Rare cell isolation and profiling on a hybrid magnetic/size-sorting chip     

Jaehoon Chung  David Issadore  Adeeti Ullal  Kyungheon Lee  Ralph Weissleder  Hakho Lee 《Biomicrofluidics》2013,7(5)

We present a hybrid magnetic/size-sorting (HMSS) chip for isolation and molecular analyses of circulating tumor cells (CTCs). The chip employs both negative and positive cell selection in order to provide high throughput, unbiased CTC enrichment. Specifically, the system utilizes a self-assembled magnet to generate high magnetic forces and a weir-style structure for cell sorting. The resulting device thus can perform multiple functions, including magnetic depletion, size-selective cell capture, and on-chip molecular staining. With such capacities, the HMSS device allowed one-step CTC isolation and single cell detection from whole blood, tested with spiked cancer cells. The system further facilitated the study of individual CTCs for heterogeneity in molecular marker expression.Circulating tumor cells (CTCs) have emerged as an important biomarker in clinical practice as well as in fundamental research.^{1, 2} CTCs, shed from primary tumors, have been shown to be an early harbinger of tumor expansion and metastasis³ and have been used to predict disease progression, response to treatment, relapse, and overall survival.^{4, 5, 6} Recent work has shown that CTCs display distinct proteomic and genetic profiles; for example, CTCs in pancreatic cancer, have increased RNA expression of Wnt, implicating this pathway in metastasis.⁷ Proteomic characterization of proliferative markers such as Ki-67, and hormonal markers such as androgen receptor in prostate cancer, also have been shown to be predictive of treatment outcome.^{8, 9}Despite such clinical potential of CTCs, their routine detection and characterization still remains a significant technical challenge.¹⁰ The task requires screening of a large number of cells (e.g., > 10⁷ cells in 10 ml blood) and enrichment of heterogeneous targets against a complex biological background. Two main methods of CTC isolation are typically used: positive and negative selection. In positive selection, CTCs are directly isolated from blood via size-based filtration^{11, 12, 13, 14, 15, 16, 17, 18, 19, 20} or antibody-based capture.^{1, 8, 21} Negative depletion reduces abundant blood cells, often by immunomagnetic separation, for downstream CTC enrichment.²² Both approaches have been used for high throughput CTC isolation from whole blood (SI Table 1).²³ Each method, however, has its own inherent limitations. Positive enrichment could be biased by its selection criteria (e.g., cell size and cell surface markers). Negative selection, albeit unbiased, often requires complex sample processing (e.g., multiple washing steps for CTC isolation) that could result in cell loss.We hypothesized that both positive and negative selection could be combined in a single platform to enable (1) highly efficient and unbiased CTC purification, and (2) in-situ molecular analyses of collected cells. As a proof-of-concept, we herein describe a hybrid magnetic/size-sorting (HMSS) system that integrates magnetic and size-based isolation into a compact microfluidic chip. The HMSS first uses a magnetic filter to deplete leukocytes through immunomagnetic capture. Samples then pass through a size-sorter region that traps individual cells at predefined locations. Since abundant leukocytes are removed by the magnetic filter, the size-sorter could have a low size cut-off (∼5 μm), which allows for the unbiased capture of even small cancer cells. Furthermore, molecular probes can be introduced to perform on-chip, multiplexed analyses at single-cell resolution. We evaluated the utility of the developed system by capturing and profiling tumor cells in whole blood. The HMSS offers the advantages of both negative and positive selection and thereby differs from the recently reported iChip system²⁴ which can operate only in either a negative or a positive selection mode.  相似文献