基于物理过滤与原位杂交技术的骨肉瘤循环肿瘤细胞检测及临床意义

目的:探寻一种有效地从骨肉瘤患者外周血中富集并鉴定循环肿瘤细胞的方法。方法:利用基于物理过滤与原位杂交结合的技术对骨肉瘤患者外周血循环肿瘤细胞分离并鉴定。采用直径8μm纳米滤膜截留外周血中体积较大的白细胞及肿瘤细胞,利用多重RNA原位杂交技术检测CD45、EpCAM、CK8、CK18、CK19、vimentin及twist基因表达,并根据结果对滤膜截留下的细胞进行鉴定并分型。结果:本研究所使用的基于物理过滤与原位杂交技术的循环肿瘤细胞检测方法可以高效地从骨肉瘤患者外周血中富集骨肉瘤循环肿瘤细胞,该方法富集细胞的效率超过90%。15例健康志愿者中1例志愿者检测结果阳性。20例纳入研究的骨肉瘤患者中19例患者外周血中检测出CTC,CTC计数范围为0-20。肿瘤转移患者外周血CTC计数为11.33±5.88,肿瘤未转移患者外周血CTC计数为4.36±2.98,差异具有统计学意义(P=0.0022)。肿瘤转移患者外周血间质型CTC比例高于肿瘤未转移患者(P=0.0031)。结论:利用基于物理过滤与原位杂交结合的技术可以有效地检测骨肉瘤患者外周血循环肿瘤细胞。CTC检测结果可以作为辅助判断肿瘤转移情况的辅助指标。     

以微小RNA作为新型标志物检测循环肿瘤细胞及其临床意义

循环肿瘤细胞（circulating tumor cell,CTC）是随血液循环一起转运的实体肿瘤细胞,与实体肿瘤的发展、转移、复发和预后等关系密切。然而,CTC数量的稀少使有效检测CTC具有较大的挑战性。微小RNA（microRNA,miRNA）作为一类新发现的基因表达调控分子,在肿瘤的发生、发展、转归等过程中起着重要的作用。CTC关联性miRNA的研究为CTC的检测和肿瘤的诊治开创了新思路。该文介绍了CTC的临床意义和主要分析方法,在CTC关联性miRNA与肿瘤诊断、治疗和预后等方面总结了这类新型肿瘤细胞标志物的研究进展。     

循环肿瘤细胞捕获与检测的纳米分析技术进展

随着纳米科学技术的发展,结构可控、表面多功能化、生物相容性良好的纳米材料在生物医药领域的各个方面都具有广泛的应用.作为一种重要的血液生物学标志物,循环肿瘤细胞(CTC)是肿瘤转移的"种子",活力较强的肿瘤细胞随着血液的流动可穿出血管在远端聚集形成微小的癌栓,对CTC的检测可用于癌症的早期诊断和转移的评估.新型纳米材料以及纳米表征测量技术的应用对CTC分析技术的进步产生了巨大的影响.近年来,基于纳米材料和微流控技术对CTC的捕获和检测已成为液体活检的研究热点,这一技术也被逐步推广到临床应用中.本文对纳米材料与纳米技术在CTC的捕获和检测中所发挥的作用进行了综述,并展望了该领域生物分析的应用前景.     

乳腺癌患者循环肿瘤细胞与癌干细胞相关性的初步研究

目的：检测乳腺癌患者外周血单核细胞（PBMC）中循环肿瘤细胞（CTC）和具有癌干细胞（CSC）标志的CTC（CSC-CTC）,探讨患者外周血微转移与CSC的相关性。方法：患者和健康者PBMC与磁珠偶联上皮细胞黏附分子单抗孵育后,用磁性分离法富集PBMC中的上皮细胞。以CK＋为患者PBMC中CTC标志,用流式细胞术（FCM）检测健康者和患者的PBMC中CK＋细胞及CK＋/CD44＋/CD24-细胞含量,并比较各组间CTC、CSC-CTC含量的差异。结果：用FCM在73.07%的患者中检测到CTC,在19例检测到CTC的患者中18例有CSC-CTC（94.74%）,CTC中CSC数量比例平均为19.01%,且患者PBMC中CTC和CSC-CTC比例与临床TNM分期相关。结论：初步建立了患者外周血CSC-CTC的检测方法,结果显示乳腺癌患者外周血微转移中有CSC-CTC的参与,临床分期越晚的患者CTC和CSC-CTC的数量越多。     

