Optical contrast agents and imaging systems for detection and diagnosis of cancer

Molecular imaging has rapidly emerged as a discipline with the potential to impact fundamental biomedical research and clinical practice. Within this field, optical imaging offers several unique capabilities, based on the ability of cells and tissues to effect quantifiable changes in the properties of visible and near-infrared light. Beyond endogenous optical properties, the development of molecularly targeted contrast agents enables disease-specific morphologic and biochemical processes to be labeled with unique optical signatures. Optical imaging systems can then provide real-time visualization of pathophysiology at spatial scales from the subcellular to whole organ levels. In this article, we review fundamental techniques and recent developments in optical molecular imaging, emphasizing laboratory and clinical systems that aim to visualize the microscopic and macroscopic hallmarks of cancer.     

Fluorescence lifetime imaging microscopy of chemotherapy‐induced apoptosis resistance in a syngenic mouse tumor model

During cancer therapy with DNA‐damaging drug‐agents, the development of secondary resistance to apoptosis can be observed. In the search for novel therapeutic approaches that can be used in these cases, we monitored chemotherapy‐induced apoptosis resistance in a syngenic mouse tumor model. For this, syngenic murine colorectal carcinoma cells, which stably expressed a FRET‐based caspase‐3 activity sensor, were introduced into animals to induce peritoneal carcinomatosis or disseminated hepatic metastases. This syngenic system allowed in vitro, in vivo and ex vivo analysis of chemotherapy induced apoptosis induction by optically monitoring the caspase‐3 sensor state in the tumor cells. Tumor tissue analysis of 5‐FU treated mice showed the selection of 5‐FU‐induced apoptosis resistant tumor cells. These and chemo‐naive fluorescent tumor cells could be re‐isolated from treated and untreated mice and propagated in cell culture. Re‐exposure to 5‐FU and second line treatment modalities in this ex‐vivo setting showed that 5‐FU induced apoptosis resistance could be alleviated by imatinib mesylate (Gleevec). We thus show that syngenic mouse systems that stably express a FRET‐based caspase‐3 sensor can be employed to analyse the therapeutic efficiency of apoptosis inducing chemotherapy.     

Evaluation of optical imaging agents in a fluorescence-guided surgical model of head and neck cancer

Background

Tumor proliferation often occurs from pathologic receptor upregulation. These receptors provide unique targets for near-infrared (NIR) probes that have fluorescence-guided surgery (FGS) applications. We demonstrate the use of three smart-targeted probes in a model of head and neck squamous cell carcinoma.

Tumor detection using folate receptor-targeted imaging agents

Folate receptors are up-regulated on a variety of human cancers, including cancers of the breast, ovaries, endometrium, lungs, kidneys, colon, brain, and myeloid cells of hematopoietic origin. This over-expression of folate receptors (FR) on cancer tissues can be exploited to target folate-linked imaging and therapeutic agents specifically to FR-expressing tumors, thereby avoiding uptake by most healthy tissues that express few if any FR. Four folate-targeted therapeutic drugs are currently undergoing clinical trials, and several folate-linked chemotherapeutic agents are in late stage preclinical development. However, because not all cancers express FR, and because only FR-expressing cancers respond to FR-targeted therapies, FR-targeted imaging agents have been required to select patients with FR-expressing tumors likely to respond to folate-targeted therapies. This review focuses on recent advances in the use of the vitamin folic acid to target PET agents, γ-emitters, MRI contrast agents and fluorescent dyes to FR⁺ cancers for the purpose of diagnosing and imaging malignant masses with improved specificity and sensitivity.     

靶向CXCR4特异性造影剂在骨肉瘤成像中的应用

目的：对骨肉瘤中趋化因子受体4（chemokine receptor 4,CXCR4）的表达进行非损伤性评价,探讨CXCR4特异性造影刺在骨肉瘤成像及早期诊断中的作用。方法：采用化学合成的方法得到了带有CXCR4特异性造影剂的l条肽段,通过近红外染料标记,进行骨肉瘤细胞SOSP-9607的体外结合试验;皮下接种裸鼠．构建骨肉瘤的异体移植模型,7d后对裸鼠进行体内光学成像,并用免疫组织化学法检测发光团组织中CXCR4的表达。结果：体外细胞结合试验证明合成的近红外造影剂能够很好的与人的骨肉瘤细胞SOSP-9607相结合。近红外光学成像表明。靶向CXCR4的特异性造影剂可用于小动物骨肉瘤的早期和晚期显影。免疫组化染色显示．发光团组织中CXCR4表达呈阳性。结论：趋化因子受体CXCR4靶标特异性分子造影剂,可用于早期和晚期骨肉瘤的显影,对骨肉瘤的诊断及其预后意义重大。     

