容积减影成像计算机自动化技术与实现

探讨在PC机上对容积CT灌注（CTP）源影像全自动生成减影CTP源影像的方法。首先在WindowsXP操作系统中使用Matlab,对4层螺旋CTP源影像进行DICOM文件格式解析,获得CT成像设备的球管每转一周扫描层数及容积减影方法需要的其它参数信息,然后经过容积的CT值集合读、容积的CT值集合减以及容积的CT值集合写等几个步骤,生成减影CTP源影像的DICOM文件序列。使用该容积减影方法得到的减影CTP源影像,在定性肉眼观察和定量CT值矩阵比对上,与使用CT影像工作站上的减影软件手工逐层半自动减影得到的结果类同。容积减影方法实现了容积CTP源影像的自动化减影处理与成像,为最终形成一个一站式的减影CTP软件奠定了基础。     

Monitoring Brain Tumor Vascular Heamodynamic following Anti-Angiogenic Therapy with Advanced Magnetic Resonance Imaging in Mice

Advanced MR imaging methods have an essential role in classification, grading, follow-up and therapeutic management in patients with brain tumors. With the introduction of new therapeutic options, the challenge for better tissue characterization and diagnosis increase, calling for new reliable non-invasive imaging methods. In the current study we evaluated the added value of a combined protocol of blood oxygen level dependent (BOLD) imaging during hyperoxic challenge (termed hemodynamic response imaging (HRI)) in an orthotopic mouse model for glioblastoma under anti-angiogenic treatment with B20-4.1.1, an anti-VEGF antibody. In glioblastoma tumors, the elevated HRI indicated progressive angiogenesis as further confirmed by histology. In the current glioblastoma model, B20-treatment caused delayed tumor progression with no significant changes in HRI yet with slightly reduced tumor vascularity as indicated by histology. Furthermore, fewer apoptotic cells and higher proliferation index were detected in the B20-treated tumors compared to control-treated tumors. In conclusion, HRI provides an easy, safe and contrast agent free method for the assessment of the brain hemodynamic function, an additionally important clinical information.     

对比剂动力学时间分辨成像及弥散加权成像技术活体动态监测兔VX2肌肉肿瘤生物学生长与血管生成

目的:应用对比剂动力学时间分辨成像(Time Resolved Imaging of Contrast Kinetics,TRICKS)技术增强磁共振血管成像(MRangiography,MRA)及弥散加权成像(Diffusion Weighted Imaging,DWI)技术活体动态监测兔VX2肌肉肿瘤生物学生长与血管生成,探讨肿瘤血管生成与肿瘤生长的关系。方法:30只新西兰白兔,每只均在右后腿肌肉内接种VX2肿瘤细胞1×107建立肿瘤模型。分别在肿瘤接种后第4、7、10、13、16天(每个时间点6只)分别进行T1WI、T2WI、DWI、TRICKS动态增强MRA及T1WI增强延迟扫描,活体监测兔VX2肌肉肿瘤血管生成,肿瘤标本HE及CD31免疫组化染色进行验证。两位医师双盲法分别测量不同生长点肿瘤的长、短径及体积,并与大体病理标本比较;测定TRICKS增强动态MRA所能显示肿瘤血管的最小直径及形态变化;观察ADC值变化与肿瘤生长的关系。结果(:1)ADC值随着肿瘤体积的长大而逐渐增大。(2)MRI活体测定肿瘤大小与病理大体标本所测算肿瘤体积的差异无显著性。(3)TRICKS增强MRA动态显示肿瘤血管的最小...     

Monitoring Prostate Tumor Growth in an Orthotopic Mouse Model Using Three-Dimensional Ultrasound Imaging Technique

Prostate cancer (CaP) is the most commonly diagnosed and the second leading cause of death from cancer in males in USA. Prostate orthotopic mouse model has been widely used to study human CaP in preclinical settings. Measurement of changes in tumor size obtained from noninvasive diagnostic images is a standard method for monitoring responses to anticancer modalities. This article reports for the first time the usage of a three-dimensional (3D) ultrasound system equipped with photoacoustic (PA) imaging in monitoring longitudinal prostate tumor growth in a PC-3 orthotopic NODSCID mouse model (n = 8). Two-dimensional and 3D modes of ultrasound show great ability in accurately depicting the size and shape of prostate tumors. PA function on two-dimensional and 3D images showed average oxygen saturation and average hemoglobin concentration of the tumor. Results showed a good fit in representative exponential tumor growth curves (n = 3; r² = 0.948, 0.955, and 0.953, respectively) and a good correlation of tumor volume measurements performed in vivo with autopsy (n = 8, r = 0.95, P < .001). The application of 3D ultrasound imaging proved to be a useful imaging modality in monitoring tumor growth in an orthotopic mouse model, with advantages such as high contrast, uncomplicated protocols, economical equipment, and nonharmfulness to animals. PA mode also enabled display of blood oxygenation surrounding the tumor and tumor vasculature and angiogenesis, making 3D ultrasound imaging an ideal tool for preclinical cancer research.     

