Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging

Early diagnosis probes that combine fluorescence, X-ray computed tomography (CT) and magnetic resonance (MR) imagings are anticipated to give three dimensional (3D) details of tissues and cells of high resolution and sensitivity. However, how to combine these three modalities together within a sub-50 nm sized structure is technically challenging. Here we report a trimodal imaging probe of PEGylated NaY/GdF₄: Yb, Er, Tm @SiO₂-Au@PEG₅₀₀₀ nanopaticles of uniform size of less than 50 nm. The as-designed nanoprobes showed (1) strong emissions ranging from the visible (Vis) to near infrared (NIR) for fluorescent imaging, (2) T₁-weighted MRI by shorting T₁ relaxation time and (3) enhanced HU value as a CT contrast agent. The structure was optimized based on a comprehensive investigation on the influence of the distance between the NaY/GdF₄: Yb, Er, Tm core and Au nanoparticles (NPs) at the surface. The potential of trimodal imaging for cancerous cells and lesions was further demonstrated both in vitro and in vivo.     

动态荧光分子成像技术研究进展

动态荧光分子成像技术能够描述荧光分子探针在生物体内吸收、分布以及排出的完整过程,是一种可以对生物体的生理、病理过程进行连续监测的动态成像技术.这项技术具有无电离辐射、成像速度快、灵敏度及特异度高、费用低廉等优点,在基础医学及临床医学研究中具有广阔的应用前景.从系统、算法和应用3个方面对动态荧光分子成像技术的研究进展进行了综述.     

Simultaneous calcium fluorescence imaging and MR of ex vivo organotypic cortical cultures: a new test bed for functional MRI

Recently, several new functional (f)MRI contrast mechanisms including diffusion, phase imaging, proton density, etc. have been proposed to measure neuronal activity more directly and accurately than blood‐oxygen‐level dependent (BOLD) fMRI. However, these approaches have proved difficult to reproduce, mainly because of the dearth of reliable and robust test systems to vet and validate them. Here we describe the development and testing of such a test bed for non‐BOLD fMRI. Organotypic cortical cultures were used as a stable and reproducible biological model of neuronal activity that shows spontaneous activity similar to that of in vivo brain cortex without any hemodynamic confounds. An open‐access, single‐sided magnetic resonance (MR) “profiler” consisting of four permanent magnets with magnetic field of 0.32 T was used in this study to perform MR acquisition. A fluorescence microscope with long working distance objective was mounted on the top of a custom‐designed chamber that keeps the organotypic culture vital, and the MR system was mounted on the bottom of the chamber to achieve real‐time simultaneous calcium fluorescence optical imaging and MR acquisition on the same specimen. In this study, the reliability and performance of the proposed test bed were demonstrated by a conventional CPMG MR sequence acquired simultaneously with calcium imaging, which is a well‐characterized measurement of neuronal activity. This experimental design will make it possible to correlate directly the other candidate functional MR signals to the optical indicia of neuronal activity in the future. Copyright © 2015 John Wiley & Sons, Ltd.     

基于荧光分子断层成像的多模成像系统研究进展

基于荧光分子断层成像（FMT）的多模成像系统已广泛应用于动物实验研究,它将现有的其他成像模态与FMT系统相融合,实现同时对被测生物解剖学结构、生理学功能和分子或细胞水平生物学活动的在体成像.首先介绍了单模FMT系统的发展历史和现状,并介绍了国内外基于FMT的多模系统的研究进展,着重介绍了文献报道的典型的FMT/CT、FMT/MRI和FMT/放射性核素成像系统的系统组成、工作原理、性能特点及实验应用.对基于FMT的多模成像系统的发展进行了展望.     