前列腺癌正电子显像剂的临床研究及应用进展

PET/CT及PET/MR近年来已被应用于前列腺癌的临床检测。通过应用不同的显像剂，PET/CT及PET/MR可以评估前列腺癌的糖代谢、脂质代谢、受体变化等生物学改变，在前列腺癌的诊断、指导治疗及预后评估等方面相对于常规检测手段有独特的优势和良好的效果。近年来，许多新的放射性标记物不断被引入临床，给前列腺癌的诊断及治疗带来更广阔的应用前景。本文主要对前列腺癌正电子显像剂的临床研究及应用进展进行综述。     

活体动物体内成像技术及其在生物医学中的应用进展

活体动物体内成像是近年来新兴的检测活体动物体内基因表达及细胞活动的光学成像技术，具有操作简便，直观性强的特点。这项技术包括生物发光成像和荧光成像，采用报告基因产生的生物发光、荧光蛋白质或染料产生的荧光作为体内生物光源，与新型冷CCD成像相结合，实时探测活体动物体内生理或病理条件下的细胞和分子事件。本文简要综述了活体动物体内成像技术的原理、应用领域及发展前景。     

Fluorescence tumor imaging by i.v. administered indocyanine green in a mouse model of colitis‐associated colon cancer

Fluorescence tumor imaging using exogenous fluorescent tumor‐targeting agents has potential to improve early tumor detection. The fluorescent contrast agent indocyanine green (ICG) is used in medical diagnostics. The aim of the present study is to investigate the tumor imaging capability and the imaging mechanism of i.v. administered ICG in a mouse model of colitis‐associated colon cancer. To do this, an azoxymethane/dextran sodium sulfate‐induced colon cancer mouse model was used. Ex vivo imaging experiments were carried out 1 hour after i.v. injection of ICG. The ICG fluorescence was observed in the colon tumor tissues, with sufficient tumor to normal tissue ratio, correlating with tumor malignancy. In the tumor tissues, ICG fluorescence was localized in the vascular interstitial tissue. Immunofluorescence microscopy revealed that tumor cells formed tight junctions normally, suggesting an inability of tumor cellular uptake of ICG. In contrast, tumor tissues increased the CD31‐immunoreactive endothelial cell area, and accumulated stromal cells immunoreactive for COX‐2 and tumor cell population immunoreactive for inducible nitric oxide synthase. In vivo vascular permeability assay revealed that prostaglandin E₂ promoted the endothelial cell permeability of ICG. In conclusion, our data indicated that fluorescence contrast‐enhanced imaging following i.v. administered ICG can be applied to the detection of colon tumors in a mouse colitis‐associated colon cancer model. The tumor tissue preference of ICG in the present model can be attributed to the enhanced vascular leakage of ICG involving inflammatory mediators, such as COX‐2 and inducible nitric oxide synthase, in conjunction with increased tumor vascularity.     

自发荧光物质在前哨淋巴结活检中的应用

前哨淋巴结活检代替腋窝清扫术已经日益成为临床工作中的标准术式。选择合适的示踪剂准确定位前哨淋巴结能够提高手术成功率、缩短学习曲线,更利于在基层医院推广。本研究探讨了自发荧光物质在前哨淋巴结活检中的应用前景。方法：使用荧光光谱仪测定美蓝溶液最佳激发光谱和发射光谱;将美蓝注射到白兔乳房皮下,通过小动物活体成像系统观察美蓝在体内向腋窝淋巴结引流并且聚集在腋窝淋巴结区域的过程。解剖腋窝区域寻找蓝染前哨淋巴结并观察其发光情况。结果：美蓝具有自发荧光的性质,其发出的近红外荧光可以在体外对腋淋巴引流及前哨淋巴结起到定位作用。结论：提示近红外荧光自发荧光物质作为前哨淋巴结示踪剂具有一定的应用前景。     

