基于微流控芯片的循环肝癌细胞检测和微量肿瘤细胞培养
|
摘要
研究背景和目的
肿瘤远处转移是指肿瘤细胞从原发灶脱落后随血液系统或淋巴系统循环到达靶器官,继而形成转移灶。侵入血液循环中的肿瘤细胞被称为循环肿瘤细胞(circulating tumor cells, CTCs)。肝切除和肝移植是目前治疗肝癌的主要方式。然而,高达40%接受肝切除术的肝癌患者及10%以上的肝移植肝癌患者术后第一年会发生复发。人们已经认识到,肝癌细胞在疾病早期可能即发生血行转移,而循环肿瘤细胞则是肝癌转移或复发的一个主要根源。因此,循环肝癌细胞检测对转移复发预测、治疗方案选择以及预后评估都具有重要的意义。
在现有众多的循环肿瘤细胞检测方法中,只有美国Veridex公司的CellSearch系统被批准用于临床检测[3]。与其他方法相类似,CellSearch System也采用基于靶向上皮细胞表面标志物EpCAM的免疫磁性分选策略分离CTCs,而EpCAM在肝癌细胞中的表达率低,不足以用来分选肝癌CTCs。
去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR)是一种专一表达于肝实质细胞表而的跨膜蛋白,能结合和内吞以半乳糖基和N-乙酰半乳糖胺基为端基的分子。根据肝细胞膜表面存在大量ASGPR的特性,我们创建了一种独特的基于ASGPR及其配体相互作用的循环肝癌细胞磁性分离方法。然而,该方法操作耗时长,且限制了被分选的肝癌细胞用于后续细胞培养和细胞生物学实验。近年发展起来的微流控芯片技术很适合用于CTCs分选,不仅可以高效分离非常稀少的CTCs,而且在细胞研究方面具有独特的优势。本研究论文将ASGPR及其配体之间相互作用的原理移植到微流控芯片分析系统,建立了一套用于循环肝癌细胞分选鉴定以及后续培养的微流控芯片实验平台。利用此平台从临床肝癌血样中分离、计数循环肝癌细胞,分析计数结果与肝癌病情之间的关系。另外设计微量细胞培养芯片,为循环肝癌细胞培养打下基础。
研究方法
1.采用L-Edit Pro8.30绘图软件设计、绘制芯片图形。
2.采用紫外光刻法及注塑法制竹PDMS芯片。
3.采用亲和素修饰芯片表面,与生物素化去唾液酸胎球蛋白相连,分离肝癌细胞。
4.采用基于广谱抗细胞角蛋白(cytokeratin, CK)抗体CK3-6H5的免疫荧光染色方法鉴定、计数分离获得的肝癌细胞。
5.通过肝癌细胞系HepG2掺入试验优化循环肝癌细胞分离芯片的设计参数及进样速度。
6.通过肝癌细胞系HepG2和乳腺癌细胞系MCF-7掺入试验分析芯片分离循环肝癌细胞的回收率和特异性。
7.采用所设计的微流控芯片检测6例不同分期的HCC患者及2例健康志愿者2mL血液样本中的CTCs。
8.设计、制作含有流速控制、配体捕获和体积捕获的循环肝癌细胞分离芯片。
9.通过HepG2肝癌细胞掺入试验分析：芯片分离循环肝癌细胞的回收率。
10.设计低密度肿瘤细胞培养芯片。
11.采用HepG2肝癌细胞稀释液检测芯片培养微量细胞的细胞成活率。
12.采用EXCEL软件对结果进行处理。各项数据通过x±SD表示。
实验结果
1.L-Edit Pro8.30绘图软件设计、绘制循环肝癌细胞分离芯片,每个芯片设有8个通道,每个通道含2254个微米柱,微米柱半径设为80μm、120μm、150μm和200μm等4个参数,PDMS制成芯片通道高为50μm。
2.细胞掺入实验测得最佳进样速度为0.8mL/h,微米柱最佳半径为80μm。
3.HepG2肝癌细胞掺入试验显示,1mL健康志愿者外周血中分别掺入100个、300个、500个和1000个HepG2细胞,在每个掺入水平,HepG2细胞的平均回收率均≥69%。
4.从6例HCC患者血液样本中均检出CTCs,2例健康人血液样本未被检出CTCs。
5.循环肝癌细胞分选芯片分为三部分：第一部分为流速控制部分；第二部分为基于配体-受体结合的循环肝癌细胞捕获部分；第三部分为基于体积循环肝癌细胞捕获部分。
6.以100个细胞/mL、500个细胞/mL和1000个细胞/mL稀释液进行检测,得到捕获率分别为79%、77%和84%。
7.细胞密度为1×103个细胞/mL时,所取12个培养小室中的细胞在第1天、第6天、第12天和第14天的计数,平均计数分别为3个、4个、6个和9个。
结论
本研究在以往工作的基础上,将原有的基于ASGPR的循环肝癌细胞捕获系统移植在微流控芯片上,与原系统相比,简化了检测步骤,缩短了检测时间,减少了样本采集量,同时在检测敏感性与特异性方面也有所提高。初步检测HCC患者与健康志愿者血样的结果表明,该微流控捕获循环肝癌细胞芯片系统具有临床应用潜能。随后进行的芯片结构改进更是大大提高了循环肝癌细胞的检测速度和捕获效率。而本研究设计的网式微流控细胞培养芯片不但可以进行少量的肿瘤细胞培养,而且可以进行后续的药敏实验,为进一步开展循环肝癌细胞的生物学研究奠定了实验基础。
Background and Objective
Hepatic resection and liver transplantation are the main modalities of curative treatment for HCC at present. However, up to40%of patients undergoing hepatectomy and more than10%of liver transplant recipients develop postoperative recurrences within the first year after surgery, which leads to death in almost all patients. It has been recognized that circulating tumor cells (CTCs), which are released into the blood circulation from either primary or metastatic lesions, are an active source of HCC metastasis or recurrence. Therefore, the enumeration and characterization of CTCs has important clinical significance in recurrence prediction and treatment monitoring in HCC patients.
The CellSearch(?) system, based on EpCAM antibody-coated magnetic beads approved by the US Food and Drug Administration has been used for detection of CTCs in breast cancer, colon cancer and prostate cancer. However, in liver neoplasia, only a small percentage (0%-20%) of HCC cases express EpCAM. Obviously, all of the EpCAM-based strategies are not appropriate for detection of HCC CTCs.
ASGPR is a transmembrane protein expressed exclusively on the surface of hepatocytes that can bind and internalize molecules with terminal galactose and N-acetylgalactosamine residues. Depended on the interaction of the ASGPR with its ligand, we have developed and validated of a sensitive and specific system to magnetically separate CTCs in HCC patients, combined liver cell-specific antibody Hep Par1immunofluorescence staining method. However, this method is a time-consuming, and limits the separated HCC CTC from subsequent cell culture and other biological experiments. As a new technology developed in recent years for CTCs isolation, the microfluidic chip not only can efficiently isolate CTCs, but also has advantages in cell research. In the current study, we further developed a high-throughput microfluidic device, which provides an enhanced platform for the HCC CTC isolation. Still Based on the interaction of the ASGPR with its ligand, combining this interaction with microfluidic chip, we developed a microfluidic CTC isolation system and a microfluidic cell culture system flowing up. It can be used to carry out the HCC CTC isolation, CTCs count and the HCC case analysis. Meanwhile HCC CTCs could be cultured in cell culture microfluidic chips, so that drug sensitivity test will be carried out.
Methods
1. Microfluidic chips were designed with L-Edit Pro8.30.
2. Microfluidic chips were fabricated following the standard protocol of UV lithography and soft lithography.
3. The CTC-chip surface was made functional with biotinylated asialofetuin for HCC CTC isolation.
4. The CTCs isolated were identified by immunofluorescence staining with the usage of the antibodies against epithelial-specific antigen cytokeratins (CKs).
5. To determine the minimum time and the most efficient micropillar radius required to achieve cell capture, we examined the cell-capture performances of the HCC CTC chip at different flow rates and different micropillar radius with HCC cell line (HepG2) spiked blood sample.
6. The recovery of the HCC CTCs isolation and detection system were determined by the HepG2cells spiked blood sample experiments.
7. The specificity of the HCC CTCs isolation and detection system were determined by the HepG2and MCF-7cells spiked blood sample experiments.
8. The established isolation and detection system for HCC CTCs was applied to examine CTCs in blood samples from6HCC patients and2healthy volunteers.
9. CTCs isolation chip contains3parts was designed with L-Edit Pro8.30.
10. The recovery of the HCC CTCs isolation was determined by the HepG2cells spiked PBS.
11. Low density cell culture chip was designed with L-Edit Pro8.30.
12. Cell survival rate and doubling time on CTC culture microfluidic chips were determined with HepG2cells.
Results
1. CTC isolation microfluidic chips were designed with8isolation channels, with2254micro pillars in each channel and a set of radius (80,120,150and200μm) high in50μm.
2. For HepG2cells, the maximal cell-capture numbers were achieved at a flow rate of0.8mL/h with radius as80um.
3. In recovery experiment,100,200,1000and2000HepG2cells were spiked into1mL blood samples from healthy volunteers. At each spiked level, the recovery is80%at the highest.
4. In specificity experiment, a set of HepG2and MCF-7cells spiked blood sample (100,300,500and1000cells mL1) were detected by CTC isolation microfluidic chips. It turned out that the recovery of HepG2cells was69%at least, however, only4%of MCF-7cells.
