title: Optimisation of immunofluorescence methods to determine MCT1 and MCT4 expression in circulating tumour cells.
abstract: BACKGROUND
  The monocarboxylate transporter-1 (MCT1) represents a novel target in rational anticancer drug design while AZD3965 was developed as an inhibitor of this transporter and is undergoing Phase I clinical trials ( http://www.clinicaltrials.gov/show/NCT01791595 ). We describe the optimisation of an immunofluorescence (IF) method for determination of MCT1 and MCT4 in circulating tumour cells (CTC) as potential prognostic and predictive biomarkers of AZD3965 in cancer patients.
  
  
  METHODS
  Antibody selectivity was investigated by western blotting (WB) in K562 and MDAMB231 cell lines acting as positive controls for MCT1 and MCT4 respectively and by flow cytometry also employing the control cell lines. Ability to detect MCT1 and MCT4 in CTC as a 4(th) channel marker utilising the Veridex™ CellSearch system was conducted in both human volunteer blood spiked with control cells and in samples collected from small cell lung cancer (SCLC) patients.
  
  
  RESULTS
  Experimental conditions were established which yielded a 10-fold dynamic range (DR) for detection of MCT1 over MCT4 (antibody concentration 6.25 μg/mL; integration time 0.4 seconds) and a 5-fold DR of MCT4 over MCT 1 (8 μg/100 μL and 0.8 seconds). The IF method was sufficiently sensitive to detect both MCT1 and MCT4 in CTCs harvested from cancer patients.
  
  
  CONCLUSIONS
  The first IF method has been developed and optimised for detection of MCT 1 and MCT4 in cancer patient CTC.
authors.affiliation: Clinical and Experimental Pharmacology Group, Manchester Cancer Research Centre, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. stephen.kershaw-2@manchester.ac.uk.
authors.forename: Stephen
authors.initials: S
authors.lastname: Kershaw
authors.affiliation: Clinical and Experimental Pharmacology Group, Manchester Cancer Research Centre, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. jeff.cummings@cruk.manchester.ac.uk.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Cummings
authors.affiliation: Clinical and Experimental Pharmacology Group, Manchester Cancer Research Centre, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. karen.morris@cruk.manchester.ac.uk.
authors.forename: Karen
authors.initials: K
authors.lastname: Morris
authors.affiliation: Clinical and Experimental Pharmacology Group, Manchester Cancer Research Centre, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. jonathan.tugwood@cruk.manchester.ac.uk.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Tugwood
authors.affiliation: Clinical and Experimental Pharmacology Group, Manchester Cancer Research Centre, Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK. caroline.dive@cruk.manchester.ac.uk.
authors.forename: Caroline
authors.initials: C
authors.lastname: Dive
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage; D045744:Cell Line, Tumor; D017321:Clinical Trials, Phase I as Topic; D005455:Fluorescent Antibody Technique; D064368:Healthy Volunteers; D006801:Humans; D027501:Monocarboxylic Acid Transporters / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000473:pathology; D011744:Pyrimidinones / Q000008:administration & dosage*; D055752:Small Cell Lung Carcinoma / Q000097:blood / Q000235:genetics* / Q000473:pathology; D027981:Symporters / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D013876:Thiophenes / Q000008:administration & dosage*; D000099233:Monocarboxylate Transport Protein 1
chemical_list: D000970:Antineoplastic Agents; D027501:Monocarboxylic Acid Transporters; D009124:Muscle Proteins; D011744:Pyrimidinones; D027981:Symporters; D013876:Thiophenes; D000099233:Monocarboxylate Transport Protein 1; C000592351:AZD3965; C406962:SLC16A4 protein, human