Alexandre Dizeux

VEGFR2-TARGETED CONTRAST-ENHANCED ULTRASOUND TO DISTINGUISH BETWEEN TWO ANTI-ANGIOGENIC TREATMENTS

The aim of this study was to evaluate the capacity of BR55, an ultrasound contrast agent specifically targeting vascular endothelial growth factor receptor 2 (VEGFR2), to distinguish the specific anti-VEGFR2 therapy effect of sunitinib from other anti-angiogenic effects of a therapy (imatinib) that does not directly inhibit VEGFR2. Sunitinib, imatinib and placebo were administered daily for 11 d (264 h) to 45 BalbC mice bearing ectopic CT26 murine colorectal carcinomas. During the course of therapy, B-mode ultrasound, contrast-enhanced ultrasound and VEGFR2-targeted contrast-enhanced ultrasound were performed to assess tumor morphology, vascularization and VEGFR2 expression, respectively. The angiogenic effects on these three aspects were characterized using tumor volume, contrast-enhanced area and differential targeted enhancement. Necrosis, microvasculature and expression of VEGFR2 were also determined by histology and immunostaining. B-Mode imaging revealed that tumor growth was significantly decreased in sunitinib-treated mice at day 11 (p < 0.05), whereas imatinib did not affect growth. Functional evaluation revealed that the contrast-enhanced area decreased significantly (p < 0.02) and by similar amounts under both anti-angiogenic treatments by day 8 (192 h): -23% for imatinib and -21% for sunitinib. No significant decrease was observed in the placebo group. Targeted contrast-enhanced imaging revealed lower differential targeted enhancement, that is, lower levels of VEGFR2 expression, in sunitinib-treated mice relative to placebo-treated mice from 24 h (p < 0.05) and relative to both placebo- and imatinib-treated mice from 48 h (p < 0.05). Histologic assessment of tumors after the final imaging indicated that necrotic area was significantly higher for the sunitinib group (21%) than for the placebo (8%, p < 0.001) and imatinib (11%, p < 0.05) groups. VEGFR2-targeted ultrasound was able to sensitively differentiate the anti-VEGFR2 effect from the reduced area of tumor with functional flow produced by both anti-angiogenic agents. BR55 molecular imaging was, thus, able both to detect early therapeutic response to sunitinib in CT26 tumors as soon as 24 h after the beginning of the treatment and to provide early discrimination (48 h) between tumor response during anti-angiogenic therapy targeting VEGFR2 expression and response during anti-angiogenic therapy not directly acting on this receptor.

Level-set segmentation of 2D and 3D ultrasound data using local gamma distribution fitting energy

Ultrasound (US) data suffer from speckle noise as well as intensity inhomogeneities due to underlying changes in acoustic properties of tissue structure and/or the effects of acoustic focusing and attenuation. This paper describes a 2D and 3D variational level-set method for segmenting such data. To deal with the local statistics of speckle noise, the data term of the level-set energy function is based on local gamma distributions which have shown an ability to model envelope data and gray-level pixel intensities of B-mode clinical images. Local statistics are estimated at a controllable scale using a smooth function with a compact support, a mollifyer, and the method of moments. Compared to manual segmentation, the investigated method provides a high Dice similarity coefficient (DSC) on 3D simulated data, an average DSC of 0.915 on 12 B-mode images of murine tumors acquired with a clinical US system, and average DSCs of 0.920, 0.806 and 0.975 for three media of 54 3D envelope data sets acquired with a high-frequency, focused transducer from dissected human lymph nodes. It also outperforms methods that employ local Gaussian statistics instead of local gamma statistics.

Quantification of tumor perfusion using dynamic contrast-enhanced ultrasound: impact of mathematical modeling.

Dynamic contrast-enhanced ultrasound has been proposed to monitor tumor therapy, as a complement to volume measurements. To assess the variability of perfusion parameters in ideal conditions, four consecutive test-retest studies were acquired in a mouse tumor model, using controlled injections. The impact of mathematical modeling on parameter variability was then investigated. Coefficients of variation (CV) of tissue blood volume (BV) and tissue blood flow (BF) based-parameters were estimated inside 32 sub-regions of the tumors, comparing the log-normal (LN) model with a one-compartment model fed by an arterial input function (AIF) and improved by the introduction of a time delay parameter. Relative perfusion parameters were also estimated by normalization of the LN parameters and normalization of the one-compartment parameters estimated with the AIF, using a reference tissue (RT) region. A direct estimation (rRTd) of relative parameters, based on the one-compartment model without using the AIF, was also obtained by using the kinetics inside the RT region. Results of test-retest studies show that absolute regional parameters have high CV, whatever the approach, with median values of about 30% for BV, and 40% for BF. The positive impact of normalization was established, showing a coherent estimation of relative parameters, with reduced CV (about 20% for BV and 30% for BF using the rRTd approach). These values were significantly lower (p  <  0.05) than the CV of absolute parameters. The rRTd approach provided the smallest CV and should be preferred for estimating relative perfusion parameters.

In Vivo Multiparametric Ultrasound Imaging of Structural and Functional Tumor Modifications during Therapy

Longitudinal imaging techniques are needed that can meaningfully probe the tumor microenvironment and its spatial heterogeneity. Contrast-enhanced ultrasound, shear wave elastography and quantitative ultrasound are ultrasound-based techniques that provide information on the vascular function and micro-/macroscopic tissue structure. Modifications of the tumor microenvironment induced by cytotoxic and anti-angiogenic molecules in ectopic murine Lewis lung carcinoma tumors were monitored. The most heterogenous structures were found in tumors treated with anti-angiogenic drug that simultaneously accumulated the highest levels of necrosis and fibrosis. The anti-angiogenic group presented the highest number of correlations between parameters related to vascular function and those related to the micro-/macrostructure of the tumor microenvironment. Results suggest how patterns of multiparametric ultrasound modifications can be related to provide a more insightful marker of changes occurring within tumors during therapy.

