As cancer mortality rates continue to rise, the global impact of the disease is beginning to overwhelm healthcare services. Improving detection methods for Circulating Tumour Cells (CTCs) can significantly help in the diagnosis of metastatic relapse, assist in the stratification of patients for therapeutic purposes, monitor response to drugs and therapies, and track tumour progression. Thus it is possible that the impact of cancer can be controlled through innovative biochemical research. Since CTCs exist in exceedingly low concentration during the early stages of the disease, highly sensitive detection methods are required. We thus investigated the potential of combining cell-SELEX- (Systematic Evolution of Ligands by Exponential Enrichment) based aptamers and microfluidic cell-affinity chromatography devices in an effort to improve detection and enrichment. This approach yielded an inexpensive, simple, sensitive and fast multiplexed cancer detection platform for detecting CTCs.
by Dr Y. Pu, Dr H. Liu, Dr J. Meekel and Dr W. Tan
Responsible for over 13% of all human deaths, the global impact of cancer has become overwhelming. If patient survival times are to be extended, our methods of responding to cancer must improve considerably. One such strategy, the earlier detection of cancer, increases the probability of a positive prognosis and decreases the cost of treatment. Furthermore, early detection has proven to decrease mortality for breast, colon, rectal and cervical cancers [1]. In response to the pressing need for earlier diagnosis, our work concentrates on the detection and enrichment of diseased cells. Our methodology focuses on immobilising cell-SELEX-based aptamers on a microfluidic surface in order to subsequently capture CTCs.
Circulating tumour cells
CTCs break away from the primary tumour and enter the bloodstream, lymphatic system, or simply settle where they fall. These metastatic CTCs travel indiscriminately throughout the body and continue to grow. These CTCs are ultimately the principal cause of death, as they are essential for cancer metastasis. Although they are exceedingly rare in the early stages of breast cancer, they increase greatly in number once the disease develops [2]. During the advanced stages of cancer, it is estimated that a tumour can shed 106 CTC cells per day into the bloodstream [3]. CTC measurement thus also serves as an adjunct to standard methods of monitoring disease status in metastatic breast cancer [4]. Thus we can not only diagnose cancer, but also monitor the status of the disease through the detection of CTCs, which also enables the most appropriate therapy
to be determined.
Limitations of CTC detection
To enable the early diagnosis of cancer, improved detection methods are necessary to measure the exceeding low concentration of CTCs present in the early stages of the disease [5, 6]. Currently, the majority of commercial CTC detection technologies rely on magnetic Epithelial Cell Adhesion Molecule (Ep-CAM) antibodies. Ep-CAM, a monomeric membrane glycoprotein, is over-expressed in a variety of human carcinomas. It can thus function as a potential therapeutic target for human solid tumours. However, the amount of Ep-CAM on tumour cells varies and is largely dependent on tumour type. Furthermore this magnetic method isolates only a small number of CTCs and the low purity of the cells prevents appropriate molecular analysis [7]. The detection technologies that we are currently developing should solve these problems and facilitate CTC measurement in the clinical laboratory.
Microfluidic devices
Microfluidic devices provide a suitable approach for CTC detection. These devices are capable of capturing rare CTCs thus permitting appropriate quantification and analysis of the cells from blood samples [7, 8]. These advantages, together with their ability to isolate CTCs in sufficient quantity and purity, makes microfluidic devices greatly superior in comparison to previous detection strategies. These devices are inexpensive and can be manufactured easily. Furthermore, only small reagent volumes are required, and rapid assays for several different biological applications are faciliated.
Aptamers and cell-based SELEX
Aptamers, or single-stranded oligonucleotide probes, selectively interact with numerous target compounds, binding to proteins, drugs, and other small molecules [9-12]. Furthermore aptamers can distinguish between homologous proteins with only minor amino acid differences [13, 14].Aptamers present an ideal alternative to antibodies for the detection and enrichment of cancerous cells. In addition, aptamers have a lower molecular weight, longer shelf life and are stable and easily modified [15]. Unlike antibodies, aptamers do not need to be manufactured in vivo, making them both cheaper and more versatile. Thus aptamers have great potential as therapeutic agents.
