Quantifying the relationship between biofilm reduction and thermal tissue damage on metal implants exposed to alternating magnetic fields.

AIM: Metal implant infections are a devastating problem due to the formation of biofilm which impairs the effectiveness of antibiotics and leads to surgical replacement as definitive treatment. Biofilm on metal implants can be reduced using heat generated by alternating magnetic fields (AMF). In this study, the relationship between implant surface biofilm reduction and surrounding tissue thermal damage during AMF exposure is investigated through numerical simulations. METHODS: Mathematical models of biofilm reduction with heat were created based on in vitro experiments. Simulations were performed to predict the spatial and temporal heating on the implant surface and surrounding tissue when exposed to AMF. RESULTS: The modeling results show that intermittent and slow heating can achieve biofilm reduction with a narrow zone of tissue damage around an implant of less than 3 mm. The results also emphasize that uniformity of implant heating is an extremely important factor impacting the effectiveness of biofilm reduction. For a knee implant, using a target temperature of 75 °C, an intermittent treatment strategy of 15 exposures (10 s to target temperature followed by cooldown) achieved a bacterial CFU reduction of 6-log10 across 25% of the implant surface with less than 3 mm of tissue damage. Alternatively, a single 60 s heating exposure to same temperature achieved a bacterial reduction of 6-log10 across 85% of the implant surface, but with 4 mm of tissue damage. CONCLUSION: Overall, this study demonstrates that with uniform heating to temperatures above 70 °C, an implant surface can be largely reduced of biofilm, with only a few mm of surrounding tissue damage.

Recent developments in dynamic contrast-enhanced ultrasound imaging of tumor angiogenesis.

Angiogenesis is a critical process for tumor growth and metastatic dissemination. There is tremendous interest in the development of noninvasive methods for imaging tumor angiogenesis, and ultrasound (US) is an emerging platform technology to address this challenge. The introduction of intravascular microbubble contrast agents not only allows real-time visualization of tumor perfusion during an US examination, but they can be functionalized with specific ligands to permit molecular US imaging of angiogenic biomarkers that are overexpressed on the tumor endothelium. In this article, we will review current concepts and developing trends for US imaging of tumor angiogenesis, including relevant preclinical and clinicsal findings.

Optical fluorescent imaging to monitor temporal effects of microbubble-mediated ultrasound therapy.

Microbubble-mediated ultrasound therapy can noninvasively enhance drug delivery to localized regions in the body. This technique can be beneficial in cancer therapy, but currently there are limitations to tracking the therapeutic effects. The purpose of this experiment was to investigate the potential of fluorescent imaging for monitoring the temporal effects of microbubble-mediated ultrasound therapy. Mice were implanted with 2LMP breast cancer cells. The animals underwent microbubble-mediated ultrasound therapy in the presence of Cy5.5 fluorescent-labeled IgG antibody (large molecule) or Cy5.5 dye (small molecule) and microbubble contrast agents. Control animals were administered fluorescent molecules only. Animals were transiently imaged in vivo at 1, 10, 30, and 60 min post therapy using a small animal optical imaging system. Tumors were excised and analyzed ex vivo. Tumors were homogenized and emulsion imaged for Cy5.5 fluorescence. Monitoring in vivo results showed significant influx of dye into the tumor (p < 0.05) using the small molecule, but not in the large molecule group (p > 0.05). However, after tumor emulsion, significantly higher dye concentration was detected in therapy group tumors for both small and large molecule groups in comparison to their control counterparts (p <0.01). This paper explores a noninvasive optical imaging method for monitoring the effects of microbubble-mediated ultrasound therapy in a cancer model. It provides temporal information following the process of increasing extravasation of molecules into target tumors.

An animal model allowing controlled receptor expression for molecular ultrasound imaging.

Reported in this study is an animal model system for evaluating targeted ultrasound (US) contrast agents binding using adenoviral (Ad) vectors to modulate cellular receptor expression. An Ad vector encoding an extracellular hemagglutinin (HA) epitope tag and a green fluorescent protein (GFP) reporter was used to regulate receptor expression. A low and high receptor density (in breast cancer tumor bearing mice) was achieved by varying the Ad dose with a low plaque forming unit (PFU) on day 1 and high PFU on day 2 of experimentation. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or IgG isotype control antibodies to the MB surface with biotin-streptavidin linkage. Targeted and control MBs were administered on both days of experimentation and contrast-enhanced US (CEUS) was performed on each mouse using MB flash destruction technique. Signal intensities from MBs retained within tumor vasculature were analyzed through a custom Matlab program. Results showed intratumoral enhancement attributable to targeted MB accumulation was significantly increased from the low Ad vector dosing and the high Ad vector dosing (p = 0.001). Control MBs showed no significant differences between day 1 and day 2 imaging (p = 0.96). Additionally, targeted MBs showed a 10.5-fold increase in intratumoral image intensity on day 1 and an 18.8-fold increase in image intensity on day 2 compared with their control MB counterparts.

Volumetric contrast-enhanced ultrasound imaging to assess early response to apoptosis-inducing anti-death receptor 5 antibody therapy in a breast cancer animal model.

