Technologies for label-free separation of circulating tumor cells: from historical foundations to recent developments.

Circulating tumor cells (CTCs) are malignant cells shed into the bloodstream from a tumor that have the potential to establish metastases in different anatomical sites. The separation and subsequent characterization of these cells is emerging as an important tool for both biomarker discovery and the elucidation of mechanisms of metastasis. Established methods for separating CTCs rely on biochemical markers of epithelial cells that are known to be unreliable because of epithelial-to-mesenchymal transition, which reduces expression for epithelial markers. Emerging label-free separation methods based on the biophysical and biomechanical properties of CTCs have the potential to address this key shortcoming and present greater flexibility in the subsequent characterization of these cells. In this review we first present what is known about the biophysical and biomechanical properties of CTCs from historical studies and recent research. We then review biophysical label-free technologies that have been developed for CTC separation, including techniques based on filtration, hydrodynamic chromatography, and dielectrophoresis. Finally, we evaluate these separation methods and discuss requirements for subsequent characterization of CTCs.

[1]  Terry J. Beveridge,et al.  Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy , 2008, Journal of bacteriology.

[2]  Ciprian Iliescu,et al.  Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives. , 2013, Biomicrofluidics.

[3]  P. Paterlini-Bréchot,et al.  Circulating tumor cells (CTC) detection: clinical impact and future directions. , 2007, Cancer letters.

[4]  Han Wei Hou,et al.  Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation. , 2011, Lab on a chip.

[5]  Tanja Fehm,et al.  Circulating Tumor Cells in Patients with Breast Cancer Dormancy , 2004, Clinical Cancer Research.

[6]  Helmuth Marcus.  Krebszellen im strömenden Blut? , 1917, Zeitschrift für Krebsforschung.

[7]  Hongshen Ma,et al.  Cell separation based on size and deformability using microfluidic funnel ratchets. , 2012, Lab on a chip.

[8]  J. Schellens,et al.  Circulating tumor cell detection in advanced non-small cell lung cancer patients by multi-marker QPCR analysis. , 2012, Lung cancer.

[9]  G. Doyle,et al.  Changes in circulating carcinoma cells in patients with metastatic prostate cancer correlate with disease status. , 2001, Urology.

[10]  Daniel A Fletcher,et al.  Force microscopy of nonadherent cells: a comparison of leukemia cell deformability. , 2006, Biophysical journal.

[11]  P. Denoix.  Mechanisms of Invasion in Cancer , 1967, UICC Monograph Series.

[12]  P. Jänne,et al.  A new device for rapid isolation by size and characterization of rare circulating tumor cells. , 2011, Anticancer research.

[13]  Yu Sun,et al.  Microfluidic approaches for cancer cell detection, characterization, and separation. , 2012, Lab on a chip.

[14]  G. Hampton,et al.  Evaluation of Circulating Tumor Cells and Circulating Tumor DNA in Non–Small Cell Lung Cancer: Association with Clinical Endpoints in a Phase II Clinical Trial of Pertuzumab and Erlotinib , 2011, Clinical Cancer Research.

[15]  Gwo-Bin Lee,et al.  High-purity and label-free isolation of circulating tumor cells (CTCs) in a microfluidic platform by using optically-induced-dielectrophoretic (ODEP) force. , 2013, Lab on a chip.

[16]  Subra Suresh,et al.  Biomechanics and biophysics of cancer cells. , 2007, Acta biomaterialia.

[17]  Kyung-A Hyun,et al.  Microfluidic flow fractionation device for label-free isolation of circulating tumor cells (CTCs) from breast cancer patients. , 2013, Biosensors & bioelectronics.

[18]  G. Sauter,et al.  Changes in Cytoskeletal Protein Composition Indicative of an Epithelial-Mesenchymal Transition in Human Micrometastatic and Primary Breast Carcinoma Cells , 2005, Clinical Cancer Research.

[19]  Li Wang,et al.  Recent Advances and Prospects in the Isolation by Size of Epithelial Tumor Cells (ISET) Methodology , 2013, Technology in cancer research & treatment.

[20]  Siyang Zheng,et al.  Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells. , 2007, Journal of chromatography. A.

