title: Isolation and characterization of living circulating tumor cells in patients by immunomagnetic negative enrichment coupled with flow cytometry.
abstract: BACKGROUND
  This study was aimed at establishing a sensitive and specific isolation, characterization, and enumeration method for living circulating tumor cells (CTCs) in patients with colorectal carcinoma.
  
  
  METHODS
  Quantitative isolation and characterization of CTCs were performed through a combination of immunomagnetic negative enrichment and fluorescence-activated cell sorting. Isolated CTCs were identified by immunofluorescence staining. The viability and purity of the sorted cells were determined by flow cytometry. Blood samples spiked with HCT116 cells (range, 3-250 cells) were used to determine specificity, recovery, and sensitivity. The method was used to enumerate, characterize, and isolate living CTCs in 10 mL of blood from patients with colorectal carcinoma.
  
  
  RESULTS
  The average recovery of HCT116 cells was 61% or more at each spiking level, and the correlation coefficient was 0.992. An analysis of samples from all 18 patients with colorectal carcinoma revealed that 94.4% were positive for CTCs with an average of 33 ± 24 CTCs per 10 mL of blood and with a diameter of 14 to 20 μm (vs 8-12 μm for lymphoma). All patients were CD47(+) , with only 4.3% to 61.2% being CD44(+) . The number of CTCs was well correlated with the patient TNM stage and could be detected in patients at an early cancer stage. The sorted cells could be recultured, and their viability was preserved.
  
  
  CONCLUSIONS
  This method provides a novel technique for highly sensitive and specific detection and isolation of CTCs in patients with colorectal carcinoma. This method complements the existing approaches for the de novo functional identification of a wide variety of CTC types. It is likely to help in predicting a patient's disease progression and potentially in selecting the appropriate treatment.
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Liang
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Yu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Xie
authors.affiliation: Surgery Department, Fuzhou General Hospital, Fuzhou, China.
authors.forename: Shuming
authors.initials: S
authors.lastname: Chen
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Dong
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Sinko
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Lian
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Xu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Jichuang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Suhong
authors.initials: S
authors.lastname: Yu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Shao
authors.affiliation: Surgery Department, Fuzhou General Hospital, Fuzhou, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yuan
authors.affiliation: Surgery Department, Fuzhou General Hospital, Fuzhou, China.
authors.forename: Lie
authors.initials: L
authors.lastname: Wang
authors.affiliation: Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, China.
authors.forename: Lee
authors.initials: L
authors.lastname: Jia
mesh_terms: D051928:CD47 Antigen / Q000378:metabolism; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D018189:Immunomagnetic Separation; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology*; D012680:Sensitivity and Specificity
chemical_list: C497483:CD44 protein, human; D051928:CD47 Antigen; C497894:CD47 protein, human; D018960:Hyaluronan Receptors
keywords: circulating tumor cell reculturing; circulating tumor cells; colorectal carcinoma; fluorescence-activated cell sorting; immunomagnetic negative enrichment