Ultrasound localization microscopy to image and assess microvasculature in a rat kidney.

The recent development of ultrasound localization microscopy, where individual microbubbles (contrast agents) are detected and tracked within the vasculature, provides new opportunities for imaging the vasculature of entire organs with a spatial resolution below the diffraction limit. In stationary tissue, recent studies have demonstrated a theoretical resolution on the order of microns. In this work, single microbubbles were localized in vivo in a rat kidney using a dedicated high frame rate imaging sequence. Organ motion was tracked by assuming rigid motion (translation and rotation) and appropriate correction was applied. In contrast to previous work, coherence-based non-linear phase inversion processing was used to reject tissue echoes while maintaining echoes from very slowly moving microbubbles. Blood velocity in the small vessels was estimated by tracking microbubbles, demonstrating the potential of this technique to improve vascular characterization. Previous optical studies of microbubbles in vessels of approximately 20 microns have shown that expansion is constrained, suggesting that microbubble echoes would be difficult to detect in such regions. We therefore utilized the echoes from individual MBs as microscopic sensors of slow flow associated with such vessels and demonstrate that highly correlated, wideband echoes are detected from individual microbubbles in vessels with flow rates below 2 mm/s

An open environment CT-US fusion for tissue segmentation during interventional guidance.

Therapeutic ultrasound (US) can be noninvasively focused to activate drugs, ablate tumors and deliver drugs beyond the blood brain barrier. However, well-controlled guidance of US therapy requires fusion with a navigational modality, such as magnetic resonance imaging (MRI) or X-ray computed tomography (CT). Here, we developed and validated tissue characterization using a fusion between US and CT. The performance of the CT/US fusion was quantified by the calibration error, target registration error and fiducial registration error. Met-1 tumors in the fat pads of 12 female FVB mice provided a model of developing breast cancer with which to evaluate CT-based tissue segmentation. Hounsfield units (HU) within the tumor and surrounding fat pad were quantified, validated with histology and segmented for parametric analysis (fat: -300 to 0 HU, protein-rich: 1 to 300 HU, and bone: HU>300). Our open source CT/US fusion system differentiated soft tissue, bone and fat with a spatial accuracy of ∼1 mm. Region of interest (ROI) analysis of the tumor and surrounding fat pad using a 1 mm(2) ROI resulted in mean HU of 68±44 within the tumor and -97±52 within the fat pad adjacent to the tumor (p<0.005). The tumor area measured by CT and histology was correlated (r(2) = 0.92), while the area designated as fat decreased with increasing tumor size (r(2) = 0.51). Analysis of CT and histology images of the tumor and surrounding fat pad revealed an average percentage of fat of 65.3% vs. 75.2%, 36.5% vs. 48.4%, and 31.6% vs. 38.5% for tumors <75 mm(3), 75-150 mm(3) and >150 mm(3), respectively. Further, CT mapped bone-soft tissue interfaces near the acoustic beam during real-time imaging. Combined CT/US is a feasible method for guiding interventions by tracking the acoustic focus within a pre-acquired CT image volume and characterizing tissues proximal to and surrounding the acoustic focus

Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was β-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that β-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of β-sitosterol was linked to mitochondrial interference. Mechanistically, β-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either β-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, β-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.publishedVersio

Magnetic Resonance Thermometry at 7T for Real-Time Monitoring and Correction of Ultrasound Induced Mild Hyperthermia

While Magnetic Resonance Thermometry (MRT) has been extensively utilized for non-invasive temperature measurement, there is limited data on the use of high field (≥7T) scanners for this purpose. MR-guided Focused Ultrasound (MRgFUS) is a promising non-invasive method for localized hyperthermia and drug delivery. MRT based on the temperature sensitivity of the proton resonance frequency (PRF) has been implemented in both a tissue phantom and in vivo in a mouse Met-1 tumor model, using partial parallel imaging (PPI) to speed acquisition. An MRgFUS system capable of delivering a controlled 3D acoustic dose during real time MRT with proportional, integral, and derivative (PID) feedback control was developed and validated. Real-time MRT was validated in a tofu phantom with fluoroptic temperature measurements, and acoustic heating simulations were in good agreement with MR temperature maps. In an in vivo Met-1 mouse tumor, the real-time PID feedback control is capable of maintaining the desired temperature with high accuracy. We found that real time MR control of hyperthermia is feasible at high field, and k-space based PPI techniques may be implemented for increasing temporal resolution while maintaining temperature accuracy on the order of 1°C

Concurrent Visualization of Acoustic Radiation Force Displacement and Shear Wave Propagation with 7T MRI.