[21]  E. Lianidou,et al.  Circulating tumor cells in breast cancer: detection systems, molecular characterization, and future challenges. , 2011, Clinical chemistry.

[22]  Dino Di Carlo,et al.  High-throughput size-based rare cell enrichment using microscale vortices. , 2011, Biomicrofluidics.

[23]  Jia Fan,et al.  Circulating tumor cells: advances in detection methods, biological issues, and clinical relevance , 2011, Journal of Cancer Research and Clinical Oncology.

[24]  S H SEAL,et al.  Silicone flotation: A simple quantitative method for the isolation of free‐floating cancer cells from the blood , 1959, Cancer.

[25]  W. Oh,et al.  The emerging role of circulating tumor cell detection in genitourinary cancer. , 2012, The Journal of urology.

[26]  Richard Superfine,et al.  Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. , 2011, Cancer research.

[27]  Hansen Bow,et al.  Microfluidics for cell separation , 2010, Medical & Biological Engineering & Computing.

[28]  Chunsheng Jiang,et al.  Microfluidics and circulating tumor cells. , 2013, The Journal of molecular diagnostics : JMD.

[29]  R. Richardson,et al.  Prominin1 marks intestinal stem cells that are susceptible to neoplastic transformation , 2008, Nature.

[30]  H. Ji,et al.  Silicon-based microfilters for whole blood cell separation , 2008, Biomedical microdevices.

[31]  E. Sackmann,et al.  Viscoelastic properties of erythrocyte membranes in high-frequency electric fields , 1984, Nature.

[32]  Jason P Beech,et al.  Sorting cells by size, shape and deformability. , 2012, Lab on a chip.

[33]  David W. Inglis,et al.  Efficient microfluidic particle separation arrays , 2009 .

[34]  Chwee Teck Lim,et al.  Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients. , 2010, Biosensors & bioelectronics.

[35]  F F Becker,et al.  Changes in Friend murine erythroleukaemia cell membranes during induced differentiation determined by electrorotation. , 1994, Biochimica et biophysica acta.

[36]  T. Imasaka,et al.  An "optical channel": a technique for the evaluation of biological cell elasticity. , 2001, Analytical chemistry.

[37]  Peter Kuhn,et al.  Cytomorphology of Circulating Colorectal Tumor Cells:A Small Case Series , 2010, Journal of oncology.

[38]  O. Eriksson.  Method for cytological detection of cancer cells in blood , 1962, Cancer.

[39]  P. Bruni,et al.  An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines , 2010, Journal of oncology.

[40]  Larry Norton,et al.  Is cancer a disease of self-seeding? , 2006, Nature Medicine.

[41]  Ion Stiharu,et al.  Interdigitated comb‐like electrodes for continuous separation of malignant cells from blood using dielectrophoresis , 2011, Electrophoresis.

[42]  H. Southwick,et al.  Carcinoma of the colon with special reference to prevention of recurrence. , 1954, Journal of the American Medical Association.

[43]  Robert H. Austin,et al.  Deterministic separation of cancer cells from blood at 10 mL/min , 2012 .

[44]  T. Scheinin,et al.  Large benign cells in circulating blood and their significance in the identification of cancer cells , 1962, Cancer.

[45]  Engell Hc.  [Cancer cells in the circulating blood; a clinical study on the occurrence of cancer cells in the peripheral blood and in venous blood draining the tumour area at operation]. , 1955 .

[46]  André A. Adams,et al.  Microsystems for the capture of low-abundance cells. , 2010, Annual review of analytical chemistry.

[47]  A. Bhagat,et al.  Continuous particle separation in spiral microchannels using Dean flows and differential migration. , 2008, Lab on a chip.

[48]  Peter Kuhn,et al.  High-definition imaging of circulating tumor cells and associated cellular events in non-small cell lung cancer patients: a longitudinal analysis , 2012, Physical biology.

[49]  C. Lim,et al.  Isolation and retrieval of circulating tumor cells using centrifugal forces , 2013, Scientific Reports.

[50]  Klaus Pantel,et al.  Circulating tumor cells: liquid biopsy of cancer. , 2013, Clinical chemistry.

