Contrast enhanced maximum intensity projection ultrasound imaging for assessing angiogenesis in murine glioma and breast tumor models: A comparative study

The purpose of this study was to prospectively compare noninvasive, quantitative measures of vascularity obtained from four contrast enhanced ultrasound (US) techniques to four invasive immunohistochemical markers of tumor angiogenesis in a large group of murine xenografts. Glioma (C6) or breast cancer (NMU) cells were implanted in 144 rats. The contrast agent Optison (GE Healthcare, Princeton, NJ) was injected in a tail vein (dose: 0.4 ml/kg). Power Doppler imaging (PDI), pulse-subtraction harmonic imaging (PSHI), flash-echo imaging (FEI), and Microflow imaging (MFI; a technique creating maximum intensity projection images over time) was performed with an Aplio scanner (Toshiba America Medical Systems, Tustin, CA) and a 7.5 MHz linear array. Fractional tumor neovascularity was calculated from digital clips of contrast US, while the relative area stained was calculated from specimens. Results were compared using a factorial, repeated measures ANOVA, linear regression and z-tests. The tortuous morphology of tumor neovessels was visualized better with MFI than with the other US modes. Cell line, implantation method and contrast US imaging technique were significant parameters in the ANOVA model (p < 0.05). The strongest correlation determined by linear regression in the C6 model was between PSHI and percent area stained with CD31 (r = 0.37, p < 0.0001). In the NMU model the strongest correlation was between FEI and COX-2 (r = 0.46, p < 0.0001). There were no statistically significant differences between correlations obtained with the various US methods (p > 0.05). In conclusion, the largest study of contrast US of murine xenografts to date has been conducted and quantitative contrast enhanced US measures of tumor neovascularity in glioma and breast cancer xenograft models appear to provide a noninvasive marker for angiogenesis; although the best method for monitoring angiogenesis was not conclusively established.     

Breast tumor vascularity identified by contrast enhanced ultrasound and pathology: initial results

Quantifiable measures of vascularity obtained from contrast enhanced color flow images were correlated with pathologic vascularity measurements in ten female patients with a solid breast mass. Each patient received Levovist Injection (Berlex Laboratories Inc., Montville, NJ). Color flow images pre- and post-contrast were obtained using an HDI 3000 unit (ATL, Bothell, WA) before removing the mass for pathologic vascularity assessments. Image-processing techniques were used to obtain both the ultrasound and pathologic vascularity measurements. Multiple linear regression found significant correlations for ultrasonic vascularity measurements post contrast and pathology (P = 0.02 and 0.06). No correlations were found between pre-contrast ultrasound and pathology. In conclusion, post-contrast ultrasonic flow measures provide a non-invasive measure of breast tumor neovascularity. However, the patient population is small, and until further patients are analyzed, these conclusions are preliminary.     

Assessment of tumor vasculature for diagnostic and therapeutic applications in a mouse model in vivo using 25-MHz power Doppler imaging

Objective

The blood flow rate in the microcirculation associated with angiogenesis plays an important role in the progression and treatment of cancer. Since the microvascular status of tumor vessels can yield useful clinical information, assessing changes in the tumor microcirculation could be particularly helpful for tumor evaluation and treatment planning.

Methods

In this study we used a self-developed 25-MHz ultrasound imaging system with a spatial resolution of 150 μm for assessing tumor-microcirculation development and the pattern of the vasculature in three tumor-bearing mice in vivo based on power Doppler images. The total Doppler power (DP) and color pixel density (CPD) revealed the presence of functional vessels distributed throughout a tumor volume. The vasculature distributions in the core and periphery were compared to the regulation of vasculature function, which facilitated determination of when the tumor grew rapidly.

Results

The data obtained from a quantified analysis of power Doppler images indicated that the tumor vascularity initially increased throughout the tumor. Both DP and CPD increased rapidly in the tumor periphery when the tumor volume exceeded 10 mm³.

Conclusion

Our preclinical findings suggest that power Doppler imaging could be useful for detecting the changes in tumor vascular perfusion and for determining the optimal treatment timing when a tumor begins its rapid volumetric growth.     

