Avinoam Bar-Zion

Co-option of Liver Vessels and Not Sprouting Angiogenesis Drives Acquired Sorafenib Resistance in Hepatocellular Carcinoma

Background: The anti-angiogenic Sorafenib is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance limits its efficacy. An emerging theory to explain intrinsic resistance to other anti-angiogenic drugs is ‘vessel co-option,’ ie, the ability of tumors to hijack the existing vasculature in organs such as the lungs or liver, thus limiting the need for sprouting angiogenesis. Vessel co-option has not been evaluated as a potential mechanism for acquired resistance to anti-angiogenic agents. Methods: To study sorafenib resistance mechanisms, we used an orthotopic human HCC model (n = 4-11 per group), where tumor cells are tagged with a secreted protein biomarker to monitor disease burden and response to therapy. Histopathology, vessel perfusion assessed by contrast-enhanced ultrasound, and miRNA sequencing and quantitative real-time polymerase chain reaction were used to monitor changes in tumor biology. Results: While sorafenib initially inhibited angiogenesis and stabilized tumor growth, no angiogenic ‘rebound’ effect was observed during development of resistance unless therapy was stopped. Instead, resistant tumors became more locally infiltrative, which facilitated extensive incorporation of liver parenchyma and the co-option of liver-associated vessels. Up to 75% (±10.9%) of total vessels were provided by vessel co-option in resistant tumors relative to 23.3% (±10.3%) in untreated controls. miRNA sequencing implicated pro-invasive signaling and epithelial-to-mesenchymal-like transition during resistance development while functional imaging further supported a shift from angiogenesis to vessel co-option. Conclusions: This is the first documentation of vessel co-option as a mechanism of acquired resistance to anti-angiogenic therapy and could have important implications including the potential therapeutic benefits of targeting vessel co-option in conjunction with vascular endothelial growth factor receptor signaling.

Lysyl oxidase-like-2 promotes tumour angiogenesis and is a potential therapeutic target in angiogenic tumours

Lysyl oxidase-like 2 (LOXL2), a secreted enzyme that catalyzes the cross-linking of collagen, plays an essential role in developmental angiogenesis. We found that administration of the LOXL2-neutralizing antibody AB0023 inhibited bFGF-induced angiogenesis in Matrigel plug assays and suppressed recruitment of angiogenesis promoting bone marrow cells. Small hairpin RNA-mediated inhibition of LOXL2 expression or inhibition of LOXL2 using AB0023 reduced the migration and network-forming ability of endothelial cells, suggesting that the inhibition of angiogenesis results from a direct effect on endothelial cells. To examine the effects of AB0023 on tumour angiogenesis, AB0023 was administered to mice bearing tumours derived from SKOV-3 ovarian carcinoma or Lewis lung carcinoma (LLC) cells. AB0023 treatment significantly reduced the microvascular density in these tumours but did not inhibit tumour growth. However, treatment of mice bearing SKOV-3-derived tumours with AB0023 also promoted increased coverage of tumour vessels with pericytes and reduced tumour hypoxia, providing evidence that anti-LOXL2 therapy results in the normalization of tumour blood vessels. In agreement with these data, treatment of mice bearing LLC-derived tumours with AB0023 improved the perfusion of the tumour-associated vessels as determined by ultrasonography. Improved perfusion and normalization of tumour vessels after treatment with anti-angiogenic agents were previously found to improve the delivery of chemotherapeutic agents into tumours and to result in an enhancement of chemotherapeutic efficiency. Indeed, treatment with AB0023 significantly enhanced the anti-tumourigenic effects of taxol. Our results suggest that inhibition of LOXL2 may prove beneficial for the treatment of angiogenic tumours.