5. CTCs were detected in all blood samples from6HCC patients while non was detected in those from2healthy volunteers.
6. CTCs isolation chip contains3parts:the first part uniformed the flow; the second part isolated CTCs depended on ligand-receptor reaction; the third part isolated CTCs on volumn.
7. At the density gradient of100,500and1000cells/ml, the recovery is79%,77%and84%separately.
8. At a cell density of1x103cells/ml, the average cell number of12wells is3,4,6and9on the1st,6th,12th and14th day.
Conclusions
Based on the ASGPR depended magnetic-activated HCC CTC separation system, we set up a new CTC isolation system on microfluidic chips. It showed concise protocol, time saving, less sample volume and also improvement in sensitivity and specificity. Through tests with HCC patients'blood samples, it showed its'possibility in HCC CTC detection, however, more clinical trials are still needed to be took out. The changes of structure of the microfluidic isolation chips improved the recovery and shorten the detection time. Rare cells could be cultured in the "net"cell culture microfluidic chips, drug sensitivity test also can be realized on it.
肿瘤远处转移是指肿瘤细胞从原发灶脱落后随血液系统或淋巴系统循环到达靶器官,继而形成转移灶。侵入血液循环中的肿瘤细胞被称为循环肿瘤细胞(circulating tumor cells, CTCs)。肝切除和肝移植是目前治疗肝癌的主要方式。然而,高达40%接受肝切除术的肝癌患者及10%以上的肝移植肝癌患者术后第一年会发生复发。人们已经认识到,肝癌细胞在疾病早期可能即发生血行转移,而循环肿瘤细胞则是肝癌转移或复发的一个主要根源。因此,循环肝癌细胞检测对转移复发预测、治疗方案选择以及预后评估都具有重要的意义。
在现有众多的循环肿瘤细胞检测方法中,只有美国Veridex公司的CellSearch系统被批准用于临床检测[3]。与其他方法相类似,CellSearch System也采用基于靶向上皮细胞表面标志物EpCAM的免疫磁性分选策略分离CTCs,而EpCAM在肝癌细胞中的表达率低,不足以用来分选肝癌CTCs。
去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR)是一种专一表达于肝实质细胞表而的跨膜蛋白,能结合和内吞以半乳糖基和N-乙酰半乳糖胺基为端基的分子。根据肝细胞膜表面存在大量ASGPR的特性,我们创建了一种独特的基于ASGPR及其配体相互作用的循环肝癌细胞磁性分离方法。然而,该方法操作耗时长,且限制了被分选的肝癌细胞用于后续细胞培养和细胞生物学实验。近年发展起来的微流控芯片技术很适合用于CTCs分选,不仅可以高效分离非常稀少的CTCs,而且在细胞研究方面具有独特的优势。本研究论文将ASGPR及其配体之间相互作用的原理移植到微流控芯片分析系统,建立了一套用于循环肝癌细胞分选鉴定以及后续培养的微流控芯片实验平台。利用此平台从临床肝癌血样中分离、计数循环肝癌细胞,分析计数结果与肝癌病情之间的关系。另外设计微量细胞培养芯片,为循环肝癌细胞培养打下基础。
研究方法
1.采用L-Edit Pro8.30绘图软件设计、绘制芯片图形。
2.采用紫外光刻法及注塑法制竹PDMS芯片。
3.采用亲和素修饰芯片表面,与生物素化去唾液酸胎球蛋白相连,分离肝癌细胞。
4.采用基于广谱抗细胞角蛋白(cytokeratin, CK)抗体CK3-6H5的免疫荧光染色方法鉴定、计数分离获得的肝癌细胞。
5.通过肝癌细胞系HepG2掺入试验优化循环肝癌细胞分离芯片的设计参数及进样速度。
6.通过肝癌细胞系HepG2和乳腺癌细胞系MCF-7掺入试验分析芯片分离循环肝癌细胞的回收率和特异性。
7.采用所设计的微流控芯片检测6例不同分期的HCC患者及2例健康志愿者2mL血液样本中的CTCs。
8.设计、制作含有流速控制、配体捕获和体积捕获的循环肝癌细胞分离芯片。
9.通过HepG2肝癌细胞掺入试验分析：芯片分离循环肝癌细胞的回收率。
10.设计低密度肿瘤细胞培养芯片。
11.采用HepG2肝癌细胞稀释液检测芯片培养微量细胞的细胞成活率。
12.采用EXCEL软件对结果进行处理。各项数据通过x±SD表示。
实验结果
1.L-Edit Pro8.30绘图软件设计、绘制循环肝癌细胞分离芯片,每个芯片设有8个通道,每个通道含2254个微米柱,微米柱半径设为80μm、120μm、150μm和200μm等4个参数,PDMS制成芯片通道高为50μm。
2.细胞掺入实验测得最佳进样速度为0.8mL/h,微米柱最佳半径为80μm。
3.HepG2肝癌细胞掺入试验显示,1mL健康志愿者外周血中分别掺入100个、300个、500个和1000个HepG2细胞,在每个掺入水平,HepG2细胞的平均回收率均≥69%。
4.从6例HCC患者血液样本中均检出CTCs,2例健康人血液样本未被检出CTCs。
5.循环肝癌细胞分选芯片分为三部分：第一部分为流速控制部分；第二部分为基于配体-受体结合的循环肝癌细胞捕获部分；第三部分为基于体积循环肝癌细胞捕获部分。
6.以100个细胞/mL、500个细胞/mL和1000个细胞/mL稀释液进行检测,得到捕获率分别为79%、77%和84%。
7.细胞密度为1×103个细胞/mL时,所取12个培养小室中的细胞在第1天、第6天、第12天和第14天的计数,平均计数分别为3个、4个、6个和9个。
结论
本研究在以往工作的基础上,将原有的基于ASGPR的循环肝癌细胞捕获系统移植在微流控芯片上,与原系统相比,简化了检测步骤,缩短了检测时间,减少了样本采集量,同时在检测敏感性与特异性方面也有所提高。初步检测HCC患者与健康志愿者血样的结果表明,该微流控捕获循环肝癌细胞芯片系统具有临床应用潜能。随后进行的芯片结构改进更是大大提高了循环肝癌细胞的检测速度和捕获效率。而本研究设计的网式微流控细胞培养芯片不但可以进行少量的肿瘤细胞培养,而且可以进行后续的药敏实验,为进一步开展循环肝癌细胞的生物学研究奠定了实验基础。
Background and Objective
Hepatic resection and liver transplantation are the main modalities of curative treatment for HCC at present. However, up to40%of patients undergoing hepatectomy and more than10%of liver transplant recipients develop postoperative recurrences within the first year after surgery, which leads to death in almost all patients. It has been recognized that circulating tumor cells (CTCs), which are released into the blood circulation from either primary or metastatic lesions, are an active source of HCC metastasis or recurrence. Therefore, the enumeration and characterization of CTCs has important clinical significance in recurrence prediction and treatment monitoring in HCC patients.
The CellSearch(?) system, based on EpCAM antibody-coated magnetic beads approved by the US Food and Drug Administration has been used for detection of CTCs in breast cancer, colon cancer and prostate cancer. However, in liver neoplasia, only a small percentage (0%-20%) of HCC cases express EpCAM. Obviously, all of the EpCAM-based strategies are not appropriate for detection of HCC CTCs.
ASGPR is a transmembrane protein expressed exclusively on the surface of hepatocytes that can bind and internalize molecules with terminal galactose and N-acetylgalactosamine residues. Depended on the interaction of the ASGPR with its ligand, we have developed and validated of a sensitive and specific system to magnetically separate CTCs in HCC patients, combined liver cell-specific antibody Hep Par1immunofluorescence staining method. However, this method is a time-consuming, and limits the separated HCC CTC from subsequent cell culture and other biological experiments. As a new technology developed in recent years for CTCs isolation, the microfluidic chip not only can efficiently isolate CTCs, but also has advantages in cell research. In the current study, we further developed a high-throughput microfluidic device, which provides an enhanced platform for the HCC CTC isolation. Still Based on the interaction of the ASGPR with its ligand, combining this interaction with microfluidic chip, we developed a microfluidic CTC isolation system and a microfluidic cell culture system flowing up. It can be used to carry out the HCC CTC isolation, CTCs count and the HCC case analysis. Meanwhile HCC CTCs could be cultured in cell culture microfluidic chips, so that drug sensitivity test will be carried out.
Methods
1. Microfluidic chips were designed with L-Edit Pro8.30.
2. Microfluidic chips were fabricated following the standard protocol of UV lithography and soft lithography.
3. The CTC-chip surface was made functional with biotinylated asialofetuin for HCC CTC isolation.
4. The CTCs isolated were identified by immunofluorescence staining with the usage of the antibodies against epithelial-specific antigen cytokeratins (CKs).
5. To determine the minimum time and the most efficient micropillar radius required to achieve cell capture, we examined the cell-capture performances of the HCC CTC chip at different flow rates and different micropillar radius with HCC cell line (HepG2) spiked blood sample.
6. The recovery of the HCC CTCs isolation and detection system were determined by the HepG2cells spiked blood sample experiments.
7. The specificity of the HCC CTCs isolation and detection system were determined by the HepG2and MCF-7cells spiked blood sample experiments.
8. The established isolation and detection system for HCC CTCs was applied to examine CTCs in blood samples from6HCC patients and2healthy volunteers.