Manual palpation is a common and very informative diagnostic tool based on estimation of changes in the stiffness of tissues that result from pathology. In the case of a small lesion or a lesion that is located deep within the body, it is difficult for changes in mechanical properties of tissue to be detected or evaluated via palpation. Furthermore, palpation is non-quantitative and cannot be used to localize the lesion. Magnetic Resonance-guided Focused Ultrasound (MRgFUS) can also be used to evaluate the properties of biological tissues non-invasively. In this study, an MRgFUS system combines high field (7T) MR and 3 MHz focused ultrasound to provide high resolution MR imaging and a small ultrasonic interrogation region (~0.5 x 0.5 x 2 mm), as compared with current clinical systems. MR-Acoustic Radiation Force Imaging (MR-ARFI) provides a reliable and efficient method for beam localization by detecting micron-scale displacements induced by ultrasound mechanical forces. The first aim of this study is to develop a sequence that can concurrently quantify acoustic radiation force displacements and image the resulting transient shear wave. Our motivation in combining these two measurements is to develop a technique that can rapidly provide both ARFI and shear wave velocity estimation data, making it suitable for use in interventional radiology. Secondly, we validate this sequence in vivo by estimating the displacement before and after high intensity focused ultrasound (HIFU) ablation, and we validate the shear wave velocity in vitro using tissue-mimicking gelatin and tofu phantoms. Such rapid acquisitions are especially useful in interventional radiology applications where minimizing scan time is highly desirable

In vitro characterization and in vivo ultrasound molecular imaging of nucleolin-targeted microbubbles

Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p < 0.001. We then set out to create a method to extract both the tumor wash-in and wash-out kinetics and tumor accumulation following a single injection of targeted MBs. In order to accomplish this, a series of ultrasound frames (a clip) was recorded at the time of injection and subsequent time points. Each pixel within this clip was analyzed for the minimum intensity projection (MinIP) and average intensity projection (AvgIP). We found that the MinIP robustly demonstrates enhanced accumulation of F3-conjugated MBs over the range of tumor diameters evaluated here (2-8 mm), and the difference between the AvgIP and the MinIP quantifies inflow and kinetics. The inflow and clearance were similar for unbound F3-conjugated MBs, control (non-targeted) and scrambled control agents. Targeted agent accumulation was confirmed by a high amplitude pulse and by a two-dimensional Fourier Transform technique. In summary, F3-conjugated MBs provide a new imaging agent for ultrasound molecular imaging of cancer vasculature, and we have validated metrics to assess performance using low mechanical index strategies that have potential for use in human molecular imaging studies

Ultrasound molecular imaging of tumor angiogenesis with a neuropilin-1-targeted microbubble.

Ultrasound molecular imaging has great potential to impact early disease diagnosis, evaluation of disease progression and the development of target-specific therapy. In this paper, two neuropilin-1 (NRP) targeted peptides, CRPPR and ATWLPPR, were conjugated onto the surface of lipid microbubbles (MBs) to evaluate molecular imaging of tumor angiogenesis in a breast cancer model. Development of a molecular imaging agent using CRPPR has particular importance due to the previously demonstrated internalizing capability of this and similar ligands. In vitro, CRPPR MBs bound to an NRP-expressing cell line 2.6 and 15.6 times more than ATWLPPR MBs and non-targeted (NT) MBs, respectively, and the binding was inhibited by pretreating the cells with an NRP antibody. In vivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the ∼6 min circulation of the CRPPR-targeted contrast agents providing high contrast images of angiogenic tumors. Approximately 67% of the initial signal from CRPPR MBs remained bound after the majority of circulating MBs had cleared (8 min), 8 and 4.5 times greater than ATWLPPR and NT MBs, respectively. Finally, at 7-21 days after the first injection, we found that CRPPR MBs cleared faster from circulation and tumor accumulation was reduced likely due to a complement-mediated recognition of the targeted microbubble and a decrease in angiogenic vasculature, respectively. In summary, we find that CRPPR MBs specifically bind to NRP-expressing cells and provide an effective new agent for molecular imaging of angiogenesis