摘要
乳腺癌是女性最常见的恶性肿瘤。不可预测的转移性复发是乳腺癌患者治疗失败、复发乃至死亡的主要原因。循环肿瘤细胞(CTC)被定义为从原发肿瘤处脱落并进入循环或淋巴系统的肿瘤细胞。研究证实,CTC的检测可以为乳腺癌的诊断、治疗策略的制定和预后评估提供重要的临床信息。作为液体活检的重要检测对象之一,CTC能够简单地通过抽取患者的血液来收集。然而,大多数CTC在循环中死亡,只有极少数存活并侵犯远处器官。数量上的稀缺、CTC的异质性以及血液中复杂成分的干扰使得CTC的准确检测成为一个巨大的挑战。针对CTC的生物和物理特性开发的各种检测方法往往需要在检测前对CTC进行分离和富集,但吸附、洗脱和转移等预处理过程不可避免地会造成CTC的损失,而耗时、操作复杂、设备昂贵等问题也限制了CTC的临床应用,因此迫切需要开发新的检测技术。以微加工结构为特征的微流控技术近年来受到了广泛关注与研究,微流控技术可以精确控制微米级的流体和细胞,因而成为一种特别适合检测稀有CTC的方法。微流控芯片具有成本低、操作简单、低耗材、高通量、实时检测等优势,其小型化的特点可将多种检测技术集成于微尺度中,为CTC的分离、鉴定和表征提供了一个高效的平台,有助于对肿瘤患者进行个体化分析与治疗。最近,三维(3D)打印技术的兴起为微流控芯片的制造提供了更高效、个性化的方式,避免了传统微流体器件制作方法步骤复杂、耗时等问题。逐层打印出的3D结构将促进微流控芯片实现更高效率和更高通量,并将推动实验室技术成功应用于临床,为肿瘤的生物学和临床研究开辟新视野,为乳腺癌的诊断治疗提供前所未有的机会。本文中,笔者分析了近年来CTC的不同检测手段的特点,阐述了微流控技术在乳腺癌CTC检测中的应用研究,以及3D打印微流控芯片技术前沿,并对3D打印微流控芯片技术在乳腺癌CTC检测中的应用前景进行了展望。
在世界范围内,乳腺癌已成为最常见的肿瘤,是女性癌症患者死亡的主要原
外周血中的CTC浓度非常低,大多数癌症患者每毫升血液中仅1~10个CT
基于CTC的生物学特征,通常利用附着在磁性物质表面的细胞表面标志物抗体等来筛选CTC。
阳性富集通过靶向CTC表面的特异性抗原来实现,通常可实现高纯度富集。CellSearch系统使用涂有上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)抗体的铁磁颗粒,对CTC进行免疫磁分离并计
技术 | 原理 | 标志物 | 检测过的癌种 | 优势 | 缺点 |
---|---|---|---|---|---|
CellSearc | 基于抗EpCAM抗体涂层的铁磁颗粒,进行免疫磁分离 | EpCAM | 乳腺癌、肺癌、胃癌、结直肠癌、前列腺癌、膀胱癌 | 敏感度高,半自动,可重复,获得FDA批准 | 无法捕获EpCAM表达水平低的CTC,无法进行后续分析 |
AdnaTes | 基于抗体组合的免疫磁分离,通过RT-PCR分析 | EpCAM、CEA、MUC-1、HER-2、EGFR、PSA等 | 乳腺癌、结肠癌、前列腺癌、卵巢癌 | 高敏感度和特异性,可进行下游分析 | WBC、核酸等污染导致假阳性结果,忽略EpCAM阴性的CTC |
MagSweeper | 基于抗EpCAM抗体靶向免疫磁珠进行分离 | EpCAM | 乳腺癌、结直肠癌、前列腺癌 | 敏感度高,CTC的纯度高,高处理量 | 昂贵,CTC被固定无法进行分析,忽略EpCAM阴性的CTC |
CanPatrol |
基于磁珠去除CD4 | CD45 | 乳腺癌、鼻咽癌、肺癌、食管癌、肝癌、结肠癌 | 不依赖EpCAM的表达,可保持CTC活性 | 特异度低,部分CTC被意外去除 |
CTC以尺寸、密度、可变形性及介电性与血细胞相区别,一些技术据此可以快速分离富集CTC而无需标记。
Drucker
基于免疫亲和力的捕获方法虽然特异度很高,但受限于CTC表面标志物的表达,迄今为止,CTC的异质性使其无法使用通用的特异性抗体来富集。而物理方法摆脱了靶向特定抗原的限制,未经抗体标记的CTC也更利于后续表征分析,但离心、稀释等预处理步骤会损失部分CTC,捕获的CTC纯度并不令人满
技术 | 原理 | 标志物 | 检测过的癌种 | 优势 | 缺点 |
---|---|---|---|---|---|
ISET | 基于细胞大小进行过滤 | — | 乳腺癌、肺癌、肝癌、结直肠癌、黑色素瘤 | 敏感度高,简单,价格低,无需标记 | 特异度低,处理慢,易出现膜堵塞 |
RosetteSep™ | 抗体标记改变细胞密度后,基于密度梯度分离CTC | CD45、CD66b、血型糖蛋白A等抗体混合物 | 乳腺癌、肝癌、胰腺癌、结直肠癌 | 敏感度高,快速,不依赖EpCAM表达 | 回收率低,易受WBC、RBC干扰 |
ScreenCell | 基于细胞大小富集CTC | — | 乳腺癌、胰腺癌、前列腺癌、黑色素瘤 | 快速,价格低,易于生产,可对捕获的CTC分析和培养 | 特异性差,无法捕获体积较小的CTC,RBC干扰 |
The Ficoll-Paqu | 基于密度梯度离心 | — | 乳腺癌、肝癌、胰腺癌、结直肠癌、肾癌、前列腺癌 | 快速方便,价格低,可保持CTC活性 | 敏感度和特异度低,受离心时间、温度影响 |
OncoQuic | 基于过滤去除RBC和WBC,并用密度梯度离心富集CTC | — | 乳腺癌、结直肠癌 | 高敏感度,高通量,价格低,CTC回收率高 | 特异度低,纯度低,有样品损失 |
ApoStrea | 基于介电泳原理 | — | 乳腺癌、胰腺癌、卵巢癌 | 无需标记,快速,可分离单个CTC,可保持CTC活性 | 通量小,纯度低 |
微流控技术在CTC检测中的应用出现于最近十几年,微流控技术以高通量和敏感度精确控制微米级的流体和细胞,旨在将所有实验室设备和功能集成到数十毫米尺寸的设备中,并以较低的成本和较短的分析时间对化合物进行微处理和微量分析。与传统方法相比,微流控芯片具有操作自动化、消耗样品少、敏感度高、通量大等优势,并可将过滤、离心、磁选等集成到微尺度