OBJECTIVES: The objective of this study was to determine whether volumetric contrast-enhanced ultrasound (US) imaging could detect early tumor response to anti-death receptor 5 antibody (TRA-8) therapy alone or in combination with chemotherapy in a preclinical triple-negative breast cancer animal model. METHODS: Animal experiments had Institutional Animal Care and Use Committee approval. Thirty breast tumor-bearing mice were administered Abraxane (paclitaxel; Celgene Corporation, Summit, NJ), TRA-8, TRA-8 + Abraxane, or saline as a controlon days 0, 3, 7, 10, 14, and 17. Volumetric contrast-enhanced US imaging was performedon days 0, 1, 3, and 7 before dosing. Changes in parametric maps of tumor perfusion were compared with the tumor volume and immunohistologic findings. RESULTS: Therapeutic efficacy was detected within 7 days after drug administration using parametric volumetric contrast-enhanced US imaging. Decreased tumor perfusion was observed in both the TRA-8-alone- and TRA-8 + Abraxane-dosed animals compared to control tumors (P = .17; P = .001, respectively). The reduction in perfusion observed in the TRA-8 + Abraxane group was matched with a corresponding regression in tumor size over the same period. Survival curves illustrate that the combination of TRA-8 + Abraxane improves drug efficacy compared to the same drugs administered alone. Immunohistologic analysis revealed increased levels of apoptotic activity in the TRA-8-dosed tumors, confirming enhanced antitumor effects. CONCLUSIONS: Preliminary results are encouraging, and volumetric contrast-enhanced US-based tumor perfusion imaging may prove clinically feasible for detecting and monitoring the early antitumor effects in response to combination TRA-8 + Abraxane therapy.

Molecular ultrasound imaging using a targeted contrast agent for assessing early tumor response to antiangiogenic therapy.

OBJECTIVES: Contrast-enhanced ultrasound (US) and targeted microbubbles have been shown to be advantageous for angiogenesis evaluation and disease staging in cancer. This study explored molecular US imaging of a multitargeted microbubble for assessing the early tumor response to antiangiogenic therapy. METHODS: Target receptor expression of 2LMP breast cancer cells was quantified by flow cytometric analysis and characterization established with antibodies against mouse α(V)ß3- integrin, P-selectin, and vascular endothelial growth factor receptor 2. Tumor-bearing mice (n = 15 per group) underwent contrast-enhanced US imaging of multitargeted microbubbles. Microbubble accumulation was calculated by destruction-replenishment techniques and time-intensity curve analysis. On day 0, mice underwent baseline imaging. Next, therapy group mice were injected with a 0.2-mg dose of bevacizumab, and controls received matched saline injections. Imaging was repeated on days 1 and 3. After imaging was completed on day 3, the mice were euthanized and tumors excised. Histologic analysis of microvessel density and intratumoral necrosis was completed on tumor sections. RESULTS: On day 3 after bevacizumab dosing, a 71.8% change in tumor vasculature was shown between the therapy and control groups (P = .01). The therapy group had a 15.4% decrease in tumor vascularity, whereas the control group had a 56.4% increase. CONCLUSIONS: Molecular US imaging of angiogenic markers can detect the early tumor response to drug therapy.

Molecular targeting of ultrasonographic contrast agent for detection of head and neck squamous cell carcinoma.

OBJECTIVE: To investigate the feasibility of ultrasonographic (US) imaging of head and neck cancer with targeted contrast agents both in vitro and in vivo. We hypothesize that conjugation of microbubble contrast agent to tumor-specific antibodies may improve US detection of head and neck squamous cell carcinoma (HNSCC). DESIGN: Preclinical blinded assessment of anti-EGFR and anti-CD147 microbubble contrast agents for US imaging of HNSCC. SETTING: Animal study. SUBJECTS: Immunodeficient mice. INTERVENTION: Injection of targeted microbubbles. MAIN OUTCOME MEASURE: Microbubble uptake in tumors as detected by US. RESULTS: In vitro assessment of anti-epidermal growth factor receptor (EGFR) and anti-CD147-targeted microbubbles in 6 head and neck cancer cell lines yielded a 6-fold improvement over normal dermal fibroblasts (P < .001). Binding of targeted agents had a positive correlation to both epidermal growth factor receptor (EGFR) (R(2) = 0.81) and CD147 (R(2) = 0.72) expression among all cell lines. In vivo imaging of flank tumors in nude mice (N = 8) yielded enhanced resolution of anti-EGFR-and anti-CD147-targeted microbubble agents over IgG control (P < .001), while dual-targeted contrast agents offered enhanced imaging over single-targeted contrast agents (P = .02 and P = .05, respectively). In a blinded in vivo assessment, targeted contrast agents increased intratumoral enhancement of flank tumors over controls. Targeted US contrast agents to both EGFR and CD147 were 100% sensitive and 87% specific in the detection of flank tumors. CONCLUSION: This preclinical study demonstrates feasibility of using molecular US to target HNSCC for contrast-enhanced imaging of HNSCC tumor in vivo.

Quantitative mapping of tumor vascularity using volumetric contrast-enhanced ultrasound.

