Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration.
Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration.
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乳腺癌的富集和分子分析使用微滤从骨髓中传播了肿瘤细胞。
DOI：
10.1371/journal.pone.0170761
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Aft RL
中科院分区：
文献类型：
--
作者：
Pillai SG;Zhu P;Siddappa CM;Adams DL;Li S;Makarova OV;Amstutz P;Nunley R;Tang CM;Watson MA;Aft RL
乳腺癌(BC)患者骨髓(BM)中播散性肿瘤细胞(DTC)的稀有性阻碍了其分子特征的研究。为了使用非抗原依赖的方法来丰富这些细胞，我们评估了一种基于大小的微滤装置，并结合几种下游的生物标记物分析。骨髓抽吸物来自健康志愿者或BC患者。将健康的BM与特定数量的BC细胞混合，通过微滤计算回收率和折叠浓缩。样品用70μm筛网进行预过滤，流出物通过CellSieve微过滤器过滤。用免疫细胞化学(ICC)、荧光原位杂交(FISH)和RNA原位杂交(RISH)检测HER-2/neu基因扩增情况。从滤器中洗脱出的细胞用于RNA提取和随后的qRT-PCR分析DTC生物标记物基因的表达。过滤平均14×106个有核骨髓细胞，可得到约17-21×103个剩余骨髓细胞。在BC细胞刺激性实验中，平均87%(范围84-92%)的肿瘤细胞在大约170-400倍的浓缩后被回收。从患者采集的BC细胞被ICC共同染色为细胞角蛋白和EpCAM，而不是CD45。经过过滤后，4毫升患者骨髓中的RNA产量平均为每1000万骨髓细胞135 ng，平均RNA完整性数(RIN)为5.3。QRT-PCR和RISH检测到DTC相关基因在过滤的尖峰或BC患者样本中均有表达，但在对照过滤的正常骨髓中未检测到。我们已经测试了一种用于浓缩BM DTCs的微滤技术。DTC的捕获效率在84.3%到92.1%之间，使用模型BC细胞可获得高达400倍的浓缩。在患者中，可以使用多种基于抗体、DNA和RNA的生物标志物分析来识别和区分恢复的DC与正常的BM细胞。
Molecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays. BM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression. Filtering an average of 14×106 nucleated BM cells yielded approximately 17–21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84–92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM. We have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.