9. CTCs isolation chip contains3parts was designed with L-Edit Pro8.30.
10. The recovery of the HCC CTCs isolation was determined by the HepG2cells spiked PBS.
11. Low density cell culture chip was designed with L-Edit Pro8.30.
12. Cell survival rate and doubling time on CTC culture microfluidic chips were determined with HepG2cells.
Results
1. CTC isolation microfluidic chips were designed with8isolation channels, with2254micro pillars in each channel and a set of radius (80,120,150and200μm) high in50μm.
2. For HepG2cells, the maximal cell-capture numbers were achieved at a flow rate of0.8mL/h with radius as80um.
3. In recovery experiment,100,200,1000and2000HepG2cells were spiked into1mL blood samples from healthy volunteers. At each spiked level, the recovery is80%at the highest.
4. In specificity experiment, a set of HepG2and MCF-7cells spiked blood sample (100,300,500and1000cells mL1) were detected by CTC isolation microfluidic chips. It turned out that the recovery of HepG2cells was69%at least, however, only4%of MCF-7cells.
5. CTCs were detected in all blood samples from6HCC patients while non was detected in those from2healthy volunteers.
6. CTCs isolation chip contains3parts:the first part uniformed the flow; the second part isolated CTCs depended on ligand-receptor reaction; the third part isolated CTCs on volumn.
7. At the density gradient of100,500and1000cells/ml, the recovery is79%,77%and84%separately.
8. At a cell density of1x103cells/ml, the average cell number of12wells is3,4,6and9on the1st,6th,12th and14th day.
Conclusions
Based on the ASGPR depended magnetic-activated HCC CTC separation system, we set up a new CTC isolation system on microfluidic chips. It showed concise protocol, time saving, less sample volume and also improvement in sensitivity and specificity. Through tests with HCC patients'blood samples, it showed its'possibility in HCC CTC detection, however, more clinical trials are still needed to be took out. The changes of structure of the microfluidic isolation chips improved the recovery and shorten the detection time. Rare cells could be cultured in the "net"cell culture microfluidic chips, drug sensitivity test also can be realized on it.
引文
[1]Mocellin S, Keilholz U, Rossi CR, Nitti D. Circulating tumor cells:the "leukemic phase" of solid cancers. Trends in molecular medicine 2006; 12:130-9.
[2]Mazzaferro V, Llovet JM, Miceli R, et al. Predicting survival after liver transplantation in patients with hepatocellular carcinoma beyond the Milan criteria:a retrospective, exploratory analysis. The lancet oncology 2009; 10:35-43.
[3]Paterlini-Brechot P, Benali NL. Circulating tumor cells (CTC) detection:clinical impact and future directions. Cancer letters 2007;253:180-204.
[4]Went PT, Lugli A, Meier S, et al. Frequent EpCam protein expression in human carcinomas. [Internet].. Human pathology 2004;35:122-128.
[5]徐文,股正丰.：去唾液酸糖蛋白受体介导的肝脏靶向性研究进展.第二军医大学学报2006;27：1002-1005.
[6]Xu W, Cao L, Chen L, et al. Isolation of circulating tumor cells in patients with hepatocellular carcinoma using a novel cell separation strategy. Clinical cancer research:an official journal of the American Association for Cancer Research 2011;17:3783-3793.
[7]Feng X, Du W, Luo Q, Liu B-F. Microfluidic chip:next-generation platform for systems biology. Analytica chimica acta 2009;650:83-97.
[8]Nagrath S, Sequist L V, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 2007;450:1235-1239.
[9]Stott SL, Hsu C-H, Tsukrov DI, et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proceedings of the National Academy of Sciences of the United States of America 2010;107:18392-18397.
[10]Adams A A, Okagbare PI, Feng J, et al.Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. Journal of the American Chemical Society 2008;130:8633-8641.
[11]Sequist L V, Nagrath S, Toner M, et al. The CTC-chip:an exciting new tool to detect circulating tumor cells in lung cancer patients. Journal of thoracic oncologyl I:official publication of the International Association for the Study of Lung Cancer 2009;4:281-283.
[12]Lenshof A, Laurell T:Continuous separation of cells and particles in microfluidic systems. Chemical Society reviews 2010;39:1203-1217.
[13]El-Ali J, Sorger PK, Jensen KF:Cells on chips. Nature 2006;442:403-411.
[14]Feng X, Du W, Luo Q, Liu B-F:Microfluidic chip:next-generation platform for systems biology. Analytica chimica acta 2009;650:83-97.
[15]Mark D, Haeberle S, Roth G, et al. Microfluidic lab-on-a-chip platforms:requirements, characteristics and applications. Chemical Society reviews 2010;39:1153-1182.
[16]Rosenberg R, Gertler R, Friederichs J, et al.:Comparison of two density gradient centrifugation systems for the enrichment of disseminated tumor cells in blood. Cytometry 2002;49:150-158.
[17]Morgan TM, Lange PH, Vessella RL. Detection and characterization of circulating and disseminated prostate cancer cells. [Internet].. Frontiers in bioscience:a journal and virtual library 2007; 12:3000-3009.
[18]Sastre J, Maestro ML, Puente J,et al. Circulating tumor cells in colorectal cancer:correlation with clinical and pathological variables. Annals of oncology:official journal of the European Society for Medical Oncology/ESMO 20080; 19:935-938.
[19]Ignatiadis M, Xenidis N, Perraki M,et al. Different prognostic value of cytokeratin-19 mRNA positive circulating tumor cells according to estrogen receptor and HER2 status in early-stage breast cancer. Journal of clinical oncologyl 1:official journal of the American Society of Clinical Oncology 2007;25:5194-5202.
[20]Klein CA:Parallel progression of primary tumours and metastases. Nature reviews Cancer 2009;9:302-312.
[21]Pocock C, Szydlo R, Davis J,et al. Stem cell transplantation for chronic myeloid leukaemia:the role of infused marrow cell dose. The hematology journal:the official journal of the European Haematology Association/EHA 2001;2:265-272.
[22]Fehm T, Sagalowsky A, Clifford E, et al. Cytogenetic evidence that circulating epithelial cells in patients with carcinoma are malignant. [Internet].. Clinical cancer research:an official journal of the American Association for Cancer Research 2002;8:2073-2084.
[23]Wlodkowic D, Skommer J, McGuinness D,et al. Chip-based dynamic real-time quantification of drug-induced cytotoxicity in human tumor cells. Analytical chemistry 2009;81:6952-6959.
[24]Komen J, Wolbers F, Franke HR,et al. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device:effect of cytostatic drugs. Biomedical microdevices 2008; 10:727-737.
[25]Li X, Ling V, Li PCH:Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip. Analytical chemistry 2008;80:4095-4102.
[26]Gates BD, Xu Q, Stewart M,et al. New approaches to nanofabrication:molding, printing, and other techniques. Chemical reviews 2005; 105:1171-1196.
[27]Thompson L. F.; Willson, C. G.; Bowden MJ:Introduction to Microlithography. American Chemical Society:Washington DC 1994;
[28]Lee, K. Y.; LaBianca, N.; Rishton, S. A.; Zolgharnain, S.; Gelorme JD. S, J.; Chang THP:In Micromachining applications of a high resolution ultrathick photoresist. The 38th International symposium on electron, ion, and photon beams, Scottsdale, Arizona (USA) 1995:3012-3016.
[29]Deng T, Wu H, Brittain S, Whitesides G:Prototyping of masks, masters, and stamps/molds for soft lithography using an office printer and photographic reduction [Internet].. Analytical chemistry 2000 Jul;72:3176-3180.
[30]Shaffer DR, Leversha MA, Danila DC, et al. Circulating tumor cell analysis in patients with progressive castration-resistant prostate cancer. Clinical cancer research:an official journal of the American Association for Cancer Research 2007; 13:2023-2029.
[31]Saias L, Autebert J, Malaquin L, Viovy J-L:Design, modeling and characterization of microfluidic architectures for high flow rate, small footprint microfluidic systems. Lab on a chip 2011; 11:822-832.
[32]Lu H, Koo LY, Wang WM,et al. Microfluidic shear devices for quantitative analysis of cell adhesion. Analytical chemistry 2004;76:5257-5264.
[33]Ranjan V, Waterbury R, Xiao Z, Diamond SL:Fluid shear stress induction of the transcriptional activator c-fos in human and bovine endothelial cells, HeLa, and Chinese hamster ovary cells. Biotechnology and bioengineering 1996;49:383-390.
[34]Usami S, Chen HH, Zhao Y,et al. Design and construction of a linear shear stress flow chamber. [Internet].. Annals of biomedical engineering 1993;21:77-83.
[35]Green J V, Kniazeva T, Abedi M,et al. Effect of channel geometry on cell adhesion in microfluidic devices. Lab on a chip 2009;9:677-685.
[36]Davis JA, Inglis DW, Morton KJ, et al. Deterministic hydrodynamics:taking blood apart. Proceedings of the National Academy of Sciences of the United States of America 2006;103:14779-14784.
[37]El-Ali J, Sorger PK, Jensen KF:Cells on chips. Nature 2006;442:403-411.
[38]Feng X, Du W, Luo Q, Liu B-F:Microfluidic chip:next-generation platform for systems biology. Analytica chimica acta 2009;650:83-97.
[39]Mark D, Haeberle S, Roth G,et al. Microfluidic lab-on-a-chip platforms:requirements, characteristics and applications. Chemical Society reviews 2010;39:1153-1182.
[40]Hung PJ, Lee PJ, Sabounchi P,et al. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnology and bioengineering 2005;89:1-8.
[41]Wlodkowic D, Skommer J, McGuinness D, et al. Chip-based dynamic real-time quantification of drug-induced cytotoxicity in human tumor cells. Analytical chemistry 2009;81:6952-6959.
