NGS中的CTC和ctDNA

1、循環(huán)腫瘤細胞循環(huán)腫瘤細胞（Circulating tumor cells(CTCs)）CTCs的概念與特點CTCs的富集與檢測CTCs的應(yīng)用與研究進展Circulating tumor cells (CTCs) are cells that have shed into the vasculature from a primary tumor and circulate in the bloodstream.2005年P(guān)achm ann將CTCs正式定義為自發(fā)或因診療操作由實體瘤或轉(zhuǎn)移灶釋放進入外周血循環(huán)的腫瘤細胞. 惡性腫瘤是我國死亡率最高的重大疾病之一，90%以上腫瘤患者的死亡都是由于腫瘤

2、轉(zhuǎn)移所致侵襲與轉(zhuǎn)移是惡性腫瘤最顯著的特征之一。 CTCs是腫瘤轉(zhuǎn)移過程中在血液循環(huán)系統(tǒng)中存活的腫瘤細胞，它的生成被認為是腫瘤發(fā)生轉(zhuǎn)移的必要前提。深入研究CTCs有助于對腫瘤轉(zhuǎn)移機制進一步了解 ， 為抗腫瘤轉(zhuǎn)移的治療提供新的依據(jù) CTCs的檢測有助于早期轉(zhuǎn)移腫瘤患者的診斷、監(jiān)測術(shù)后患者腫瘤的復(fù)發(fā)與轉(zhuǎn)移有助于評估抗腫瘤藥物的敏感性與患者預(yù)后以及選擇個體化的治療策略 CTCs并沒有顯著的特異性同其它血細胞明確區(qū)分且不同組織學(xué)類型和分子表型的腫瘤分別表達不同的標志物。 CTCs在外周血中數(shù)量稀少，一般在106-107個白細胞中僅含有1個。無顯著特異性數(shù)量少非特異性富集方法特異性富集方法方法CTCs-芯

3、片富集法納米粒富集法免疫磁珠富集法密度梯度離心富集法細胞大小 富集法細胞變形性富集法細胞電學(xué)特征富集法Ficoll法和Oncoquick法上皮腫瘤細胞大小分離(ISET)和微濾芯片技術(shù)雙向電泳-場流分離法（DEP-FFF）磁性活細胞分選法(MACS)、 AdnaTest法、 Cellsearch法Using size-based enrichment techniques, diluted whole blood is passed through a filtration device with specific sized pores (typically 8 m). CTCs are c

4、aptured 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 ho

5、les. (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.(A)Schematic of a pool-dam chip. The decreasing width of gaps between successive pools allows the separation of CTCs

6、 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. (C) Isolation of cancer cells by crescent-shaped traps. The distanc

7、e 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. Plasma:血漿Mono

8、nuclear cell：單核細胞（包括淋巴細胞、 單核細胞、 上皮細胞和腫瘤細胞）Ficoll：密度梯度液RBCs：紅細胞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 havin

9、g 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).Immunomagnetic separation involves the use of iron-

10、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.whole blood is slowly passed across a chip-based surface and isolated using either CTC targeted antibody-coated microposts or

11、dielectrophoresis （DEP）流式細胞儀分析(flow cytometry FCM)免疫細胞化學(xué)技術(shù) ICC（immunocytochemistry）鑒定標本中細胞抗原性和形態(tài)學(xué)特征，能使富集的目的細胞維持細胞形態(tài)并保持細胞活力 ICC是用能與顯色劑結(jié)合的單抗與 CTCs特異性結(jié)合后，通過顯色劑顯色從而對CTCs進行鑒定的技術(shù)通過分析上皮細胞或腫瘤細胞的正常起源組織特異的候選基因的表達來檢測 CTC, 靈敏度非常高但是壞死的癌細胞、上皮細胞污染等都可以造成假陽性結(jié)果，此法無法檢測CTCs細胞形態(tài)，在臨床應(yīng)用上有局限性。反轉(zhuǎn)錄-聚合酶鏈反應(yīng)RT-PCRCellsearchFDA唯

12、一批準的檢測CTC的方法。一種半自動技術(shù)，通過載有抗EpCAM抗體的鐵磁流體富集CTC；隨后CTC用CK和DAPI熒光抗體染色，其余血細胞用CD45對比染色，CK+DAPI+CD45-細胞使用自動熒光顯微鏡進行計數(shù)一種基于酶聯(lián)免疫吸附測定原理的免疫學(xué)分析方法。選擇性去除CD45陽性細胞富集CXCR4陽性細胞后，測定特異性蛋白如cathepsin-D、musin-1來計數(shù)能分泌蛋白的存活CTCEPISPOT一 種 更 為 先 進 的 檢 測 C T C 的 技 術(shù) 。 設(shè)備載有78000個包被EpCAM抗體的微柱，當血液樣品流經(jīng)時，EpCAM陽性細胞被粘附于微柱的表面CTC-chipCircul

13、ating 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 Prostate cancermicrosieve chipFlow cytometry size-

14、based filtration (ISET) microvortex herringbone-chipCell searchhigh-definition fluorescence scanning microscopyCirculating tumor materials (CTMat): damaged cells, fragmented cells, cellular debris, microparticles, and clump-like aggregatesprovide flexibility in prognosis due totheir high abundancele

15、ss stringent target identification, easier enumeration, and fully automated image processingCTDNA correlated with the malignancy status and the therapy responseAdvantageTelomerase has been found activated in the majority of cancer types and is known to be associated with malignant propertiesAssessin

16、g 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 destroying all intactCTCsadvantagedisadvantageAptamers： 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