Department of Bioengineering, University of California, Los Angeles, 410 Westwood Plaza, Los Angeles, California 90095, USA.
Lab Chip. 2017 Apr 11;17(8):1452-1461. doi: 10.1039/c7lc00038c.
Isolation and enumeration of circulating tumor cells (CTCs) from blood is important for determining patient prognosis and monitoring treatment. Methods based on affinity to cell surface markers have been applied to both purify (via immunoseparation) and identify (via immunofluorescence) CTCs. However, variability of cell biomarker expression associated with tumor heterogeneity and evolution and cross-reactivity of antibody probes have long complicated CTC enrichment and immunostaining. Here, we report a truly label-free high-throughput microfluidic approach to isolate, enumerate, and characterize the biophysical properties of CTCs using an integrated microfluidic device. Vortex-mediated deformability cytometry (VDC) consists of an initial vortex region which enriches large CTCs, followed by release into a downstream hydrodynamic stretching region which deforms the cells. Visualization and quantification of cell deformation with a high-speed camera revealed populations of large (>15 μm diameter) and deformable (aspect ratio >1.2) CTCs from 16 stage IV lung cancer samples, that are clearly distinguished by increased deformability compared to contaminating blood cells and rare large cells isolated from healthy patients. The VDC technology demonstrated a comparable positive detection rate of putative CTCs above healthy baseline (93.8%) with respect to standard immunofluorescence (71.4%). Automation allows full enumeration of CTCs from a 10 mL vial of blood within <1 h after sample acquisition, compared with 4+ hours with standard approaches. Moreover, cells are released into any collection vessel for further downstream analysis. VDC shows potential for accurate CTC enumeration without labels and confirms the unique highly deformable biophysical properties of large CTCs circulating in blood.
从血液中分离和计数循环肿瘤细胞（CTCs）对于确定患者预后和监测治疗非常重要。基于细胞表面标志物亲和力的方法已被用于纯化（通过免疫分离）和鉴定（通过免疫荧光） CTCs。然而，与肿瘤异质性和进化相关的细胞生物标志物表达的可变性以及抗体探针的交叉反应性长期以来一直使 CTC 富集和免疫染色复杂化。在这里，我们报告了一种真正无标记的高通量微流控方法，该方法使用集成的微流控设备来分离、计数和表征 CTC 的生物物理特性。涡旋介导的变形细胞术（VDC）由初始涡旋区域组成，该区域富集大的 CTC，然后释放到下游的流体动力拉伸区域，使细胞变形。高速摄像机可视化和量化细胞变形，从 16 个 IV 期肺癌样本中揭示了大（> 15 μm 直径）和可变形（纵横比> 1.2）CTC 群体，与污染的血细胞和从健康患者中分离的罕见大细胞相比，这些细胞的变形性明显增加。VDC 技术与标准免疫荧光（71.4%）相比，在健康基线以上具有类似的阳性检测率（93.8%）。自动化允许在采集样品后<1 小时内对来自 10 mL 小瓶的血液中的 CTC 进行全面计数，而标准方法则需要 4 个多小时。此外，细胞被释放到任何收集容器中，以进行进一步的下游分析。VDC 显示出无需标记即可准确计数 CTC 的潜力，并证实了在血液中循环的大 CTC 独特的高度可变形生物物理特性。