Tumor Volume Can Be Used as a Parameter Indicating the Severity of Disease in Parathyroid Cancer

ObjectiveTo determine whether tumor volume and tumor size are related to disease severity in parathyroid cancer (PC).MethodsPatients treated for PC at our institution were retrospectively identified. Data were collected about clinical and pathologic characteristics, laboratory parameters, tumor volume, recurrence, metastasis, and mortality. Correlation analysis was applied to laboratory parameters, tumor volume, and tumor size in PC patients.ResultsThe study included 20 patients diagnosed with PC at our center. The median follow-up was 33 months. Serum calcium (median, 12.5 mg/dL), serum parathormone (PTH) (median, 743 pg/mL), and serum alkaline phosphatase (ALP) (median, 298 U/L) levels were found to be increased, and 25-hydroxyvitamin D (25[0H)D) (median, 12.3 ng/mL) and serum phosphorus (median, 2.1 mg/dL) levels were decreased. Magnesium level was within normal limits (median, 1.9 mg/dL). The median tumor volume was 5.7 mL and median tumor size was 2.5 cm. Significant positive correlations were found between tumor volume and calcium, ALP, and PTH levels. A significant negative correlation was found between tumor volume and 25(OH)D level. There were no significant correlations between tumor size and calcium, ALP, PTH, and 25(OH)D.ConclusionThese results found that the tumor volume affected PTH, calcium, ALP, and 25(OH)D levels. The morbidity and mortality associated with PC were usually associated with PTH secretion and hypercalcemia. Therefore, tumor volume may be a more effective parameter than tumor size when evaluating the severity of disease.     

Tumor Volume as an Alternative Response Measurement for Imatinib Treated GIST Patients

Background

Assessment of tumor size changes is crucial in clinical trials and patient care. We compared imatinib-induced volume changes of liver metastases (LM) from gastro-intestinal stromal tumors (GIST) to RECIST and Choi criteria and their association with overall survival (OS).

Methods

LM from 84 GIST patients (training and validation set) were evaluated using manual and semi-automated Computed Tomography measurements at baseline, after 3, 6 and 12 months of imatinib. The ability of uni-dimensional (1D) and three-dimensional (3D) measurements to detect size changes (increase/decrease) ≥20% was evaluated. Volumetric response cut-offs were derived from minimally relevant changes (+20/−30%) by RECIST, considering lesions as spherical or ellipsoidal.

Results

3D measurements detected size changes ≥20% more frequently than 1D at every time-point (P≤0.008). 3D and Choi criteria registered more responses than RECIST at 3 and 6 months for 3D-spheres (P≤0.03) and at all time-points for 3D-ellipsoids and Choi criteria (P<0.001). Progressive disease by 3D criteria seems to better correlate to OS at late time-points than other criteria.

Conclusion

Volume criteria (especially ellipsoids) classify a higher number of patients as imatinib-responders than RECIST. Volume discriminates size changes better than diameter in GIST and constitutes a feasible and robust method to evaluate response and predict patient benefit.     

Cyanine 5.5 Conjugated Nanobubbles as a Tumor Selective Contrast Agent for Dual Ultrasound-Fluorescence Imaging in a Mouse Model

Nanobubbles and microbubbles are non-invasive ultrasound imaging contrast agents that may potentially enhance diagnosis of tumors. However, to date, both nanobubbles and microbubbles display poor in vivo tumor-selectivity over non-targeted organs such as liver. We report here cyanine 5.5 conjugated nanobubbles (cy5.5-nanobubbles) of a biocompatible chitosan–vitamin C lipid system as a dual ultrasound-fluorescence contrast agent that achieved tumor-selective imaging in a mouse tumor model. Cy5.5-nanobubble suspension contained single bubble spheres and clusters of bubble spheres with the size ranging between 400–800 nm. In the in vivo mouse study, enhancement of ultrasound signals at tumor site was found to persist over 2 h while tumor-selective fluorescence emission was persistently observed over 24 h with intravenous injection of cy5.5-nanobubbles. In vitro cell study indicated that cy5.5-flurescence dye was able to accumulate in cancer cells due to the unique conjugated nanobubble structure. Further in vivo fluorescence study suggested that cy5.5-nanobubbles were mainly located at tumor site and in the bladder of mice. Subsequent analysis confirmed that accumulation of high fluorescence was present at the intact subcutaneous tumor site and in isolated tumor tissue but not in liver tissue post intravenous injection of cy5.5-nanobubbles. All these results led to the conclusion that cy5.5-nanobubbles with unique crosslinked chitosan–vitamin C lipid system have achieved tumor-selective imaging in vivo.     