Comparison of fundamental and wideband harmonic contrast imaging of liver tumors

Wideband harmonic imaging (with phase inversion for improved tissue suppression) was compared to fundamental imaging in vivo. Four woodchucks with naturally occurring liver tumors were injected with Imagent (Alliance Pharmaceutical Corp., San Diego, CA). Randomized combinations of dose (0.05, 0.2 and 0.4 ml/kg) and acoustic output power (AO; 5, 25 and 63% or MI < or = 0.9) were imaged in gray scale using a Sonoline Elegra scanner (Siemens Medical Systems, Issaquah, WA). Tumor vascularity, conspicuity and contrast enhancement were rated by three independent observers. Imagent produced marked tumor enhancement and improved depiction of neovascularity at all dosages and AO settings in both modes. Tumor vascularity and enhancement correlated with mode, dose and AO (P < 0.002). Fundamental imaging produced more enhancement (P < 0.05), but tumor vascularity and conspicuity were best appreciated in harmonic mode (P < 0.05). Under the conditions studied here, the best approach was wideband harmonic imaging with 0.2 ml/kg of Imagent at an AO of 25%.     

Contrast enhanced vascular three-dimensional ultrasound imaging

In other imaging modalities three-dimensional (3D) data displays are well established; not so in ultrasound. Due to the real-time requirements of ultrasound the time available to compute 3D displays is limited, particularly when flow data is acquired with Doppler techniques. Consequently, it is only recently that improvements in computer processing power have resulted in useful vascular 3D ultrasound scans. Many manufacturers have now implemented free-hand 3D power Doppler capabilities on their scanners. However, to obtain flow signals from smaller vessels associated e.g., with tumor neovascularity, may very well require the introduction of a microbubble based ultrasound contrast agent into the blood stream. Given the up to 30 dB enhancement of Doppler signals produced by the contrast microbubbles quite spectacular vascular 3D images are feasible. Moreover, new contrast imaging techniques, such as harmonic imaging, have now permitted 3D vascular information to be acquired and displayed in grayscale with the associated improvement in resolution. In this paper we will review different aspects of contrast enhanced vascular 3D ultrasound imaging including implementation, contrast specific techniques and in vivo imaging.     

On dynamic tumor eradication conditions under combined chemical/anti-angiogenic therapies

In this paper ultimate dynamics of the five-dimensional cancer tumor growth model at the angiogenesis phase is studied. This model elaborated by Pinho et al. in 2014 describes interactions between normal/cancer/endothelial cells under chemotherapy/anti-angiogenic agents in tumor growth process. The author derives ultimate upper bounds for normal/tumor/endothelial cells concentrations and ultimate upper and lower bounds for chemical/anti-angiogenic concentrations. Global asymptotic tumor clearance conditions are obtained for two versions: the use of only chemotherapy and the combined application of chemotherapy and anti-angiogenic therapy. These conditions are established as the attraction conditions to the maximum invariant set in the tumor free plane, and furthermore, the case is examined when this set consists only of tumor free equilibrium points.     

Reduced capillary perfusion and permeability in human tumour xenografts treated with the VEGF signalling inhibitor ZD4190: an in vivo assessment using dynamic MR imaging and macromolecular contrast media

We describe the use of perfusion-permeability magnetic resonance imaging (ppMRI) to study hemodynamic parameters in human prostate tumor xenografts, following treatment with the vascular endothelial growth factor-A (VEGF) receptor tyrosine kinase inhibitor, ZD4190. Using a macromolecular contrast agent (P792), a fast MR imaging protocol and a compartmental data analysis, we were able to demonstrate a significant simultaneous reduction in tumor vascular permeability, tumor vascular volume and tumor blood flow (43%, 30% and 42%, respectively) following ZD4190 treatment (100 mg/kg orally, 24 h and 2 h prior to imaging). This study indicates that MR imaging can be used to measure multiple hemodynamic parameters in tumors, and that tumor vascular permeability, volume and flow, can change in response to acute treatment with a VEGF signaling inhibitor.     

