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.

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.

Ultrasound-stimulated microbubble radiation enhancement of tumors: Single-dose and fractionated treatment evaluation.

The use of ultrasound-stimulated microbubble therapy has successfully been used to target tumor vasculature and enhance the effects of radiation therapy in tumor xenografts in mice. Here, we further investigate this treatment using larger, more clinically relevant tumor models. New Zealand white rabbits bearing prostate tumor (PC3) xenografts received a single treatment of either ultrasound-stimulated microbubbles (USMB), ionizing radiation (XRT; 8Gy), or a combination of both treatments (USMB+XRT). Treatment outcome was evaluated 24 hours after treatment using histopathology, immunolabeling, 3D Doppler ultrasound and photoacoustic imaging. A second cohort of rabbits received multiple treatments over a period of three weeks, where USMB treatments were delivered twice weekly with daily XRT treatments to deliver a fractionated 2Gy dose five days per week. A significant decrease in vascular function, observed through immunolabeling of vascular endothelial cells, was observed in tumors receiving the combined treatment (USMB+XRT) compared to control and single treatment groups. This was associated with an increase in cell death as observed through in situ end labeling (ISEL), a decrease in vascular index measured by Power Doppler imaging, and a decrease in oxygen saturation. In rabbits undergoing the long-term fractionated combined treatment, a significant growth delay was observed after 1 week and a significant reduction in tumor size was observed after 3 weeks with combined therapy. Results demonstrated an enhancement of radiation effect and superior anti-tumor effect of the combination of USMB+XRT compared to the single treatments alone. Tumor growth was maximally inhibited with fractionated radiotherapy combined with the ultrasound-stimulated microbubble-based therapy.

Erratum: Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo.

[This corrects the article DOI: 10.18632/oncoscience.299.].

Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo.

Acoustically stimulated microbubbles have been demonstrated to perturb endothelial cells of the vasculature resulting in biological effects. In the present study, vascular and tumor response to ultrasound-stimulated microbubble and radiation treatment was investigated in vivo to identify effects on the blood vessel endothelium. Mice bearing breast cancer tumors (MDA-MB-231) were exposed to ultrasound after intravenous injection of microbubbles at different concentrations, and radiation at different doses (0, 2, and 8 Gy). Mice were sacrificed 12 and 24 hours after treatment for histopathological analysis. Tumor growth delay was assessed for up to 28 days after treatment. The results demonstrated additive antitumor and antivascular effects when ultrasound stimulated microbubbles were combined with radiation. Results indicated tumor cell apoptosis, vascular leakage, a decrease in tumor vasculature, a delay in tumor growth and an overall tumor disruption. When coupled with radiation, ultrasound-stimulated microbubbles elicited synergistic anti-tumor and antivascular effects by acting as a radioenhancing agent in breast tumor blood vessels. The present study demonstrates ultrasound driven microbubbles as a novel form of targeted antiangiogenic therapy in a breast cancer xenograft model that can potentiate additive effects to radiation in vivo.

Enhancing laser therapy using PEGylated gold nanoparticles combined with ultrasound and microbubbles.

BACKGROUND: Gold nanorod (AuNR) laser therapy (LT) is a non-invasive method of increasing the temperature of a target tissue using near infrared light. In this study, the effects of ultrasound and microbubbles (USMB) with AuNR and LT were investigated on cell viability. METHODS: MDA-MB-231 cells in suspension were treated with three different treatment combinations of AuNR, LT and USMB (Pneg=0.6 or 1.0 MPa): (1) AuNR with USMB followed by LT, (2) AuNR and LT followed by USMB, and (3) USMB followed by AuNR and LT. Cells were also exposed to USMB and LT without AuNR. The USMB conditions were: 500 kHz frequency, 16 cycles, 1kHz pulse repetition frequency for 1 min in the presence of Definity microbubbles (1.7% v/v). AuNR and LT conditions were: mPEG coated AuNR at 3×10(11) np/mL and 1.9 W/cm(2) for 3 min. Following the treatment, cell viability was assessed using propidium iodide (PI) fluorescent marker and flow cytometry (VPI), and colony assay (VCA). Cell viabilities were compared using a non-parametric Mann-Whitney U-test and synergism was assessed using the Bliss Independence Model. RESULTS AND DISCUSSION: USMB improved cell death when combined with AuNR and LT. VPI of 17±2% (at 0.6 MPa) and 11±4% (at 1.0 MPa) were observed with combined treatment of AuNR and USMB followed by LT compared to VPI of 60±2% (at 0.6 MPa) and 42±3% (at 1.0 MPa) with USMB alone and VPI of 22±3% for AuNR and LT. The combined effect of AuNR and LT with USMB was additive regardless of treatment order. VCA results agreed with the additive effect caused by combining AuNR and LT with USMB for all treatment orders. In the absence of AuNR, samples exposed to LT prior to USMB at 0.6 MPa increased VPI by 13% (p<0.01) showing a protective effect. CONCLUSION: Combining AuNR and LT with USMB resulted in an additive effect on cell viability compared to AuNR and LT, or USMB. In addition, cells exposed to low intensity NIR light appear to be protected against USMB exposure.

Enhancing laser thermal-therapy using ultrasound-microbubbles and gold nanorods of in vitro cells.

Gold nanorods (GNRs) are being exploited for their absorption properties to improve thermal therapy. However, a key challenge is delivering sufficient concentration of GNRs to induce a therapeutic effect. In this study, ultrasound and microbubbles (USMBs) were used to enhance intracellular uptake of GNRs. AML-5 cells in suspension (0.6 mL) were exposed to ultrasound (1.3 and 1.7 MPa peak negative pressure) and definity microbubbles (1.7% v/v) for 1 min at varying GNR concentrations (0-2.5×10(11) per mL). Following ultrasound-microbubble treatment, cells were centrifuged twice and treated with an 810 nm laser at an average fluence rate of 3.6 W/cm(2) for 5 min. In addition, cells were incubated with GNRs for 12 h prior to laser treatment. Following the treatment, cell viability (V(PI)) was assessed using propidium iodide (PI) and flow cytometry. Cell viability decreased by ∼4-folds with the combined treatment of USMB+GNR+Laser (V(PI)=17%) compared to cells incubated with GNR+Laser (V(PI)=68%). This effect depended on ultrasound pressure and GNR concentration. Higher cell death was achieved at higher GNR concentration and 1.3 MPa peak negative pressure. Cell viability decreased from 92% to 29% with increasing GNR concentration from 1×10(11) to 1.5×10(11) GNR/mL. In addition, higher temperatures were observed using a thermal camera with the combined treatment (USMB+GNR+Laser) of 59±1°C compared to 54±0.9°C for cells incubated with GNRs. The combined treatment of ultrasound-microbubble and gold nanorod laser induced thermal-therapy improved treatment response of in vitro cells.