宫颈癌患者外周血循环肿瘤细胞检测的意义

目的:通过分析宫颈癌患者的临床资料与循环肿瘤细胞(CTC)之间的关系,评估其在宫颈癌患者诊疗过程中的临床意义。方法:回顾性分析我院2017年3月至2018年6月行机器人辅助下宫颈癌根治术的病例共82例,术前术后分别行CTC检测,分析CTC阳性率及CTC水平与临床参数的相关性。结果:不同病理类型、分化程度、脉管侵犯、淋巴转移及临床分期宫颈癌患者术前及术后CTC阳性率及CTC水平比较差异均无统计学意义(P0.05)。当宫颈癌浸润深度≥5 mm时,CTC的阳性率及CTC水平明显升高,CTC阳性率的差异仅在术前有统计学意义(P0.05);而CTC水平的差异在术前及术后均有统计学意义(P0.05)。结论:CTC的阳性表达及水平与肿瘤的浸润深度密切相关。检测宫颈癌患者CTC水平可为宫颈癌的诊治及预后评估提供重要参考价值。     

乳腺癌患者循环肿瘤细胞检测的研究进展

血行播散是乳腺癌转移的重要途径,检测腋窝淋巴结已经不能完全准确判断乳腺癌患者是否存在转移。肿瘤细胞进入外周血是肿瘤远处转移的前提,对乳腺癌患者循环肿瘤细胞的检测将有助于判断预后,指导治疗,监测治疗效果。简要综述了乳腺癌循环肿瘤细胞及其检测方法的研究进展。     

肺癌循环肿瘤细胞的单细胞EGFR基因突变检测

循环肿瘤细胞（Circulating tumor cells,CTCs）是从肿瘤原发病灶脱落并侵入外周血循环的肿瘤细胞。由于CTCs存在较大的异质性,其与癌症发展转移密切相关,但目前尚缺乏有效的CTCs单细胞异质性检测方法。鉴于此,本文发展了在单细胞层面对CTCs进行基因突变的检测方法并用于单个肺癌CTC的EGFR（Epidermal growth factor receptor）基因突变检测。首先用集成式微流控系统完成血液中稀有CTCs的捕获,接着将CTCs释放入含有多个微孔的微阵列芯片中,得到含有单个CTC的微孔,通过显微操作将单个CTC转入PCR管内完成单细胞基因组的放大,并进行单细胞的EGFR基因突变检测。以非小细胞肺癌细胞系A549、NCI-H1650和NCI-H1975为样本,通过芯片与毛细管修饰、引物扩增条件（复性温度、循环次数）的优化,结果显示在复性温度59℃、30个循环次数的条件下,引物扩增效果最优。利用该方法成功地对非小细胞肺癌（Non-small cell lung cancer, NSCLC）患者的血液样本进行了测试。从患者2 mL血液中获取5个CTCs,分别对其EGFR基因的第18、19、20、21外显子进行测序,发现该患者CTCs均为EGFR野生型。研究结果证明此检测方法可以灵敏地用于单个CTC基因突变的检测,在临床研究上具有重要的指导意义。     

循环表皮细胞的检测

事实上,很早以前——早在1869年——科学家就发现癌症细胞可以在血液中消失。20世纪50年代中期,一些研究者认为这些所谓的循环肿瘤细胞（CTC）可以帮助临床学家更早的捕获癌症或者检测治疗的效果。一些研究人员更专业的倾向于称之为循环表皮细胞——而不一定是循环肿瘤细胞本身——然而CTC的称谓仍然广泛使用。抛开这些术语不管,     

循环肿瘤细胞水平与卵巢癌患者临床病理特征及生存率的关系研究

目的:研究循环肿瘤细胞(CTC)水平与卵巢癌患者临床病理特征及生存率的关系。方法:选择从2012年6月到2014年12月在我院就诊的卵巢癌患者94例记为观察组。根据所有患者CTC水平将其分成高CTC组(CTC水平≥7个/5mL)57例及低CTC组(CTC水平7个/5 m L)37例,另选同期在我院体检的卵巢良性囊肿者90例记为对照组,分析CTC阳性与患者病理特征的关系、CTC水平与患者生存率和复发转移的关系以及CTC水平与患者病理特征、生存率、复发转移率的相关性。结果:对照组90例患者均未检测出CTC阳性。观察组94例患者中检测出68例CTC阳性,总阳性率为72.34%;观察组患者临床分期为II期和III期的CTC阳性率均明显高于I期,有淋巴结转移的CTC阳性率明显高于无淋巴结转移(P0.05)。高CTC组的1年、2年、3年生存率与低CTC组相比,差异均无统计学意义(P0.05)。高CTC组的1年、2年、3年复发转移率均高于低CTC组(P0.05)。Spearman相关性分析结果显示,CTC水平与患者的临床分期、淋巴结转移以及复发转移率均呈正相关(P0.05),而与年龄、病理分型、病理分级及生存率均无明显相关性(P0.05)。结论:卵巢癌患者临床分期为Ⅱ期和Ⅲ期和有淋巴结转移的CTC阳性率较高,且CTC水平与复发转移率关系密切,临床通过检测CTC水平,可能有助于评估患者的预后。     

