Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release.

Cancer cells release high levels of lactate that has been correlated to increased metastasis and tumor recurrence. Single-cell measurements of lactate release can identify malignant cells and help decipher metabolic cancer pathways. We present here a novel droplet microfluidic method that allows the fast and quantitative determination of lactate release in many single cells. Using passive forces, droplets encapsulated cells are positioned in an array. The single-cell lactate release rate is determined from the increase in droplet fluorescence as the lactate is enzymatically converted to a fluorescent product. The method is used to measure the cell-to-cell variance of lactate release in K562 leukemia and U87 glioblastoma cancer cell lines and under the chemical inhibition of lactate efflux. The technique can be used in the study of cancer biology, but more broadly in cell biology, to capture the full range of stochastic variations in glycolysis activity in heterogeneous cell populations in a repeatable and high-throughput manner.

[1]  Christoph A. Merten,et al.  Functional single-cell hybridoma screening using droplet-based microfluidics , 2012, Proceedings of the National Academy of Sciences.

[2]  H. Stone,et al.  Formation of dispersions using “flow focusing” in microchannels , 2003 .

[3]  Dale E Taylor,et al.  Glucose and lactate biosensors for scanning electrochemical microscopy imaging of single live cells. , 2008, Analytical chemistry.

[4]  A. deMello,et al.  Quantitative detection of protein expression in single cells using droplet microfluidics. , 2007, Chemical communications.

[5]  Franziska Hirschhaeuser,et al.  Lactate: a metabolic key player in cancer. , 2011, Cancer research.

[6]  Godfrey L. Smith,et al.  Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform. , 2006, Lab on a chip.

[7]  V. Ganapathy,et al.  Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond. , 2009, Pharmacology & therapeutics.

[8]  D. Armbruster,et al.  Limit of blank, limit of detection and limit of quantitation. , 2008, The Clinical biochemist. Reviews.

[9]  M A Arnold,et al.  Fiber-optic biosensors based on the fluorometric detection of reduced nicotinamide adenine dinucleotide. , 1988, Analytical chemistry.

[10]  Stefan Walenta,et al.  Lactate: mirror and motor of tumor malignancy. , 2004, Seminars in radiation oncology.

[11]  G. Stephanopoulos,et al.  Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption , 2014, Nature Biotechnology.

[12]  Hong Bin Yang,et al.  Optical detection of single cell lactate release for cancer metabolic analysis. , 2010, Analytical chemistry.

[13]  John L Cleveland,et al.  Targeting lactate metabolism for cancer therapeutics. , 2013, Journal of Clinical Investigation.

[14]  A. Abate,et al.  Ultrahigh-throughput screening in drop-based microfluidics for directed evolution , 2010, Proceedings of the National Academy of Sciences.

[15]  B. Jena,et al.  Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles. , 2006, Analytical chemistry.

[16]  M. Honavar,et al.  Monocarboxylate transporters (MCTs) in gliomas: expression and exploitation as therapeutic targets. , 2013, Neuro-oncology.

[17]  Mehmet Toner,et al.  Controlled encapsulation of single-cells into monodisperse picolitre drops. , 2008, Lab on a chip.

[18]  Christoph A. Merten,et al.  Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms. , 2008, Chemistry & biology.

[19]  Helen Song,et al.  Reactions in droplets in microfluidic channels. , 2006, Angewandte Chemie.

[20]  Charles N. Baroud,et al.  Rails and anchors: guiding and trapping droplet microreactors in two dimensions. , 2011, Lab on a chip.

[21]  D. Weitz,et al.  Droplet microfluidics for high-throughput biological assays. , 2012, Lab on a chip.

[22]  Alejandro San Martín,et al.  A Genetically Encoded FRET Lactate Sensor and Its Use To Detect the Warburg Effect in Single Cancer Cells , 2013, PloS one.

[23]  Wouter Olthuis,et al.  Lactate biosensors: current status and outlook , 2013, Analytical and Bioanalytical Chemistry.

[24]  D. Weitz,et al.  Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. , 2009, Lab on a chip.

[25]  M. Dewhirst,et al.  Targeting the Lactate Transporter MCT1 in Endothelial Cells Inhibits Lactate-Induced HIF-1 Activation and Tumor Angiogenesis , 2012, PloS one.

[26]  Min Wu,et al.  Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. , 2007, American journal of physiology. Cell physiology.

[27]  L. Mazutis,et al.  Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. , 2011, Lab on a chip.

[28]  Jean-Louis Viovy,et al.  Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells , 2008, Proceedings of the National Academy of Sciences.

[29]  M. Casal,et al.  Role of monocarboxylate transporters in human cancers: state of the art , 2012, Journal of Bioenergetics and Biomembranes.

[30]  D R Walt,et al.  A fiber-optic lactate sensor based on bacterial cytoplasmic membranes. , 2001, Biosensors & bioelectronics.

[31]  P. Pittet,et al.  Fast prototyping using a dry film photoresist: microfabrication of soft-lithography masters for microfluidic structures , 2007 .

[32]  Julien Verrax,et al.  Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. , 2008, The Journal of clinical investigation.

[33]  N. Perrimon,et al.  Droplet microfluidic technology for single-cell high-throughput screening , 2009, Proceedings of the National Academy of Sciences.

[34]  M. Dewhirst,et al.  Tumor metabolism of lactate: the influence and therapeutic potential for MCT and CD147 regulation. , 2010, Future oncology.

[35]  O. Warburg.  [Origin of cancer cells]. , 1956, Oncologia.

[36]  Dan Bratton,et al.  Static microdroplet arrays: a microfluidic device for droplet trapping, incubation and release for enzymatic and cell-based assays. , 2009, Lab on a chip.

[37]  Bradley E. Enerson,et al.  Molecular features, regulation, and function of monocarboxylate transporters: implications for drug delivery. , 2003, Journal of pharmaceutical sciences.

[38]  S. Quake,et al.  Dynamic pattern formation in a vesicle-generating microfluidic device. , 2001, Physical review letters.

[39]  A. Tsirigos,et al.  Ketones and lactate increase cancer cell “stemness,” driving recurrence, metastasis and poor clinical outcome in breast cancer , 2011, Cell cycle.

[40]  A. Lee,et al.  Droplet microfluidics for amplification-free genetic detection of single cells. , 2012, Lab on a chip.

[41]  J. Haveman,et al.  The relevance of tumour pH to the treatment of malignant disease. , 1984, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[42]  Saroj P. Mathupala,et al.  METABOLIC REMODELING OF MALIGNANT GLIOMAS FOR ENHANCED SENSITIZATION DURING RADIOTHERAPY: AN IN VITRO STUDY , 2006, Neurosurgery.

[43]  T. Mak,et al.  Regulation of cancer cell metabolism , 2011, Nature Reviews Cancer.

[44]  Pierre Sonveaux,et al.  Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-κB/IL-8 pathway that drives tumor angiogenesis. , 2011, Cancer research.