AngioSense(R) 750EX和Hypoxisense 680对体内肿瘤新生血管和缺氧微环境荧光成像效果检测

目的 检测以新生血管渗漏区域为靶标(AngioSense(R)750EX,Ang750)和以缺氧肿瘤细胞表面的碳酸酐酶9为靶标Hypoxisense 680,Hyp680两种新试剂对活体肿瘤的荧光成像效果.方法 应用Ang750和Hyp680两种荧光试剂分别对原发性肝肿瘤小鼠(H-ras12V转基因肝癌小鼠)、腋下移植瘤小鼠(S180,HepG2,H-ras12V转基因小鼠肝肿瘤组织)、以及背部皮下移植瘤小鼠(S180)进行活体成像,观察这两种试剂对活体肿瘤新生血管和缺氧微环境的指示效果.结果 对于原发性肝肿瘤,Ang750试剂可在尾静脉注射后6h对肝肿瘤新生血管进行有效的指示,12 h达到最佳检测效果.对于腋下移植瘤,Ang750试剂分别在尾静脉注射后10和6h对S180移植瘤及HepG2和H-ras12V转基因小鼠肝肿瘤组织的移植瘤的新生血管进行有效指示,24 h达到最佳检测效果.对于背部皮下移植瘤,Ang750试剂可在尾静脉注射后10h对S180移植瘤新生血管进行有效的指示,24 h达到最佳检测效果,但其荧光强度显著低于原发性肝肿瘤和腋下移植瘤.Hyp680试剂不能对本实验中活体肿瘤的缺氧微环境进行有效指示.结论 以新生血管为靶标的荧光指示剂Ang750能够对体内肿瘤新生血管微环境进行有效的指示,但不能有效确认相邻近多发性肿瘤的大小和个数.以缺氧为靶标Hyp680对体内肿瘤缺氧微环境的指示作用没有普适性.     

量子点荧光成像在肿瘤诊治应用研究进展

量子点(quantum dot,QD),又名“人造原子”,是一种用原子人工合成的纳米材料,其在紫外光激发下可发出不同波长的荧光.QD直径小、发出的荧光峰窄、荧光亮度持久,具有取代有机染料用于肿瘤诊治的潜能.总结近年QD在荧光成像及肿瘤诊治方面的研究进展.方法 应用PubMed及中国知网(CNKI)数据库检索系统,以“quantum dots,fluorescence imaging,oncotherapy,tumor therapy,量子点,肿瘤治疗,肿诊断,荧光成像”为关键词,检索2005-01-2016-03的相关文献391篇.纳入标准:(1)可用于生物荧光成像的低毒化QD;(2)靶向识别的功能化QD.根据纳入标准最终分析31篇文献.结果 低毒化QD在细胞成像、活体靶向成像、淋巴结成像、活体肿瘤成像、活体肿瘤细胞示踪等荧光成像方面的研究,使正确定位淋巴结、利用QD深成像能力发现极小的肿瘤、乃至彻底切除肿瘤组织成为可能.功能化QD在肿瘤早期诊断和治疗上发挥更大作用,QD的药物靶向和QD介导的光热治疗肿瘤也有很好的临床应用前景.结论 QD在生物荧光成像及肿瘤诊疗中的潜力巨大,具有进一步探索开发利用于临床的价值.     

Near infrared thoracoscopy of tumoral protease activity for improved detection of peripheral lung cancer

Improvement in tumor detection using "smart" probes in combination with microcatheter fluorescence thoracoscopy was evaluated in a mouse model. These imaging probes increase in fluorescence intensity after protease activation; cathepsin B is a major activator of the probes used in this study. Lewis lung carcinoma cells were orthotopically implanted in the subpleural lung parenchyma. Two activatable near infrared (NIR) probes with different excitation and emission wavelength were administered intravenously to determine whether wavelength would modulate target to background ratio (TBR). Mice were selectively intubated and thoracoscopy performed. A 0.8 mm outer diameter imaging catheter was used to record simultaneous white-light (anatomic) and NIR (protease expression) images. At both wavelength pairs evaluated (680/700 and 750/780 nm excitation/emission), the intrinsic luminosity differences between tumors and normal lung in uninjected animals was low (p > 0.3 and p = 0.4, respectively and TBR near 1). In mice receiving protease probes IV, tumors were significantly more fluorescent than adjacent lung (p < 0.0005 for 680/700 and p < 0.006 for 750/780) and TBR increased to approximately 9-fold. Confirmatory fluorescence microscopy and immunohistochemistry were similar and revealed that normal lung had very low levels when compared to tumors of cathepsin B and probe fluorescence. In conclusion, protease sensitive imaging probes selective for cathepsin B, imaged with NIR microcatheters, significantly increase the TBR, making small peripheral lung tumors more readily apparent. Such an approach may be a useful adjunct in staging or restaging patients with lung cancer to find minimal disease in the pleural and subpleural space.     

