title: Fluorescence-expressing viruses allow rapid identification and separation of rare tumor cells in spiked samples of human whole blood.
abstract: BACKGROUND
  Finding and isolating rare tumor cells in blood allows for diagnosis of disseminated cancer and for molecular profiling to direct the choice of biologic therapy. We explored whether the candidate gene therapy virus NV1066-designed to specifically infect cancer cells and express green fluorescence protein (GFP)-can be used for rapid infection, identification, and isolation of rare circulating tumor cells (CTC) in human whole blood.
  
  
  METHODS
  Mixtures of human cancer cell lines and human whole blood were exposed to NV1066 or heat-inactivated virus, incubated, and then examined for GFP expression by fluorescence microscopy and flow cytometry. Fluorescence-assisted cell sorting (FACS) was used to determine the efficiency of virally assisted tumor cell isolation. Sorted cells were subsequently stained for carcinoembryonic antigen (CEA) to determine if cells isolated in this way would maintain sufficient cellular integrity for molecular characterization.
  
  
  RESULTS
  In our study, there was 100% specificity for detection of cancer cells. Detection was consistent even at the highest dilution tested (10 cancer cells in 10 ml whole blood). The processing involved simple incubation without the technical demands of immunohistochemistry. FACS allowed for rapid isolation of GFP-expressing cells. Cells isolated by this method can subsequently undergo molecular characterization.
  
  
  CONCLUSIONS
  Oncolytic herpes simplex virus mediated green fluorescence in combination with FACS is a novel technique for the identification and isolation of cancer cells in an experimental model of blood-borne metastases. This procedure is a promising method for improving our diagnosis, staging, and molecular profiling of cancer.
authors.affiliation: Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Sandra M B
authors.initials: SM
authors.lastname: Fong
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Lee
authors.forename: Prasad S
authors.initials: PS
authors.lastname: Adusumilli
authors.forename: Kaitlyn J
authors.initials: KJ
authors.lastname: Kelly
mesh_terms: D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology*; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins