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— Detecting circulating tumor cells in blood
Ali Asgar. S. Bhagat1 and Chwee Teck Lim
Clearbridge BioMedics Pte Ltd, Singapore
Department of Biomedical Engineering, National University of Singapore, Singapore
Mechanobiology Institute, Singapore

Microfluidics, the physics of fluid flow in micron sized channels, is a well-established field with earliest work reported just over two decades ago by Andreas Manz and co-workers. Although initial progress was slow, the introduction of novel fabrication techniques such as soft lithography towards the end of the 1990's saw a burgeoning in the development of devices and applications of microfluidics in the area of biomedical, chemical and environmental . However, translating these devices to industry use has been rather challenging as most attempts have been towards replacing existing conventional techniques. For example, although microfluidics-based immunoassays for biomarker identification have high sensitivity, these devices have historically struggled to gain a strong foothold in the industry over traditional lateral flow immunoassays strips in terms of cost, scalability and ease of use. Both academics and industry have in the past attempted to find that niche ‘killer application’ where only microfluidics can offer the most probable disruptive technology to an existing problem. We propose that cell separation/detection for disease diagnosis, and especially cancer diagnosis, is that ‘killer application’. Microfluidics offers an attractive solution for this application as the channel sizes are comparable to cell size (~tens of microns) thus allowing for greater control on individual cell trajectory. The behavior of fluids at the microscale is predominantly laminar in nature and this allows for precise manipulation of cells and reagents at the single cell level for rare-cell detection and disease diagnosis. In this article we will review how microfluidics-based solutions have been developed for rare-cell sorting for the detection of circulating tumor cells for cancer diagnosis. Many of these techniques have led to successful spin-outs, and are currently used in cancer related clinical trials around the world.

Cancer metastasis, the leading cause of cancer related mortality, is the spread of cancer cells from one organ to another in the human body. Using blood vessels for transport, individual cells detach from the primary tumor, travel through the vascular system and seed at distant organs to form secondary tumors. These cells, aptly termed ‘circulating tumor cells’, thus play a vital role in metastasis and their presence in blood is an important marker for cancer diagnosis. Compared to invasive tissue biopsy, this minimally invasive “liquid biopsy” (just a simple blood draw) can be used as a disease as well as treatment monitoring tool for cancer management. In 2004, a seminal paper by Cristofanili et al. convincingly showed that the count of circulating tumor cells (CTCs) in the blood of patients with metastatic breast cancer can be used as a prognostic biomarker for predicting patient outcome. The technology, CellSearch® by Janssen Diagnostics (https://www.cellsearchctc.com/), uses antibody-coated magnetic particles to isolate cells expressing epithelial marker (EpCAM – Epithelial Cell Adhesion Molecule) and confirm using immuno-fluorescence detection. Currently, this is the only United States Food and Drug Administration (FDA) cleared CTC test for metastatic breast, prostate and colorectal cancer.

Circulating tumor cells holds much promise as a reliable minimally invasive biomarker to aid clinical decision making in our war against cancer. Apart from just counting and detection, a current limitation of the CellSearch® technology, molecular characterization of these cells for mutations as well as other cancer signatures can pave the way for targeted therapy and personalized medicine. In blood, typically CTCs are found at very low concentrations, about 1 in 108-9 blood cells, making their detection exceedingly challenging. To address this, many cell separation technologies have been developed using microfluidics to find these proverbial “needles in a haystack”, with quite a number of them that have already been or are in the process of being commercialized. These techniques often rely on (a) immuno-capture methods or (b) physical filtration methods to isolate the CTCs from other blood cells.

Microfluidics immuno-capture separation methods target specific molecules expressed on the surface of CTCs to isolate them from blood cells. These techniques are mainly inspired from the FDA-approved CellSearch® system, relying on cell surface markers such as EpCAM for CTC isolation. For example, the IsoFlux system by Fluxion Biosciences (https://fluxion-biosciences.squarespace.com/isoflux/) uses magnetic particles with antibodies against EpCAM to magnetically tag the CTCs similar to the CellSearch® method. The blood mixture is then pumped through a microfluidic channel with a magnet placed at the top to isolate the magnetically tagged CTCs from blood. Taking advantage of the high surface to volume ratio of microfluidic channels, the On-q-ity system (http://www.on-q-ity.com/) covalently functionalizes anti-EpCAM antibody on an micropillar array to capture CTCs from blood. Other groups have tried similar techniques using antibodies against other cancer cell surface markers (eg. prostate specific membrane antigens) to capture CTCs in blood using microfluidic biochips. However, these techniques are limited in their application as they heavily rely on the expression of a single surface marker for immuno-capture of cancer cells. To overcome this limitation, the OncoCEE system by Biocept (http://www.biocept.com/) uses a cocktail of various antibodies against cancer cell markers to capture these cells in microfluidic channels. Taking advantage of highly specific streptavidin-biotin conjugation chemistry, the OncoCEE system tags the cancer cells with biotin conjugated antibodies. The blood mixture is then passed through a microfluidic chip patterns with an array of micropost functionalized with streptavidin to capture all biotin tagged CTCs. Since the initial biotin conjugated mixture targets multiple surface markers expressed on cancer cells (eg. anti-EpCAM, anti-Her2, anti-EGFR), this tests can capture a more heterogeneous CTC population. While immuno-capture methods offer very high specificity, it often suffers from (a) low blood volume sampling due to the low flowrates required for efficient CTC binding, (b) high cost associated with the antibodies, and finally (c) ability to retrieve the native CTCs from the channels and without damage.