Assessing microvessels and angiogenesis in human breast cancer, using VE-cadherin

AIMS: Vascular endothelial (VE)-cadherin, also known as cadherin-5 and CD144, is an adhesion molecule uniquely expressed in endothelial cells. We hypothesized that VE-cadherin may be a useful marker for assessing microvessels and angiogenesis in human breast cancer and sought to determine whether a correlation exists between levels of VE-cadherin, angiogenic markers factor VIII and platelet endothelial cell adhesion molecule (PECAM)-1 and patient outcome in breast cancer. METHODS AND RESULTS: Frozen sections from breast cancer primary tumours (tumour n = 114, background n = 30) were immunostained with VE-cadherin, factor VIII and PECAM-1 antibodies and microvessel number was assessed. RNA was reverse transcribed and analysed by quantitative polymerase chain reaction (Q-PCR). VE-cadherin immunostaining showed a significant difference in microvessel number in tumour compared with background. There was no significant difference in the number of microvessels stained with PECAM-1 or factor VIII; there was increased staining of other structures within the sample and higher general background staining. Q-PCR revealed elevated levels of VE-cadherin and PECAM-1 in tumour samples compared with background tissue and in patients with a poor prognosis, as determined by the Nottingham Prognostic Index. There was no difference in levels with factor VIII. Both VE-cadherin and PECAM-1 had significantly reduced expression in lobular compared with ductal carcinomas: there was no difference with factor VIII. CONCLUSION: Higher levels of angiogenic marker molecules in breast cancer may have an association with poor prognosis in patients. Moreover, VE-cadherin appears to be a preferable marker for such analysis.     

肝原位移植瘤裸鼠模型的建立及活体荧光成像检测

目的 建立能够通过活体荧光成像系统实时监测肿瘤进展的肝原位移植瘤裸鼠模型.方法 重组质粒pcDNA3.1-Luc转染细胞构建稳定表达荧光素酶的PLC/PRF/5肝癌细胞株,将该细胞注入裸鼠肝脏实质内,应用活体成像技术动态监测肿瘤进展情况,解剖荧光信号阳性裸鼠观察肿瘤组织生长情况.免疫荧光检测肿瘤组织中HBsAg表达.结果 对裸鼠肝原位移植瘤应用活体荧光系统成像,在肿瘤细胞注射部位检测到荧光信号,解剖动物后可在肝脏组织中观察到异质细胞团块.免疫荧光证实移植瘤中有HBsAg表达.结论 肝脏原位移植瘤裸鼠模型建立成功,为抗肝癌药物的研发提供了工具.     

Cancer metabolism in a snapshot: MRS(I)

The contribution of MRS(I) to the in vivo evaluation of cancer‐metabolism‐derived metrics, mostly since 2016, is reviewed here. Increased carbon consumption by tumour cells, which are highly glycolytic, is now being sampled by ¹³C magnetic resonance spectroscopic imaging (MRSI) following the injection of hyperpolarized [1‐¹³C] pyruvate (Pyr). Hot‐spots of, mostly, increased lactate dehydrogenase activity or flow between Pyr and lactate (Lac) have been seen with cancer progression in prostate (preclinical and in humans), brain and pancreas (both preclinical) tumours. Therapy response is usually signalled by decreased Lac/Pyr ¹³C‐labelled ratio with respect to untreated or non‐responding tumour. For therapeutic agents inducing tumour hypoxia, the ¹³C‐labelled Lac/bicarbonate ratio may be a better metric than the Lac/Pyr ratio. ³¹P MRSI may sample intracellular pH changes from brain tumours (acidification upon antiangiogenic treatment, basification at fast proliferation and relapse). The steady state tumour metabolome pattern is still in use for cancer evaluation. Metrics used for this range from quantification of single oncometabolites (such as 2‐hydroxyglutarate in mutant IDH1 glial brain tumours) to selected metabolite ratios (such as total choline to N‐acetylaspartate (plain ratio or CNI index)) or the whole ¹H MRSI(I) pattern through pattern recognition analysis. These approaches have been applied to address different questions such as tumour subtype definition, following/predicting the response to therapy or defining better resection or radiosurgery limits.     

