An integrated on-chip platform for negative enrichment of tumour cells
Introduction
Many studies suggest that circulating tumour cells (CTCs) are responsible for the metastatic spread of cancer from the primary tumour to other parts of the human body [1], [2] and their significance for clinical cancer management has been widely recognized [3], [4], [5], [6]. A good understanding of CTC biology together with the progress of more advanced technologies could enable real-time examination of CTCs probing for non-invasive screening of cancer [7]. Many efforts have been made to analyze CTCs; these results, in turn, enabled the development of several technologies for isolating such rare cells from blood samples [8], [9]. However, despite many efforts to develop methods for isolation of CTCs, it has been noted that an ideal technology optimized for this purpose is still unavailable. CellSearch, the only US Food and Drug Administration (FDA) cleared CTC isolation method for prognostic application, uses an immunomagnetic enrichment technique [10], [11].
Current know-how for CTC isolation has been well reviewed [12], [13], [14], [15], [16], [17]. The most challenging aspect in CTC isolation is the rare occurrence of cancer cells in the sample, which makes an effective separation challenging. Approaches such as flow cytometry [18], [19] and magnetic separation [20], [21], [22], [23] have been commonly employed for CTC enrichment. CTC enrichment has then been proposed, mainly based on the physical properties of cells such as size [24], [25], [26], [27], [28], [29], deformability [30], [31], [32] and density [33], [34], [35]. Other methodologies include cell-affinity chromatography [36], separation procedures based on the differences between the dielectric properties of various cell types [37], [38], separation exploiting hydrodynamic forces [39] and genetic-based approach [40]. Miniaturization of biomedical systems with the integration of nanotechnologies has a significant impact on biomedical research [41]. Therefore, microfluidics-based cell separation methods on Lab-on-chip platforms have recently been proposed and extensively developed lately because they are feasible cost effective methods easily amenable for CTCs isolation [42], [43] and also because of their well-known demonstrated effectiveness in biotechnology [44]. Several Lab-on-chip CTC isolation devices have been developed and commercialized [45] such as immunomagnetic-based CellSearch™ System (Veridex), label-free CTC enrichment platform ClearCell® System (Clearbridge BioMedics, Singapore), OncoCEE-BR™ (Biocept, Inc.) and dielectrophoretic based Apostream™ (Apocell).
The heterogeneous and mutagenic characteristics of tumour cells could affect their biological and physical properties [46], and therefore, an ideal CTC platform should not depend on subjective markers such as antigen expression or physical parameters. For instance, the CTC isolation platform based on antibody-antigen interaction using Epithelial Cell Adhesion Molecule (EpCAM) as a common surface marker [36], [47]. However, it has also been reported that the EpCAM-based techniques could potentially miss CTCs since these cells may undergo an Epithelial to Mesenchymal Transition (EMT) [1], [10], [48], [49]. Therefore, this approach has an inherent fundamental limitation in separating CTCs of non-epithelial origin. The implication is that relying exclusively on the presence of one or more cell surface proteins can lead to a high risk of losing CTC subpopulations which do not have appropriate protein expression. Isolating CTCs from blood based on immunomagnetic separation via cell-surface antigen recognition also has this drawback [9]. In addition, targeting CTCs directly by antibody or magnetic particle binding could adversely affect the cell health [50].
In the case of a size-based isolation of CTCs, the objective is to allow the passage of all normal blood cells while capturing the tumour cells, typically relying on cell size as the key separation criteria. However, this approach is vulnerable to many shortcomings, due to the overlapping of size and density between the CTCs and white blood cells (WBCs), as well as with other cell types. Consequently, this method is characterized by a recovery/purity trade-off, which is a critical drawback [51]. Furthermore, the cells that are undergoing EMT have been reported to adopt normal blood-cell like features, in terms of size and deformability. Such characteristics would also make size-based separation techniques highly susceptible to the loss of CTCs [52], [53], [54].