Detection procedures of ultrasound contrast agents

In the early days, it was believed that ultrasound contrast agents (UCA) could be sufficiently detected and imaged with the conventional imaging methods nowadays referred to as fundamental imaging. Newer imaging techniques proved to be more sensitive and are based on specific properties of the UCA. In general, these new characteristics involve non-linear and transient characteristics of contrast agents that appear at the high end of the diagnostic acoustic intensity. Imaging modalities used today for UCA are, besides fundamental imaging, second harmonic imaging, power Doppler, harmonic power Doppler, pulse inversion and pulse inversion Doppler, multi-pulse imaging and subharmonic imaging. Although the results of conventional second harmonic imaging are still not optimal for perfusion imaging applications, in combination with Doppler techniques (colour Doppler, power Doppler) it is one of the most sensitive techniques currently available in terms of agent-to-tissue ratio. Further improvements in current and future detection methods demand a complete understanding of the ultrasound-UCA interaction.     

Leveraging re-chargeable nanobubbles on amine-functionalized ZnO nanocrystals for sustained ultrasound cavitation towards echographic imaging

Nanoparticles able to promote inertial cavitation when exposed to focused ultrasound have recently gained much attention due to their vast range of possible applications in the biomedical field, such as enhancing drug penetration in tumor or supporting ultrasound contrast imaging. Due to their nanometric size, these contrast agents could penetrate through the endothelial cells of the vasculature to target tissues, thus enabling higher imaging resolutions than commercial gas-filled microbubbles. Herein, Zinc Oxide NanoCrystals (ZnO NCs), opportunely functionalized with amino-propyl groups, are developed as novel nanoscale contrast agents that are able, for the first time, to induce a repeatedly and over-time sustained inertial cavitation as well as ultrasound contrast imaging. The mechanism behind this phenomenon is investigated, revealing that re-adsorption of air gas nanobubbles on the nanocrystal surface is the key factor for this re-chargeable cavitation. Moreover, inertial cavitation and significant echographic signals are obtained at physiologically relevant ultrasound conditions (MI < 1.9), showing great potential for low side-effects in in-vivo applications of the novel nanoscale agent from diagnostic imaging to gas-generating theranostic nanoplatforms and to drug delivery.     

New multi-volume rendering technique for three-dimensional power Doppler imaging

In this paper, we present a new multi-volume rendering technique (i.e., progressive fusion) to combine 3D anatomical structures from B-mode imaging and flow information from power Doppler imaging. A post-fusion technique, in which B-mode and power Doppler volumes are independently rendered and then fused based on alpha blending, is typically used in 3D power Doppler imaging. However, it has limitations in preserving the spatial relationship (i.e., depth order) between tissue structure and vasculature since they are rendered independently and then merged. With the proposed progressive fusion, B-mode and power Doppler volumes are composited together while rendering by sharing the opacity values. After compositing, two rendered frames are blended by utilizing a 2D color lookup table designed to fuse two properties (i.e., tissues and blood flows). We have evaluated the progressive-fusion multi-volume rendering method with the phantom and in vivo data acquired using a commercial ultrasound machine (EUB-8500, Hitachi Medical Corporation, Japan) with a 3.5 MHz mechanical probe. From the preliminary study, we have found that the new progressive-fusion method can better retain and display the spatial relationship between tissue structure, vasculature and their corresponding depth order.     

AG-013736, a novel inhibitor of VEGF receptor tyrosine kinases, inhibits breast cancer growth and decreases vascular permeability as detected by dynamic contrast-enhanced magnetic resonance imaging

Dynamic contrast-enhanced MRI (DCE-MRI) was used to noninvasively evaluate the effects of AG-03736, a novel inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, on tumor microvasculature in a breast cancer model. First, a dose response study was undertaken to determine the responsiveness of the BT474 human breast cancer xenograft to AG-013736. Then, DCE-MRI was used to study the effects of a 7-day treatment regimen on tumor growth and microvasculature. Two DCE-MRI protocols were evaluated: (1) a high molecular weight (MW) contrast agent (albumin-(GdDTPA)(30)) with pharmacokinetic analysis of the contrast uptake curve and (2) a low MW contrast agent (GdDTPA) with a clinically utilized empirical parametric analysis of the contrast uptake curve, the signal enhancement ratio (SER). AG-013736 significantly inhibited growth of breast tumors in vivo at all doses studied (10-100 mg/kg) and disrupted tumor microvasculature as assessed by DCE-MRI. Tumor endothelial transfer constant (K(ps)) measured with albumin-(GdDTPA)(30) decreased from 0.034+/-0.005 to 0.003+/-0.001 ml min(-1) 100 ml(-1) tissue (P<.0022) posttreatment. No treatment-related change in tumor fractional plasma volume (fPV) was detected. Similarly, in the group of mice studied with GdDTPA DCE-MRI, AG-013736-induced decreases in tumor SER measures were observed. Additionally, our data suggest that 3D MRI-based volume measurements are more sensitive than caliper measurements for detecting small changes in tumor volume. Histological staining revealed decreases in tumor cellularity and microvessel density with treatment. These data demonstrate that both high and low MW DCE-MRI protocols can detect AG-013736-induced changes in tumor microvasculature. Furthermore, the correlative relationship between microvasculature changes and tumor growth inhibition supports DCE-MRI methods as a biomarker of VEGF receptor target inhibition with potential clinical utility.     