[51]  R. Goswami,et al.  Quantitative detection of circulating epithelial cells by Q-RT-PCR , 2007, Breast Cancer Research and Treatment.

[52]  R. Tompkins,et al.  Continuous inertial focusing, ordering, and separation of particles in microchannels , 2007, Proceedings of the National Academy of Sciences.

[53]  A. I. Spriggs,et al.  THE DIFFERENTIAL DIAGNOSIS OF TUMOUR CELLS IN CIRCULATING BLOOD , 1960, Journal of clinical pathology.

[54]  Kyung-A Hyun,et al.  Continual collection and re-separation of circulating tumor cells from blood using multi-stage multi-orifice flow fractionation. , 2013, Biomicrofluidics.

[55]  Peter R. C. Gascoyne,et al.  Correlations between the dielectric properties and exterior morphology of cells revealed by dielectrophoretic field‐flow fractionation , 2013, Electrophoresis.

[56]  J. Rao,et al.  Nanomechanical analysis of cells from cancer patients. , 2007, Nature nanotechnology.

[57]  Andrew J Armstrong,et al.  Angiogenesis , Metastasis , and the Cellular Microenvironment Circulating Tumor Cells from Patients with Advanced Prostate and Breast Cancer Display Both Epithelial and Mesenchymal Markers , 2011 .

[58]  G. Ward.  THE BLOOD IN CANCER WITH BONE METASTASES. , 1913 .

[59]  F F Becker,et al.  Dielectric properties of human leukocyte subpopulations determined by electrorotation as a cell separation criterion. , 1999, Biophysical journal.

[60]  Mehmet Toner,et al.  Circulating tumor cells: approaches to isolation and characterization , 2011, The Journal of cell biology.

[61]  M. Keeney,et al.  Circulating Tumor Cell Analysis: Technical and Statistical Considerations for Application to the Clinic , 2009, Journal of oncology.

[62]  Subra Suresh,et al.  Nanomedicine: elastic clues in cancer detection. , 2007, Nature nanotechnology.

[63]  G. Doyle,et al.  Significance of Circulating Tumor Cells Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer , 2009, Journal of oncology.

[64]  Mo Chao Huang,et al.  Microsieve lab-chip device for rapid enumeration and fluorescence in situ hybridization of circulating tumor cells. , 2012, Lab on a chip.

[65]  Fei Huang,et al.  Rapid isolation of cancer cells using microfluidic deterministic lateral displacement structure. , 2013, Biomicrofluidics.

[66]  J. Nieva,et al.  Case study of the morphologic variation of circulating tumor cells. , 2007, Human pathology.

[67]  F. Bidard,et al.  Microfluidic: an innovative tool for efficient cell sorting. , 2012, Methods.

[68]  T. Molina,et al.  Preoperative Circulating Tumor Cell Detection Using the Isolation by Size of Epithelial Tumor Cell Method for Patients with Lung Cancer Is a New Prognostic Biomarker , 2010, Clinical Cancer Research.

[69]  F. Bischoff,et al.  FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE™ platform. , 2011, Cancer genetics.

[70]  Hongshen Ma,et al.  Chromatographic behaviour of single cells in a microchannel with dynamic geometry. , 2011, Lab on a chip.

[71]  K. Schütze,et al.  Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells. , 2000, The American journal of pathology.

[72]  Bo Lu,et al.  Parylene membrane slot filter for the capture, analysis and culture of viable circulating tumor cells , 2010, 2010 IEEE 23rd International Conference on Micro Electro Mechanical Systems (MEMS).

[73]  John W M Martens,et al.  Circulating tumour cell detection on its way to routine diagnostic implementation? , 2007, European journal of cancer.

[74]  S. Groshen,et al.  Portable Filter-Based Microdevice for Detection and Characterization of Circulating Tumor Cells , 2010, Clinical Cancer Research.

[75]  B. Lim,et al.  Enrichment, detection and clinical significance of circulating tumor cells. , 2013, Lab on a chip.

[76]  Daniela Massi,et al.  Application of a filtration- and isolation-by-size technique for the detection of circulating tumor cells in cutaneous melanoma. , 2010, The Journal of investigative dermatology.

[77]  Chao Liu,et al.  Double spiral microchannel for label-free tumor cell separation and enrichment. , 2012, Lab on a chip.