The limitations in directly targeting the CTCs for their immediate isolation can be overcome by a methodology called negative selection, where normal cells from blood are removed and thereby enriching CTCs. Hence, the negative selection ideally consists of separating all non-target cells and eluting all target cells. Therefore, unlabeled target cells can be collected in an undamaged form. Usually, the negative selection approach involves removing both the WBCs and RBCs which requires multiple complex processing steps, such as density gradient centrifugation and chemical lysis. These processing steps can contribute to the risk of losing CTCs and harmfully affect them [55], [56], [57], [58], [59], [60]. Tong et al. [59] and Lara et al. [60] reported that, RBC lysis and density gradient centrifugation steps can lead to 10% and 30% cell loss of spiked tumour cells respectively. Hence, a methodology with minimum processing steps which avoids labelling the CTCs or defining their size has been recommended as an optimal approach [56].
This paper proposes and characterizes a novel design in which the isolation of CTCs is achieved through depletion of hematopoietic cell population by combining both a centrifugation-free WBC and a chemical-free RBC depletion. The device consists of two modules: WBCs are depleted in first module, and then tumour cells are enriched by removing RBCs and platelets in the second module (Fig. 1). The first module uses microfluidic immunomagnetic separation to remove more than 99.9% of WBCs from the whole blood. The second module employs a size-based separation which selectively allows only the passage of RBCs and platelets, retaining nucleated cells including tumour cells. The entire design is capable of separating tumour cells from the whole blood at a high flow rate of 500 μL/min, which results in a higher throughput than that reported previously for other developed techniques [36], [61], [62], [63], [64], [65], [66]. Hence, our chip employs a negative selection approach which avoids multiple sample-handling steps and thereby minimizes the risk of rare cell loss. This method achieves an average tumour cell isolation efficiency of >80% across multiple cell lines with an average of 3.94 log WBC depletion.
Section snippets
Design of module-1: immunomagnetic chamber
The immunomagnetic separation module consists of a microfluidic chamber created inside the chip where magnetically tagged WBCs in the blood can be manipulated. Two arrays of permanent magnets were placed on the top and bottom surfaces of the chip, which created a large magnetic field gradient inside the chamber. The Module-1 design employs microfluidics-based magnetophoresis, which combines the benefits of both immunomagnetic assays and microfluidic devices. The main design factors considered
Reliability of the microslit membrane
To assess the reliability of the microslit membrane to retain nucleated cells, the capture efficiency has been evaluated for four cancer cell lines, namely NCI-H1975, SW48, PC3 and MCF-7. As a surrogate for WBCs, Jurkat cells were also used. Each cell line (50 cells) was suspended in 4 mL of PBS and flowed through the device at a flow rate of 500 μL/min. Experiments were repeated for three iterations and after screening, the chip was transferred to an upright fluorescence microscope (BX63,
Conclusions
We demonstrated a unique, simple negative enrichment protocol for CTC isolation on a microfluidic platform. In our previous papers [51], [80], we tested our idea of negative enrichment by using upstream immunomagnetic WBC depletion, but with some limitations such as low purity and lack of integrated lab-chip platform. This CTC isolation platform achieved a higher WBC depletion of 99.98% compared to the previous platform (99.5%) and therefore improved purity. The system also integrated both the
Acknowledgement
This work was supported by the Science and Engineering Research Council of A*STAR (Agency for Science, Technology and Research), Singapore.
References (85)
- et al.
Circulating tumor cells (CTC) detection: clinical impact and future directions
Cancer Lett.
(2007) - et al.
The evolving war on cancer
Cell
(2011) - et al.
Circulating tumor cells: approaches to isolation and characterization
J. Cell Biol.
(2011) - et al.
Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells
J. Chromatogr. A
(2007) - et al.
Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulatingtumor cells
Am. J. Pathol.
(2000) - et al.
Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients
Biosens. Bioelectron.
(2010) - et al.
Isolation of tumor cells using size and deformation
J. Chromatogr. A
(2009) - et al.
Electroanalysis in micro- and nano-scales
J. Electroanal. Chem.
(2013) - et al.
Pitfalls in the detection of disseminated non-hematological tumor cells
Ann. Oncol.
(2000) - et al.
Enrichment of rare cancer cells through depletion of normal cells using density and flow-through, immunomagnetic cell separation
Exp. Hematol.
(2004)