Subharmonic imaging of contrast agents

Ultrasound contrast agents promise to improve the sensitivity and specificity of diagnostic ultrasound imaging. It is of great importance to adapt ultrasound equipment for optimal use with contrast agents e.g., by exploiting the nonlinear properties of the contrast microbubbles. Harmonic imaging is one technique that has been extensively studied and is commercially available. However, harmonic imaging is associated with problems, due to second harmonic generation and accumulation within the tissue itself. Given the lack of subharmonic generation in tissue, one alternative is the creation of subharmonic images by transmitting at the fundamental frequency (fo) and receiving at the subharmonic (fo/2). Subharmonic imaging should have a much better lateral resolution and may be suitable for scanning deep-lying structures owing to the higher transmit frequency and the much smaller attenuation of scattered subharmonic signals. In this paper, we will review different aspects of subharmonic imaging including implementation, in-vitro gray-scale imaging and subharmonic aided pressure estimation.     

Dynamic contrast-enhanced MRI of vascular changes induced by the VEGF-signalling inhibitor ZD4190 in human tumour xenografts

Dynamic contrast-enhanced magnetic resonance imaging (DCEMRI) was used to examine the acute effects of treatment with an inhibitor of vascular endothelial growth factor (VEGF) signaling. ZD4190 is an orally bioavailable inhibitor of VEGF receptor-2 (KDR) tyrosine kinase activity, which elicits broad-spectrum antitumour activity in preclinical models following chronic once-daily dosing. Nude mice, bearing established (0.5-1.0 mL volume) human prostate (PC-3), lung (Calu-6) and breast (MDA-MB-231) tumor xenografts, were dosed with ZD4190 (p.o.) using a 1 day (0 and 22 h) or 7 day (0, 24, 48, 72, 96,120,144, and 166 h) treatment regimen. DCEMRI was employed 2 h after the last dose of ZD4190, using the contrast agent gadopentetate dimeglumine. Dynamic data were fit to a compartmental model to obtain voxelwise K(trans), the transfer constant for gadopentetate into the tumor. K(trans) was averaged over the entire tumor, and a multi-threshold histogram analysis was also employed to account for tumor heterogeneity. Reductions in K(trans) reflect reductions in flow, in endothelial surface area, and/or in vascular permeability. A vascular input function was obtained for each mouse simultaneously with the tumor DCEMRI data. ZD4190 treatment produced a dose-dependent (12.5-100 mg x kg(-1) per dose) reduction in K(trans) in PC-3 prostate tumors. At 100 mg x kg(-1), the largest concentration examined, ZD4190 reduced K(trans) in PC-3 tumors by 31% following 2 doses (1 day treatment regimen; p < 0.001) and by 53% following 8 doses (7 day regimen; p < 0.001). Comparative studies in the three models using a showed similar reductions in K(trans) for the lung and breast tumors using the histogram analysis, although the statistical significance was lost when K(trans) was averaged over the entire tumor. Collectively these studies suggest that DCEMRI using gadopentetate may have potential clinically, for monitoring inhibition of VEGF signaling in solid tumors.     