[42]Komen J, Wolbers F, Franke HR,et al. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device:effect of cytostatic drugs. Biomedical microdevices; 10:727-737.
[43]Li X, Ling V, Li PCH:Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip. Analytical chemistry 2008;80:4095-4102.
[44]Ostrovidov S, Jiang J, Sakai Y, Fujii T:Membrane-based PDMS microbioreactor for perfused 3D primary rat hepatocyte cultures. Biomedical microdevices 2004;6:279-287.
[45]Wang L, Ni X-F, Luo C-X,et al. Self-loading and cell culture in one layer microfluidic devices. Biomedical microdevices 2009;11:679-684.
[1]Hashimoto M, Barany F, Xu F, et al. Serial processing of biological reactions using flow-through microfluidic devices:coupled PCR/LDR for the detection of low-abundant DNA point mutations. Analyst,2007,132:913-921.
[2]Liu P, Seo T S, Beyor N, et al. Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing. Anal Chem,2007, 79:1881-1889.
[3]Kim S M, Burns M A, Hasselbrink E F. Electrokinetic protein preconcentration using a simple glass/poly microfluidic chip. Anal Chem,2006,78:4779-4785.
[4]Emrich C A, Medintz I L, Chu W K, et al. Microfabricated Two-Dimensional Electrophoresis Device for Differential Protein Expression Profiling. Anal Chem,2007,79:7360-7366.
[5]Sun Y, Yin XF. Novel muti-depth microfluidic chip for single cell analysis. J Chromatography A, 2006,1117:228-233.
[6]VanDijken J, Kaigala G V, Lauzon J, et al. Microfluidic chips for detecting the t(4;14) translocation and monitoring disease during treatment using RT-PCR analysis of IgH-MMSET hybrid transcript. J Mol Diagn,2007,9:358-367.
[7]Feng X, Du W, Luo Q, et al. Microfluidic chip:next-generation platform for systems biology. Anal Chim Acta.2009,650:83-97.
[8]Jamil El-Ali, Sorger PK, Jensen KF. Cells on chips. Nature,2006,442:403-411.
[9]Feng X, Du W, Luo Q, et al. Microfluidic chip:next-generation platform for systems biology. Anal Chim Acta,2009,650:83-97.
[10]Mark D, Haeberle S, Roth G, et al. Microfluidic lab-on-a-chip platforms:requirements, characteristics and applications. Chem Soc Rev,2010,39:1153-1182.
[11]Longo D, Hasty J. Dynamics of single-cell gene expression. Mol Syst Biol 2006,2:64.
[12]Friesen C,Fulda S,Debatin KM,et al.Induction of CD95 ligand and apoptosis by doxorubicin is modulated by the redox state in chemosensitive and drug resistant tumor cells.Cell Death Differ,1999,6:471-480.
[13]Lindstrom S, Mori K, Ohashi T, Andersson-Svahn H. A microwell array device with integrated microfluidic components for enhanced single-cell analysis. Electrophoresis,2009, 12:4166-4171.
[14]Mocellin S, Keilholz U, Rossi C R, et al. Circulating tumor cells:the'leukemic phase'of solid cancers. Trends Mol Med,2006,12:130-139.
[15]Rosenberg R, Gertler R, Friederichs J, et al.Comparison of two density gradient centrifugation systems for the enrichment of disseminated tumor cells in blood. Cytometry,2002,49:150-158.
[16]Vona G, Estepa L, Bdroud C, et al. Impact of cytomorphological detection of circulating tumor cells in patients with liver cancer. Hepatology,2004,39:792-797.
[17]Paterlini-Brechot P, Benali N L. Circulating tumor cells (CTC) detection:clinical impact and future direction. Cancer Lett,2007,253:180-204.
[18]Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature,2007,450:1235-1239.
[19]Allard WJ, Matera J, Miller MC, et al. Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases. Clin Cancer Res, 2004,10:6897-6904.
[20]Adams A A, Okagbare PI, Juan Feng, et al. Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. Am Chem Soc,2008,130:8633-8641.
[21]Tan SJ, Yobas L, Lee GYH. Et al Microdevice for the isolation and enumeration of cancer cells from blood. Biomed Microdevices,2009,11:883-892.
[22]Hung PJ, Lee PJ, Sabounchi P, et al. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnol Bioeng,2005,89:1-8.
[23]Popovtzer R, Neufeld T, Popovtzer A, et al. Electrochemical lab on a chip for high-throughput analysis of anticancer drugs efficiency. Nanomedicine,2008,4:121-126.
[24]Wlodkowic D, Skommer J, McGuinness D, et al. Chip-based dynamic real-time quantification of drug-induced cytotoxicity in human tumor cells. Anal Chem,2009,81:6952-6959.
[25]Li X, Ling V, Li PC. Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip. Anal Chem,2008,80:4095-4102.
[26]Di Carlo D, Wu LY, Lee LP. Dynamic single cell culture array. Lab on Chip,2006,6:1445-1449.
[27]Komen J, Wolbers F, Franke HR, et al. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device:effect of cytostatic drugs. Biomed Microdevices,2008,10:727-737.
[28]Ye NN, Qin JH, Shi WW, et al. Cell-Based high content screening using an integrated microfluidic device. Lab on Chip,2007,7:1696-1704.
[1]Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet.2003;362:1907-17.
[2]Shariff MI, Cox IJ, Gomaa AI, et al. Hepatocellular carcinoma:current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics. Expert Rev Gastroenterol Hepatol.2009;3:353-67.
[3]Mazzaferro V, Llovet JM, Miceli R, et al. Predicting survival after liver transplantation in patients with hepatocellular carcinoma beyond the Milan criteria:a retrospective, exploratory analysis. Lancet Oncol.2009; 10:35-43.
[4]Scatton O, Chiappini F, Riou P, et al. Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma. Oncogene.2006;25:4067-75.
[5]Pantel K, Alix-Panabieres C. Circulating tumour cells in cancer patients:challenges and perspectives. Trends Mol Med.2010; 16:398-406.
[6]Jacob K, Sollier C, Jabado N. Circulating tumor cells:detection, molecular profiling and future prospects. Expert Rev Proteomics.2007;4:741-56.
[7]Attard G, de Bono JS. Utilizing circulating tumor cells:challenges and pitfalls. Curr Opin Genet Dev.2011;21:50-8.
[8]Gertler R, Rosenberg R, Fuehrer K, et al. Detection of circulating tumor cells in blood using an optimized density gradient centrifugation. Recent Results Cancer Res.2003; 162:149-55.
[9]Vona G, Sabile A, Louha M, et al. Isolation by size of epithelial tumor cells:a new method for the immunomorphological and molecular characterization of circulatingtumor cells. Am J Pathol. 2000; 156:57-63.
[10]Lin HK, Zheng S, Williams AJ, et al. Portable filter-based microdevice for detection and characterization of circulating tumor cells. Clin Cancer Res.2010; 16:5011-8.
[11]Xu T, Lu B, Tai YC, et al. A cancer detection platform which measures telomerase activity from live circulating tumor cells captured on a microfilter. Cancer Res.2010;70:6420-6.
[12]Zheng S, Lin HK, Lu B, et al.3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood. Biomed Microdevices.2011; 13:203-13.
[13]McNiece I, Briddell R, Stoney G, et al. Large-scale isolation of CD34+ cells using the Amgen cell selection device results in high levels of purity and recovery. J Hematother.1997;6:5-11.
[14]Neurauter AA, Bonyhadi M, Lien E, et al. Cell isolation and expansion using Dynabeads. Adv Biochem Eng Biotechnol.2007;106:41-73.
[15]Naume B, Borgen E, T(?)ssvik S, et al. Detection of isolated tumor cells in peripheral blood and in BM:evaluation of a new enrichment method. Cytotherapy.2004;6:244-52.
[16]Talasaz AH, Powell AA, Huber DE, et al. Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc Natl Acad Sci USA.2009; 106:3970-5.
[17]Andreopoulou E, Yang LY, Rangel KM, et al. Comparison of assay methods for detection of circulating tumor cells (CTCs) in metastatic breast cancer (MBC):AdnaGen AdnaTest BreastCancer Select/Detect versus Veridex CellSearch system. Int J Cancer.2012; 130:1590-7.
[18]Miller MC, Doyle GV, Terstappen LW. Significance of circulating tumor cells detected by the CellSearch system in patients with metastatic breast colorectal and prostate cancer. J Oncol. 2010;2010:617421.
[19]Radisky DC, LaBarge MA. Epithelial-mesenchymal transition and the stem cell phenotype. Cell Stem Cell.2008;2:511-2.
[20]Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature.2007;450:1235-9.
[21]Stott SL, Hsu CH, Tsukrov DI, et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci USA.2010; 107:18392-7.
[22]Adams AA, Okagbare PI, Feng J, et al. Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. J Am Chem Soc.2008; 130:8633-41.
[23]Zheng X, Cheung LS, Schroeder JA, et al. A high-performance microsystem for isolating circulating tumor cells. Lab Chip.2011;11:3269-76.
[24]Saliba AE, Saias L, Psychari E, et al. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays. Proc Natl Acad Sci USA.2010; 107:14524-9.
[25]Hoshino K, Huang YY, Lane N, et al. Microchip-based immunomagnetic detection of circulating tumor cells. Lab Chip.2011; 11:3449-57.
[26]Wang S, Owens GE, Tseng HR. Nano "fly paper" technology for the capture of circulating tumor cells. Methods Mol Biol.2011;726:141-50.
