基于微流体技术分选循环肿瘤细胞的方法研究
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摘要
循环肿瘤细胞(Circulating Tumor Cells, CTCs)主要来源于肿瘤组织自发脱落的外周血液中,其对恶性肿瘤传播转移具有重要影响,已逐渐被认为是肿瘤远处转移的标志。对外周血中极微量的具有特异性、敏感性的CTCs进行分选、富集及检测,不仅有利于肿瘤的早期诊断、疗效评价及复发转移监控,还可以为后续的CTCs鉴定和下游单细胞基因组和转录组测序提供良好基础,为肿瘤靶向治疗提供新策略,在临床上个性化医疗等领域有重要意义。微流控技术可在微米尺度下整合物理、化学及生物学方法,实现对微量CTCs的高通量、高效率以及低成本分选富集,该技术发展迅速并已被广泛研究应用。本文对微流控芯片进行CTCs分选富集及捕获的最新研究进展进行综述,阐述无标记、有标记分选方法的原理及应用实例,分析各种技术的优缺点,对现阶段该领域存在的问题进行讨论,对微流控CTCs分选芯片的应用前景和未来发展趋势进行展望。
Circulating tumor cells(CTCs) are mainly derived from the peripheral blood of tumor tissue that is spontaneously shedding. CTCs have an important influence on the spread and metastasis of malignant tumors and have gradually been considered as a marker of distant metastasis of tumors. The selection, enrichment and detection of a very small amount of specific and sensitive CTCs in peripheral blood are not only helpful for the early diagnosis of tumors, the evaluation of efficacy and the monitoring of recurrence and metastasis, but also conducive to the identification of CTCs and the genome and transcriptome sequencings of downstream single cells, providing a new strategy for targeted therapy of tumors, which is of great significance in personalized medicine. Microfluidics can integrate physical, chemical and biological methods at the microscale to achieve high-throughput, high-efficiency, low-cost sorting and enrichment of a small amount of CTCs. This technology has been rapidly developed in recent years and has been widely applied. Herein, the latest research progresses on microfluidic chip systems in the sorting, enrichment and capture of CTCs are summarized. Moreover, the principle and application examples of the unmarked or labeled sorting method are expounded, and the advantages and disadvantages of various technologies are analyzed. The problems existing at the stage are also discussed. Finally, the applications and future development trends of microfluidic CTCs sorting chips are prospected.
Circulating tumor cells(CTCs) are mainly derived from the peripheral blood of tumor tissue that is spontaneously shedding. CTCs have an important influence on the spread and metastasis of malignant tumors and have gradually been considered as a marker of distant metastasis of tumors. The selection, enrichment and detection of a very small amount of specific and sensitive CTCs in peripheral blood are not only helpful for the early diagnosis of tumors, the evaluation of efficacy and the monitoring of recurrence and metastasis, but also conducive to the identification of CTCs and the genome and transcriptome sequencings of downstream single cells, providing a new strategy for targeted therapy of tumors, which is of great significance in personalized medicine. Microfluidics can integrate physical, chemical and biological methods at the microscale to achieve high-throughput, high-efficiency, low-cost sorting and enrichment of a small amount of CTCs. This technology has been rapidly developed in recent years and has been widely applied. Herein, the latest research progresses on microfluidic chip systems in the sorting, enrichment and capture of CTCs are summarized. Moreover, the principle and application examples of the unmarked or labeled sorting method are expounded, and the advantages and disadvantages of various technologies are analyzed. The problems existing at the stage are also discussed. Finally, the applications and future development trends of microfluidic CTCs sorting chips are prospected.
引文
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[12]HOSOKAWA M, KENMOTSU H, KOH Y, et al. Size-based isolationof circulating tumor cells in lung cancer patients using a microcavityarray system[J]. PLoS One, 2013, 8(6):e67466.
[13]TAN S J, YOBAS L, LEE G Y H, et al. Microdevice for the isolationand enumeration of cancer cells from blood[J]. Biomed Microdevices,2009, 11(4):883-890.
[14]LüP, TANG Z, LIANG X, et al. Spatially gradated segregation andrecovery of circulating tumor cells from peripheral blood of cancerpatients[J]. Biomicrofluidics, 2013, 7(3):180-204.
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[16]黄笛,项楠,唐文来,等.基于微流控技术的循环肿瘤细胞分选研究[J].化学进展, 2015, 27(7):882-912.HUANG D, XIANG N, TANG W L, et al. Microfluidics-basedcirculating tumor cells separation[J]. Progress in Chemistry, 2015, 27(7):882-912.
[17]CHOI Y S, LEE S J. Holographic analysis of three-dimensional inertialmigration of spherical particles in micro-scale pipe flow[J].Microfluid Nanofluidics, 2010, 9(4-5):819-829.
[18]CHEN H. A triplet parallelizing spiral microfluidic chip for continuousseparation of tumor cells[J]. Sci Rep, 2018, 8(1):4042.