Dynamic contrast-enhanced MRI of gastric cancer: Correlations of the pharmacokinetic parameters with histological type,Lauren classification,and angiogenesis

PurposeTo compare the pharmacokinetic parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in gastric cancers of different histological type and Lauren classification, and to investigate whether DCE-MRI parameters correlate with vascular endothelial growth factor (VEGF) expression levels in gastric cancer.MethodsIncluded were 32 patients with gastric cancer who underwent DCE-MRI of the upper abdomen before tumor resection. DCE-MRI parameters including the volume transfer coefficient (K^trans), reverse reflux rate constant (K_ep), and extracellular extravascular volume fraction (V_e) were calculated from the tumor region. Post-operative specimens were used for determination of histological differentiation (i.e., non-mucinous, mucinous, or signet-ring-cell adenocarcinoma) as well as Lauren classification (intestinal type or diffuse type). VEGF expression was examined for assessing angiogenesis. DCE-MRI parameters with different histological type and Lauren classification were compared using independent samples t-test and analysis of variance, respectively. Correlations between DCE-MRI parameters and VEGF expression grades were tested using Spearman correlation analysis.ResultsAmong gastric adenocarcinomas of three different histological types, mucinous adenocarcinomas showed a higher V_e and lower K^trans than the others (P < 0.01). Between the two Lauren classifications, the diffuse type showed a higher V_e than the intestinal type (P < 0.001). The mean K^trans showed a significantly positive correlation with VEGF (r = 0.762, P < 0.001).ConclusionDCE-MRI permits noninvasive prediction of tumor histological type and Lauren classification and estimation of tumor angiogenesis in gastric cancer. DCE-MRI parameters can be used as imaging biomarkers to predict the biologic aggressiveness of a tumor as well as patient prognosis.     

超声分子成像进展

超声分子成像在超声医学成像的基础上,利用靶向超声造影剂为分子探针,以可视化和定量获取活体组织细胞的分子信息为目标的影像术。它不用进行手术活检,不仅可以给出病灶的空间信息,而且能确定它的性质,进行针对性的治疗和对疗效进行评估。本文对现有的核医学分子成像,磁共振分子成像,光学分子成像和光声分子成像技术作了简单介绍,着重讨论了超声分子成像技术和应用的进展。     

3D spin-lock imaging of human gliomas

We investigated whether the simultaneous use of paramagnetic contrast medium and 3D on-resonance spin lock (SL) imaging could improve the contrast of enhancing brain tumors at 0.1 T. A phantom containing serial concentrations of gadopentetate dimeglumine (Gd-DTPA) in cross-linked bovine serum albumin (BSA) was imaged. Eleven patients with histologically verified glioma were also studied. T₁-weighted 3D gradient echo images with and without SL pulse were acquired before and after a Gd-DTPA injection. SL effect, contrast, and contrast-to-noise ratio (CNR) were calculated for each patient. In the glioma patients, the SL effect was significantly smaller in the tumor than in the white and gray matter both before (p = 0.001, p = 0.025, respectively), and after contrast medium injection (p < 0.001, p < 0.001, respectively). On post-contrast images, SL imaging significantly improved tumor contrast (p = 0.001) whereas tumor CNR decreased slightly (p = 0.024). The combined use of SL imaging and paramagnetic Gd-DTPA contrast agent offers a modality for improving tumor contrast in magnetic resonance imaging (MRI) of enhancing brain tumors. 3D gradient echo SL imaging has also shown potential to increase tissue characterization properties of MR imaging of human gliomas.     

Monitoring tissue inflammation and responses to drug treatments in early stages of mice bone fracture using 50 MHz ultrasound

