NGS中的CTC和ctDNA.ppt

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1、循环肿瘤细胞循环肿瘤细胞（Circulatingtumorcells(CTCs)）目录CTCs的概念与特点CTCs的富集与检测CTCs的应用与研究进展概念Circulating tumor cells(CTCs)arecellsthathaveshedintothevasculaturefromaprimarytumorandcirculateinthebloodstream.2005年Pachm ann将CTCs正式定义为自发或因诊疗操作由实体瘤或转移灶释放进入外周血循环的肿瘤细胞.概念 恶性肿瘤是我国死亡率最高的重大疾病之一，90%以上肿瘤患者的死亡都是由于肿瘤转移所致侵袭与转移是恶性肿瘤

2、最显著的特征之一。CTCs是肿瘤转移过程中在血液循环系统中存活的肿瘤细胞，它的生成被认为是肿瘤发生转移的必要前提。特点研究意义深入研究CTCs有助于对肿瘤转移机制进一步了解，为抗肿瘤转移的治疗提供新的依据 CTCs的检测有助于早期转移肿瘤患者的诊断、监测术后患者肿瘤的复发与转移有助于评估抗肿瘤药物的敏感性与患者预后以及选择个体化的治疗策略研究难点 CTCs并没有显著的特异性同其它血细胞明确区分且不同组织学类型和分子表型的肿瘤分别表达不同的标志物。CTCs在外周血中数量稀少，一般在106-107个白细胞中仅含有1个。无显著特异性数量少CTC分析的三个主要步骤非特异性富集方法特异性富集方法方法CT

3、Cs-芯片富集法纳米粒富集法免疫磁珠富集法CTCs的富集密度梯度离心富集法细胞大小 富集法细胞变形性富集法细胞电学特征富集法Ficoll法和Oncoquick法上皮肿瘤细胞大小分离(ISET)和微滤芯片技术双向电泳-场流分离法（DEP-FFF）磁性活细胞分选法(MACS)、AdnaTest法、Cellsearch法CTCs的富集CTCs细胞大小富集方法Using size-based enrichment techniques,diluted whole blood is passed through a filtration device with specific sized pores(

4、typically 8 m).CTCs are captured based on differences in cell size between CTCs(typically 8 m)and white blood cells(WBCs;typically 8 m)Scanning electron microscopy(SEM)images of:(A)commercial track-etched membranes.The random distribution of pores results in the formation of larger holes by fusion o

5、f neighboring holes.(B)A microfabricated hole in a parylene membrane filter.(C)Captured cells on a parylene membrane with controlled distribution and size of holes.(D)SEM view of a captured CTC.Track-etched polycarbonate filters(A)Schematicofapool-damchip.Thedecreasingwidthofgapsbetweensuccessivepoo

6、lsallowstheseparationofCTCsinthefirstpool,followedbyWBCsandRBCsinthesubsequentpools(B)Schematicofarraysofobstacleswithsuccessivelydecreasingdistances.LargerCTCsaretrappedbetweentheobstacles,whilesmallerbloodcellsmovefreely.Microfluidic setting(C)Isolationofcancercellsbycrescent-shapedtraps.Thedistan

7、cebetweeneachofthethreemicropillarsis5mwhichpresumablyresultsinthecaptureoflargercancercellsandnotbloodcells.Density-basedenrichmentutilizesFicoll(orsimilardensitygradientmedium)toenrichformononuclearcells(includingCTCs)fromotherbloodcomponents.CTCs密度梯度离心富集法Plasma:血浆Mononuclearcell：单核细胞（包括淋巴细胞、单核细胞、

8、上皮细胞和肿瘤细胞）Ficoll：密度梯度液RBCs：红细胞The DEP/G-FFF principle for cell separation.Different cells are levitated at different heights determined by the collective effect of DEP and gravity(FDEPz and Fgrav).After cells reach their equilibriums,a parabolic laminar flow having different velocity profiles at dif

