An integrated enrichment system to facilitate isolation and molecular characterization of single cancer cells from whole blood.
An integrated enrichment system to facilitate isolation and molecular characterization of single cancer cells from whole blood.
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DOI：
10.1002/cyto.a.23599
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发表时间：
2018-12
期刊：
影响因子：
--
通讯作者：
Carpenter EL
中科院分区：
文献类型：
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作者：
Yu L;Sa S;Wang L;Dulmage K;Bhagwat N;Yee SS;Sen M;Pletcher CH Jr;Moore JS;Saksena S;Dixon EP;Carpenter EL
循环肿瘤细胞（CTC）携带有价值的生物学信息。虽然外周血中CTC的计数是FDA批准的转移性前列腺癌和其他癌症中存活的预后指标，但需要分析CTC表型和基因组标志物来鉴定癌症起源并阐明可以指导个性化药物的治疗选择的途径。鉴于单细胞mRNA测序技术的出现，需要一种方法来分离具有高灵敏度和特异性以及与下游基因组分析相容性的CTC。流式细胞术是分析和分选单细胞的强大工具，但由于血液中CTC的频率极低（数十亿个血细胞中的一个），因此在流式分选之前需要预富集以有效分离CTC。虽然目前的富集技术通常需要许多步骤并导致回收率低下，但我们展示了磁性分离器和声学微流体聚焦芯片集成系统，该系统可与FACS™（荧光激活细胞分选）和单细胞测序一起富集稀有细胞。该系统分析、分离和索引分选单细胞，直接进入含有单细胞分子索引（MI）和转录谱分析试剂的96孔板。通过使用集成富集-FACS系统的优化工作流程，我们用外周血中的掺入的前列腺癌细胞进行了概念验证实验，并实现了：（i）从裂解的全血中分离稀有癌细胞的快速一步法;（ii）富集后平均92%的癌细胞回收率（R2 = 0.9998），而传统台式工作流程的回收率为55%;和（iii）在单细胞水平检测差异表达的基因，其与报道的细胞-前列腺癌细胞的类型依赖性表达特征。这些模型系统结果为将我们的方法应用于来自前列腺和其他癌症患者的人类血液样品奠定了基础，并支持富集-FACS系统作为用于癌症诊断的CTC分离和表征的灵活解决方案。
Circulating tumor cells (CTCs) carry valuable biological information. While enumeration of CTCs in peripheral blood is an FDA-approved prognostic indicator of survival in metastatic prostate and other cancers, analysis of CTC phenotypic and genomic markers is needed to identify cancer origin and elucidate pathways that can guide therapeutic selection for personalized medicine. Given the emergence of single-cell mRNA sequencing technologies, a method is needed to isolate CTCs with high sensitivity and specificity as well as compatibility with downstream genomic analysis. Flow cytometry is a powerful tool to analyze and sort single cells, but pre-enrichment is required prior to flow sorting for efficient isolation of CTCs due to the extreme low frequency of CTCs in blood (one in billions of blood cells). While current enrichment technologies often require many steps and result in poor recovery, we demonstrate a magnetic separator and acoustic microfluidic focusing chip integrated system that enriches rare cells in-line with FACS™ (fluorescent activated cell sorting) and single-cell sequencing. This system analyzes, isolates, and index sorts single cells directly into 96-well plates containing reagents for Molecular Indexing (MI) and transcriptional profiling of single cells. With an optimized workflow using the integrated enrichment-FACS system, we performed a proof-of-concept experiment with spiked prostate cancer cells in peripheral blood and achieved: (i) a rapid one-step process to isolate rare cancer cells from lysed whole blood; (ii) an average of 92% post-enrichment cancer cell recovery (R2 = 0.9998) as compared with 55% recovery for a traditional benchtop workflow; and (iii) detection of differentially expressed genes at a single cell level that are consistent with reported cell-type dependent expression signatures for prostate cancer cells. These model system results lay the groundwork for applying our approach to human blood samples from prostate and other cancer patients, and support the enrichment-FACS system as a flexible solution for isolation and characterization of CTCs for cancer diagnosis.
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影响因子：
45.3
作者：
Cristofanilli, M;Hayes, DF;Terstappen, LWMM
通讯作者：
Terstappen, LWMM
影响因子：
7.4
作者：
Fu, Glenn K.;Wilhelmy, Julie;Stern, David;Fan, H. Christina;Fodor, Stephen P. A.
通讯作者：
Fodor, Stephen P. A.
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
3.7
作者：
Coumans FA;van Dalum G;Beck M;Terstappen LW
通讯作者：
Terstappen LW
影响因子：
46.9
作者：
通讯作者：
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