[78]  Massimo Cristofanilli,et al.  Molecular mechanisms of metastasis in breast cancer—clinical applications , 2010, Nature Reviews Clinical Oncology.

[79]  Peter Ulz,et al.  Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing. , 2013, Cancer research.

[80]  S H SEAL,et al.  A sieve for the isolation of cancer cells and other large cells from the blood , 1964, Cancer.

[81]  A. Ashkin,et al.  Optical trapping and manipulation of single cells using infrared laser beams , 1987, Nature.

[82]  S. Yie,et al.  Detection of Survivin-expressing circulating cancer cells in the peripheral blood of breast cancer patients by a RT-PCR ELISA , 2006, Clinical & Experimental Metastasis.

[83]  Joshua M. Kunken,et al.  Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers , 2012, Physical biology.

[84]  Jonathan W. Uhr,et al.  Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases , 2004, Clinical Cancer Research.

[85]  P. Black,et al.  Highly selective biomechanical separation of cancer cells from leukocytes using microfluidic ratchets and hydrodynamic concentrator. , 2013, Biomicrofluidics.

[86]  H. Jung,et al.  Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP). , 2011, Lab on a chip.

[87]  K. Gravdal,et al.  A Switch from E-Cadherin to N-Cadherin Expression Indicates Epithelial to Mesenchymal Transition and Is of Strong and Independent Importance for the Progress of Prostate Cancer , 2007, Clinical Cancer Research.

[88]  N. Kröger,et al.  Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro. , 1998, British Journal of Cancer.

[89]  Dino Di Carlo,et al.  A mechanical biomarker of cell state in medicine. , 2012 .

[90]  Hongshen Ma,et al.  Microfluidic micropipette aspiration for measuring the deformability of single cells. , 2012, Lab on a chip.

[91]  F F Becker,et al.  Cell separation on microfabricated electrodes using dielectrophoretic/gravitational field-flow fractionation. , 1999, Analytical chemistry.

[92]  P. Gascoyne,et al.  Antibody-independent isolation of circulating tumor cells by continuous-flow dielectrophoresis. , 2013, Biomicrofluidics.

[93]  H. Amini,et al.  Label-free cell separation and sorting in microfluidic systems , 2010, Analytical and bioanalytical chemistry.

[94]  A. Sabile,et al.  Liver resection and needle liver biopsy cause hematogenous dissemination of liver cells , 1999, Hepatology.

[95]  S. Digumarthy,et al.  Isolation of rare circulating tumour cells in cancer patients by microchip technology , 2007, Nature.

[96]  J. Sturm,et al.  Continuous Particle Separation Through Deterministic Lateral Displacement , 2004, Science.

[97]  Daniel T Chiu,et al.  Deformability considerations in filtration of biological cells. , 2010, Lab on a chip.

[98]  R. Weinberg,et al.  Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits , 2009, Nature Reviews Cancer.

[99]  W. Cole,et al.  Technique and results of isolation of cancer cells from the circulating blood. , 1958, A.M.A. archives of surgery.

[100]  Oscar Lin,et al.  Fluorescence In situ Hybridization Analysis of Circulating Tumor Cells in Metastatic Prostate Cancer , 2009, Clinical Cancer Research.

[101]  P. Gessner,et al.  Electromanipulation of mammalian cells: fundamentals and application , 2000 .

[102]  Geert W. Schmid-Schönbein,et al.  Biomechanical interactions of cancer cells with the microvasculature during metastasis , 1989, Cell Biophysics.

[103]  Young-Ho Cho,et al.  A continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process , 2005 .

[104]  Jason P. Gleghorn,et al.  Rare Cell Capture in Microfluidic Devices. , 2011, Chemical engineering science.

[105]  Stefan Schinkinger,et al.  Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. , 2005, Biophysical journal.

[106]  Peter Kuhn,et al.  High speed detection of circulating tumor cells. , 2006, Biosensors & bioelectronics.

[107]  Bo Lu,et al.  3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood , 2011, Biomedical microdevices.

[108]  F. Becker,et al.  Isolation of rare cells from cell mixtures by dielectrophoresis , 2009, Electrophoresis.