Pillai Sreeraj G, Zhu Peixuan, Siddappa Chidananda M, Adams Daniel L, Li Shuhong, Makarova Olga V, Amstutz Pete, Nunley Ryan, Tang Cha-Mei, Watson Mark A, Aft Rebecca L
Washington University School of Medicine, Dept. of Surgery, St. Louis, Missouri, United States of America.
Creatv MicroTech, Inc., Rockville, Maryland, United States of America.
PLoS One. 2017 Jan 27;12(1):e0170761. doi: 10.1371/journal.pone.0170761. eCollection 2017.
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.
乳腺癌（BC）患者骨髓（BM）中播散肿瘤细胞（DTCs）的分子特征因细胞稀少而受到阻碍。为了使用不依赖抗原的方法富集这些细胞，我们评估了一种基于尺寸的微滤装置，并结合了几种下游生物标志物检测方法。
从健康志愿者或BC患者中采集骨髓抽吸物。将健康骨髓与特定数量的BC细胞混合，以计算微滤的回收率和富集倍数。标本先用70μm筛网预过滤，流出物再通过细胞筛微滤器过滤。捕获的细胞通过免疫细胞化学（ICC）、用于HER-2/neu基因扩增状态的荧光原位杂交（FISH）和RNA原位杂交（RISH）进行分析。从过滤器洗脱的细胞用于RNA分离以及随后对DTC生物标志物基因表达的定量逆转录聚合酶链反应（qRT-PCR）分析。
过滤约14×10⁶个有核骨髓细胞可产生约17 - 21×10³个残留骨髓细胞。在BC细胞加标实验中，平均87%（范围84% - 92%）的肿瘤细胞被回收，富集倍数约为170至400倍。通过ICC检测，从患者捕获的BC细胞共染细胞角蛋白和上皮细胞黏附分子（EpCAM），但不表达CD45。过滤后，每4ml患者骨髓的RNA产量平均为每1000万个过滤的骨髓细胞135ng，平均RNA完整性数值（RIN）为5.3。在过滤的加标或BC患者标本中，通过qRT-PCR和RISH均检测到了与DTC相关的基因表达，但在对照过滤的正常骨髓中未检测到。
我们测试了一种用于富集骨髓DTCs的微滤技术。使用BC模型细胞系，DTC捕获效率显示在84.3%至92.1%之间，富集倍数高达400倍。在患者中，使用多种基于抗体、DNA和RNA的生物标志物检测方法，可以识别回收的DTCs并将其与正常骨髓细胞区分开来。
