In vivo photoacoustic imaging of mouse embryos

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.     

Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy

The effect of anti-vascular agents on the growth of experimental tumors is well studied. Their impact on tumor vasculature, the primary therapeutic target of these agents, is not as well characterized, even though this primarily determines treatment outcome. Hypothesizing that the response of vessels to therapy is influenced by their stage of maturation, we studied vascular development and the vascular effects of therapy in several transplanted murine tumor models. Based on size, perfusion, endothelial cell (EC) proliferation, and the presence of pericytes, tumor vessels segregated into three categories. Least mature were highly proliferative, nonperfused EC sprouts emanating from functional vessels. Intermediate were small, perfused vessels which, like the angiogenic sprouts, were not covered by pericytes. Most mature were larger vessels, which were predominantly pericyte-covered with quiescent ECs and few associated sprouts. Thus, a developmental order, similar to that described during physiological neovascularization, was evident among vessels in growing tumors. This order markedly influenced tumor vessel response to anti-vascular therapy with recombinant interleukin-12. Therapy reduced tumor vessel density, which was attributable to a decrease in angiogenic sprouts and induction of EC apoptosis in pericyte-negative vessels. Although the great majority of vessels in growing tumors lacked pericyte coverage, selective loss of less mature vessels with therapy significantly increased the fraction of pericyte-positive vessels after therapy. These data indicate that the therapeutic susceptibility of tumor vasculature to recombinant murine IL-12 and, potentially, other anti-vascular agents is limited by its level of maturation. An implication is that tumor susceptibility is similarly limited, making pericyte coverage of tumor vasculature a potential indicator of tumor responsiveness.     

基于滤波反投影算法的血管内光声图像重建

将目前在光声断层（PAT）成像中得到广泛应用的滤波反投影（FBP）重建算法应用到血管内光声（IVPA）成像中,提出一种简单快速的二维图像重建方法。首先,对组织产生的光声信号进行滤波、逆卷积和时域一阶求导的预处理;然后,针对IVPA在血管腔内封闭成像的特殊性,采用权重法将预处理后的光声信号数据对导管以外的成像区域沿弧线进行反投影,得到成像平面内每个网格点处的初始光声压。最后,得到反映血管壁组织结构形态的横截面灰阶图像。对仿真血管模型的实验表明,采用所提出的方法重建IVPA图像的结构,相似性指标（SSIM）可达到 0.571 7。合理选择滤波函数、滤波截止频率以及测量位置数,可以提高IVPA重建图像的质量;对光声信号进行时域一阶求导处理,能有效地突出重建图像中的组织结构信息。该方法为后续图像重建算法的优化奠定基础。     

Multifunctional nanocarriers for mammographic quantification of tumor dosing and prognosis of breast cancer therapy

Nanoscale therapeutic interventions are increasingly important elements in the portfolio of cancer therapeutics. The efficacy of nanotherapeutics is dictated, in part, by the access they have to tumors via the leaky tumor vasculature. Yet, the extent of tumor vessel leakiness in individual tumors varies widely resulting in a correspondingly wide tumor dosing and resulting range of responses to therapy. Here we report the design of a multifunctional nanocarrier that simultaneously encapsulates a chemotherapeutic and a contrast agent which enables a personalized nanotherapeutic approach for breast cancer therapy by permitting tracking of the nanocarrier distribution by mammography, a widely used imaging modality. Following systemic administration in a rat breast tumor model, imaging demonstrated a wide range of intratumoral deposition of the nanocarriers, indicating variable tumor vessel leakiness. Notably, specific tumors that exhibited high uptake of the nanocarrier as visualized by imaging were precisely the animals that responded best to the treatment as quantified by low tumor growth and prolonged survival.     

Deformation-compensated averaging for clutter reduction in epiphotoacoustic imaging in vivo

Photoacoustic imaging, based on ultrasound detected after laser irradiation, is an extension to diagnostic ultrasound for imaging the vasculature, blood oxygenation and the uptake of optical contrast media with promise for cancer diagnosis. For versatile scanning, the irradiation optics is preferably combined with the acoustic probe in an epi-style arrangement avoiding acoustically dense tissue in the acoustic propagation path from tissue irradiation to acoustic detection. Unfortunately epiphotoacoustic imaging suffers from strong clutter, arising from optical absorption in tissue outside the image plane, and from acoustic backscattering. This limits the imaging depth for useful photoacoustic image contrast to typically less than one centimeter. Deformation-compensated averaging (DCA), which takes advantage of clutter decorrelation induced by palpating the tissue with the imaging probe, has previously been proposed for clutter reduction. We demonstrate for the first time that DCA results in reduced clutter in real-time freehand clinical epiphotoacoustic imaging. For this purpose, combined photoacoustic and pulse-echo imaging at 10-Hz frame rate was implemented on a commercial scanner, allowing for ultrasound-based motion tracking inherently coregistered with photoacoustic frames. Results from the forearm and the neck confirm that contrast is improved and imaging depth increased by DCA.     