基于标记的方法依赖免疫亲和原理,微流控芯片精确控制流体的流动速度和方向,通过影响CTC表面抗原与抗体的相互作用从而直接影响捕获效率。针对不同的细胞表面抗原,此类方法可以进一步分为正向富集或负向富集。
基于CTC的抗原表达,正向富集法最常用的是EpCAM。Nagrath
负向富集靶向并去除背景细胞来实现CTC的分离。Mishra
无标记法旨在减少使用特异性抗体而导致的误差,既不影响细胞活力和基因表达谱,更有利于CTC的下游分析,并有望实现更高的捕获
主动分选主要通过施加外力操纵细胞来分离CTC。基于细胞的电生理特性,DEP利用介电粒子与电场之间的相互作用将CTC分离、捕
在一项多中心临床试
无标记策略不会影响细胞活力及基因表达谱,更快的样品处理确保了较大通量,然而CTC的异质性以及与背景细胞的重叠,导致其回收率和纯度并不令人满意。
目前,对CTC的研究已经不限于单纯枚举数量,已有研究将基于各种原理的检测手段集成在微流控芯片中,结合各自的优势并实现互补,在有效分离和富集CTC后对其进行分子表征,据此可以帮助阐明肿瘤转移机制、寻找治疗靶点及监测药物效应
Wang
传统的微流控设备基于光刻的制造工艺,复杂的制造步骤、昂贵的材料成本、耗时的制作过程均限制了微流控设备从实验室到实际临床实施的转
Che
3D打印微流控芯片可以为CTC检测和早期监测肿瘤转移开辟新机会,可以预测,3D打印技术在未来将会成为微流控芯片制造领域最为重要方法之一,为CTC检测广泛应用于临床提供有益的见解。
CTC的检测可以为乳腺癌的进展提供有价值的临床见解,监测患者在治疗期间的反应并为治疗方案的制定提供参考。微流控技术以其成本低廉、操作简单、试剂消耗少、小型化、快速等独特优势,越来越多地应用于CTC分离、鉴定和表征的各项研究中。3D打印技术凭借设计灵活性,克服了微流控设备功能表面积有限的不足,为微流控芯片制造提出了一种更优方案。在未来,期望微流控芯片将常规用于CTC的即时检测,这对研究乳腺癌及其转移机制的研究具有重要意义,将在癌症的早期诊断、预后的观察,以及患者的个性化治疗方面发挥关键作用。
作者贡献声明
尉卓凡负责调研整理文献,设计论文框架,论文撰写,根据修改意见进行论文修订;宁智文负责调研整理文献,对论文中微流控等专业领域部分做出修改解释;胡波负责协助确定研究选题,提供相关研究材料支持,论文修订,对微流控领域相关知识提供见解与指导;袁时芳提出并确定研究选题,确定论文结构框架,明确论文撰写内容,论文修订,给予指导性意见。
利益冲突
所有作者均声明不存在利益冲突。
参考文献
Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3):209-249. doi: 10.3322/caac.21660. [百度学术]
Fridrichova I, Kalinkova L, Ciernikova S. Clinical relevancy of circulating tumor cells in breast cancer: epithelial or mesenchymal characteristics, single cells or clusters?[J]. Int J Mol Sci, 2022, 23(20):12141. doi: 10.3390/ijms232012141. [百度学术]
Ramos-Medina R, López-Tarruella S, del Monte-Millán M, et al. Technical challenges for CTC implementation in breast cancer[J]. Cancers, 2021, 13(18):4619. doi: 10.3390/cancers13184619. [百度学术]
Zhuang J, Xia L, Zou Z, et al. Recent advances in integrated microfluidics for liquid biopsies and future directions[J]. Biosens Bioelectron, 2022, 217:114715. doi: 10.1016/j.bios.2022.114715. [百度学术]
Yang YP, Giret TM, Cote RJ. Circulating tumor cells from enumeration to analysis: current challenges and future opportunities[J]. Cancers, 2021, 13(11):2723. doi: 10.3390/cancers13112723. [百度学术]
Dirix L, Buys A, Oeyen S, et al. Circulating tumor cell detection: a prospective comparison between CellSearch® and RareCyte® platforms in patients with progressive metastatic breast cancer[J]. Breast Cancer Res Treat, 2022, 193(2):437-444. doi: 10.1007/s10549-022-06585-5. [百度学术]
Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer[J]. N Engl J Med, 2004, 351(8):781-791. doi: 10.1056/NEJMoa040766. [百度学术]