Fast Vascular Ultrasound Imaging With Enhanced Spatial Resolution and Background Rejection

Ultrasound super-localization microscopy techniques presented in the last few years enable non-invasive imaging of vascular structures at the capillary level by tracking the flow of ultrasound contrast agents (gas microbubbles). However, these techniques are currently limited by low temporal resolution and long acquisition times. Super-resolution optical fluctuation imaging (SOFI) is a fluorescence microscopy technique enabling sub-diffraction limit imaging with high temporal resolution by calculating high order statistics of the fluctuating optical signal. The aim of this work is to achieve fast acoustic imaging with enhanced resolution by applying the tools used in SOFI to contrast-enhance ultrasound (CEUS) plane-wave scans. The proposed method was tested using numerical simulations and evaluated using two in-vivo rabbit models: scans of healthy kidneys and VX-2 tumor xenografts. Improved spatial resolution was observed with a reduction of up to 50% in the full width half max of the point spread function. In addition, substantial reduction in the background level was achieved compared to standard mean amplitude persistence images, revealing small vascular structures within tumors. The scan duration of the proposed method is less than a second while current super-localization techniques require acquisition duration of several minutes. As a result, the proposed technique may be used to obtain scans with enhanced spatial resolution and high temporal resolution, facilitating flow-dynamics monitoring. Our method can also be applied during a breath-hold, reducing the sensitivity to motion artifacts.

Stable Support Recovery of Stream of Pulses With Application to Ultrasound Imaging

This paper considers the problem of estimating the delays of a weighted superposition of pulses, called stream of pulses, in a noisy environment. We show that the delays can be estimated using a tractable convex optimization problem with a localization error proportional to the square root of the noise level. Furthermore, all false detections produced by the algorithm have small amplitudes. Numerical and in-vitro ultrasound experiments corroborate the theoretical results and demonstrate their applicability for the ultrasound imaging signal processing.

Functional Flow Patterns and Static Blood Pooling in Tumors Revealed by Combined Contrast-Enhanced Ultrasound and Photoacoustic Imaging

Alterations in tumor perfusion and microenvironment have been shown to be associated with aggressive cancer phenotypes, raising the need for noninvasive methods of tracking these changes. Dynamic contrast-enhanced ultrasound (DCEUS) and photoacoustic (PA) imaging serve as promising candidates-one has the ability to measure tissue perfusion, whereas the other can be used to monitor tissue oxygenation and hemoglobin concentration. In this study, we investigated the relationship between the different functional parameters measured with DCEUS and PA imaging, using two morphologically different hind-limb tumor models and drug-induced alterations in an orthotopic breast tumor model. Imaging results showed some correlation between perfusion and oxygen saturation maps and the ability to sensitively monitor antivascular treatment. In addition, DCEUS measurements revealed different vascular densities in the core of specific tumors compared with their rims. Noncorrelated perfusion and hemoglobin concentration measurements facilitated discrimination between blood lakes and necrotic areas. Taken together, our results illustrate the utility of a combined contrast-enhanced ultrasound method with photoacoustic imaging to visualize blood flow patterns in tumors. Cancer Res; 76(15); 4320-31. ©2016 AACR.

Towards sub-Nyquist tissue Doppler imaging using non-uniformly spaced stream of pulses

Tissue Doppler ultrasound imaging enables quantification of the heart function by mapping the velocity of the tissue in different regions within the left ventricle. This estimation is performed by transmitting a series of evenly spaced pulses at a specific direction and analyzing phase shifts in the returning echoes. Traditionally the received signals are sampled at the Nyquist rate. However, high sampling rates produce high volumes of data. In addition, the number of consecutive pulses needed for reliable velocity estimation at each orientation in the presence of clutter limits the size of the sectors that can be imaged simultaneously. In this work, compressed sensing techniques that were suggested for solving similar problems in radar signal processing are adapted to tissue Doppler ultrasound imaging. The proposed method reduces the amount of data propagated through the scanner in two ways: A non-uniformly spaced stream of pulses is transmitted and each received echo is sampled at a sub-Nyquist rate. In addition, the time needed for each velocity estimation is reduced. The proposed method was evaluated using realistic synthetic echocardiographic sequences.

Fast and background free super-resolution ultrasound angiography

Spatial resolution in classic contrast-enhanced ultrasound (CEUS) is limited by acoustic diffraction. Ultrafast ultrasound localization microscopy (uULM) has enabled a sub-diffraction spatial resolution of tens of micrometers in-vivo, although at the expense of a scan duration of tens of seconds. In contrast, by exploiting the statistical properties of CEUS, super resolution optical fluctuation imaging (SOFI) recently demonstrated temporal resolution of 90ms in-vivo with a spatial resolution two times better than the diffraction limit. Here, we report on a method which achieves a spatial resolution similar to that of uULM, but with temporal resolution of CEUS SOFI, termed sparsity based ultrasonic super resolution hemodynamic imaging (SUSHI).