Microfluidic filtration methods rely on the difference in physical properties (size and/or stiffness) between CTCs and other blood cells (red blood cells and leukocytes) for isolation. As CTCs are larger and stiffer compared to blood cells (CTCs ~15-20 µm; red blood cells (RBCs) ~7-8 µm; leukocytes ~7-15 µm), microfluidic channels incorporate either pillar or weir like structures to either impede the flow of larger CTCs while allowing the blood cells to sieve through [20, 21]. The critical gap between the pillars and the weirs are accurately controlled such that blood cells can flow though while the larger CTCs are retained. Currently, two methods – one based on pillars and the other based on weir features are being commercialized for CTCs isolation from blood. The ClearCell® CX system currently sold by Clearbridge Biomedics (http://www.clearbridgebiomedics.com/) for the isolation and retrieval of CTCs from blood, use crescent shaped isolation traps consisting of three micropillars placed within microchannels to filter out the CTCs from blood. The Parsortix system sold by ANGLE plc (http://www.angleplc.com/), use gradually reducing weir structures to isolate CTCs from blood. Both the ClearCell® CX and the Parsortix system allow immuno-staining the captured cells on chip using antibodies. The cells are typically fluorescently stained for cytokeratin (a cancer cell marker) and CD45 (a leukocyte marker) to differentiate blood cells from CTCs. Apart from staining for CTC counts, the advantage of these two systems over other existing CTC isolation techniques lies in the ability to retrieve viable CTCs post isolation from the microfluidic chip for numerous downstream applications, including molecular analysis and cell culture. Recently, new microfluidic devices based on the principles of inertial focusing of cells have been developed for CTC isolation. For example, using just a simple spiral patterned microchannel and unique physics emergent at the microscale, CTCs can be isolated with high efficiency. Unlike the pillars and weir based devices, these inertial microfluidic devices do not physically trap cells within them, thus allowing for continuous collection of CTCs during blood processing and reducing the processing time by eliminating the need for a retrieval step. This technology is currently being developed by Clearbridge Biomedics and will soon be commercially available under the name ClearCell® FX. Physical filtration methods are typically preferred over immuno-capture methods, as they are label-free and do not present any bias to the separation technique. Also, since they do not require any antibodies, these methods are typically cheaper.

The use of microfluidics in detecting and isolating CTCs is fast gaining a strong foothold among researchers both in the academia and industry. For now, cell separation in general seems to be the ‘killer application’ that researchers have been searching for where microfluidics can make significant impact. Increasing efforts are being dedicated towards development of microfluidic biochips for efficient cell separation ranging from disease diagnosis (eg. malaria, cancer, leukemia) to screening of fetal cells in maternal blood for prenatal testing. It is hoped that such microfluidic technologies will eventually deliver on its promise and further advance the various fields of clinical research and applications. This will not only aid in combating the various diseases such as cancer, but will also directly benefit the patients, the ultimate aim of medical technology development.

Ali Asgar S. Bhagat received his M.S. (2006) and Ph.D. (2009) in Electrical Engineering from the University of Cincinnati, USA. He is currently the Technical Director at Clearbridge BioMedics Pte Ltd, leading the development of next generation microfluidic based cancer diagnostic solutions. Prior to joining Clearbridge, he was a research scientist in the BioSystems and Micromechanics (BioSyM) group at the Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore and a visiting scientist at Massachusetts Institute of Technology, USA.

Professor Lim Chwee Teck is a Professor of Biomedical Engineering and Mechanical Engineering at the National University of Singapore. He has developed a suite of Cancer Microfluidic Biochips that is one of the world's first in being able to isolate viable cancer cells from patient's blood without using antibodies.

Professor Lim co-founded Clearbridge BioMedics Pte Ltd in 2009 to commercialize this technology. He and his team have received numerous awards including the Wall Street Journal Asian Innovation Awards 2012 (Gold), Credit Suisse Technopreneur of the Year Award 2012, Asian Enterpreneurship Award 2012 (First Prize), InnovFest Promising Startup Award 2012, President's Technology Award 2011, IES Prestigious Engineering Achievement Award 2010 and the Tan Kah Kee Young Inventors Award 2009.

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