Lin D, Shen L, Luo M, et al. Circulating tumor cells: biology and clinical significance[J]. Signal Transduct Target Ther, 2021, 6(1): 404. doi: 10.1038/s41392-021-00817-8. [百度学术]
Russo GI, Musso N, Romano A, et al. The role of dielectrophoresis for cancer diagnosis and prognosis[J]. Cancers, 2021, 14(1):198. doi: 10.3390/cancers14010198. [百度学术]
Descamps L, Le Roy D, Deman AL. Microfluidic-based technologies for CTC isolation: a review of 10 years of intense efforts towards liquid biopsy[J]. Int J Mol Sci, 2022, 23(4):1981. doi: 10.3390/ijms23041981. [百度学术]
Wu SY, Liu ZM, Liu SY, et al. Enrichment and enumeration of circulating tumor cells by efficient depletion of leukocyte fractions[J]. Clin Chem Lab Med, 2014, 52(2):243-251. doi: 10.1515/cclm-2013-0558 [百度学术]
Bittner AK, Keup C, Hoffmann O, et al. Molecular characterization of circulating tumour cells identifies predictive markers for outcome in primary, triple-negative breast cancer patients[J]. J Cell Mol Med, 2020, 24(15):8405-8416. doi: 10.1111/jcmm.15349. [百度学术]
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[J]. Proc Natl Acad Sci U S A, 2009, 106(10):3970-3975. doi: 10.1073/pnas.0813188106. [百度学术]
Drucker A, Teh EM, Kostyleva R, et al. Comparative performance of different methods for circulating tumor cell enrichment in metastatic breast cancer patients[J]. PLoS One, 2020, 15(8):e0237308. doi: 10.1371/journal.pone.0237308. [百度学术]
Rosenberg R, Gertler R, Friederichs J, et al. Comparison of two density gradient centrifugation systems for the enrichment of disseminated tumor cells in blood[J]. Cytometry, 2002, 49(4):150-158. doi: 10.1002/cyto.10161. [百度学术]
Le Du F, Fujii T, Kida K, et al. EpCAM-independent isolation of circulating tumor cells with epithelial-to-mesenchymal transition and cancer stem cell phenotypes using ApoStream® in patients with breast cancer treated with primary systemic therapy[J]. PLoS One, 2020, 15(3):e0229903. doi: 10.1371/journal.pone.0229903. [百度学术]
Zhang Y, Li YF, Tan ZC. A review of enrichment methods for circulating tumor cells: from single modality to hybrid modality[J]. Analyst, 2021, 146(23):7048-7069. doi: 10.1039/d1an01422f. [百度学术]
Guo LH, Liu C, Qi ML, et al. Recent progress of nanostructure-based enrichment of circulating tumor cells and downstream analysis[J]. Lab Chip, 2023, 23(6):1493-1523. doi: 10.1039/d2lc00890d [百度学术]
Zeng HL, Veeramootoo JS, Ma G, et al. Clinical value and feasibility of ISET in detecting circulating tumor cells in early breast cancer[J]. Transl Cancer Res TCR, 2020, 9(7):4297-4305. doi: 10.21037/tcr-19-2662. [百度学术]