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body’s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).     

Engineering Agatoxin,a Cystine-Knot Peptide from Spider Venom,as a Molecular Probe for In Vivo Tumor Imaging

Background

Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor.

Methodology/Principal Findings

We used a rational loop-grafting approach to engineer AgTx variants that bound to α_vβ₃ integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants.Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI.Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins.

Conclusions/Significance

In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.     

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.     

Familial Hyperaldosteronism Type 3 with a Rapidly Growing Adrenal Tumor: An In Situ Aldosterone Imaging Study

Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel KCNJ5. Germ line mutations of KCNJ5 cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic β-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings.     

Multimodality Multiparametric Imaging of Early Tumor Response to a Novel Antiangiogenic Therapy Based on Anticalins

Anticalins are a novel class of targeted protein therapeutics. The PEGylated Anticalin Angiocal (PRS-050-PEG40) is directed against VEGF-A. The purpose of our study was to compare the performance of diffusion weighted imaging (DWI), dynamic contrast enhanced magnetic resonance imaging (DCE)-MRI and positron emission tomography with the tracer [¹⁸F]fluorodeoxyglucose (FDG-PET) for monitoring early response to antiangiogenic therapy with PRS-050-PEG40. 31 mice were implanted subcutaneously with A673 rhabdomyosarcoma xenografts and underwent DWI, DCE-MRI and FDG-PET before and 2 days after i.p. injection of PRS-050-PEG40 (n = 13), Avastin (n = 6) or PBS (n = 12). Tumor size was measured manually with a caliper. Imaging results were correlated with histopathology. In the results, the tumor size was not significantly different in the treatment groups when compared to the control group on day 2 after therapy onset (P = 0.09). In contrast the imaging modalities DWI, DCE-MRI and FDG-PET showed significant differences between the therapeutic compared to the control group as early as 2 days after therapy onset (P<0.001). There was a strong correlation of the early changes in DWI, DCE-MRI and FDG-PET at day 2 after therapy onset and the change in tumor size at the end of therapy (r = −0.58, 0.71 and 0.67 respectively). The imaging results were confirmed by histopathology, showing early necrosis and necroptosis in the tumors. Thus multimodality multiparametric imaging was able to predict therapeutic success of PRS-050-PEG40 and Avastin as early as 2 days after onset of therapy and thus promising for monitoring early response of antiangiogenic therapy.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG_2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.     

Quantitative Multi-Parametric Magnetic Resonance Imaging of Tumor Response to Photodynamic Therapy

ObjectiveThe aim of this study was to characterize response to photodynamic therapy (PDT) in a mouse cancer model using a multi-parametric quantitative MRI protocol and to identify MR parameters as potential biomarkers for early assessment of treatment outcome.MethodsCT26.WT colon carcinoma tumors were grown subcutaneously in the hind limb of BALB/c mice. Therapy consisted of intravenous injection of the photosensitizer Bremachlorin, followed by 10 min laser illumination (200 mW/cm²) of the tumor 6 h post injection. MRI at 7 T was performed at baseline, directly after PDT, as well as at 24 h, and 72 h. Tumor relaxation time constants (T₁ and T₂) and apparent diffusion coefficient (ADC) were quantified at each time point. Additionally, Gd-DOTA dynamic contrast-enhanced (DCE) MRI was performed to estimate transfer constants (K^trans) and volume fractions of the extravascular extracellular space (v_e) using standard Tofts-Kermode tracer kinetic modeling. At the end of the experiment, tumor viability was characterized by histology using NADH-diaphorase staining.ResultsThe therapy induced extensive cell death in the tumor and resulted in significant reduction in tumor growth, as compared to untreated controls. Tumor T₁ and T₂ relaxation times remained unchanged up to 24 h, but decreased at 72 h after treatment. Tumor ADC values significantly increased at 24 h and 72 h. DCE-MRI derived tracer kinetic parameters displayed an early response to the treatment. Directly after PDT complete vascular shutdown was observed in large parts of the tumors and reduced uptake (decreased K^trans) in remaining tumor tissue. At 24 h, contrast uptake in most tumors was essentially absent. Out of 5 animals that were monitored for 2 weeks after treatment, 3 had tumor recurrence, in locations that showed strong contrast uptake at 72 h.ConclusionDCE-MRI is an effective tool for visualization of vascular effects directly after PDT. Endogenous contrast parameters T₁, T₂, and ADC, measured at 24 to 72 h after PDT, are also potential biomarkers for evaluation of therapy outcome.