Bone fracture induces moderate inflammatory responses that are regulated by cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) for initiating tissue repair and bone formation. Only a handful of non-invasive techniques focus on monitoring acute inflammation of injured bone currently exists. In the current study, we monitored in vivo inflammation levels during the initial 2 weeks of the inflammatory stage after mouse bone fracture utilizing 50 MHz ultrasound. The acquired ultrasonic images were correlated well with histological examinations. After the bone fracture in the tibia, dynamic changes in the soft tissue at the medial-posterior compartment near the fracture site were monitored by ultrasound on the days of 0, 2, 4, 7, and 14. The corresponding echogenicity increased on the 2nd, 4th, and 7th day, and subsequently declined to basal levels after the 14th day. An increase of cell death was identified by the positive staining of deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and was consistent with ultrasound measurements. The increases of both COX-2 and Leukotriene B4 receptor 1 (BLT₁, 5-LO-relative receptor), which are regulators for tissue inflammation, in the immunohistochemistry staining revealed their involvement in bone fracture injury. Monitoring the inflammatory response to various non-steroidal anti-inflammatory drugs (NSAIDs) treatments was investigated by treating injured mice with a daily oral intake of aspirin (Asp), indomethacin (IND), and a selective COX-2 inhibitor (SC-236). The Asp treatment significantly reduced fracture-increased echogenicity (hyperechogenicity, p < 0.05) in ultrasound images as well as inhibited cell death, and expression of COX-2 and BLT₁. In contrast, treatment with IND or SC-236 did not reduce the hyperechogenicity, as confirmed by cell death (TUNEL) and expression levels of COX-2 or BLT₁. Taken together, the current study reports the feasibility of a non-invasive ultrasound method capable of monitoring post-fracture tissue inflammation that positively correlates with histological findings. Results of this study also suggest that this approach may be further applied to elucidate the underlying mechanisms of inflammatory processes and to develop therapeutic strategies for facilitating fracture healing.     

自建光声超声双模态成像系统的肿瘤成像分析

现有B超成像可以提供基于声阻抗差异的组织解剖结构信息,而近年来研究发现,光声成像可以提供标记组织成分的分布和功能信息。本文基于商用B超仪和脉冲激光系统建立了光声超声双模态成像系统,实现了超声组织结构成像和光声生物功能的同时成像。首先基于血红蛋白在某些波段的较强吸光性,实现了肿瘤内部组织血管灌注图像;其次用链接有靶向抗体的纳米颗粒作为靶向光声造影剂,对恶性肿瘤边缘和内部的血管以及血管附近的肿瘤组织进行了成像。最终,通过超声和光声的融合图像提供的肿瘤结构信息与光声图像提供的肿瘤功能信息,可以准确识别肿瘤组织。     

超声动态向量血流成像的产品化实现

传统超声彩色多普勒成像测量的是血流沿超声传播方向上的速度分量,故无法得到垂直于超声传播方向的血流。向量血流成像是一种更加先进的超声血流成像技术。它不受角度限制,可以直接计算出血流速度的大小和方向。本文总结了现有多种超声向量血流成像技术的特点和发展情况,并从产品化实现的角度分析了各项技术的优缺点。从超声系统发射接收、血流成像、向量速度方向合成、显示等几个方面详述了迈瑞超声向量血流成像技术产品化实现过程中遇到的主要问题及解决方案。实验采用了中科院声学所研制的超声多普勒仿血流体模,通过向量血流成像和脉冲多普勒成像分别测量体模的仿血流速度。将向量血流成像直接计算出来的速度值与脉冲多普勒经过角度校正得到的速度进行对比。在不同条件下,经过多次测量,二者的平均相对误差均在10%以内。     

Polymeric contrast agent with targeting potential

The Arg-Gly-Asp (RGD) peptide sequence was conjugated to poly (lactid acid), (PLA), microcapsules. These hollow, biodegradable PLGA microcapsules were developed in our laboratory for use as ultrasound contrast agents. By modifying the surface of the agent with a targeting ligand, it can be targeted to a specific address within the body. This application is ideal for both targeted imaging and/or targeted drug delivery. Integrins are membrane-spanning proteins in cells that play a vital role in cell attachment and many other processes. The RGD peptide sequence targets integrins expressed during angiogenesis, alphavbeta3 and alphavbeta5. The integrins specific to angiogenesis are more active during cancer and can be used as receptors for the RGD-conjugated contrast agents. Although the generic RGD sequence is not specific to only alphavbeta3 and alphavbeta5 integrins, it is an excellent candidate for proof of concepts studies such as described here. Preliminary in vitro results indicate that the modified capsules remain highly echogenic (maximum enhancement of 20 dB in vitro) and adhere specifically to a breast cancer cell line MDA-MB-231 in static experiments. However, no adherence is seen with either unmodified capsules (negative control), or when cells that have been pre-saturated with RGD ligand are contacted with modified capsules (positive control). Specific targeting of ultrasound contrast agents could lead the way to imaging as a method for discrimination of malignant from benign.