Noninvasive imaging of the functional effects of anti-VEGF therapy on tumor cell extravasation and regional blood volume in an experimental brain metastasis model

Brain metastasis has become an increasing cause of morbidity and mortality in cancer patients as the treatment of systemic disease has improved. Brain metastases frequently are highly vascularized, a process driven primarily by VEGF. VEGF mediates numerous changes within the vasculature including endothelial cell retraction and increased permeability, vasodilation, and new vessel formation. Here we describe a xenograft brain metastasis model that mimics the critical steps of metastasis including tumor cell dissemination and vascular adhesion, tumor growth and tumor associated angiogenesis. Magnetic resonance (MR) imaging was used to evaluate two aspects of the functional response of brain metastasis to the anti-VEGF receptor therapeutic, AZD2171 (Cediranib, RECENTIN™). MR tracking of individual cells demonstrated that cediranib did not impede tumor cell extravasation into the brain parenchyma despite evidence that anti-VEGF treatment decreases the permeability of the blood brain barrier. In a second assay, blood volume imaging using ultrasmall superparamagnetic iron oxide revealed that treatment of well-developed brain metastasis with cediranib for 7 days led to a heterogeneous response with respect to individual tumors. Overall, there was a significant average decrease in the tumor vascular bed volume. The majority of large tumors demonstrated substantially reduced central blood volumes relative to normal brain while retaining a rim of elevated blood volume at the tumor brain interface. Small tumors or occasional large tumors displayed a static response. Models and assays such as those described here will be important for designing mechanism-based approaches to the use of anti-angiogenesis therapies for the treatment of brain metastasis.     

Noninvasive photoacoustic imaging of the developing vasculature during early tumor growth

In this study, we monitor the progress of vasculature in early tumor growth using photoacoustic imaging over a 20 day period after subcutaneous inoculation of breast cancer tumor cells in a mouse. With 532 nm laser pulses employed as an irradiation source, the photoacoustic images were obtained through the photoacoustic signals received by a hydrophone in orthogonal mode. The morphological characteristics of vasculature in tumor region are clearly resolved in the photoacoustic images, and the change in structure as well as the increase in density can be identified. Moreover, the average photoacoustic signal strength of vasculature in tumor region, which is highly correlated with the total hemoglobin concentration of blood, is enhanced during early tumor growth. These results indicate the feasibility of detecting early stage tumor and monitoring the progress of anti-angiogenic therapy by photoacoustic imaging.     

MicroCT scanner performance and considerations for vascular specimen imaging

Obtaining three-dimensional geometrical data of vascular systems is of major importance to a number of research areas in medicine and biology. Examples are the characterization of tumor vasculature, modeling blood flow, or genetic effects on vascular development. The performance of the General Electric Medical Systems MS8 microCT scanner is examined in the context of these applications. The system is designed to acquire high-resolution images of specimens up to 5 cm in diameter. A maximum resolution of 38 lp/mm at the 10% modulation transfer function level or 22 microm full width at half maximum of the plane spread function can be achieved with 8.5 microm voxels and a 17 mm field of view. Three different contrast agents are discussed and applied for imaging of small animal vasculature: corrosion casting material Batson's No. 17 with an added lead pigment, silicon rubber MICROFIL MV122, and a suspension of barium sulfate (Baritop) in gelatin. Contrast for all of these agents was highly variable in different vessels as well as within the same vessel. Imaging of PMMA tubing filled with MICROFIL shows that even vessels below 20 microm in diameter are detectable and that diameter estimation of vessels based on thresholding is possible with a precision of 2-3 pixels.     

Mechanistic insights on the inhibition of tumor angiogenesis

Angiogenesis, the growth of new vasculature, is an absolute requirement for the maintenance and progression of the overwhelming majority of the solid tumors. Unraveling the mechanisms that govern this complex biological process has become a central issue not only for understanding of the molecular basis of cancer but also for developing new therapeutic approaches that interfere with neovascularization of the tumor mass. Here we discuss the survival and apoptosis of endothelial cells in the context of vessel formation and regression in response to mediators of angiogenesis produced by tumors. It is the balance between proangiogenic and antiangiogenic molecules in the microenvironment of a vessel in vivo that determines whether the existing vasculature will expand, remain the same, or regress. Here we propose that the vascular endothelial cells themselves interpret and respond to these environmental cues by integrating the activities of the survival and apoptotic pathways within the cell. Thus it is the survival or death of the vulnerable cells that venture out to form new vessels that is the ultimate arbiter of whether neovascularization, as well as the growth of a malignancy that depends on it, succeeds or fails.     