He ST, Wei JL, Ding L, et al. State-of-the-arts techniques and current evolving approaches in the separation and detection of circulating tumor cell[J]. Talanta, 2022, 239:123024. doi: 10.1016/j.talanta.2021.123024. [百度学术]
Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology[J]. Nature, 2007, 450(7173):1235-1239. doi: 10.1038/nature06385. [百度学术]
Lin ZJ, Luo GY, Du WX, et al. Recent advances in microfluidic platforms applied in cancer metastasis: circulating tumor cells' (CTCs) isolation and tumor-on-A-chip[J]. Small, 2020, 16(9):e1903899. doi: 10.1002/smll.201903899. [百度学术]
Stroock AD, Dertinger SKW, Ajdari A, et al. Chaotic mixer for microchannels[J]. Science, 2002, 295(5555):647-651. doi: 10.1126/science.1066238. [百度学术]
Wang S, Thomas A, Lee E, et al. Highly efficient and selective isolation of rare tumor cells using a microfluidic chip with wavy-herringbone micro-patterned surfaces[J]. Analyst, 2016, 141(7):2228-2237. doi: 10.1039/C6AN00236F. [百度学术]
Wang C, Xu Y, Li SN, et al. Designer tetrahedral DNA framework-based microfluidic technology for multivalent capture and release of circulating tumor cells[J]. Mater Today Bio, 2022, 16:100346. doi: 10.1016/j.mtbio.2022.100346. [百度学术]
Wang ZL, Wu ZE, Sun N, et al. Antifouling hydrogel-coated magnetic nanoparticles for selective isolation and recovery of circulating tumor cells[J]. J Mater Chem B, 2021, 9(3):677-682. doi: 10.1039/d0tb02380a. [百度学术]
Wu X, Lin Z, Zhao C, vet al. Neutrophil membrane-coated immunomagnetic nanoparticles for efficient isolation and analysis of circulating tumor cells[J]. Biosens Bioelectron, 2022, 213:114425. doi: 10.1016/j.bios.2022.114425. [百度学术]
Sieuwerts AM, Kraan J, Bolt J, et al. Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[J]. J Natl Cancer Inst, 2009, 101(1):61-66. doi: 10.1093/jnci/djn419. [百度学术]
Mishra A, Dubash TD, Edd JF, et al. Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells[J]. Proc Natl Acad Sci U S A, 2020, 117(29):16839-16847. doi: 10.1073/pnas.2006388117. [百度学术]
Civelekoglu O, Frazier AB, Sarioglu AF. The origins and the current applications of microfluidics-based magnetic cell separation technologies[J]. Magnetochemistry, 2022, 8(1):10. doi: 10.3390/magnetochemistry8010010. [百度学术]
Topa J, Grešner P, Żaczek AJ, et al. Breast cancer circulating tumor cells with mesenchymal features-an unreachable target?[J]. Cell Mol Life Sci, 2022, 79(2):81. doi: 10.1007/s00018-021-04064-6. [百度学术]