Cytotoxic T-cells as imaging probes for detecting glioma

Tumor vaccination using tumor-associated antigen-primed dendritic cells (DCs) is in clinical trials. Investigators are using patients' own immune systems to activate T-cells against recurrent or metastatic tumors. Following vaccination of DCs or attenuated tumor cells, clinical as well as radiological improvements have been noted due to migration and accumulation of cytotoxic T-cells (CTLs). CTLs mediated tumor cell killing resulted in extended survival in clinical trails and in preclinical models. Besides administration of primed DCs or attenuated or killed tumors cells to initiate the generation of CTLs, investigators have started making genetically altered T-cells (CTLs) to target specific tumors and showed in vivo migration and accumulation in the implanted or recurrent tumors using different imaging modalities. Our groups have also showed the utilization of both in vivo and in vitro techniques to make CTLs against glioma and used them as imaging probes to determine the sites of tumors. In this short review, the current status of vaccination therapy against glioma and utilization of CTLs as in vivo imaging probes to determine the sites of tumors and differentiate recurrent glioma from radiation necrosis will be discussed.     

Complementary use of fluorescence and magnetic resonance imaging of metastatic esophageal cancer in a novel orthotopic mouse model

We describe the development of an aggressive orthotopic metastatic model of esophageal cancer, which is visualized in real time with combined magnetic resonance imaging (MRI) and fluorescence imaging. The aim of the study was to describe the development of a novel model of metastatic tumor disease of esophageal carcinoma and use this model to evaluate fluorescence and MRI in early detection of local and metastatic disease. The human esophageal adenocarcinoma cell line PT1590 was stably transfected with green fluorescent protein (GFP). Nude mice were orthotopically implanted with PT1590‐GFP cells. Orthotopic tumor growth as well as metastatic spread was examined by fluorescence imaging and high‐resolution MRI at defined intervals after orthotopic implantation. Highly aggressive novel fluorescent cell lines were isolated from metastatic tissues and put into culture. After implantation of these cells, 100% of the animals developed orthotopic primary tumors. In 83% of animals, metastatic spread to liver, lung and lymph nodes was observed. Primary tumor growth could be visualized with fluorescence imaging and with MRI with high correlation between the 2 methods. Fluorescence imaging allows fast, sensitive, and economical imaging of the primary and metastatic tumor without anesthesia. With MRI, anatomical structures are visualized more precisely and tumors can be more accurately localized to specific organs. This model should prove highly useful to understand esophageal carcinoma and to identify novel therapeutics for this treatment‐resistant disease.     

In vivo optical imaging of cancer cell function and tumor microenvironment

In vivo optical imaging using fluorescence and bioluminescence is superior to other methods in terms of spatiotemporal resolution and specificity, and represents a new technology for comprehensively studying living organisms in a less invasive way. Nowadays, it is an indispensable technology for studying many aspects of cancer biology, including dynamic invasion and metastasis. In observations of fluorescence or bioluminescence signals in a living body, various problems were caused by optical characteristics such as absorption and scattering and, therefore, observation of deep tissue was difficult. Recent developments in techniques for observation of the deep tissues of living animals overcame this difficulty by improving bioluminescent proteins, fluorescent proteins, and fluorescent dyes, as well as detection technologies such as two‐photon excitation microscopy. In the present review, we introduce these technological developments and in vivo application of bioluminescence and fluorescence imaging, and discuss future perspectives on the use of in vivo optical imaging technology in cancer research.     