Noninvasive monitoring of orthotopic glioblastoma therapy response using RGD-conjugated iron oxide nanoparticles

Noninvasive imaging techniques have been considered important strategies in the clinic to monitor tumor early response to therapy. In the present study, we applied RGD peptides conjugated to iron oxide nanoparticles (IONP-RGD) as contrast agents in magnetic resonance imaging (MRI) to noninvasively monitor the response of a vascular disrupting agent VEGF(121)/rGel in an orthotopic glioblastoma model. RGD peptides were firstly coupled to IONPs coated with a crosslinked PEGylated amphiphilic triblock copolymer. In vitro binding assays confirmed that cellular uptake of particles was mainly dependent on the interaction between RGD and integrin α(v)β(3) of human umbilical vein endothelial cells (HUVEC). The tumor targeting of IONP-RGD was observed in an orthotopic U87 glioblastoma model. Finally, noninvasive monitoring of the tumor response to VEGF(121)/rGel therapy at early stages of treatment was successfully accomplished using IONP-RGD as a contrast agent for MRI, a superior method over common anatomical approaches which are based on tumor size measurements. This preclinical study can accelerate anticancer drug development and promote clinical translation of nanoprobes.     

Imaging tumor angiogenesis with fluorescent proteins

We have developed three unique mouse models to image angiogenesis with fluorescent proteins, which are described in this review. First, we have adapted the surgical orthotopic implantation (SOI) model to image angiogenesis of human tumors labeled with green fluorescent protein (GFP) transplanted in nude mice. The nonluminous induced capillaries are clearly visible by contrast against the very bright tumor fluorescence examined either intravitally or by whole-body imaging in real time. Intravital images of an SOI model of human pancreatic tumors expressing GFP visualized angiogenic capillaries at both primary and metastatic sites. Whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period in an SOI model of human breast cancer expressing GFP. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold and enabling vessels to be externally visualized in GFP-expressing tumors growing on internal organs. The second model utilizes dual-color fluorescence imaging, effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice that express GFP in all cells. This dual-color model visualizes with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis. The GFP-expressing tumor vasculature, both nascent and mature, are readily distinguished interacting with the RFP-expressing tumor cells. Using a spectral imaging system based on liquid crystal tunable filters, we were able to separate individual spectral species on a pixel-by-pixel basis. Such techniques non-invasively visualized the presence of host GFP-expressing vessels within an RFP-labeled orthotopic human breast tumor by real-time whole-body imaging. The third model involves a transgenic mouse in which the regulatory elements of the stem cell marker nestin drive GFP. The nestin-GFP mouse expresses GFP in areas of the brain, hair follicle stem cells, and in a network of blood vessels in the skin interconnecting hair follicles. RFP-expressing tumors transplanted to nestin-GFP mice enable specific visualization of nascent vessels in skin-growing tumors such as melanoma. Thus, fluorescent proteins expressed in vivo offer very high resolution and sensitivity for real-time imaging of angiogenesis.     

Angiopoietin-1 promotes tumor angiogenesis in a rat glioma model

Angiopoietins have been implicated in playing an important role in blood vessel formation, remodeling, maturation, and maintenance. However, the role of angiopoietins in tumor angiogenesis remains uncertain. In this study, expression of human angiopoietin-1 (hAng-1) and angiopoietin (hAng-2) was amplified in the rat glioma cell line GS9L by stable transfection using an inducible tet-off system. Transfected cells were implanted intracerebrally into syngenic Fischer 344 rats. We demonstrated by means of magnetic resonance imaging that increased hAng-1 expression promoted a significant in vivo growth of intracerebral gliomas in rats. Overexpression of hAng-1 resulted in more numerous, more highly branched vessels, which were covered by pericytes. On the other hand, tumors derived from hAng-2-overexpressing cells were smaller than empty-plasmid control tumors. The tumor vasculature in these tumors was composed of aberrant small vascular cords, which were associated with few mural cells. Our results indicate that in the presence of hAng-1, tumors induce a more functional vascular network, which led to better tumor perfusion and growth. On the other hand, overexpression of hAng-2 led to less intact tumor vessels, inhibited capillary sprouting, and impaired tumor growth.     