应用逆转录聚合酶链反应技术检测原发肝癌患者外周血中肿瘤细胞

大量临床资料表明,肿瘤的转移和复发是影响肿瘤患者预后的重要因素。血行播散可能是原发肝癌转移和复发的主要原因,因此及时发现外周血的肿瘤细胞不仅对肿瘤的转移、复发判断具有重要价值,而且对指导临床制定治疗措施亦有重要意义。但由于肿瘤脱落至循环中的细胞数很少,现有的细胞形态学及免疫细胞化学技术敏感性较低,使其在检测外周血中少数肿瘤细胞方面的应用受到限制。应用逆转     

Circulating tumor cells: detection,molecular profiling and future prospects

Disseminated malignancy is responsible for the vast majority of cancer-related deaths. During this process, circulating tumor cells (CTC) are generated, spread from the primary tumor, colonize distant organs and lead to overt metastatic disease. CTC are essential for establishing metastasis; however, they are not sufficient as this process is highly inefficient and most will fail to grow in target sites. Several CTC die during migration while others remain dormant for several years and very few grow into macrometastases. CTC have been well documented in the bloodstream of cancer patients; however, the clinical relevance of this detection is still the subject of controversies and their biology is poorly understood. Indeed, available markers fail to distinguish between subgroups of CTC, and several current methods lack sensitivity, specificity or reproducibility in CTC characterization and detection. The advent of more precise technologies is renewing the interest in CTC biology. We will review herein recent findings on CTC biology, on the role of host–tumor interactions in CTC shedding and implantation, available methods of CTC detection and future perspectives for the molecular characterization of the CTC subset(s) responsible for the development of metastasis. Ultimately, understanding CTC biology and host–tumor ‘complementarities’ will help define metastasis-related biomarkers providing formidable and tailored novel therapeutic targets.     

Circulating tumor cells: detection, molecular profiling and future prospects

Disseminated malignancy is responsible for the vast majority of cancer-related deaths. During this process, circulating tumor cells (CTC) are generated, spread from the primary tumor, colonize distant organs and lead to overt metastatic disease. CTC are essential for establishing metastasis; however, they are not sufficient as this process is highly inefficient and most will fail to grow in target sites. Several CTC die during migration while others remain dormant for several years and very few grow into macrometastases. CTC have been well documented in the bloodstream of cancer patients; however, the clinical relevance of this detection is still the subject of controversies and their biology is poorly understood. Indeed, available markers fail to distinguish between subgroups of CTC, and several current methods lack sensitivity, specificity or reproducibility in CTC characterization and detection. The advent of more precise technologies is renewing the interest in CTC biology. We will review herein recent findings on CTC biology, on the role of host-tumor interactions in CTC shedding and implantation, available methods of CTC detection and future perspectives for the molecular characterization of the CTC subset(s) responsible for the development of metastasis. Ultimately, understanding CTC biology and host-tumor 'complementarities' will help define metastasis-related biomarkers providing formidable and tailored novel therapeutic targets.     

X-ray enabled detection and eradication of circulating tumor cells with nanoparticles

The early detection and eradication of circulating tumor cells (CTCs) play an important role in cancer metastasis management. This paper describes a new nanoparticle-enabled technique for integrated enrichment, detection and killing of CTCs by using magnetic nanoparticles and bismuth nanoparticles, X-ray fluorescence spectrometry, and X-ray radiation. The nanoparticles are modified with tumor targeting agents and conjugated with tumor cells through folate receptors over-expressed on cancer cells. A permanent micro-magnet is used to collect CTCs suspended inside a flowing medium that contains phosphate buffered saline (PBS) or whole blood. The characteristic X-ray emissions from collected bismuth nanoparticles, upon excitation with collimated X-rays, are used to detect CTCs. Results show that the method is capable of selectively detecting CTCs at concentrations ranging from 100-100,000cells/mL in the buffer solution, with a detection limit of ～100CTCs/mL. Moreover, the dose of primary X-rays can be enhanced to kill the localized CTCs by radiation induced DNA damage, with minimal invasiveness, thus making in vivo personalized CTC management possible.     

Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.     

Circulating tumor cells in the early detection of human cancers

Cancer is a severe disease with high morbidity and mortality globally. Thus, early detection is emerging as an important topic in modern oncology. Although the strategies for early detection have developed rapidly in recent decades, they remain challenging due to the subtle symptoms in the initial stage of the primary tumor. Currently, tumor biomarkers, imaging, and specific screening tests are widely used in various cancer types; however, each method has limitations. The harms are even overweight against the benefits in some cases. Therefore, early detection approaches should be improved urgently. Liquid biopsy, for now, is a convenient and non-invasive way compared to the traditional tissue biopsy in screening and early diagnosis. Circulating tumor cells (CTCs) are vital in liquid biopsy and play a central role in tumor dissemination and metastases. They have promising potential as cancer biomarkers in early detection. This review updates the knowledge of the biology of CTC; it also highlights the CTC enrichment technologies and their applications in the early detection of several human cancers.     

Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.     

Classification of Circulating Tumor Cells by Epithelial-Mesenchymal Transition Markers

In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107(65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.