Comparison of tumor microvasculature assessment via Ultrafast Doppler Tomography and Dynamic Contrast Enhanced Ultrasound

Assessing structural organization and dynamics of tumor blood supply is of highest interest since numerous cancer treatments rely on antiangiogenic effects. Dynamic Contrast Enhanced Ultrasound (DCE-US) is widely used in cancer research to extract quantitative parameters reflecting tumor perfusion. However, the underlying vascular structure is not revealed and local spatial fluctuations in DCE parametric maps remain unexplained. The recently introduced Ultrafast Doppler Tomography (UFD-T) has been shown able to reconstruct the 3D vascular network of a rat brain with a 100μm resolution. It is applied here to tumor vasculature imaging and quantitative hemodynamics assessment along with DCE-US to show the complementarity between the techniques.

Regularized linear resolution of a one-compartment model to improve the reproducibility of perfusion parameters in CEUS

Contrast-enhanced ultrasound (CEUS) has been proposed to monitor tumor therapy, in complement to size measurements. Estimating reliable perfusion parameters from CEUS studies is essential in order to propose adapted therapy options according to the parameter values. The variability of these parameters was assessed in an ideal case of consecutive test-retest CEUS studies, in a mouse tumor model. The impact of mathematical modeling on parameter variability was investigated on these data. Four models were compared in 32 tumor sub-regions: the log-normal model (LN), the relative LN model (rLN) where parameters of LN are normalized by the parameters estimated inside a reference tissue (RT) region, a linear resolution of a one-compartment model based on the RT (rLin), a modified version of rLin implementing regularization (rLinReg) to ensure coherent results between the different sub-regions of the tumor. Results show that LN model had highest coefficients of variation. The positive impact of normalization using RT (rLN) was established, showing reduced coefficients of variation. The rLin approach showed large variations especially for flow parameters. Its regularization version, rLinReg, greatly improved parameter reproducibility while providing coherent results between the sub-regions. In conclusion,the rLinReg approach provided the smallest coefficients of variations and should be preferred for estimating perfusion parameters in CEUS.

A multiplicative model to improve microvascular flow evaluation in the context of dynamic contrast-enhanced ultrasound (DCE-US)

Estimation of perfusion parameters from dynamic contrast-enhanced ultrasound (DCE-US) data relies on locally fitting mathematical models to the time-echo-power curves derived from a sequence. The least-squares method generally used to fit a parametric perfusion model to experimental data is optimal only under the hypothesis of an additive Gaussian noise. Due to the nature of the DCE-US signal, this hypothesis is disputable. A maximum likelihood estimator based on a multiplicative noise model is proposed and tested. Results on simulated data show improvements of the precision and accuracy of commonly estimated perfusion parameters. We also analyzed the perfusion of a rather homogeneous in vivo tissue, the renal cortex of an healthy mouse. The new method leads to more homogeneous parametric maps. These improvements should contribute to a more robust estimation of perfusion parameters and an improved resolution of DCE-US parametric images.

Functional ultrasound imaging in awake non-human primates performing voluntary saccade

Functional ultrasound (fUS) is a novel technique for in vivo neuroimaging which allows to map subtle changes of the Cerebral Blood Volume (CBV) due to neurovascular coupling with a very high sensitivity, and thus map dynamics of brain activity [1]. In this study, we apply fUS to awake and behaving non-human primate to investigate the monitoring of saccadic eye movement by the supplementary eye field (SEF) brain region [2]. This represents the first fUS images in non-human primate, doing complex tasks, which generally remains very challenging to perform in an MRI environment in awake and behaving conditions.

Comparing QUS parameters to structural and functional changes in tumors during therapy

Tumor microstructure can potentially be evaluated with quantitative ultrasound (QUS) during therapy, but parameters are influenced by a complex combination of components and modifications in the tumor. To better understand how QUS parameters reflect tumor response to therapy, we estimated effective scatterer diameter (ESD, μm) and effective acoustic concentration (EAC, dB/cm) from ultrasound backscatter measurements in a murine Lewis Lung Carcinoma model on days 7 to 13 after the start of therapy by administration of: anti-angiogenic (AA) agent to inhibit new blood vessels (n = 21); a cytotoxic agent (CA) to damage tumor cells (n = 24) and placebo (n = 24). At D7, there was no significant difference between ESD and EAC for the different therapy groups. At D13, the AA group had higher mean ESD than for CA and placebo (p <; 0.005). At D13, the placebo group exhibited a significantly higher EAC compared to the AA (p <; 0.005) and was elevated compared to the CA group. Histology showed higher (p <; 0.05) necrosis and fibrosis in the AA group at D13 as compared to other groups but no significant differences at D7. Higher mean ESD and lower EAC in the AA group at D13 are consistent with thicker, more sparsely distributed fibrotic structures in the corresponding histology.

Spatiotemporal response of rat visual cortex during moving stimuli using Functional Ultrasound (fUS) imaging

The spatiotemporal study of the visual system of the rodent is a challenging area that conventional functional imaging modalities, despite fundamental achievements, still struggle to study nowadays. In the other hand Functional Ultrasound (fUS) offers an unprecedented combination of spatiotemporal resolution. By coupling fUS with an adapted visual stimulation device, we mapped the vision-evoked activity in the rodent brain. We then optimized the visual-stimulus toward the spatiotemporal study of the rodent visual pathway using fUS.