Paper-Based MicroRNA Expression Profiling from Plasma and Circulating Tumor Cells.

BACKGROUND Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. METHODS Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA)® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. RESULTS We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancer patients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. CONCLUSIONS Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses.

[1]  G. Yousef,et al.  Direct Comparison of Metastasis-Related miRNAs Expression Levels in Circulating Tumor Cells, Corresponding Plasma, and Primary Tumors of Breast Cancer Patients. , 2016, Clinical chemistry.

[2]  A. Schneeweiss,et al.  Heterogeneity of miR-10b expression in circulating tumor cells , 2015, Scientific Reports.

[3]  Nicolò Manaresi,et al.  Molecular profiling of single circulating tumor cells with diagnostic intention , 2014, EMBO molecular medicine.

[4]  S. Raguz,et al.  The miR-106b∼25 cluster promotes bypass of doxorubicin-induced senescence and increase in motility and invasion by targeting the E-cadherin transcriptional activator EP300 , 2013, Cell Death and Differentiation.

[5]  R. Askeland,et al.  MiR-93 enhances angiogenesis and metastasis by targeting LATS2 , 2012, Cell cycle.

[6]  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.

[7]  R. Bast,et al.  Modulation of MicroRNA-194 and Cell Migration by HER2-Targeting Trastuzumab in Breast Cancer , 2012, PloS one.

[8]  Gregory J Tsongalis,et al.  KRAS Detection in Colonic Tumors by DNA Extraction From FTA Paper: The Molecular Touch-Prep , 2011, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

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

[10]  G. Calin,et al.  MicroRNAs in body fluids—the mix of hormones and biomarkers , 2011, Nature Reviews Clinical Oncology.

[11]  Edith A Perez,et al.  MicroRNA signatures: clinical biomarkers for the diagnosis and treatment of breast cancer. , 2011, Trends in molecular medicine.

[12]  John W M Martens,et al.  mRNA and microRNA Expression Profiles in Circulating Tumor Cells and Primary Tumors of Metastatic Breast Cancer Patients , 2011, Clinical Cancer Research.

[13]  S. Sleijfer,et al.  Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients , 2011, Expert review of molecular diagnostics.

[14]  T. Fehm,et al.  HER2 status of circulating tumor cells in patients with metastatic breast cancer: a prospective, multicenter trial , 2010, Breast Cancer Research and Treatment.

[15]  H. Too,et al.  High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers. , 2010, RNA.

[16]  W. Filipowicz,et al.  Regulation of mRNA translation and stability by microRNAs. , 2010, Annual review of biochemistry.

[17]  Wei Xiong,et al.  MicroRNA-125b Confers the Resistance of Breast Cancer Cells to Paclitaxel through Suppression of Pro-apoptotic Bcl-2 Antagonist Killer 1 (Bak1) Expression* , 2010, The Journal of Biological Chemistry.

[18]  T. Fehm,et al.  Detection and HER2 Expression of Circulating Tumor Cells: Prospective Monitoring in Breast Cancer Patients Treated in the Neoadjuvant GeparQuattro Trial , 2010, Clinical Cancer Research.

[19]  Alexander van Oudenaarden,et al.  Genome-wide dissection of microRNA functions and cotargeting networks using gene set signatures. , 2010, Molecular cell.

[20]  D. Haber,et al.  Circulating tumor cells: a window into cancer biology and metastasis. , 2010, Current opinion in genetics & development.

[21]  John A. Foekens,et al.  Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR , 2009, Breast Cancer Research and Treatment.

[22]  Tanja Fehm,et al.  Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients , 2009, Breast Cancer Research.

[23]  Marilyn E Morris,et al.  MicroRNA-328 Negatively Regulates the Expression of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Cancer Cells , 2009, Molecular Pharmacology.

[24]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[25]  Tyler E. Miller,et al.  MicroRNA-221/222 Confers Tamoxifen Resistance in Breast Cancer by Targeting p27Kip1*♦ , 2008, Journal of Biological Chemistry.

[26]  Olga Kovalchuk,et al.  Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin , 2008, Molecular Cancer Therapeutics.

[27]  H. Allgayer,et al.  MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer , 2008, Oncogene.

[28]  A. Terzic,et al.  Translating MicroRNA discovery into clinical biomarkers in cancer. , 2007, JAMA.

[29]  H. Shah,et al.  Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards. , 2006, Journal of microbiological methods.

[30]  C. Croce,et al.  MicroRNA-cancer connection: the beginning of a new tale. , 2006, Cancer research.

[31]  F. Slack,et al.  Oncomirs — microRNAs with a role in cancer , 2006, Nature Reviews Cancer.

[32]  K. Pienta,et al.  Global gene expression profiling of circulating tumor cells. , 2005, Cancer research.

[33]  Alison Stopeck,et al.  Circulating tumor cells, disease progression, and survival in metastatic breast cancer. , 2004, The New England journal of medicine.

[34]  W. Al-Soud,et al.  Purification and Characterization of PCR-Inhibitory Components in Blood Cells , 2001, Journal of Clinical Microbiology.

[35]  L. Whitaker,et al.  ON THE POISSON LAW OF SMALL NUMBERS , 1914 .

[36]  Patel Jignal,et al.  Forensic Conception: DNA typing of FTA Spotted Samples - , 2014 .

[37]  Massimo Cristofanilli,et al.  Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition , 2013 .

[38]  L. Tanoue,et al.  Detection of Mutations in EGFR in Circulating Lung-Cancer Cells , 2010 .

[39]  L. Schwartz,et al.  New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). , 2009, European journal of cancer.