It is generally accepted that most cancer-related mortalities result from metastatic disease.1
While the process of metastasis is not well understood, a number of chemical, physical, and molecular events occur that ultimately result in the dissemination and deposition of tumor cells into targeted organs using the circulatory system and/or bone marrow as the carrier(s). This “seed and soil” process was proposed as early as 1889 by Paget and was later modified with the caveat that shed tumor cells consist of a heterogeneous population with subpopulations possessing different metastatic potentials. The fundamental entities primarily responsible for spawning metastatic disease are circulating tumor cells (CTCs), which can be produced during early stages of tumorigenesis.4
Elucidating the quantity of CTCs in peripheral blood or bone marrow can serve as an indicator for the clinical management of several cancer-related diseases by providing information on the success/failure of therapeutic intervention and disease stage forecasting.5
, The isolation and enumeration of exfoliated CTCs in peripheral blood or bone marrow for a variety of cancer-related diseases has already been reported for a variety of cancer-related diseases, such as breast,79
and nonsmall cell lung14
cancers. As an example of the clinical utility of CTC information, Cristofanilli et al. recently reported a study of 177 breast cancer patients using the amount of CTCs in peripheral blood as an indicator of survival.7
Patients with ≥5 CTCs per 7.5 mL of whole blood possessed a median progression-free survival of 2.7 months versus 7.0 months for those patients containing <5 CTCs in 7.5 mL of their peripheral blood.
The major issue with securing viable clinical information via quantification of CTC levels is the extremely low abundance or rare-event nature of these cells among a high number of spectator cells in peripheral blood.6,15–18
For example, it is clinically useful to quantitatively enumerate 0–10 CTCs in whole blood composed of >109
erythrocytes and >106
leukocytes per mL.7
For sampling rare events in a large population, three important metrics must be assessed: (1) throughput, the number of cell identification or sorting steps per unit time; (2) recovery, an indicator of the fraction of target cells collected from the input sample; and (3) purity, which depends on the number of “interfering” cells excluded from the analysis.19
In addition to these three metrics, highly efficient quantification of the number of enriched cells must be provided as well.
The approaches used to date to enrich CTCs from clinical samples have provided lower-than-desired recoveries with high purity, relatively poor purity but with high recoveries, or, in other cases, highly specialized sample processing and handling whose success is laboratory dependent.20–25
For example, immunomagnetic approaches for CTC enrichment using ferromagnetic micrometer-sized particles coated with molecular recognition elements specific for antigenic-bearing target cells can interrogate diluted blood samples typically yield modest recoveries (~70%) but extremely favorable purity.22
In the case of size-based separations employing nuclear tracked membranes, polycarbonate membranes with varying pore sizes (8–14 μ
m) can filter large volumes (9.0–18 mL) of blood and recover nearly 85% of the CTCs, but significant numbers of leukocytes are also retained (i.e., low purity) potentially complicating the enumeration process.26
Investigations utilizing reverse-transcription PCR, in which mRNAs are used as surrogates to report CTC levels, have the ability to detect one CTC in an excess of 106
However, these assays are prone to high interlaboratory variability and require extensive sample handling and manipulation.
Most of the CTC isolation/sorting tools currently in use possess some common procedural characteristics that make them prohibitively difficult to implement, such as the ability to sort only the mononucleated fraction of whole blood requiring density gradient centrifugation prior to enrichment and the use of either flow cytometry or fluorescence microscopy following cell staining to enumerate the enriched CTCs. These additional steps require sample handling and transfer, which can induce cell loss or contamination that can dramatically affect the assay result, especially when dealing with low numbers of targets.
Microfluidics provides a venue for producing integrated systems that can process clinical samples in closed architectures to minimize sample contamination and loss. However, high throughput sampling of relatively large volumes (>1 mL) has not been a mainstay for microfluidics due to the macro-to-micro dilemma resulting from the small dimensional features associated with these devices. For example, exhaustively sampling a 1.0 mL volume input using a microchannel of 30 μ
m × 30 μ
m at a linear velocity of 1.0 mm s−1
would require 309 h (12.9 days). This sampling bottleneck was recently addressed by studies with a glass-based microfluidic device fabricated using deep reactive ion etching.28
The high-surface area immunological capture bed consisted of microposts (100 μ
m diameter × 100 μ
m tall) arranged in an equilateral triangular format; the device was capable of capturing ~60% of CTCs from untreated whole blood with enumeration achieved by cell staining and microscopic visualization.
Herein we report our efforts aimed toward the development of a self-contained system capable of meticulously separating intact CTCs from peripheral blood and directly quantifying the CTC level upon isolation and enrichment. As a result of careful fluidic design rules and system integration methods, we have successfully employed a microfluidic system capable of exhaustively and rapidly interrogating >1.0 mL of unprocessed whole blood possibly harboring low-abundant CTCs. At the heart of the system were carefully engineered, exceedingly efficient high-aspect ratio capture beds decorated with mABs specific for antigenic integral membrane proteins expressed in CTCs of epithelial origin and a label-free, highly specific single-cell conductivity sensor. The device operational characteristics were achieved by tailoring the dominant CTC capture dynamics, specific device architecture, and suspension linear velocity in a high throughput microsampling unit (HTMSU) containing high-aspect ratio microstructures replicated in a polymeric substrate. Direct single-cell counting of the captured cells was made possible by their release as a result of enzymatic digestion of cell-antigen/antibody-surface complexes. Quantitative assessment of CTC numbers was accomplished using an integrated conductivity sensor capable of specifically detecting CTCs via their electrical signatures without requiring cell staining or microscopic visualization.