OBJECTIVE: The goal of this research project was to develop a volumetric strategy for real-time monitoring and characterization of tumor blood flow using microbubble contrast agents and ultrasound (US) imaging. MATERIALS AND METHODS: Volumetric contrast-enhanced US (VCEUS) imaging was implemented on a SONIX RP US system (Ultrasonix Medical Corp, Richmond, BC) equipped with a broadband 4DL14-5/38 probe. Using a microbubble-sensitive harmonic imaging mode (transducer transmits at 5 MHz and receives at 10 MHz), acquisition of postscan-converted VCEUS data was achieved at a volume rate of 1 Hz. After microbubble infusion, custom data processing software was used to derive microbubble time-intensity curve-specific parameters, namely, blood volume (IPK), transit time (T1/2PK), flow rate (SPK), and tumor perfusion (AUC). RESULTS: Using a preclinical breast cancer animal model, it is shown that millimeter-sized deviations in transducer positioning can have profound implications on US-based blood flow estimators, with errors ranging from 6.4% to 40.3% and dependent on both degree of misalignment (offset) and particular blood flow estimator. These errors indicate that VCEUS imaging should be considered in tumor analyses, because they incorporate the entire mass and not just a representative planar cross-section. After administration of an antiangiogenic therapeutic drug (bevacizumab), tumor growth was significantly retarded compared with control tumors (P > 0.03) and reflects observed changes in VCEUS-based blood flow measurements. Analysis of immunohistologic data revealed no differences in intratumoral necrosis levels (P = 0.70), but a significant difference was found when comparing microvessel density counts in control with therapy group tumors (P = 0.05). CONCLUSIONS: VCEUS imaging was shown to be a promising modality for monitoring changes in tumor blood flow. Preliminary experimental results are encouraging, and this imaging modality may prove clinically feasible for detecting and monitoring the early antitumor effects in response to cancer drug therapy.

Model system using controlled receptor expression for evaluating targeted ultrasound contrast agents.

This report details a model system for evaluating targeted ultrasound (US) contrast agents using adenoviral (Ad) vectors to regulate target receptor expression. Receptor density in vitro was modulated in breast cancer cells by varying the multiplicity of infection (MOI) from 0 to 100. Target receptors were induced using a green fluorescent protein (GFP) reporter Ad vector for gene transfer and expression of the hemagglutinin (HA) tag. These reporter genes were under the control of the ubiquitous cytomegalovirus (CMV) promoter. Subsequently, receptor expression and anti-HA antibody (Ab) binding was examined with flow cytometry. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or isotype control Ab to the surface of biotin coated MBs via a streptavidin bridge. Targeted MBs were incubated with Ad infected 2LMP cells to evaluate in vitro MB binding. Experimental results found GFP expression to be directly correlated with Ad MOI (r² = 0.96). Increasing the Ad MOI produced a corresponding increase in binding and accumulation of anti-HA Ab on the cell surface (p < 0.01). However, no difference was found between Cy5-labeled anti-HA Ab exposed cell groups at an MOI of 0 (p > 0.29). Additionally, no difference was found between the isotype control Ab group (p > 0.44) indicating minimal nonspecific binding. No difference was found between cell groups incubated with isotype-targeted MBs (p > 0.42) regardless of receptor density. However, cells exposed to HA-targeted MBs showed increased levels of cell binding proportional to induced receptor expression levels (p < 0.02).

A triple-targeted ultrasound contrast agent provides improved localization to tumor vasculature.

OBJECTIVES: Actively targeting ultrasound contrast agents to tumor vasculature improves contrast-enhanced sonography of tumor angiogenesis. This report summarizes an evaluation of multitargeted microbubbles, comparing single-, dual-, and triple-targeted motifs. METHODS: Microbubbles were avidin-biotin linked to antibodies against mouse α(V)ß(3)-integrin, P-selectin, and vascular endothelial growth factor receptor 2. These receptors are constitutively overexpressed in tumor vasculature. Binding comparisons between targeted microbubble groups were evaluated on mouse SVR angiosarcoma endothelial cells. Levels of the targeted receptors were characterized with flow cytometry. Targeted microbubble groups were administered to human MDA-MB-231 breast cancer tumor-bearing mice (n = 3) followed by contrast-enhanced sonography in a microbubble-sensitive harmonic imaging mode implemented on an ultrasound scanner equipped with a linear array transducer (5 MHz transmit and 10 MHz receive) to evaluate differences in microbubble accumulation in the tumor vasculature. RESULTS: In vitro analysis showed a 50% increase (P < .001) in triple-targeted microbubble binding over dual-targeted microbubble groups in mouse SVR cells. Mice bearing MDA-MB-231 tumors showed a 40% increase in tumor image intensity after dosing with triple-targeted microbubbles compared with single- and dual-targeted microbubbles (P = .006). Histologic staining confirmed the presence of α(V)ß(3)-integrin, P-selectin, and vascular endothelial growth factor receptor 2 in the tumors. CONCLUSIONS: Microbubble accumulation in the tumor vasculature was improved using a triple-targeted microbubble approach.