducing blood flow shutdown and necrosis in a range of tissue types in an approach termed antivascular ultrasound or nonthermal ablation. In oncology, this approach has demonstrated tumor growth inhibition, and profound synergistic antitumor effects when combined with traditional platforms of chemo-, radiation- and immune-therapies. However, the exposure schemes employed have been broad and underlying mechanisms remain unclear with fundamental questions about exposures, vessel types and sizes involved, and the nature of bubble behaviors and their acoustic emissions resulting in vascular damage - impeding the establishment of standard protocols. Methods: Here, ultrasound transmitters and receivers are integrated into a murine dorsal window chamber tumor model for intravital microscopy studies capable of real-time visual and acoustic monitoring during antivascular ultrasound. Vessel type (normal and tumor-affected), caliber, and viability are assessed under higher pressure conditions (1, 2, and 3 MPa), and cavitation signatures are linked to the biological effects. Results: Vascular events occurred preferentially in tumor-affected vessels with greater incidence in smaller vessels and with more severity as a function of increasing pressure. Vascular blood flow shutdown was found to be due to a combination of focal disruption events and network-related flow changes. Acoustic emissions displayed elevated broadband noise and distinct sub- and ultra-harmonics and their associated third-order peaks with increasing pressure. Conclusions: The observed vascular events taken collectively with identified cavitation signatures provide an improved mechanistic understanding of antivascular ultrasound at the microscale, with implications for establishing a specific treatment protocol and control platform.

Theranostics in the vasculature: bioeffects of ultrasound and microbubbles to induce vascular shutdown.

Ultrasound-triggered microbubbles destruction leading to vascular shutdown have resulted in preclinical studies in tumor growth delay or inhibition, lesion formation, radio-sensitization and modulation of the immune micro-environment. Antivascular ultrasound aims to be developed as a focal, targeted, non-invasive, mechanical and non-thermal treatment, alone or in combination with other treatments, and this review positions these treatments among the wider therapeutic ultrasound domain. Antivascular effects have been reported for a wide range of ultrasound exposure conditions, and evidence points to a prominent role of cavitation as the main mechanism. At relatively low peak negative acoustic pressure, predominantly non-inertial cavitation is most likely induced, while higher peak negative pressures lead to inertial cavitation and bubbles collapse. Resulting bioeffects start with inflammation and/or loose opening of the endothelial lining of the vessel. The latter causes vascular access of tissue factor, leading to platelet aggregation, and consequent clotting. Alternatively, endothelium damage exposes subendothelial collagen layer, leading to rapid adhesion and aggregation of platelets and clotting. In a pilot clinical trial, a prevalence of tumor response was observed in patients receiving ultrasound-triggered microbubble destruction along with transarterial radioembolization. Two ongoing clinical trials are assessing the effectiveness of ultrasound-stimulated microbubble treatment to enhance radiation effects in cancer patients. Clinical translation of antivascular ultrasound/microbubble approach may thus be forthcoming.

AMPK is required for recovery from metabolic stress induced by ultrasound microbubble treatment

Ultrasound-stimulated microbubble (USMB) treatment is a promising strategy for cancer therapy. USMB promotes drug delivery by sonoporation and enhanced endocytosis, and also impairs cell viability. However, USMB elicits heterogeneous effects on cell viability, with apparently minimal effects on a subset of cells. This suggests that mechanisms of adaptation following USMB allow some cells to survive and/or proliferate. Herein, we used several triple negative breast cancer cells to identify the molecular mechanisms of adaptation to USMB-induced stress. We found that USMB alters steady-state levels of amino acids, glycolytic intermediates, and citric acid cycle intermediates, suggesting that USMB imposes metabolic stress on cells. USMB treatment acutely reduces ATP levels and stimulates the phosphorylation and activation of AMP-activated protein kinase (AMPK). AMPK is required to restore ATP levels and support cell proliferation post-USMB treatment. These results suggest that AMPK and metabolic perturbations are likely determinants of the antineoplastic efficacy of USMB treatment.