[27]Mikolajczyk SD, Millar LS, Tsinberg P, et al. Detection of EpCAM-Negative and cytokeratin-negative circulating tumor cells in peripheral blood. J Oncol.2011;2011:252361.
[28]Tan SJ, Lakshmi RL, Chen P, et al. Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients. Biosens Bioelectron.2010;26:1701-5.
[29]Bhagat AA, Hou HW, Li LD, et al. Pinched flow coupled shear-modulateed inertial microfluidics for high-throughputrare bloodcell separation. Lab Chip.2011;11:1870-8.
[30]Hur SC, Henderson-MacLennan NK, McCabe ER, et al. Deformability-based cell classification and enrichment using inertial microfluidics. Lab Chip.2011;11:912-20.
[31]Gascoyne PR, Noshari J, Anderson TJ, et al. Isolation of rare cells from cell mixtures by dielectrophoresis. Electrophoresis.2009;30:1-11.
[32]Moon HS, Kwon K, Kim SI, et al. Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP). Lab Chip. 2011;11:1118-25.
[33]Paterlini-Brdchot P, Vona G, Brechot C. Circulating tumorous cells in patients with hepatocellular carcinoma. Clinical impact and future directions. Semin Cancer Biol.2000; 10:241-9.
[34]Ring AE, Zabaglo L, Ormerod MG, et al. Detection of circulating epithelial cells in the blood of patients with breast cancer:comparison of three techniques. Br J Cancer.2005;92:906-12.
[35]Takao M, Takeda K. Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination-free flow cytometry. Cytometry A.2011;79:107-17.
[36]Muthu MS, Feng SS. Targeted nanomedicine for detection and treatment of circulating tumor cells. Nanomedicine.2011;6:579-81.
[37]He W, Wang H, Hartmann LC, et al. In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry. Proc Natl Acad Sci USA.2007;104:11760-5.
[38]Galanzha El, Shashkov EV, Spring PM, et al. In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser. Cancer Res.2009;69:7926-34.
[39]Galanzha EI, Kokoska MS, Shashkov EV, et al. In vivo fiber-based multicolor photoacoustic detection and photothermal purging of metastasis in sentinel lymph nodes targeted by nanoparticles. J Biophoton.2009;2:528-39.
[40]Nedosekin DA, Sarimollaoglu M, Ye JH, et al. In vivo ultra-fast photoacoustic flow cytometry of circulating human melanoma cells using near-infrared high-pulse rate lasers. Cytometry A. 2011;79:825-33.
[41]Hsieh HB, Marrinucci D, Bethel K, et al. High speed detection of circulating tumor cells. Biosens Bioelectron.2006;21:1893-9.
[42]Scholtens TM, Schreuder F, Ligthart ST, et al. CellTracks TDI:an image cytometer for cell characterization. Cytometry A.2011;79:203-13.
[43]Henriksen M, Miller B, Newmark J, et al. Laser scanning cytometry and its applications:a pioneering technology in the field of quantitative imaging cytometry. Methods Cell Biol. 2011;102:161-205.
[44]Deng G, Herrler M, Burgess D, et al. Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients. Breast Cancer Res.2008;10:R69.
[45]Alix-Panabieres C, Rebillard X, Brouillet JP, et al. Detection of circulating prostate-specific antigen-secreting cells in prostate cancer patients. Clin Chem.2005;51:1538-41.
[46]Lu J, Fan T, Zhao Q, et al. Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients. Int J Cancer.2010;126:669-83.
[47]Kyo S, Takakura M, Fujiwara T, et al. Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers. Cancer Sci.2008;99:1528-38.
[48]Waguri N, Suda T, Nomoto M, et al. Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation. Clin Cancer Res.2003;9:3004-11.
[49]Min AL, Choi JY, Woo HY, et al. High expression of Snail mRNA in blood from hepatocellular carcinoma patients with extra-hepatic metastasis. Clin Exp Metastasis.2009;26:759-67.
[50]Zhang XF, Lai EC, Kang XY, et al. Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein as a marker of prognosis and a monitor of recurrence of hepatocellular carcinoma after curative liver resection. Ann Surg Oncol.2011; 18:2218-23.
[51]Yang ZF, Ho DW, Ng MN, et al. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell.2008;13:153-66.
[52]Yang ZF, Ngai P, Ho DW, et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology.2008;47:919-28.
[53]Fan ST, Yang ZF, Ho DW, et al. Prediction of posthepatectomy recurrence of hepatocellular carcinoma by circulating cancer stem cells:a prospective study. Ann Surg.2011;254:569-76.
[54]Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res.2005;65:10946-51.
[55]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer.2006;5:67.
[56]de Boer CJ, van Krieken JH, Janssen-van Rhijn CM, et al. Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J Pathol.1999; 188:201-6.
[57]Schmelzer E, Wauthier E, Reid LM. The phenotypes of pluripotent human hepatic progenitors. Stem Cells.2006;24:1852-8.
[58]Proca DM, Niemann TH, Porcell AI, et al. MOC31 immunoreactivity in proimary and metastatic carcinoma of the liver. Report findings and review of other utilized markers. Appl Immunohistochem Mol Morphol.2000;8:120-5.
[59]Porcell AI, De Young BR, Proca DM, et al. Immunohistochemical analysis of hepatocellular and adenocarcinoma in the liver:mOC31 compares favorably with other putative markers. Mod Pathol. 2000; 13:773-8.
[60]Went PT, Lugli A, Meier S, et al. Frequent EpCam protein expression in human carcinomas. Hum Pathol.2004;35:122-8.
[61]Xu W, Cao L, Chen L, et al. Isolation of circulating tumor cells in patients with hepatocellular carcinoma using a novel cell separation strategy. Clin Cancer Res.2011;17:3783-93.
[62]Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531-54.
[63]Spiess M. The asialoglycoprotein receptor:a model for endocytic transport receptors. Biochemistry. 1990;29:10009-18.
[64]Wong IH, Lau WY, Leung T, et al. Hematogenous dissemination of hepatocytes and tumor cells after surgical resection of hepatocellular carcinoma:a quantitative analysis. Clin Cancer Res. 1999;5:4021-7.
[65]Zhou L, Rui JA, Ye DX, et al. Edmondson-Steiner grading increases the predictive efficiency of TNM staging for longterm survival of patients with hepatocellular carcinoma after curative resection. World J Surg.2008;32:1748-56.
[66]Polakis P. The oncogenic activation of beta-catenin. Curr Opin Genet Dev.1999;9:15-21.
[67]Bressac B, Kew M, Wands J, et al. Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature.1991;350:429-31.
[68]Ross JS, Fletcher JA, Linette GP, et al. The Her-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. Oncologist.2003;8:307-25.
[69]Bacaksiz A, Sahin FI, Bilezikci B, et al. Determination of HER-2/Neu status in hepatocellular carcinoma cases. Genet Test.2008; 12:211-4.
[70]Liu J, Ahiekpor A, Li L, et al. Increased expression of ErbB-2 in liver is associated with hepatitis B x antigen and shorter survival in patients with liver cancer. Int J Cancer.2009; 125:1894-901.
[71]Shackleton M, Quintana E, Fearon ER, et al. Heterogeneity in cancer:cancer stem cells versus clonal evolution. Cell.2009; 138:822-9.
[72]Hermann PC, Huber SL, Heeschen C. Metastatic cancer stem cells:a new target for anti-cancer therapy? Cell Cycle.2008;7:188-93.
[73]Mikhail S, He AR. Liver cancer stem cells. Int J Hepatol.2011;2011:486954.
[74]Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal transitions and circulating tumor cells. J Mammary Gland Biol Neoplasia.2010; 15:261-73.
[75]Ma S, Chan KW, Hu L, et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology.2007; 132:2542-56.
[76]Yamashita T, Ji J, Budhu A, et al. Ep-CAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology.2009; 136:1012-24.
[77]Haraguchi N, Ishii H, Mimori K, et al. CD 13 is a therapeutic target in human liver cancer stem cells. J Clin Invest.2010; 120:3326-39.
[78]Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology.2006;44:240-51.
[79]Shi GM, Xu Y, Fan J, et al. Identification of side population cells in human hepatocellular carcinoma cell lines with stepwise metastatic potentials. J Cancer Res Clin Oncol. 2008;134:1155-63.
[80]Cao L, Zhou Y, Zhai B, et al. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines. BMC Gastroenterol.2011; 11:71.
[81]Uchida Y, Tanaka S, Aihara A, et al. Analogy between sphere forming ability and stmness of human hepatoma cells. Oncol Rep.2010;24:1147-51.
[82]Hiraiwa K, Takeuchi H, Hasegawa H, et al. Clinical significance of circulating tumor cells in blood from patients with gastrointestinal cancers. Ann Surg Oncol.2008; 15:3092-100.
[83]Tanaka K, Yano M, Motoori M, et al. CEA-antigen and SCC-antigen mRNA expression in peripheral blood predict hematogenous recurrence after resection in patients with esophageal cancer. Ann Surg Oncol.2010; 17:2779-86.
[84]Peach G, Kim C, Zacharakis E, et al. Prognostic significance of circulating tumour cells following surgical resection of colorectal cancers:a systematic review. Br J Cancer.2010;102:1327-34.
[85]Salnikov AV, Kusumawidjaja G, Rausch V, et al. Cancer stem cell marker expression in hepatocellular carcinoma and liver metastases is not sufficient as single prognostic parameter. Cancer Lett.2009;275:185-93.