[19]KIM T H, YOON H J, STELLA P, et al. Cascaded spiral microfluidicdevice for deterministic and high purity continuous separation ofcirculating tumor cells[J]. Biomicrofluidics, 2014, 8(6):449.
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[21]LOUTHERBACK K, D'SILVA J, LIU L, et al. Deterministic separationof cancer cells from blood at 10 mL/min[J]. AIP Adv, 2012, 2(4):042107.
[22]LIU Z, HUANG F, DU J, et al. Rapid isolation of cancer cells usingmicrofluidic deterministic lateral displacement structure[J].Biomicrofluidics, 2013, 7(1):11801.
[23]HOLMES D, GREEN N G, MORGAN H. Microdevices fordielectrophoretic flow-through cell separation[J]. IEEE Eng Med BiolMag, 2003, 22(6):85-90.
[24]WU L, YUNG L Y, LIM K M. Dielectrophoretic capture voltagespectrum for measurement of dielectric properties and separation ofcancer cells[J]. Biomicrofluidics, 2012, 6(1):14113. DOI:10.1063/1.3690470.
[25]ALSHAREEF M, METRAKOS N, PEREZ E J, et al. Separation oftumor cells with dielectrophoresis-based microfluidic chip[J].Biomicrofluidics, 2013, 7(1):011803.
[26]CHEN W, WENG S, ZHANG F, et al. Nanoroughened surfaces forefficient capture of circulating tumor cells without using captureantibodies[J]. ACS Nano, 2012, 7(1):566-575.
[27]吕晓庆,李雷,陈红梅,等.微流控芯片技术在循环肿瘤细胞分离中的研究进展[J].生物化学与生物物理进展, 2015, 42(4):301-312.LüX Q, LI L, CHEN H M, et al. Advances in isolating circulatingtumor cells with microfluidic chips[J]. Progress in Biochemistry andBiophysics, 2015, 42(4):301-312.
[28]HE R, ZHAO L, LIU Y, et al. Biocompatible Ti O2nanoparticle-basedcell immunoassay for circulating tumor cells capture and identificationfrom cancer patients[J]. Biomed Microdevices, 2013, 15(4):617-626.
[29]SHENG W, OGUNWOBI O O, CHEN T, et al. Capture, release andculture of circulating tumor cells from pancreatic cancer patients usingan enhanced mixing chip[J]. Lab Chip, 2014, 14(1):89-98.
[30]KANG J H, KRAUSE S, TOBIN H, et al. A combinedmicromagnetic microfluidic device for rapid capture and culture ofrare circulating tumor cells[J]. Lab Chip, 2012, 12(12):2175-2181.
[31]HYUN K A, LEE T Y, LEE S H, et al. Two-stage microfluidic chip forselective isolation of circulating tumor cells(CTCs)[J]. BiosensBioelectron, 2015, 67:86-92.
[32]HOU H W, WARKIANI M E, KHOO B L, et al. Isolation and retrievalof circulating tumor cells using centrifugal forces[J]. Sci Rep, 2013,3(7):1259.
[2] ESMAEILSABZALI H, BEISCHLAG T V, COX M E, et al. Detectionand isolation of circulating tumor cells:principles and methods[J].Biotechnol Adv, 2013, 31(7):1063-1084.
[3] CRNIC I, CHRISTOFORI G. Novel technologies and recent advancesin metastasis research[J]. Int J Dev Biol, 2004, 48(5-6):573-581.
[4] KRIVACIC R T, LADANYI A, CURRY D N, et al. A rare-cell detectorfor cancer[J]. Proc Natl Acad Sci USA, 2004, 101(29):10501-10504.
[5] ADALSTEINSSON V A, LOVE J C. Toward engineered processes forsequencing-based analysis of single circulating tumor cells[J]. CurrOpin Chem Eng, 2014, 4:97-104.
[6]贾海明,米泰宇,杨雯,等. Cell Search系统检测胃癌患者循环肿瘤细胞及临床意义[J].卫生职业教育, 2017, 35(17):149-151.JIA H M, MI T Y, YANG W, et al. CellSearch system for detectingcirculating tumor cells in patients with gastric cancer and its clinicalsignificance[J]. Health Vocational Education, 2017, 35(17):149-151.
[7] LARA O, TONG X, ZBOROWSKI M, et al. Enrichment of rare cancercells through depletion of normal cells using density and flow-through,immunomagnetic cell separation[J]. Exp Hematol, 2004, 32(10):891-904.
[8] WHITESIDES G M. The origins and the future of microfluidics[J].Nature, 2006, 442(7101):368-373.
[9] SCHWARTZ M. Molecular characterization of CTCs[J]. Genet EngBiotechnol News, 2013, 33:36-37.
[10]GLEGHORN J P, PRATT E D, GLEGHORN D D, et al. Capture ofcirculating tumor cells from whole blood of prostate cancer patientsusing geometrically enhanced differential immunocapture(GEDI)anda prostate-specific antibody[J]. Lab Chip, 2010, 10(1):27-29.