Ultrasound-Stimulated Microbubbles Enhance Radiation-Induced Cell Killing.

Recent in vivo studies using ultrasound-stimulated microbubbles as a localized radiosensitizer have had impressive results. While in vitro studies have also obtained similar results using human umbilical vein endothelial cells (HUVEC), studies using other cell lines have had varying results. This study was aimed at investigating any increases in radiation-induced cell killing in vitro using two carcinoma lines not previously investigated before (metastatic follicular thyroid carcinoma cells [FTC-238] and non-small cell lung carcinoma cells [NCI-H727]), in addition to HUVEC. Cells were treated using a combination of 1.6% (v/v) microbubbles, ∼90 s of 2-MHz ultrasound (mechanical index=0.8) and 0-6 Gy of kilovolt or MV X-rays. Cell viability assays obtained 72 h post-treatment were normalized to untreated controls, and analysis of variance was used to determine statistical significance. All cells treated with combined ultrasound-stimulated microbubbles and radiation exhibited decreased normalized survival, with statistically significant effects observed for the NCI-H727 cells. No statistically significant differences in effects were observed using kV compared with MV radiation. Further studies using increased microbubble concentrations may be required to achieve statistically significant results for the FTC-238 and HUVEC lines.

Stable Cavitation-Mediated Delivery of miR-126 to Endothelial Cells.

In endothelial cells, microRNA-126 (miR-126) promotes angiogenesis, and modulating the intracellular levels of this gene could suggest a method to treat cardiovascular diseases such as ischemia. Novel ultrasound-stimulated microbubbles offer a means to deliver therapeutic payloads to target cells and sites of disease. The purpose of this study was to investigate the feasibility of gene delivery by stimulating miR-126-decorated microbubbles using gentle acoustic conditions (stable cavitation). A cationic DSTAP microbubble was formulated and characterized to carry 6 µg of a miR-126 payload per 109 microbubbles. Human umbilical vein endothelial cells (HUVECs) were treated at 20-40% duty cycle with miR-126-conjugated microbubbles in a custom ultrasound setup coupled with a passive cavitation detection system. Transfection efficiency was assessed by RT-qPCR, Western blotting, and endothelial tube formation assay, while HUVEC viability was monitored by MTT assay. With increasing duty cycle, the trend observed was an increase in intracellular miR-126 levels, up to a 2.3-fold increase, as well as a decrease in SPRED1 (by 33%) and PIK3R2 (by 46%) expression, two salient miR-126 targets. Under these ultrasound parameters, HUVECs maintained >95% viability after 96 h. The present work describes the delivery of a proangiogenic miR-126 using an ultrasound-responsive cationic microbubble with potential to stimulate therapeutic angiogenesis while minimizing endothelial damage.

Optimal treatment occasion for ultrasound stimulated microbubbles in promoting gemcitabine delivery to VX2 tumors

Ultrasound stimulated microbubbles (USMB) is a widely used technology that can promote chemotherapeutic delivery to tumors yet the best treatment occasion for USMB is unknown or ignored. We aimed to determine the optimal treatment occasion for USMB treatment to enhance tumor chemotherapy to achieve the highest drug concentration in tumors. Experiments were conducted on VX2 tumors implanted in 60 rabbits. Gemcitabine (GEM) was intravenously infused as a chemotherapeutic agent and USMB was administered before, during or after chemotherapy. USMB was conducted with a modified diagnostic ultrasound at 3 MHz employing short bursts (5 cycles and 0.125% duty cycle) at 0.26 MPa in combination with a lipid microbubble. Subsequently, tumor blood perfusion quantitation, drug concentration detection, and fluorescence microscopy were performed. The results showed that the group that received USMB treatment immediately after GEM infusion had the highest drug concentration in tumors, which was 2.83 times that of the control group. Fifteen tumors were then treated repeatedly with the optimal USMB-plus-GEM combination, and along with the GEM and the control groups, were studied for tumor growth, tumor cell proliferation, apoptosis, and related cytokine contents. The combined treatment significantly inhibited tumor growth and promoted apoptosis. The levels of related cytokines, including HIF-1α, decreased after six combination therapies. These results suggest that the optimal treatment occasion for USMB occurs immediately after chemotherapy and tumor hypoxia improves after multiple combination therapies.