[86]Toso C, Mentha G, Majno P. Liver transplantation for hepatocellular carcinoma:five steps to prevent recurrence. Am J Transplant.2011;11:2031-5.
[87]Wicha MS, Hayes DF. Circulating tumor cells:not all detected cells are bad and not all bad cells aredetected. J Clin Oncol.2011;29:1508-11.
[88]Baeuerle PA, Gires O. EpCAM (CD326) finding its role in cancer. Br J Cancer.2007;96:417-23.
[89]Colombo F, Baldan F, Mazzucchelli S, et al. Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma. PLoS One. 2011;6:e21369.
[90]Raimondi C, Gradilone A, Naso G, et al. Epithelial-mesenchymal transition and sternness features in circulating tumorcells from breast cancer patients. Breast Cancer Res Treat.2011;130:449-55.
[91]Navin N, Hichs J. Future medical applications of single-cell sequencing in cancer. Genome Med. 2011;3:31.
[92]Liu LL, Fu D, Ma Y, et al. The power and the promise of liver cancer stem cell markers. Stem Cells Dev.2011;20:2023-30.
[2]Mazzaferro V, Llovet JM, Miceli R, et al. Predicting survival after liver transplantation in patients with hepatocellular carcinoma beyond the Milan criteria:a retrospective, exploratory analysis. The lancet oncology 2009; 10:35-43.
[3]Paterlini-Brechot P, Benali NL. Circulating tumor cells (CTC) detection:clinical impact and future directions. Cancer letters 2007;253:180-204.
[4]Went PT, Lugli A, Meier S, et al. Frequent EpCam protein expression in human carcinomas. [Internet].. Human pathology 2004;35:122-128.
[5]徐文,股正丰.：去唾液酸糖蛋白受体介导的肝脏靶向性研究进展.第二军医大学学报2006;27：1002-1005.
[6]Xu W, Cao L, Chen L, et al. Isolation of circulating tumor cells in patients with hepatocellular carcinoma using a novel cell separation strategy. Clinical cancer research:an official journal of the American Association for Cancer Research 2011;17:3783-3793.
[7]Feng X, Du W, Luo Q, Liu B-F. Microfluidic chip:next-generation platform for systems biology. Analytica chimica acta 2009;650:83-97.
[8]Nagrath S, Sequist L V, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 2007;450:1235-1239.
[9]Stott SL, Hsu C-H, Tsukrov DI, et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proceedings of the National Academy of Sciences of the United States of America 2010;107:18392-18397.
[10]Adams A A, Okagbare PI, Feng J, et al.Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. Journal of the American Chemical Society 2008;130:8633-8641.
[11]Sequist L V, Nagrath S, Toner M, et al. The CTC-chip:an exciting new tool to detect circulating tumor cells in lung cancer patients. Journal of thoracic oncologyl I:official publication of the International Association for the Study of Lung Cancer 2009;4:281-283.
[12]Lenshof A, Laurell T:Continuous separation of cells and particles in microfluidic systems. Chemical Society reviews 2010;39:1203-1217.
[13]El-Ali J, Sorger PK, Jensen KF:Cells on chips. Nature 2006;442:403-411.
[14]Feng X, Du W, Luo Q, Liu B-F:Microfluidic chip:next-generation platform for systems biology. Analytica chimica acta 2009;650:83-97.
[15]Mark D, Haeberle S, Roth G, et al. Microfluidic lab-on-a-chip platforms:requirements, characteristics and applications. Chemical Society reviews 2010;39:1153-1182.
[16]Rosenberg R, Gertler R, Friederichs J, et al.:Comparison of two density gradient centrifugation systems for the enrichment of disseminated tumor cells in blood. Cytometry 2002;49:150-158.
[17]Morgan TM, Lange PH, Vessella RL. Detection and characterization of circulating and disseminated prostate cancer cells. [Internet].. Frontiers in bioscience:a journal and virtual library 2007; 12:3000-3009.
[18]Sastre J, Maestro ML, Puente J,et al. Circulating tumor cells in colorectal cancer:correlation with clinical and pathological variables. Annals of oncology:official journal of the European Society for Medical Oncology/ESMO 20080; 19:935-938.
[19]Ignatiadis M, Xenidis N, Perraki M,et al. Different prognostic value of cytokeratin-19 mRNA positive circulating tumor cells according to estrogen receptor and HER2 status in early-stage breast cancer. Journal of clinical oncologyl 1:official journal of the American Society of Clinical Oncology 2007;25:5194-5202.
[20]Klein CA:Parallel progression of primary tumours and metastases. Nature reviews Cancer 2009;9:302-312.
[21]Pocock C, Szydlo R, Davis J,et al. Stem cell transplantation for chronic myeloid leukaemia:the role of infused marrow cell dose. The hematology journal:the official journal of the European Haematology Association/EHA 2001;2:265-272.
[22]Fehm T, Sagalowsky A, Clifford E, et al. Cytogenetic evidence that circulating epithelial cells in patients with carcinoma are malignant. [Internet].. Clinical cancer research:an official journal of the American Association for Cancer Research 2002;8:2073-2084.
[23]Wlodkowic D, Skommer J, McGuinness D,et al. Chip-based dynamic real-time quantification of drug-induced cytotoxicity in human tumor cells. Analytical chemistry 2009;81:6952-6959.
[24]Komen J, Wolbers F, Franke HR,et al. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device:effect of cytostatic drugs. Biomedical microdevices 2008; 10:727-737.
[25]Li X, Ling V, Li PCH:Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip. Analytical chemistry 2008;80:4095-4102.
[26]Gates BD, Xu Q, Stewart M,et al. New approaches to nanofabrication:molding, printing, and other techniques. Chemical reviews 2005; 105:1171-1196.
[27]Thompson L. F.; Willson, C. G.; Bowden MJ:Introduction to Microlithography. American Chemical Society:Washington DC 1994;
[28]Lee, K. Y.; LaBianca, N.; Rishton, S. A.; Zolgharnain, S.; Gelorme JD. S, J.; Chang THP:In Micromachining applications of a high resolution ultrathick photoresist. The 38th International symposium on electron, ion, and photon beams, Scottsdale, Arizona (USA) 1995:3012-3016.
[29]Deng T, Wu H, Brittain S, Whitesides G:Prototyping of masks, masters, and stamps/molds for soft lithography using an office printer and photographic reduction [Internet].. Analytical chemistry 2000 Jul;72:3176-3180.
[30]Shaffer DR, Leversha MA, Danila DC, et al. Circulating tumor cell analysis in patients with progressive castration-resistant prostate cancer. Clinical cancer research:an official journal of the American Association for Cancer Research 2007; 13:2023-2029.
[31]Saias L, Autebert J, Malaquin L, Viovy J-L:Design, modeling and characterization of microfluidic architectures for high flow rate, small footprint microfluidic systems. Lab on a chip 2011; 11:822-832.
[32]Lu H, Koo LY, Wang WM,et al. Microfluidic shear devices for quantitative analysis of cell adhesion. Analytical chemistry 2004;76:5257-5264.
[33]Ranjan V, Waterbury R, Xiao Z, Diamond SL:Fluid shear stress induction of the transcriptional activator c-fos in human and bovine endothelial cells, HeLa, and Chinese hamster ovary cells. Biotechnology and bioengineering 1996;49:383-390.
[34]Usami S, Chen HH, Zhao Y,et al. Design and construction of a linear shear stress flow chamber. [Internet].. Annals of biomedical engineering 1993;21:77-83.
[35]Green J V, Kniazeva T, Abedi M,et al. Effect of channel geometry on cell adhesion in microfluidic devices. Lab on a chip 2009;9:677-685.
[36]Davis JA, Inglis DW, Morton KJ, et al. Deterministic hydrodynamics:taking blood apart. Proceedings of the National Academy of Sciences of the United States of America 2006;103:14779-14784.
[37]El-Ali J, Sorger PK, Jensen KF:Cells on chips. Nature 2006;442:403-411.
[38]Feng X, Du W, Luo Q, Liu B-F:Microfluidic chip:next-generation platform for systems biology. Analytica chimica acta 2009;650:83-97.
[39]Mark D, Haeberle S, Roth G,et al. Microfluidic lab-on-a-chip platforms:requirements, characteristics and applications. Chemical Society reviews 2010;39:1153-1182.
[40]Hung PJ, Lee PJ, Sabounchi P,et al. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnology and bioengineering 2005;89:1-8.
[41]Wlodkowic D, Skommer J, McGuinness D, et al. Chip-based dynamic real-time quantification of drug-induced cytotoxicity in human tumor cells. Analytical chemistry 2009;81:6952-6959.
[42]Komen J, Wolbers F, Franke HR,et al. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device:effect of cytostatic drugs. Biomedical microdevices; 10:727-737.
[43]Li X, Ling V, Li PCH:Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip. Analytical chemistry 2008;80:4095-4102.
[44]Ostrovidov S, Jiang J, Sakai Y, Fujii T:Membrane-based PDMS microbioreactor for perfused 3D primary rat hepatocyte cultures. Biomedical microdevices 2004;6:279-287.
[45]Wang L, Ni X-F, Luo C-X,et al. Self-loading and cell culture in one layer microfluidic devices. Biomedical microdevices 2009;11:679-684.
[1]Hashimoto M, Barany F, Xu F, et al. Serial processing of biological reactions using flow-through microfluidic devices:coupled PCR/LDR for the detection of low-abundant DNA point mutations. Analyst,2007,132:913-921.
[2]Liu P, Seo T S, Beyor N, et al. Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing. Anal Chem,2007, 79:1881-1889.