Small molecule fluorophore and copolymer RGD peptide conjugates for ex vivo two-photon fluorescence tumor vasculature imaging

We report the use of small molecule and block copolymer RGD peptide conjugates for deep ex?vivo imaging of tumor vasculature in "whole" excised tumors using two-photon fluorescence microscopy (2PFM). The fluorescent probes were administered to mice via tail-vein injection, after which the tumors were excised, fixed, and imaged without further sample preparation. Both RGD conjugates demonstrated specific targeting to tumor blood vessels, and this selectivity imparted excellent contrast in 2PFM micrographs that captured high-resolution 3-D images of the tumor vasculature up to depths of 830?μm in Lewis Lung Carcinoma (LLC) tumors. 2PFM ex?vivo fluorescence micrographs clearly revealed tumor vessels, while differences in the sensitivity of tumor vessel imaging were apparent between the small molecule and block copolymer conjugates. Both the small molecule and polymer-based two-photon absorbing probe conjugate are valuable for deep tissue tumor microvasculature imaging.     

In vivo photoacoustic imaging of chemotherapy-induced apoptosis in squamous cell carcinoma using a near-infrared caspase-9 probe

Anti-cancer drugs typically exert their pharmacological effect on tumors by inducing apoptosis, or programmed cell death, within the cancer cells. However, no tools exist in the clinic for detecting apoptosis in real time. Microscopic examination of surgical biopsies and secondary responses, such as morphological changes, are used to verify efficacy of a treatment. Here, we developed a novel near-infrared dye-based imaging probe to directly detect apoptosis with high specificity in cancer cells by utilizing a noninvasive photoacoustic imaging (PAI) technique. Nude mice bearing head and neck tumors received cisplatin chemotherapy (10 mg/kg) and were imaged by PAI after tail vein injection of the contrast agent. In vivo PAI indicated a strong apoptotic response to chemotherapy on the peripheral margins of tumors, whereas untreated controls showed no contrast enhancement by PAI. The apoptotic status of the mouse tumor tissue was verified by immunohistochemical techniques staining for cleaved caspase-3 p11 subunit. The results demonstrated the potential of this imaging probe to guide the evaluation of chemotherapy treatment.     

Coronary artery wall imaging in mice using osmium tetroxide and micro-computed tomography (micro-CT)

The high spatial resolution of micro-computed tomography (micro-CT) is ideal for 3D imaging of coronary arteries in intact mouse heart specimens. Previously, micro-CT of mouse heart specimens utilized intravascular contrast agents that hardened within the vessel lumen and allowed a vascular cast to be made. However, for mouse coronary artery disease models, it is highly desirable to image coronary artery walls and highlight plaques. For this purpose, we describe an ex vivo contrast-enhanced micro-CT imaging technique based on tissue staining with osmium tetroxide (OsO(4) ) solution. As a tissue-staining contrast agent, OsO(4) is retained in the vessel wall and surrounding tissue during the fixation process and cleared from the vessel lumens. Its high X-ray attenuation makes the artery wall visible in CT. Additionally, since OsO(4) preferentially binds to lipids, it highlights lipid deposition in the artery wall. We performed micro-CT of heart specimens of 5- to 25-week-old C57BL/6 wild-type mice and 5- to 13-week-old apolipoprotein E knockout (apoE(-/-) ) mice at 10 μm resolution. The results show that walls of coronary arteries as small as 45 μm in diameter are visible using a table-top micro-CT scanner. Similar image clarity was achieved with 1/2000th the scan time using a synchrotron CT scanner. In 13-week-old apoE mice, lipid-rich plaques are visible in the aorta. Our study shows that the combination of OsO(4) and micro-CT permits the visualization of the coronary artery wall in intact mouse hearts.     

Photoacoustic and high-frequency power Doppler ultrasound biomicroscopy: a comparative study

Both photoacoustic imaging and power Doppler ultrasound are capable of producing images of the vasculature of living subjects, however, the contrast mechanisms of the two modalities are very different. We present a quantitative and objective comparison of the two methods using phantom data, highlighting relative merits and shortcomings. An imaging system for combined photoacoustic and high-frequency power Doppler ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and power Doppler ultrasound images can be coregistered. Experiments are performed on flow phantoms with various combinations of vessel size, flow velocity, and optical wavelength. For the task of blood volume detection, power Doppler is seen to be advantageous for large vessels and high flow speeds. For small vessels with low flow speeds, photoacoustic imaging is seen to be more effective than power Doppler at the detection of blood as quantified by receiver operating characteristic analysis. A combination of the two modes could provide improved estimates of fractional blood volume in comparison with either mode used alone.     