Involvement of Ceramide Signalling in Radiation-Induced Tumour Vascular Effects and Vascular-Targeted Therapy.

Sphingolipids are well-recognized critical components in several biological processes. Ceramides constitute a class of sphingolipid metabolites that are involved in important signal transduction pathways that play key roles in determining the fate of cells to survive or die. Ceramide accumulated in cells causes apoptosis; however, ceramide metabolized to sphingosine promotes cell survival and angiogenesis. Studies suggest that vascular-targeted therapies increase endothelial cell ceramide resulting in apoptosis that leads to tumour cure. Specifically, ultrasound-stimulated microbubbles (USMB) used as vascular disrupting agents can perturb endothelial cells, eliciting acid sphingomyelinase (ASMase) activation accompanied by ceramide release. This phenomenon results in endothelial cell death and vascular collapse and is synergistic with other antitumour treatments such as radiation. In contrast, blocking the generation of ceramide using multiple approaches, including the conversion of ceramide to sphingosine-1-phosphate (S1P), abrogates this process. The ceramide-based cell survival “rheostat” between these opposing signalling metabolites is essential in the mechanotransductive vascular targeting following USMB treatment. In this review, we aim to summarize the past and latest findings on ceramide-based vascular-targeted strategies, including novel mechanotransductive methodologies.

Application of Ultrasound Combined with Microbubbles for Cancer Therapy.

At present, cancer is one of the leading causes of death worldwide. Treatment failure remains one of the prime hurdles in cancer treatment due to the metastatic nature of cancer. Techniques have been developed to hinder the growth of tumours or at least to stop the metastasis process. In recent years, ultrasound therapy combined with microbubbles has gained immense success in cancer treatment. Ultrasound-stimulated microbubbles (USMB) combined with other cancer treatments including radiation therapy, chemotherapy or immunotherapy has demonstrated potential improved outcomes in various in vitro and in vivo studies. Studies have shown that low dose radiation administered with USMB can have similar effects as high dose radiation therapy. In addition, the use of USMB in conjunction with radiotherapy or chemotherapy can minimize the toxicity of high dose radiation or chemotherapeutic drugs, respectively. In this review, we discuss the biophysical properties of USMB treatment and its applicability in cancer therapy. In particular, we highlight important preclinical and early clinical findings that demonstrate the antitumour effect combining USMB and other cancer treatment modalities (radiotherapy and chemotherapy). Our review mainly focuses on the tumour vascular effects mediated by USMB and these cancer therapies. We also discuss several current limitations, in addition to ongoing and future efforts for applying USMB in cancer treatment.

Investigating the resonance response of a system of two ultrasound-driven lipid encapsulated microbubbles

Ultrasound-stimulated microbubbles are clinical imaging agents and being developed for therapeutic applications. Here, we aim to understand the behaviour of two individual phospholipid-encapsulated microbubbles in close proximity to each other, typically the case given clinical doses. We developed a finite element model to study the radial resonance response of each microbubble within a two-microbubble system from 1–8 MHz with bubble diameters ranging from 2 to 4 μm, bubble center-to-center distances h = 8–24 μm, and peak-negative pressures of 30–45 kPa. For two identical microbubbles, our results show the frequency of maximum response (fMR) decreases (7%–10%) and the amplitude of maximum response (AMR) increases (9%–11%) as the microbubbles approach one another. For a two-bubble system of different microbubble sizes, the larger microbubble shows no change in fMR and a slight shift of AMR (2–3%). However, the smaller microbubble exhibits an increase in fMR (7–11%) and a significant decrease of AMR (38–52%). Furthermore, when in very close proximity (h = 8 μm), smaller bubbles exhibit a secondary resonance peak corresponding to the fMR of the larger bubble with amplitudes comparable to its primary resonance peak. Our work suggests that microbubble resonance behaviour is greatly affected by the presence of nearby bubbles, which has implications in imaging and therapy.

