Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation
Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation
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DOI：
10.1371/journal.pone.0138032
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发表时间：
2015-09-23
期刊：
影响因子：
3.7
通讯作者：
Lu, Yong-Jie
中科院分区：
文献类型：
--
作者：
Xu, Lei;Mao, Xueying;Lu, Yong-Jie
从外周血中分离循环肿瘤细胞（CTC）具有提供比肿瘤组织活检更容易的“液体活检”的潜力，以在疾病进展期间和响应于治疗监测肿瘤细胞群体。已经开发了许多CTC分离技术。我们优化了Parsortix系统，这是一种用于CTC分离的表位独立的、基于大小和可压缩性的平台，使得可以以与标准CellSearch系统相当的速度和样品体积收获CTC。我们使用该系统捕获了来自不同癌细胞系的一半以上的癌细胞，这些癌细胞在健康供体的血液样本中被掺入。转移和固定在载玻片上的细胞的免疫染色期间的细胞损失是分析稀有细胞样品的主要问题。我们开发了一种新的细胞转移和固定方法，在免疫荧光过程后将>90%的细胞保留在载玻片上，而不影响信号强度和特异性。使用这种优化的方法，我们评估了Parsortix系统在前列腺癌患者中的CTC收获，并与基于免疫珠的CTC分离系统IsoFlux和CellSearch进行了比较。我们使用Parsortix和IsoFlux从10名前列腺癌患者的7.5 mL血液样品中收获了相似数量（p = 0.33）的细胞角蛋白（CK）阳性CTC（平均值分别为33.8和37.6）。通过Parsortix收获的CTC的纯度为3.1%，显著高于IsoFlux的纯度1.0%（p = 0.02）。在七个前列腺癌患者样品中，Parsortix比CellSearch（p = 0.04）收获了显著更多的CK阳性CTC，其中使用了两种系统（平均值分别为32.1和10.1）。我们还使用Parsortix捕获CTC集群。使用四色免疫荧光，我们发现85.8%的PC 3细胞表达EpCAM，91.7%表达CK，2.5%的细胞缺乏两种上皮标记。有趣的是，95.6%的PC 3细胞表达波形蛋白，包括那些缺乏上皮标记物表达的细胞，表明上皮向间充质转化。在5名前列腺癌患者中的4名中也观察到CK阳性/波形蛋白阳性/CD 45阴性和CK阴性/波形蛋白阳性/CD 45阴性细胞，但在3名健康对照中很少观察到，表明Parsortix收获具有上皮和间充质特征的CTC。我们还使用PC 3和DU 145加标实验证明，Parsortix收获的细胞可用于细胞培养。
Isolation of circulating tumor cells (CTCs) from peripheral blood has the potential to provide a far easier "liquid biopsy" than tumor tissue biopsies, to monitor tumor cell populations during disease progression and in response to therapies. Many CTC isolation technologies have been developed. We optimized the Parsortix system, an epitope independent, size and compressibility-based platform for CTCs isolation, making it possible to harvest CTCs at the speed and sample volume comparable to standard CellSearch system. We captured more than half of cancer cells from different cancer cell lines spiked in blood samples from healthy donors using this system. Cell loss during immunostaining of cells transferred and fixed on the slides is a major problem for analyzing rare cell samples. We developed a novel cell transfer and fixation method to retain >90% of cells on the slide after the immunofluorescence process without affecting signal strength and specificity. Using this optimized method, we evaluated the Parsortix system for CTC harvest in prostate cancer patients in comparison to immunobead based CTC isolation systems IsoFlux and CellSearch. We harvested a similar number (p = 0.33) of cytokeratin (CK) positive CTCs using Parsortix and IsoFlux from 7.5 mL blood samples of 10 prostate cancer patients (an average of 33.8 and 37.6 respectively). The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02). Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively). We also captured CTC clusters using Parsortix. Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers. Interestingly, 95.6% of PC3 cells expressed Vimentin, including those cells that lacked both epithelial marker expression, indicating epithelial-to-mesenchymal transition. CK-positive/Vimentin-positive/CD45-negative, and CK-negative/Vimentin-positive/CD45-negative cells were also observed in four of five prostate cancer patients but rarely in three healthy controls, indicating that Parsortix harvests CTCs with both epithelial and mesenchymal features. We also demonstrated using PC3 and DU145 spiking experiment that Parsortix harvested cells were viable for cell culture.