[3]Kim S M, Burns M A, Hasselbrink E F. Electrokinetic protein preconcentration using a simple glass/poly microfluidic chip. Anal Chem,2006,78:4779-4785.
[4]Emrich C A, Medintz I L, Chu W K, et al. Microfabricated Two-Dimensional Electrophoresis Device for Differential Protein Expression Profiling. Anal Chem,2007,79:7360-7366.
[5]Sun Y, Yin XF. Novel muti-depth microfluidic chip for single cell analysis. J Chromatography A, 2006,1117:228-233.
[6]VanDijken J, Kaigala G V, Lauzon J, et al. Microfluidic chips for detecting the t(4;14) translocation and monitoring disease during treatment using RT-PCR analysis of IgH-MMSET hybrid transcript. J Mol Diagn,2007,9:358-367.
[7]Feng X, Du W, Luo Q, et al. Microfluidic chip:next-generation platform for systems biology. Anal Chim Acta.2009,650:83-97.
[8]Jamil El-Ali, Sorger PK, Jensen KF. Cells on chips. Nature,2006,442:403-411.
[9]Feng X, Du W, Luo Q, et al. Microfluidic chip:next-generation platform for systems biology. Anal Chim Acta,2009,650:83-97.
[10]Mark D, Haeberle S, Roth G, et al. Microfluidic lab-on-a-chip platforms:requirements, characteristics and applications. Chem Soc Rev,2010,39:1153-1182.
[11]Longo D, Hasty J. Dynamics of single-cell gene expression. Mol Syst Biol 2006,2:64.
[12]Friesen C,Fulda S,Debatin KM,et al.Induction of CD95 ligand and apoptosis by doxorubicin is modulated by the redox state in chemosensitive and drug resistant tumor cells.Cell Death Differ,1999,6:471-480.
[13]Lindstrom S, Mori K, Ohashi T, Andersson-Svahn H. A microwell array device with integrated microfluidic components for enhanced single-cell analysis. Electrophoresis,2009, 12:4166-4171.
[14]Mocellin S, Keilholz U, Rossi C R, et al. Circulating tumor cells:the'leukemic phase'of solid cancers. Trends Mol Med,2006,12:130-139.
[15]Rosenberg R, Gertler R, Friederichs J, et al.Comparison of two density gradient centrifugation systems for the enrichment of disseminated tumor cells in blood. Cytometry,2002,49:150-158.
[16]Vona G, Estepa L, Bdroud C, et al. Impact of cytomorphological detection of circulating tumor cells in patients with liver cancer. Hepatology,2004,39:792-797.
[17]Paterlini-Brechot P, Benali N L. Circulating tumor cells (CTC) detection:clinical impact and future direction. Cancer Lett,2007,253:180-204.
[18]Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature,2007,450:1235-1239.
[19]Allard WJ, Matera J, Miller MC, et al. Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases. Clin Cancer Res, 2004,10:6897-6904.
[20]Adams A A, Okagbare PI, Juan Feng, et al. Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. Am Chem Soc,2008,130:8633-8641.
[21]Tan SJ, Yobas L, Lee GYH. Et al Microdevice for the isolation and enumeration of cancer cells from blood. Biomed Microdevices,2009,11:883-892.
[22]Hung PJ, Lee PJ, Sabounchi P, et al. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnol Bioeng,2005,89:1-8.
[23]Popovtzer R, Neufeld T, Popovtzer A, et al. Electrochemical lab on a chip for high-throughput analysis of anticancer drugs efficiency. Nanomedicine,2008,4:121-126.
[24]Wlodkowic D, Skommer J, McGuinness D, et al. Chip-based dynamic real-time quantification of drug-induced cytotoxicity in human tumor cells. Anal Chem,2009,81:6952-6959.
[25]Li X, Ling V, Li PC. Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip. Anal Chem,2008,80:4095-4102.
[26]Di Carlo D, Wu LY, Lee LP. Dynamic single cell culture array. Lab on Chip,2006,6:1445-1449.
[27]Komen J, Wolbers F, Franke HR, et al. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device:effect of cytostatic drugs. Biomed Microdevices,2008,10:727-737.
[28]Ye NN, Qin JH, Shi WW, et al. Cell-Based high content screening using an integrated microfluidic device. Lab on Chip,2007,7:1696-1704.
[1]Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet.2003;362:1907-17.
[2]Shariff MI, Cox IJ, Gomaa AI, et al. Hepatocellular carcinoma:current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics. Expert Rev Gastroenterol Hepatol.2009;3:353-67.
[3]Mazzaferro V, Llovet JM, Miceli R, et al. Predicting survival after liver transplantation in patients with hepatocellular carcinoma beyond the Milan criteria:a retrospective, exploratory analysis. Lancet Oncol.2009; 10:35-43.
[4]Scatton O, Chiappini F, Riou P, et al. Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma. Oncogene.2006;25:4067-75.
[5]Pantel K, Alix-Panabieres C. Circulating tumour cells in cancer patients:challenges and perspectives. Trends Mol Med.2010; 16:398-406.
[6]Jacob K, Sollier C, Jabado N. Circulating tumor cells:detection, molecular profiling and future prospects. Expert Rev Proteomics.2007;4:741-56.
[7]Attard G, de Bono JS. Utilizing circulating tumor cells:challenges and pitfalls. Curr Opin Genet Dev.2011;21:50-8.
[8]Gertler R, Rosenberg R, Fuehrer K, et al. Detection of circulating tumor cells in blood using an optimized density gradient centrifugation. Recent Results Cancer Res.2003; 162:149-55.
[9]Vona G, Sabile A, Louha M, et al. Isolation by size of epithelial tumor cells:a new method for the immunomorphological and molecular characterization of circulatingtumor cells. Am J Pathol. 2000; 156:57-63.
[10]Lin HK, Zheng S, Williams AJ, et al. Portable filter-based microdevice for detection and characterization of circulating tumor cells. Clin Cancer Res.2010; 16:5011-8.
[11]Xu T, Lu B, Tai YC, et al. A cancer detection platform which measures telomerase activity from live circulating tumor cells captured on a microfilter. Cancer Res.2010;70:6420-6.
[12]Zheng S, Lin HK, Lu B, et al.3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood. Biomed Microdevices.2011; 13:203-13.
[13]McNiece I, Briddell R, Stoney G, et al. Large-scale isolation of CD34+ cells using the Amgen cell selection device results in high levels of purity and recovery. J Hematother.1997;6:5-11.
[14]Neurauter AA, Bonyhadi M, Lien E, et al. Cell isolation and expansion using Dynabeads. Adv Biochem Eng Biotechnol.2007;106:41-73.
[15]Naume B, Borgen E, T(?)ssvik S, et al. Detection of isolated tumor cells in peripheral blood and in BM:evaluation of a new enrichment method. Cytotherapy.2004;6:244-52.
[16]Talasaz AH, Powell AA, Huber DE, et al. Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc Natl Acad Sci USA.2009; 106:3970-5.
[17]Andreopoulou E, Yang LY, Rangel KM, et al. Comparison of assay methods for detection of circulating tumor cells (CTCs) in metastatic breast cancer (MBC):AdnaGen AdnaTest BreastCancer Select/Detect versus Veridex CellSearch system. Int J Cancer.2012; 130:1590-7.
[18]Miller MC, Doyle GV, Terstappen LW. Significance of circulating tumor cells detected by the CellSearch system in patients with metastatic breast colorectal and prostate cancer. J Oncol. 2010;2010:617421.
[19]Radisky DC, LaBarge MA. Epithelial-mesenchymal transition and the stem cell phenotype. Cell Stem Cell.2008;2:511-2.
[20]Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature.2007;450:1235-9.
[21]Stott SL, Hsu CH, Tsukrov DI, et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci USA.2010; 107:18392-7.
[22]Adams AA, Okagbare PI, Feng J, et al. Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. J Am Chem Soc.2008; 130:8633-41.
[23]Zheng X, Cheung LS, Schroeder JA, et al. A high-performance microsystem for isolating circulating tumor cells. Lab Chip.2011;11:3269-76.
[24]Saliba AE, Saias L, Psychari E, et al. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays. Proc Natl Acad Sci USA.2010; 107:14524-9.
[25]Hoshino K, Huang YY, Lane N, et al. Microchip-based immunomagnetic detection of circulating tumor cells. Lab Chip.2011; 11:3449-57.
[26]Wang S, Owens GE, Tseng HR. Nano "fly paper" technology for the capture of circulating tumor cells. Methods Mol Biol.2011;726:141-50.
[27]Mikolajczyk SD, Millar LS, Tsinberg P, et al. Detection of EpCAM-Negative and cytokeratin-negative circulating tumor cells in peripheral blood. J Oncol.2011;2011:252361.
[28]Tan SJ, Lakshmi RL, Chen P, et al. Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients. Biosens Bioelectron.2010;26:1701-5.
[29]Bhagat AA, Hou HW, Li LD, et al. Pinched flow coupled shear-modulateed inertial microfluidics for high-throughputrare bloodcell separation. Lab Chip.2011;11:1870-8.
[30]Hur SC, Henderson-MacLennan NK, McCabe ER, et al. Deformability-based cell classification and enrichment using inertial microfluidics. Lab Chip.2011;11:912-20.
[31]Gascoyne PR, Noshari J, Anderson TJ, et al. Isolation of rare cells from cell mixtures by dielectrophoresis. Electrophoresis.2009;30:1-11.
[32]Moon HS, Kwon K, Kim SI, et al. Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP). Lab Chip. 2011;11:1118-25.