Ultrasound-assisted membrane permeabilization of endothelial cells under flow conditions

Ultrasound-stimulated microbubbles have been shown a feasible approach for localized therapeutic delivery. As applications of this technique span many anatomical sites, so too do the local fluid dynamics experienced by the circulating microbubbles and the adjacent endothelial cells. Our objective was to assess the relative effectiveness of endothelial cell sonoporation as a function of flow conditions. Human umbilical vein (HUVECs) or human brain endothelial cells (HBECs) were cultured as a monolayer in flow chamber slides connected to a fluidic system and placedupon an acoustically-coupled microscope. A suspension of diluted lipid-encapsulated microbubbles and propidium iodide (PI), used as a sonoporation marker, was constantly perfused over the monolayer at either 5 or 30 ml/min. Cells were treated with 1 MHz ultrasound (PRI= 1 ms, 20 cycles, duration = 2 s), and the video-microscopy data were quantified offline to assess the number of PI-positive cells. Our results demonstrate a marked increase in sonoporation efficiency at 30 ml/min as compared to 5 ml/min in both endothelial cell lines under identical acoustic conditions (9.7-fold increase and 2.3-fold increase for HUVECs and HBECs respectively, p < 0.001). Our results suggest the local fluid flow environment plays a role in US-mediated endothelial perforation efficiency and can modulate treatment strategies.

Ultrasound-stimulated, drug-loaded bubbles for cancer therapy

Non-invasive, focused ultrasound in combination with microbubbles have previously been used for targeted drug delivery. Focused ultrasound-stimulated microbubbles can increase local tumour blood vessel permeability such that co-injected chemotherapeutics can diffuse preferentially into tumour tissue. Focused ultrasound and microbubbles have also been used to initiate other local bioeffects, for example, to damage tumour vessels for mechanically induced vascular disruption therapy, which can profoundly increase the efficacy of a variety of anticancer drugs. In this talk, the development of new acoustically-active bubbles that are capable of carrying and releasing therapeutic concentrations of hydrophobic and hydrophilic chemotherapeutic drugs will be introduced. Agents that are entirely composed of FDA-approved components are preferred as they may permit expedited translation into clinical use. Trade-offs among size, stability, drug-loading, and in vivo performance between bubble variants will be overviewed. Challenges and opportunities identified through recent efforts towards achieving cancer therapy potentiation in small animal cancer models will also be discussed.

Effect of Ultrasound-Stimulated Microbubbles and Hyperthermia on Tumor Vasculature of Breast Cancer Xenograft.

The objective of the present study was to investigate the treatment effects of ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) on breast tumor vasculature. Tumor-bearing mice with breast cancer xenografts (MDA-MB-231), were exposed to different treatment conditions consisting of control (no treatment), USMB alone, HT alone, USMB with HT exposures of 10 and 50minutes. Quantitative 3D Doppler ultrasound and photoacoustic imaging were used to detect tumor blood flow and oxygen saturation, respectively. In addition, histopathological analysis including TUNEL staining for cell death, and CD31 staining for the vessel count, was performed to complement the results of power Doppler and photoacoustic imaging. Results demonstrated a decrease in tumor blood flow as well as oxygenation level following 50minutes HT treatment either alone or combined with USMB. In contrast, 10minutes HT alone or combined with USMB had minimal effects on blood flow and tumor oxygenation level. Treatment with HT for 50 minutes caused drops in tumor oxygenation, which were not evident with USMB treatment alone. Additionally, results revealed an increase in cell death after 10minutes HT with or without USMB and a decrease in vessel count compared to control. Unlike previous studies which demonstrated synergistic treatment effects combining USMB with other modalities such as radiation or chemotherapy, USMB and HT effects were not synergistic in the present study. The results here demonstrated HT and USMB both alone or together resulted in a significant reduction in tumor blood flow, tumor oxygenation, and vessel count with observed increases in cell death response.