Szczerba BM, Castro-Giner F, Vetter M, et al. Neutrophils escort circulating tumour cells to enable cell cycle progression[J]. Nature, 2019, 566(7745):553-557. doi: 10.1038/s41586-019-0915-y. [百度学术]
Lu N, Tay HM, Petchakup C, et al. Label-free microfluidic cell sorting and detection for rapid blood analysis[J]. Lab Chip, 2023, 23(5):1226-1257. doi: 10.1039/D2LC00904H. [百度学术]
Farshchi F, Hasanzadeh M. Microfluidic biosensing of circulating tumor cells (CTCs): recent progress and challenges in efficient diagnosis of cancer[J]. Biomed Pharmacother, 2021, 134:111153. doi: 10.1016/j.biopha.2020.111153. [百度学术]
Al-Ali A, Waheed W, Abu-Nada E, et al. A review of active and passive hybrid systems based on Dielectrophoresis for the manipulation of microparticles[J]. J Chromatogr A, 2022, 1676:463268. doi: 10.1016/j.chroma.2022.463268. [百度学术]
Jahangiri M, Ranjbar-Torkamani M, Abadijoo H, et al. Low frequency stimulation induces polarization-based capturing of normal, cancerous and white blood cells: a new separation method for circulating tumor cell enrichment or phenotypic cell sorting[J]. Analyst, 2020, 145(23):7636-7645. doi: 10.1039/d0an01033b. [百度学术]
Varmazyari V, Habibiyan H, Ghafoorifard H, et al. A dielectrophoresis-based microfluidic system having double-sided optimized 3D electrodes for label-free cancer cell separation with preserving cell viability[J]. Sci Rep, 2022, 12(1):12100. doi: 10.1038/s41598-022-16286-0. [百度学术]
Zhang Y, Zhang ZA, Zheng DB, et al. Label-free separation of circulating tumor cells and clusters by alternating frequency acoustic field in a microfluidic chip[J]. Int J Mol Sci, 2023, 24(4):3338. doi: 10.3390/ijms24043338. [百度学术]
Witek M, Freed IM, Soper S. Cell separations and sorting[J]. Anal Chem, 2020,92(1):105-131. doi: 10.1021/acs.analchem.9b05357. [百度学术]
Hu X, Zhu D, Chen M, et al. Precise and non-invasive circulating tumor cell isolation based on optical force using homologous erythrocyte binding[J]. Lab Chip, 2019, 19(15):2549-2556. doi: 10.1039/c9lc00361d. [百度学术]
Cohen EN, Jayachandran G, Moore RG, et al. A Multi-Center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsorti
Akbarnataj K, Maleki S, Rezaeian M, et al. Novel size-based design of spiral microfluidic devices with elliptic configurations and trapezoidal cross-section for ultra-fast isolation of circulating tumor cells[J]. Talanta, 2023, 254:124125. doi: 10.1016/j.talanta.2022.124125. [百度学术]
Zhu Z, Wu D, Li S, et al. A polymer-film inertial microfluidic sorter fabricated by jigsaw puzzle method for precise size-based cell separation[J]. Anal Chimica Acta, 2021, 1143:306-314. doi: 10.1016/j.aca.2020.11.001. [百度学术]