Cell-based SELEX allows the selection of a panel of aptamers, which bind to disease-specific membrane proteins. During the SELEX process [Figure 1], a large pool of oligonucleotides is purified to remove unbound and weakly bound sequences (thereby retaining only the high affinity sequences). The bound sequences are then eluted in denaturing conditions such as high temperatures, which causes oligonucleotides to lose their ability to bind. Subsequently 20-30 rounds of SELEX are performed to ensure appropriate enrichment of the pool from which the aptamers are selected. The cell-based SELEX technology permits selection of a nucleic acid aptamer panel that binds with high specificity and sensitivity to disease-specific membrane proteins. Using the cell-based SELEX process, our research has revealed significant benefits of aptamers for the treatment of cancerous cells [13]. Cell-SELEX aptamers are created without explicit knowledge of the molecular differences between cancer cells and healthy cells, so aptamers can be used for detection and enrichment before a corresponding antibody can be developed.
Enrichment and identification of CTCs
To achieve an inexpensive, simple, sensitive and fast multiplexed cancer detection platform, we combined the advantages of DNA aptamers generated by in vitro cell-SELEX with microfluidic cell-affinity chromatography devices. We used cell-based SELEX to select the aptamers for the target CTCs and then immobilised these aptamers on microfluidic surfaces to capture and purify cancer cells from a background of non-target cells [Figure 2], [15].
To extend the aptamer platform, we developed a mechanism that incorporates aptamers in separate regions of the device. The “S-Channel Design” compartmentalised the cell-capture zones; three cell-capture regions were created via walls at each bend. The S shape and the channel’s 40µm height and 4 mm width allowed a greater surface area to be contained in a smaller device, thus increasing cell capture efficiency [Figure 3]. Since this device has no limit to the number of different regions that can be added, the design is ideal for multiplexed detection. When batch-sorting aptamers using the S-Channel Design, different types of cancer cells (from a heterogeneous cancer cell mixture) can be simultaneously enriched and detected.
By blocking different combinations of wells, we were able to immobilise three different aptamers in three different regions of the S-Channel device. The S design allowed cell mixture profilling within the device. When a cell mixture passed through the device, each region captured and enriched different cells according to their aptamer-binding properties. Following the cell-capture assay, the device was examined in order to detect the captured cells using confocal microscopy. To facilitate cell release, an air bubble was introduced into a specific region of the device and the meniscus formed then collected the captured cells. We found capture efficiencies and purities similar to those reported for microfluidic cell-affinity chromatographic devices [8, 15].
Our data show that the aptamer-based device captures target cells with > 97% purity and > 80% efficiency. Consequently, we can enrich and detect CTCs, whilst identifying different cancer types simultaneously [14, 16]. Since the assay is completed within minutes and requires no cell pre-treatment, the enrichment and detection of multiple rare cancer cells can be carried out, with significant medical benefits. For example, the device can be used to diagnose an early metastatic relapse, monitor response to drugs and therapies, stratify patients for therapeutic purposes, track tumour progression, and gain a deeper understanding of the biology of CTCs. We aim to further develop our device in order to create a “gold standard” assay for CTCs detection.
Conclusion
Combining cell-SELEX-based aptamers with microfluidics greatly improved the sensitivity of CTCs detection. Highly sensitive enrichment and identification will enable CTCs to play a critical role in clinical diagnosis and therapy. This technology will permit the use of such assays into routine clinical care. We also expect to increase our knowledge of the cancer cell invasion process and to generate new CTC data, which we anticipate will improve the medical care and life expectancy of cancer patients.
ACKNOWLEDGMENT:
We thank our coworkers for help and discussion. This work is supported by NIH and Moffitt grants.
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The authors
Ying Pu†‡, Huixia Liu†, Jennifer Meekel‡ and Weihong Tan‡
† P.O. Box 190, Xiangya Hospital, Central South University
Changsha,
Hunan 410008,
China
e-mail: lhx900@yahoo.com.cn
‡Department of Chemistry and Department of Physiology and Functional Genomics
Shands Cancer Center and Center for Research at Bio/nano Interface
Moffitt Cancer Center and
McKnight Brain Institute
University of Florida
Gainesville
FL 32611, USA
tan@chem.ufl.edu