MR and fluorescence imaging of gadobutrol-induced optical clearing of red fluorescent protein signal in an in vivo cancer model

Multimodality registration of optical and MR images in the same tissue volume in vivo may be enabled by MR contrast agents with an optical clearing (OC) effect. The goals of this study were to (a) investigate the effects of clinical MR contrast agent gadobutrol (GB) and its combinations as a potential OC agent assisting in fluorescence intensity (FI) imaging in vivo and (b) evaluate MRI as a tool for imaging of topical or systemic application of GB for the purpose of OC. Subcutaneous tumor xenografts expressing red fluorescent marker protein were used as disease models. MRI was performed at 1 T ¹H MRI using T₁-weighted 3D gradient-echo (T1w-3D GRE) sequences to measure time-dependent MR signal intensity changes by region of interest analysis after image segmentation. Topical application of 1.0 M or 0.7 M GB-containing OC mixture with water and dimethyl sulfoxide showed similar 30–40% increases of tumor FI during the initial 15 min. Afterwards, the OC effect of GB on FI and tumor/background FI ratio showed a decrease over time in the case of 1.0 M GB, unlike the 0.7 M GB mixture, which resulted in a steady increase of FI and tumor/background ratio for 15–60 min. The use of T1w-3D GRE MR pulse sequences showed that concentrated 1.0 M GB resulted in MR signal loss of the skin due to high magnetic susceptibility and that signal loss coincided with the OC effect. Intravenous injection of 0.3 mmol GB/kg resulted in a rapid but transient 40% increase of FI of the tumors. Overall, 1 T MRI enabled tracking of GB-containing OC compositions on the skin surface and tumor tissue, supporting the observation of a time-dependent FI increase in vivo.     

3.0T超导型MRI灌注加权成像联合动态增强扫描技术在乳腺早期良恶性病变鉴定中的研究

目的探讨3.0 T超导型MRI灌注加权成像(PWI)联合动态增强扫描(DCE)在乳腺早期良恶性病变鉴定中的价值。方法选择术后经病理确诊为良恶性的乳腺早期病变女性患者61例,年龄24~65岁,平均年龄30.12岁。所有患者均经3.0 T超导型MRI PWI常规T2加权成像(T2WI)和T1加权成像(T1WI)平扫后行三维(3D)动态增强扫描技术,并根据病理结果分为恶性病变和良性病变,对比病变形态学变化、时间-信号强度曲线(TIC)及表观弥散系数(ADC)值,并分析PWI联合DCE对乳腺早期良恶性病变鉴别诊断价值。结果病理结果为恶性病变27例,良性病变34例;DCE-MRI扫描结果为恶性病变患者20例,良性病变患者26例,病变检出率75.41%;PWI扫描结果为恶性病变患者21例,良性病变患者27例,病变检出率78.69%。乳腺早期良性病变形态以类圆形(76.5%)、边缘以光滑(70.6%)为主,乳腺早期恶性病变形态以分叶形(63.0%)、边缘以毛刺征(59.3%)为主;乳腺早期良恶性病变DCE-MRI扫描形态学特征对比,差异有显著统计学意义(χ^2=43.557、37.459,P=0.000、0.000)。乳腺早期良性病变TIC形态以Ⅰ型(61.8%)为主,乳腺早期恶性病变TIC形态以Ⅲ型(77.8%)为主,两者比较,差异有显著统计学意义(χ^2=121.852,P=0.000);22例(81.5%)恶性病变患者ADC值≤1.195×10-3 mm2/s,28例(82.4%)良性病变患者ADC值>1.195×10-3 mm2/s,两者差异有显著统计学意义(χ2=26.148,P=0.000)。二者联合鉴别诊断乳腺早期良恶性病变的灵敏度、特异度及准确度与DCE-MRI、PWI单一诊断更高(P<0.05)。结论 3.0 T超导型MRI PWI联合DCE在乳腺早期良恶性病变鉴定中具有较高的临床价值。     