Focused Ultrasound Stimulation of Microbubbles in Combination With Radiotherapy for Acute Damage of Breast Cancer Xenograft Model.

Objective: Several studies have focused on the use of ultrasound-stimulated microbubbles (USMB) to induce vascular damage in order to enhance tumor response to radiation. Methods: In this study, power Doppler imaging was used along with immunohistochemistry to investigate the effects of combining radiation therapy (XRT) and USMB using an ultrasound-guided focused ultrasound (FUS) therapy system in a breast cancer xenograft model. Specifically, MDA-MB-231 breast cancer xenograft tumors were induced in severe combined immuno-deficient female mice. The mice were treated with FUS alone, ultrasound and microbubbles (FUS + MB) alone, 8 Gy XRT alone, or a combined treatment consisting of ultrasound, microbubbles, and XRT (FUS + MB + XRT). Power Doppler imaging was conducted before and 24 h after treatment, at which time mice were sacrificed and tumors assessed histologically. The immunohistochemical analysis included terminal deoxynucleotidyl transferase dUTP nick end labeling, hematoxylin and eosin, cluster of differentiation-31 (CD31), Ki-67, carbonic anhydrase (CA-9), and ceramide labeling. Results: Tumors receiving treatment of FUS + MB combined with XRT demonstrated significant increase in cell death (p = 0.0006) compared to control group. Furthermore, CD31 and Power Doppler analysis revealed reduced tumor vascularization with combined treatment indicating (P < .0001) and (P = .0001), respectively compared to the control group. Additionally, lesser number of proliferating cells with enhanced tumor hypoxia, and ceramide content were also reported in group receiving a treatment of FUS + MB + XRT. Conclusion: The study results demonstrate that the combination of USMB with XRT enhances treatment outcomes.

Combination therapy with low-frequency ultrasound irradiation and radiofrequency ablation as a synergistic treatment for pancreatic cancer

ABSTRACT We aim to evaluate the efficacies of combination therapy with low-frequency ultrasound-stimulated microbubbles (USMB) and radiofrequency ablation (RFA) on suppressing the proliferation of pancreatic cancer cell and treating Panc02 subcutaneous xenograft mice. The proliferation of HPDE6-C7 and Panc02 cells after the treatment of USMB and RFA alone or combination were evaluated by CCK-8 assay. Scratch test was performed to assess the cell migration capability. Panc02-bearing mice were received 14-day treatment of USMB and RFA alone or combination. Tumor size and survival rate were recorded once two days. The serum levels of immune-related factors and changes of apoptosis- and autophagy-related factors were detected by ELISA and western blotting methods. As a result, CKK-8 assays revealed significant inhibition on Panc02 cell proliferation in combination therapy with USMB and RFA relative to other groups (all p < 0.05). Strong synergistic effect of USMB combined with RFA was confirmed via the calculated combination index (CI) <0.4. In addition, combination therapy of USMB and RFA significantly inhibited the migration of Panc02 cells. Moreover, combined treatment remarkably inhibited the size and width of xenograft and improved the survival in Panc02-bearing mice. Furthermore, 14-day combination therapy of USMB and RFA in Panc02-bearing mice significantly facilitated the apoptosis and autophagy of tumor cells. In summary, combination therapy of USMB and RFA showed synergistic anti-tumor efficacies on Panc02 cells attributing to the promotion on apoptosis and autophagy in Panc02 subcutaneous xenograft mice.