吲哚菁绿荧光成像在全腹腔镜远端胃癌根治术中的应用

目的:探讨吲哚菁绿(indocyanine green,ICG)荧光成像技术在全腹腔镜远端胃癌根治术中肿瘤定位及淋巴结清扫方面的应用价值。方法:回顾性收集2017年8月至2019年8月北京大学肿瘤医院收治的经胃镜及病理确诊的126例远端胃癌患者临床病理资料。其中62例患者在ICG标记近红外荧光腹腔镜下完成手术(观察组),64例行常规腹腔镜手术(对照组)。评估对比术前ICG标记与术中内镜定位的效果;对比两组淋巴结清扫与检获情况。结果:T1～2期观察组患者术中标本测量近端切缘距离(4.92±1.65)cm与对照组(4.76±1.66)cm相比差异无统计学意义(P=0.671)。T3～4期观察组患者第二站淋巴结清扫数目为(11.09±6.19)枚,高于对照组(8.89±4.35)枚(P=0.049);观察组破损淋巴结检出数量(0.74±0.46)枚少于对照组(1.27±1.22)枚(P=0.009)。结论:T1～2期胃癌患者行术前ICG标记可精准判断肿瘤边界,其肿瘤定位效果与术中胃镜相当,全腹腔镜手术时可指导选择合适的切除线。ICG在T3～4期胃癌的淋巴导航作用可指导术中更精细、完整、彻底地...     

新型染料IR-808靶向肿瘤近红外荧光成像的体内研究

目的 探讨近红外荧光染料IR-808在移植瘤动物模型中特异性的肿瘤靶向示踪作用。方法建立人肺癌细胞系NCI-H460、结肠癌细胞系HCT-116及鼠源肝癌细胞系Hepa 1-6皮下移植瘤裸小鼠模型,未接种癌细胞的裸小鼠作对照。尾静脉注射同等剂量染料IR-808,分别在不同时间点对裸小鼠正常组织及肿瘤组织进行近红外荧光成像,追踪染料在裸小鼠体内的分布,评价其肿瘤靶向作用。结果 在未接种的裸小鼠体内,IR-808染料首先在肝脏、心脏聚集,随后信号减弱,48h后基本代谢排出体外;在荷瘤裸小鼠模型体内,IR 808染料注入24h后开始靶向肿瘤,72h后荧光信号仍很稳定;取荷瘤裸小鼠组织进行近红外荧光成像,发现仅肿瘤组织有信号,其他组织无信号。全部实验小鼠状态良好,未见明显毒性反应。结论 近红外荧光染料IR 808能特异性靶向肿瘤成像,且信号稳定,无明显毒性,具有重要的应用前景     

Preferential tumor cellular uptake and retention of indocyanine green for in vivo tumor imaging

Indocyanine green (ICG) is a fluorescent agent approved for clinical applications by the Food and Drug Administration and European Medicines Agency. This study examined the mechanism of tumor imaging using intravenously administered ICG. The in vivo kinetics of intravenously administered ICG were determined in tumor xenografts using microscopic approaches that enabled both spatio‐temporal and high‐magnification analyses. The mechanism of ICG‐based tumor imaging was examined at the cellular level in six phenotypically different human colon cancer cell lines exhibiting different grades of epithelioid organization. ICG fluorescence imaging detected xenograft tumors, even those < 1 mm in size, based on their preferential cellular uptake and retention of the dye following its rapid tissue‐non‐specific delivery, in contrast to its rapid clearance by normal tissue. Live‐cell imaging revealed that cellular ICG uptake is temperature‐dependent and occurs after ICG binding to the cellular membrane, a pattern suggesting endocytic uptake as the mechanism. Cellular ICG uptake correlated inversely with the formation of tight junctions. Intracellular ICG was entrapped in the membrane traffic system, resulting in its slow turnover and prolonged retention by tumor cells. Our results suggest that tumor‐specific imaging by ICG involves non‐specific delivery of the dye to tissues followed by preferential tumor cellular uptake and retention. The tumor cell‐preference of ICG is driven by passive tumor cell‐targeting, the inherent ability of ICG to bind to cell membranes, and the high endocytic activity of tumor cells in association with the disruption of their tight junctions.     

基于适配子的分子探针在肿瘤分子影像学中的应用

理想的分子影像探针应具有一定的特异性、敏感性、安全性。适配子是可与靶分子高特异性和高亲和力结合的单链 DNA 或 RNA 分子,具有靶分子广泛、分子小、易生产和修饰、免疫原性低、渗透性强等特点,是一种新型的分子影像探针。目前基于适配子的肿瘤分子影像学研究越来越多,为肿瘤的诊断和治疗提供了新的技术和工具。