细胞标记与体内追踪成像：在动物和人类中应用的最新进展

文题释义：细胞治疗：采用生物工程方法获取细胞,再通过体外扩增、特殊培养等处理后,使这些细胞具有增强免疫、杀死病原体和肿瘤细胞、促进组织器官再生和机体康复等治疗功效,从而达到治疗疾病的目的。 分子影像学：运用影像学对活体状态下的生物过程进行细胞和分子水平的定性和定量研究。以药物分子作为探针进行成像,能够得到药物的药代动力学、生物分布、靶向性、毒性等众多信息,从而帮助缩短药物研发周期、降低失败风险。 背景：随着国内外多个细胞治疗产品获得临床试验批准,细胞治疗例如干细胞治疗、肿瘤过继免疫疗法得到广泛关注。体内细胞实时观察与成像可以直观显示细胞的分布,追踪细胞的活动,监测细胞的活性,观察细胞的迁移和生长。目前的许多成像技术可以使体内细胞可视化,例如超声、光学、磁共振成像以及核成像技术,这些方法均需对应不同的标记和检测策略,每种策略都有其固有的优点和缺点。 目的：结合最新的研究动态,将对不同细胞追踪方法的原理、发展和这些方法在动物和人类中应用的最新进展进行综述。 方法：第一作者以“cell tracking,in vivo cell tracking,PET imaging,MRI,optical imaging”为关键词在PubMed、Google Scholar、Web of Science、中国知网等数据库进行检索,重点关注过去5-10年的相关文章。文章内容主要描述不同细胞追踪方法的原理以及在动物模型和临床患者体内的细胞追踪应用。 结果与结论：在过去的二十几年中,细胞追踪已经发展成为一个多方面的学科,不仅在动物模型中建立了多种稳健的方法,并且在人类的一些研究中证明了临床转化的可行性。尤其是以PET、MRI成像技术为代表的无创检测,新型对比剂的研发,为细胞治疗在临床与科研的应用提供了强有力的支持。 ORCID: 0000-0002-3764-0154(徐梦欣) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Antibody-directed neutralization of annexin II (ANX II) inhibits neoangiogenesis and human breast tumor growth in a xenograft model

Activation of the fibrinolytic pathway has long been associated with human breast cancer. Plasmin is the major end product of the fibrinolytic pathway and is critical for normal physiological functions. The mechanism by which plasmin is generated in breast cancer is not yet fully described. We previously identified annexin II (ANX II), a fibrinolytic receptor, in human breast tumor tissue samples and observed a strong positive correlation with advanced stage cancer (Sharma et al., 2006a). We further demonstrated that tissue plasminogen activator (tPA) binds to ANX II in invasive breast cancer MDA-MB231cells, which leads to plasmin generation (Sharma et al., 2010). We hypothesize that ANX II-dependent plasmin generation in breast tumor is necessary to trigger the switch to neoangiogenesis, thereby stimulating a more aggressive cancer phenotype. Our immunohistochemical studies of human breast tumor tissues provide compelling evidence of a strong positive correlation between ANX II expression and neoangiogenesis, and suggest that ANX II is a potential target to slow or inhibit breast tumor growth by inhibiting neoangiogenesis. We now report that administration of anti-ANX II antibody potently inhibits the growth of human breast tumor in a xenograft model. Inhibition of tumor growth is at least partly due to attenuation of neoangiogenic activity within the tumor. In vitro studies demonstrate that anti-ANX II antibody inhibits angiogenesis on three dimensional matrigel cultures by eliciting endothelial cell (EC) death likely due to apoptosis. Taken together, these data suggest that selective disruption of the fibrinolytic activity of ANX II may provide a novel strategy for specific inhibition of neoangiogenesis in human breast cancer.     

Near-infrared fluorescence heptamethine carbocyanine dyes mediate imaging and targeted drug delivery for human brain tumor

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood–brain and blood–tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.     