Dual imaging-guided photothermal/photodynamic therapy using micelles

We report a type of photosensitizer (PS)-loaded micelles integrating cyanine dye as potential theranostic micelles for precise anatomical tumor localization via dual photoacoustic (PA)/near-infrared fluorescent (NIRF) imaging modalities, and simultaneously superior cancer therapy via sequential synergistic photothermal therapy (PTT)/photodynamic therapy (PDT). The micelles exhibit enhanced photostability, cell internalization and tumor accumulation. The dual NIRF/PA imaging modalities of the micelles cause the high imaging contrast and spatial resolution of tumors, which provide precise anatomical localization of the tumor and its inner vasculature for guiding PTT/PDT treatments. Moreover, the micelles can generate severe photothermal damage on cancer cells and destabilization of the lysosomes upon PTT photoirradiation, which subsequently facilitate synergistic photodynamic injury via PS under PDT treatment. The sequential treatments of PTT/PDT trigger the enhanced cytoplasmic delivery of PS, which contributes to the synergistic anticancer efficacy of PS. Our strategy provides a dual-modal cancer imaging with high imaging contrast and spatial resolution, and subsequent therapeutic synergy of PTT/PDT for potential multimodal theranostic application.     

生物光声层析成像中超声探测器特性对图像质量的影响及解决方法

生物光声层析成像(PAT)是一种多物理场耦合的新型复合功能成像方法,对肿瘤和心血管疾病等的早期检测和准确诊断具有极高的研究价值。在PAT图像重建中,为了简化问题,通常假设超声探测器是具有全向响应的理想点探测器,在目标周围形成一个连续完整的测量面,且不考虑探测器的空间脉冲响应(SIR)和电脉冲响应(EIR)对成像质量的影响。但在实际应用中,这一理想假设会导致成像分辨率和图像质量的下降。从有限孔径效应、SIR和EIR、方向性、扫描半径、有限测量角度和有限带宽以及位置不确定性等6个方面,就超声探测器特性对图像重建的影响,综述相应的解决方法,对比不同方法的优缺点以及适用范围,并展望未来可能的发展方向。     

Intussusceptive microvascular growth in human glioma

Intussusceptive microvascular growth (IMG), which occurs by splitting of the existing vasculature by transluminal pillars or transendothelial bridges, has been demonstrated in several tumors such as colon and mammary carcinomas, melanoma and B-cell non-Hodgkin’s lymphomas. In this study, we have correlated in human glioma the extent of angiogenesis, evaluated as microvascular density, the immunoreactivity of tumor cells to vascular endothelial growth factor (VEGF), vessel diameter and IMG to the tumor stage. Results demonstrate for the first time a relationship in human glioma progression between angiogenesis, VEGF immunoreactivity of tumor cells, vessel diameter and the number of connections of intraluminal tissue folds with the opposite vascular wall, expression of IMG and suggest that IMG could be a mechanism of compensatory vascular growth occurring in human glioma. The advantages are that (1) blood vessels are generated more rapidly; (2) it is energetically and metabolically more economic; (3) the capillaries thereby formed are less leaky.     

An imaging-based stochastic model for simulation of tumour vasculature

A mathematical model which reconstructs the structure of existing vasculature using patient-specific anatomical, functional and molecular imaging as input was developed. The vessel structure is modelled according to empirical vascular parameters, such as the mean vessel branching angle. The model is calibrated such that the resultant oxygen map modelled from the simulated microvasculature stochastically matches the input oxygen map to a high degree of accuracy (R(2)?≈ 1). The calibrated model was successfully applied to preclinical imaging data. Starting from the anatomical vasculature image (obtained from contrast-enhanced computed tomography), a representative map of the complete vasculature was stochastically simulated as determined by the oxygen map (obtained from hypoxia [(64)Cu]Cu-ATSM positron emission tomography). The simulated microscopic vasculature and the calculated oxygenation map successfully represent the imaged hypoxia distribution (R(2)?= 0.94). The model elicits the parameters required to simulate vasculature consistent with imaging and provides a key mathematical relationship relating the vessel volume to the tissue oxygen tension. Apart from providing an excellent framework for visualizing the imaging gap between the microscopic and macroscopic imagings, the model has the potential to be extended as a tool to study the dynamics between the tumour and the vasculature in a patient-specific manner and has an application in the simulation of anti-angiogenic therapies.