[33]Paterlini-Brdchot P, Vona G, Brechot C. Circulating tumorous cells in patients with hepatocellular carcinoma. Clinical impact and future directions. Semin Cancer Biol.2000; 10:241-9.
[34]Ring AE, Zabaglo L, Ormerod MG, et al. Detection of circulating epithelial cells in the blood of patients with breast cancer:comparison of three techniques. Br J Cancer.2005;92:906-12.
[35]Takao M, Takeda K. Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination-free flow cytometry. Cytometry A.2011;79:107-17.
[36]Muthu MS, Feng SS. Targeted nanomedicine for detection and treatment of circulating tumor cells. Nanomedicine.2011;6:579-81.
[37]He W, Wang H, Hartmann LC, et al. In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry. Proc Natl Acad Sci USA.2007;104:11760-5.
[38]Galanzha El, Shashkov EV, Spring PM, et al. In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser. Cancer Res.2009;69:7926-34.
[39]Galanzha EI, Kokoska MS, Shashkov EV, et al. In vivo fiber-based multicolor photoacoustic detection and photothermal purging of metastasis in sentinel lymph nodes targeted by nanoparticles. J Biophoton.2009;2:528-39.
[40]Nedosekin DA, Sarimollaoglu M, Ye JH, et al. In vivo ultra-fast photoacoustic flow cytometry of circulating human melanoma cells using near-infrared high-pulse rate lasers. Cytometry A. 2011;79:825-33.
[41]Hsieh HB, Marrinucci D, Bethel K, et al. High speed detection of circulating tumor cells. Biosens Bioelectron.2006;21:1893-9.
[42]Scholtens TM, Schreuder F, Ligthart ST, et al. CellTracks TDI:an image cytometer for cell characterization. Cytometry A.2011;79:203-13.
[43]Henriksen M, Miller B, Newmark J, et al. Laser scanning cytometry and its applications:a pioneering technology in the field of quantitative imaging cytometry. Methods Cell Biol. 2011;102:161-205.
[44]Deng G, Herrler M, Burgess D, et al. Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients. Breast Cancer Res.2008;10:R69.
[45]Alix-Panabieres C, Rebillard X, Brouillet JP, et al. Detection of circulating prostate-specific antigen-secreting cells in prostate cancer patients. Clin Chem.2005;51:1538-41.
[46]Lu J, Fan T, Zhao Q, et al. Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients. Int J Cancer.2010;126:669-83.
[47]Kyo S, Takakura M, Fujiwara T, et al. Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers. Cancer Sci.2008;99:1528-38.
[48]Waguri N, Suda T, Nomoto M, et al. Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation. Clin Cancer Res.2003;9:3004-11.
[49]Min AL, Choi JY, Woo HY, et al. High expression of Snail mRNA in blood from hepatocellular carcinoma patients with extra-hepatic metastasis. Clin Exp Metastasis.2009;26:759-67.
[50]Zhang XF, Lai EC, Kang XY, et al. Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein as a marker of prognosis and a monitor of recurrence of hepatocellular carcinoma after curative liver resection. Ann Surg Oncol.2011; 18:2218-23.
[51]Yang ZF, Ho DW, Ng MN, et al. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell.2008;13:153-66.
[52]Yang ZF, Ngai P, Ho DW, et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology.2008;47:919-28.
[53]Fan ST, Yang ZF, Ho DW, et al. Prediction of posthepatectomy recurrence of hepatocellular carcinoma by circulating cancer stem cells:a prospective study. Ann Surg.2011;254:569-76.
[54]Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res.2005;65:10946-51.
[55]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer.2006;5:67.
[56]de Boer CJ, van Krieken JH, Janssen-van Rhijn CM, et al. Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J Pathol.1999; 188:201-6.
[57]Schmelzer E, Wauthier E, Reid LM. The phenotypes of pluripotent human hepatic progenitors. Stem Cells.2006;24:1852-8.
[58]Proca DM, Niemann TH, Porcell AI, et al. MOC31 immunoreactivity in proimary and metastatic carcinoma of the liver. Report findings and review of other utilized markers. Appl Immunohistochem Mol Morphol.2000;8:120-5.
[59]Porcell AI, De Young BR, Proca DM, et al. Immunohistochemical analysis of hepatocellular and adenocarcinoma in the liver:mOC31 compares favorably with other putative markers. Mod Pathol. 2000; 13:773-8.
[60]Went PT, Lugli A, Meier S, et al. Frequent EpCam protein expression in human carcinomas. Hum Pathol.2004;35:122-8.
[61]Xu W, Cao L, Chen L, et al. Isolation of circulating tumor cells in patients with hepatocellular carcinoma using a novel cell separation strategy. Clin Cancer Res.2011;17:3783-93.
[62]Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531-54.
[63]Spiess M. The asialoglycoprotein receptor:a model for endocytic transport receptors. Biochemistry. 1990;29:10009-18.
[64]Wong IH, Lau WY, Leung T, et al. Hematogenous dissemination of hepatocytes and tumor cells after surgical resection of hepatocellular carcinoma:a quantitative analysis. Clin Cancer Res. 1999;5:4021-7.
[65]Zhou L, Rui JA, Ye DX, et al. Edmondson-Steiner grading increases the predictive efficiency of TNM staging for longterm survival of patients with hepatocellular carcinoma after curative resection. World J Surg.2008;32:1748-56.
[66]Polakis P. The oncogenic activation of beta-catenin. Curr Opin Genet Dev.1999;9:15-21.
[67]Bressac B, Kew M, Wands J, et al. Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature.1991;350:429-31.
[68]Ross JS, Fletcher JA, Linette GP, et al. The Her-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. Oncologist.2003;8:307-25.
[69]Bacaksiz A, Sahin FI, Bilezikci B, et al. Determination of HER-2/Neu status in hepatocellular carcinoma cases. Genet Test.2008; 12:211-4.
[70]Liu J, Ahiekpor A, Li L, et al. Increased expression of ErbB-2 in liver is associated with hepatitis B x antigen and shorter survival in patients with liver cancer. Int J Cancer.2009; 125:1894-901.
[71]Shackleton M, Quintana E, Fearon ER, et al. Heterogeneity in cancer:cancer stem cells versus clonal evolution. Cell.2009; 138:822-9.
[72]Hermann PC, Huber SL, Heeschen C. Metastatic cancer stem cells:a new target for anti-cancer therapy? Cell Cycle.2008;7:188-93.
[73]Mikhail S, He AR. Liver cancer stem cells. Int J Hepatol.2011;2011:486954.
[74]Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal transitions and circulating tumor cells. J Mammary Gland Biol Neoplasia.2010; 15:261-73.
[75]Ma S, Chan KW, Hu L, et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology.2007; 132:2542-56.
[76]Yamashita T, Ji J, Budhu A, et al. Ep-CAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology.2009; 136:1012-24.
[77]Haraguchi N, Ishii H, Mimori K, et al. CD 13 is a therapeutic target in human liver cancer stem cells. J Clin Invest.2010; 120:3326-39.
[78]Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology.2006;44:240-51.
[79]Shi GM, Xu Y, Fan J, et al. Identification of side population cells in human hepatocellular carcinoma cell lines with stepwise metastatic potentials. J Cancer Res Clin Oncol. 2008;134:1155-63.
[80]Cao L, Zhou Y, Zhai B, et al. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines. BMC Gastroenterol.2011; 11:71.
[81]Uchida Y, Tanaka S, Aihara A, et al. Analogy between sphere forming ability and stmness of human hepatoma cells. Oncol Rep.2010;24:1147-51.
[82]Hiraiwa K, Takeuchi H, Hasegawa H, et al. Clinical significance of circulating tumor cells in blood from patients with gastrointestinal cancers. Ann Surg Oncol.2008; 15:3092-100.
[83]Tanaka K, Yano M, Motoori M, et al. CEA-antigen and SCC-antigen mRNA expression in peripheral blood predict hematogenous recurrence after resection in patients with esophageal cancer. Ann Surg Oncol.2010; 17:2779-86.
[84]Peach G, Kim C, Zacharakis E, et al. Prognostic significance of circulating tumour cells following surgical resection of colorectal cancers:a systematic review. Br J Cancer.2010;102:1327-34.
[85]Salnikov AV, Kusumawidjaja G, Rausch V, et al. Cancer stem cell marker expression in hepatocellular carcinoma and liver metastases is not sufficient as single prognostic parameter. Cancer Lett.2009;275:185-93.
[86]Toso C, Mentha G, Majno P. Liver transplantation for hepatocellular carcinoma:five steps to prevent recurrence. Am J Transplant.2011;11:2031-5.
[87]Wicha MS, Hayes DF. Circulating tumor cells:not all detected cells are bad and not all bad cells aredetected. J Clin Oncol.2011;29:1508-11.
[88]Baeuerle PA, Gires O. EpCAM (CD326) finding its role in cancer. Br J Cancer.2007;96:417-23.
[89]Colombo F, Baldan F, Mazzucchelli S, et al. Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma. PLoS One. 2011;6:e21369.
[90]Raimondi C, Gradilone A, Naso G, et al. Epithelial-mesenchymal transition and sternness features in circulating tumorcells from breast cancer patients. Breast Cancer Res Treat.2011;130:449-55.
[91]Navin N, Hichs J. Future medical applications of single-cell sequencing in cancer. Genome Med. 2011;3:31.
[92]Liu LL, Fu D, Ma Y, et al. The power and the promise of liver cancer stem cell markers. Stem Cells Dev.2011;20:2023-30.