Carbon nanotube lipid drug approach for targeted delivery of a chemotherapy drug in a human breast cancer xenograft animal model

Carbon nanotube (CNT) possesses excellent properties as a drug carrier. To overcome the challenge of drug functionalization with CNT, we have developed a lipid-drug approach for efficient drug loading onto CNT, in which a long chain lipid molecule is conjugated to the drug molecule so that the lipid-drug can be loaded directly onto CNT through binding of the lipid ‘tail’ in the drug molecule to CNT surfaces via hydrophobic interactions. In a proof-of-concept study, drug paclitaxel (PTX) was conjugated with a non-toxic lipid molecule docosanol for functionalization with CNT. Folic acid was also conjugated to CNT for targeted drug delivery. High level of drug loading onto SWNT could be achieved by lipid-drug approach. Conjugation of FA to SWNT-lipid-PTX led to an increase in cell penetration capacity, and the targeted SWNT-lipid-PTX showed much improved drug efficacy in vitro in comparison to free drug Taxol and non-targeted SWNT-lipid-PTX at 48 h (78.5% vs. 31.6% and 59.1% in cytotoxicity respectively, p < 0.01). In vivo analysis using a human breast cancer xenograft mice model also confirmed the improved drug efficacy. The targeted SWNT-lipid-PTX was found non-toxic as evaluated by biochemical analysis using blood samples, and by histological analysis of major organs.     

Optimization of arterial spin labeling MRI for quantitative tumor perfusion in a mouse xenograft model

Perfusion is an important biomarker of tissue function and has been associated with tumor pathophysiology such as angiogenesis and hypoxia. Arterial spin labeling (ASL) MRI allows noninvasive and quantitative imaging of perfusion; however, the application in mouse xenograft tumor models has been challenging due to the low sensitivity and high perfusion heterogeneity. In this study, flow‐sensitive alternating inversion recovery (FAIR) ASL was optimized for a mouse xenograft tumor. To assess the sensitivity and reliability for measuring low perfusion, the lumbar muscle was used as a reference region. By optimizing the number of averages and inversion times, muscle perfusion as low as 32.4 ± 4.8 (mean ± standard deviation) ml/100 g/min could be measured in 20 min at 7 T with a quantification error of 14.4 ± 9.1%. Applying the optimized protocol, heterogeneous perfusion ranging from 49.5 to 211.2 ml/100 g/min in a renal carcinoma was observed. To understand the relationship with tumor pathology, global and regional tumor perfusion was compared with histological staining of blood vessels (CD34), hypoxia (CAIX) and apoptosis (TUNEL). No correlation was observed when the global tumor perfusion was compared with these pathological parameters. Regional analysis shows that areas of high perfusion had low microvessel density, which was due to larger vessel area compared with areas of low perfusion. Nonetheless, these were not correlated with hypoxia or apoptosis. The results suggest that tumor perfusion may reflect certain aspect of angiogenesis, but its relationship with other pathologies needs further investigation. Copyright © 2015 John Wiley & Sons, Ltd.     

Longitudinal absolute metabolite quantification of white and gray matter regions in healthy controls using proton MR spectroscopic imaging

The purpose of this study was to evaluate quality parameters, metabolite concentrations and concentration ratios, and to investigate the reproducibility of quantitative proton magnetic resonance spectroscopic imaging (¹H‐MRSI) of selected white and gray matter regions of healthy adults. 2D‐quantitative short‐T_E ¹H‐MRSI spectra were obtained at 1.5T from the healthy human brain. Subjects (n = 12) were scanned twice with an interval of six months. Absolute metabolite concentrations were obtained based on coil loading, taking into account differences in sensitivity of the phased‐array head coil. Spectral quality parameters, absolute metabolite concentrations, concentration ratios, and their reproducibility were determined and compared between time‐points using a repeated measures general linear model. The quality of the spectra of selected brain areas was good, as determined by a mean spectral linewidth between 4.8 and 7.3 Hz (depending on the region). No significant differences between the two time‐points were observed for spectral quality, concentrations, or concentration ratios. The mean intrasubject coefficient of variation (CoV) varied between 4.0 and 8.5% for total N‐acetylaspartate, 7.2 and 10.8% for total creatine, 5.9 and 9.8% for myo‐inositol, and 8.0 and 13.3% for choline, and remained below 20% for glutamate. CoV was generally lower when concentration ratios were considered. The study shows that longitudinal quantitative short‐T_E ¹H‐MRSI generates reproducible absolute metabolite concentrations in healthy human white and gray matter. This may serve as a background for longitudinal clinical studies in adult patients. Copyright © 2014 John Wiley & Sons, Ltd.