[11]WARKIANI M E, GUAN G, LUAN K B, et al. Slanted spiralmicrofluidics for the ultra-fast, label-free isolation of circulating tumorcells[J]. Lab Chip, 2014, 14(1):128-137.
[12]HOSOKAWA M, KENMOTSU H, KOH Y, et al. Size-based isolationof circulating tumor cells in lung cancer patients using a microcavityarray system[J]. PLoS One, 2013, 8(6):e67466.
[13]TAN S J, YOBAS L, LEE G Y H, et al. Microdevice for the isolationand enumeration of cancer cells from blood[J]. Biomed Microdevices,2009, 11(4):883-890.
[14]LüP, TANG Z, LIANG X, et al. Spatially gradated segregation andrecovery of circulating tumor cells from peripheral blood of cancerpatients[J]. Biomicrofluidics, 2013, 7(3):180-204.
[15]JIN C, MCFAUL S M, MA H, et al. Continuous flow cell separationsystem using microfluidic ratchets[C]. 17th International Conferenceon Miniatnrized Systems for Chemistry and Life Sciences October 27-31, 2013, Freiburg, Germany.
[16]黄笛,项楠,唐文来,等.基于微流控技术的循环肿瘤细胞分选研究[J].化学进展, 2015, 27(7):882-912.HUANG D, XIANG N, TANG W L, et al. Microfluidics-basedcirculating tumor cells separation[J]. Progress in Chemistry, 2015, 27(7):882-912.
[17]CHOI Y S, LEE S J. Holographic analysis of three-dimensional inertialmigration of spherical particles in micro-scale pipe flow[J].Microfluid Nanofluidics, 2010, 9(4-5):819-829.
[18]CHEN H. A triplet parallelizing spiral microfluidic chip for continuousseparation of tumor cells[J]. Sci Rep, 2018, 8(1):4042.
[19]KIM T H, YOON H J, STELLA P, et al. Cascaded spiral microfluidicdevice for deterministic and high purity continuous separation ofcirculating tumor cells[J]. Biomicrofluidics, 2014, 8(6):449.
[20]SUN J, LIU C, LI M, et al. Size-based hydrodynamic rare tumor cellseparation in curved microfluidic channels[J]. Biomicrofluidics, 2013,7(1):11802.
[21]LOUTHERBACK K, D'SILVA J, LIU L, et al. Deterministic separationof cancer cells from blood at 10 mL/min[J]. AIP Adv, 2012, 2(4):042107.
[22]LIU Z, HUANG F, DU J, et al. Rapid isolation of cancer cells usingmicrofluidic deterministic lateral displacement structure[J].Biomicrofluidics, 2013, 7(1):11801.
[23]HOLMES D, GREEN N G, MORGAN H. Microdevices fordielectrophoretic flow-through cell separation[J]. IEEE Eng Med BiolMag, 2003, 22(6):85-90.
[24]WU L, YUNG L Y, LIM K M. Dielectrophoretic capture voltagespectrum for measurement of dielectric properties and separation ofcancer cells[J]. Biomicrofluidics, 2012, 6(1):14113. DOI:10.1063/1.3690470.
[25]ALSHAREEF M, METRAKOS N, PEREZ E J, et al. Separation oftumor cells with dielectrophoresis-based microfluidic chip[J].Biomicrofluidics, 2013, 7(1):011803.
[26]CHEN W, WENG S, ZHANG F, et al. Nanoroughened surfaces forefficient capture of circulating tumor cells without using captureantibodies[J]. ACS Nano, 2012, 7(1):566-575.
[27]吕晓庆,李雷,陈红梅,等.微流控芯片技术在循环肿瘤细胞分离中的研究进展[J].生物化学与生物物理进展, 2015, 42(4):301-312.LüX Q, LI L, CHEN H M, et al. Advances in isolating circulatingtumor cells with microfluidic chips[J]. Progress in Biochemistry andBiophysics, 2015, 42(4):301-312.
[28]HE R, ZHAO L, LIU Y, et al. Biocompatible Ti O2nanoparticle-basedcell immunoassay for circulating tumor cells capture and identificationfrom cancer patients[J]. Biomed Microdevices, 2013, 15(4):617-626.
[29]SHENG W, OGUNWOBI O O, CHEN T, et al. Capture, release andculture of circulating tumor cells from pancreatic cancer patients usingan enhanced mixing chip[J]. Lab Chip, 2014, 14(1):89-98.
[30]KANG J H, KRAUSE S, TOBIN H, et al. A combinedmicromagnetic microfluidic device for rapid capture and culture ofrare circulating tumor cells[J]. Lab Chip, 2012, 12(12):2175-2181.
[31]HYUN K A, LEE T Y, LEE S H, et al. Two-stage microfluidic chip forselective isolation of circulating tumor cells(CTCs)[J]. BiosensBioelectron, 2015, 67:86-92.
[32]HOU H W, WARKIANI M E, KHOO B L, et al. Isolation and retrievalof circulating tumor cells using centrifugal forces[J]. Sci Rep, 2013,3(7):1259.