NGS中的CTC和ctDNA专题培训课件.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,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)are cells that have shed into the vasculature from a primary tumor and circulate in thebloodstream.,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,10,6,-10,7,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,CTCs-,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(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 of 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,Schematic,of a pool-dam chip.The decreasing width of gaps between successive pools allows the separation of CTCs in the first pool,followed by WBCs and RBCs in the subsequent pools,(B)Schematic of arrays of obstacles with successively decreasing distances.Larger CTCs are trapped between the obstacles,while smaller blood cells move freely.,Microfluidic setting,(C)Isolation of cancer cells by crescent-shaped traps.The distance between each of the three micropillars is 5 m which presumably results in the capture of larger cancer cells and not blood cells.,Density-based enrichment utilizes Ficoll(or similar density gradient medium)to enrich for mononuclear cells(including CTCs)from other blood components.,CTCs,密度梯度离心富集法,Plasma:,血浆,Mononuclear cell,：单核细胞（包括淋巴细胞、单核细胞、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 different 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 toward 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,（,DEP,）,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,-,聚合酶链反应,RT-PCR,Cellsearch,FDA,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,CTC,EPISPOT,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,阳性细胞被粘附于微柱的表面,CTC-chip,CTCs,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),CTC,CTM,CTM are composed of at least two tumor cells,and occasionally,normal blood cells.,CTM could be an indication of higher metastatic potential,Metastatic lung,cancer,Colorecta,cancer,Liver,cancer,Renal,cancer,Breast,cancer,Prostate,cancer,microsieve chip,Flow cytometry,size-based filtration(ISET),microvortex herringbone-chip,Cell search,high-definition fluores,cence scanning microscopy,CTM,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 aggregates,provide flexibility in prognosis due totheir high abundance,less stringent target identification,easier enumeration,and fully automated image processing,CTDNA,correlated with the,malignancy status and the therapy response,Advantage,CtDNA,as an alternative to CTC analysis,CTC,与,CTDNA,的分析比较,CTC,的研发趋势,端粒酶的活性与,CTC,的检测,Telomerase has been found activated in the majority of cancer types and is known to be associated with malignant properties,Assessing telomerase activity may as a method to accurately detect entire CTC populations,blood sample has to be,Cells were lysed in order to measure the enzyme activity,thereby destroying all intactCTCs,advantage,disadvantage,CTC,的研发趋势,Aptamer technology in CTC Detection,Aptamers,：,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 characterization,Aptamer production,:,SELEX technique,less expensive and much faster,due to small size,simple structure,and no use of animal cells,CTC,的功能研究,Thanks!,