Improved Contrast-Enhanced Power Doppler Using a Coherence-Based Estimator

While plane-wave imaging can improve the performance of power Doppler by enabling much longer ensembles than systems using focused beams, the long-ensemble averaging of the zero-lag autocorrelation R(0) estimates does not directly decrease the mean noise level, but only decreases its variance. Spatial variation of the noise due to the time-gain compensation and the received beamforming aperture ultimately limits sensitivity. In this paper, we demonstrate that the performance of power Doppler imaging can be improved by leveraging the higher lags of the autocorrelation [e.g., R(1), R(2),…] instead of the signal power (R(0)). As noise is completely uncorrelated from pulse-to-pulse while the flow signal remains correlated significantly longer, weak signals just above the noise floor can be made visible through the reduction of the noise floor. Finally, as coherence decreases proportionally with respect to velocity, we demonstrate how signal coherence can be targeted to separate flows of different velocities. For instance, we show how long-time-range coherence of microbubble contrast-enhanced flow specifically isolates slow capillary perfusion (as opposed to conduit flow).

Abstract 4197: Combined contrast enhanced ultrasound and photoacoustic imaging reveals both functional flow patterns and dysfunctional vascular pooling in tumor models

The tumor vasculature and its hypoxic microenvironment are constantly undergoing changes. These alterations are key attributes associated with aggressive cancer phenotypes, raising the need for non-invasive methods to track these changes. Similarly, in many cases, various cancer treatments also affect tumor vasculature, and preferably – should be monitored. Dynamic contrast-enhance ultrasound (DCEUS) and photoacoustic (PA) imaging are two promising candidates. DCEUS has the ability to measure functional tissue perfusion, whereas multispectral PA imaging can be used to evaluate tissue oxygenation related parameters. This study investigates the relationship between blood perfusion, oxygen saturation levels and hemoglobin concentration in two hind-limb tumor models, and evaluates the ability of these two modalities to image vascular structures and functions. Xenograft tumors were induced in SHO mice using either LS174T human colorectal cancer cells (n = 6), or PC3 human prostate cancer cells (n = 6). Tumors were grown to a depth of 4-6 mm before imaging was performed using a laser integrated high-frequency ultrasound system (Vevo®LAZR, VisualSonics Inc.). Contrast enhanced images were collected after a 50μL bolus injection of MicroMarker ultrasound contrast agents (VisualSonics Inc.) using non-linear contrast imaging. Perfusion parameters were quantified after applying wavelet denoising to the DCEUS clips. PA images were acquired using a 21MHz linear array transducer with fiber optical bundles integrated to each side, used to deliver light from a 680-970 nm tunable laser. Oxygen saturation levels and hemoglobin concentration were estimated from the PA measurements using spectral un-mixing. Tumor vascularity and hypoxia were confirmed with immunohistochemistry staining for CD31 and CA9. Reasonable correlations were found between corresponding pixels in the DCEUS perfusion maps and oxygen saturation maps (R = 0.63 and R = 0.5 for LS174T and PC3 respectively). In contrast, the correlation between blood perfusion and hemoglobin concentration was nil for LS174T tumors (R = -0.1), and low for PC3 tumors (R = 0.34). This discrepancy was explained by the presence of blood pools in LS174T tumors, observed in tumor histology. The presence of hemoglobin inside regions of hemorrhage together with the limited capability to separate hypoxic and necrotic regions, impeded the ability of PA imaging to detect blood vessels inside tumors. Compared to PA imaging, DECUS provides better detection of functional vasculature and enables the visualization of single blood vessels around the tumor core, without including blood pools. This study demonstrates that a multi-modality imaging scheme combining DCEUS and PA imaging can provide both distinctive and complementary information on tumor microenvironment in experimental animal studies. Citation Format: Melissa Yin, Avinoam Bar-Zion, Dan Adam, Stuart Foster. Combined contrast enhanced ultrasound and photoacoustic imaging reveals both functional flow patterns and dysfunctional vascular pooling in tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4197.