Ring A, Nguyen-Sträuli BD, Wicki A, et al. Biology, vulnerabilities and clinical applications of circulating tumour cells[J]. Nat Rev Cancer, 2023, 23(2):95-111. doi: 10.1038/s41568-022-00536-4. [百度学术]
Wang SB, Hong SL, Cai SJ, et al. Negative depletion mediated brightfield circulating tumour cell identification strategy on microparticle-based microfluidic chip[J]. J Nanobiotechnol, 2020, 18(1):70. doi: 10.1186/s12951-020-00623-4. [百度学术]
Lee J, Kwak B. Simultaneous on-chip isolation and characterization of circulating tumor cell sub-populations[J]. Biosens Bioelectron, 2020, 168:112564. doi: 10.1016/j.bios.2020.112564. [百度学术]
Green BJ, Marazzini M, Hershey B, et al. PillarX: a microfluidic device to profile circulating tumor cell clusters based on geometry, deformability, and epithelial state[J]. Small, 2022, 18(17):2106097. doi: 10.1002/smll.202106097. [百度学术]
Schwab FD, Scheidmann MC, Ozimski LL, et al. MyCTC chip: microfluidic-based drug screen with patient-derived tumour cells from liquid biopsies[J]. Microsyst Nanoeng, 2022, 8:130. doi: 10.1038/s41378-022-00467-y. [百度学术]
Lv SW, Zheng D, Chen ZX, et al. Near-infrared light-responsive size-selective lateral flow chip for single-cell manipulation of circulating tumor cells[J]. Anal Chem, 2023, 95(2):1201-1209. doi: 10.1021/acs.analchem.2c03947. [百度学术]
Bhat MP, Thendral V, Uthappa UT, et al. Recent advances in microfluidic platform for physical and immunological detection and capture of circulating tumor cells[J]. Biosensors, 2022, 12(4):220. doi: 10.3390/bios12040220. [百度学术]
Safhi AY. Three-dimensional (3D) printing in cancer therapy and diagnostics: current status and future perspectives[J]. Pharmaceuticals, 2022, 15(6):678. doi: 10.3390/ph15060678. [百度学术]
Chen ZZ, Zhao L, Wei LJ, et al. River meander-inspired cross-section in 3D-printed helical microchannels for inertial focusing and enrichment[J]. Sens Actuat B Chem, 2019, 301:127125. doi: 10.1016/j.snb.2019.127125. [百度学术]
Yin PJ, Zhao L, Chen ZZ, et al. Simulation and practice of particle inertial focusing in 3D-printed serpentine microfluidic chips via commercial 3D-printers[J]. Soft Matter, 2020, 16(12): 3096-3105. doi: 10.1039/d0sm00084a. [百度学术]
Chu CH, Liu RX, Ozkaya-Ahmadov T, et al. Negative enrichment of circulating tumor cells from unmanipulated whole blood with a 3D printed device[J]. Sci Rep, 2021, 11:20583. doi: 10.1038/s41598-021-99951-0. [百度学术]