9、ferent heights(VFFF2 N VFFF1)collects the cells at the same output but at different times.Cells within thefaster profile(VFFF2)are collected first,followed by the cells lie within the slower profile(VFFF1).细胞电学特征富集法Immunomagnetic separation involves the use of iron-conjugated antibodies targeted tow

10、ard CTCs(e.g.,EpCAM;positive selection)or contaminating blood cells(e.g.,CD45;negative selection)and incubation in a magnetic field.CTCs免疫磁珠富集法CTCs微流体技术富集法whole blood is slowly passed across a chip-based surface and isolated using either CTC targeted antibody-coated microposts or dielectrophoresis（D

11、EP）CTCs各种富集方法的优缺点CTCs的富集CTCs的检测CTCs部分检测方法CTCs的检测流式细胞仪分析(flow cytometry FCM)免疫细胞化学技术 ICC（immunocytochemistry）鉴定标本中细胞抗原性和形态学特征，能使富集的目的细胞维持细胞形态并保持细胞活力 ICC是用能与显色剂结合的单抗与 CTCs特异性结合后，通过显色剂显色从而对CTCs进行鉴定的技术通过分析上皮细胞或肿瘤细胞的正常起源组织特异的候选基因的表达来检测 CTC,灵敏度非常高但是坏死的癌细胞、上皮细胞污染等都可以造成假阳性结果，此法无法检测CTCs细胞形态，在临床应用上有局限性。反转录-聚合

12、酶链反应RT-PCRCellsearchFDA唯一批准的检测CTC的方法。一种半自动技术，通过载有抗EpCAM抗体的铁磁流体富集CTC；随后CTC用CK和DAPI荧光抗体染色，其余血细胞用CD45对比染色，CK+DAPI+CD45-细胞使用自动荧光显微镜进行计数一种基于酶联免疫吸附测定原理的免疫学分析方法。选择性去除CD45阳性细胞富集CXCR4阳性细胞后，测定特异性蛋白如cathepsin-D、musin-1来计数能分泌蛋白的存活CTCEPISPOT一 种 更 为 先 进 的 检 测 CTC的 技 术。设备载有78000个包被EpCAM抗体的微柱，当血液样品流经时，EpCAM阳性细胞被粘附于

13、微柱的表面CTC-chipCTCs富集和检测相结合的技术CTC的研发趋势CTM的检测Circulating tumor microemboli(CTM)CTCCTMCTM are composed of at least two tumor cells,and occasionally,normal blood cells.CTM could be an indication of higher metastatic potential Metastatic lung cancerColorectacancer Liver cancerRenalcancerBreastcancer Prost

14、ate cancermicrosieve chipFlowcytometrysize-based filtration(ISET)microvortex herringbone-chipCell searchhigh-definition fluorescence scanning microscopyCTM的检测CTC的研发趋势CTMat的检测Circulating tumor materials(CTMat):damaged cells,fragmented cells,cellular debris,microparticles,and clump-like aggregatesprov

15、ide flexibility in prognosis due totheir high abundanceless stringent target identification,easier enumeration,and fully automated image processingCTDNA correlated with the malignancy status and the therapy responseAdvantageCtDNA as an alternative to CTC analysisCTC与CTDNA的分析比较CTC的研发趋势端粒酶的活性与CTC的检测Te

16、lomerase has been found activated in the majority of cancer types and is known to be associated with malignant propertiesAssessing telomerase activity may as a method to accurately detect entire CTC populationsblood sample has to be Cells were lysed in order to measure the enzyme activity,thereby de

17、stroying all intactCTCsadvantagedisadvantageCTC的研发趋势Aptamer technology in CTC DetectionAptamers：single-stranded RNA or DNA molecules，molecular weight is about 8 to 15 kDa,leading to rapid tumor penetration andblood clearance.Incorporation of aptamer technology into microdevices has the most potential for the development of a fast,easy,and accurate method for cell isolation,enrichment and related purposes such as detection,enumeration,and characterizationAptamer production:SELEX techniqueless expensive and much faster due to small size,simple structure,and no use of animal cellsCTC的功能研究