title: Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells.
abstract: BACKGROUND
  Circulating tumor cells (CTCs) are often undetected through the immunomagnetic epithelial cell adhesion molecule (EpCAM)-based CellSearch(®) System in breast and colorectal cancer (CRC) patients treated with bevacizumab (BEV), where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro.
  
  
  METHODS
  The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch(®), untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch(®).
  
  
  RESULTS
  Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins (CK), while no evidence of epithelial to mesenchymal transition (EMT) in treated cells was observed. The recovery rate of cells through CellSearch(®) was gradually reduced in course of treatment with BEV, being 84%, 70% and 40% at 1, 2 and 3 months, respectively.
  
  
  CONCLUSIONS
  We hypothesize that BEV may prevent CellSearch(®) from capturing CTCs through altering EpCAM isoforms.
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Nicolazzo
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Massimi
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Lavinia V
authors.initials: LV
authors.lastname: Lotti
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Vespa
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Raimondi
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Fabio Maria
authors.initials: FM
authors.lastname: Pulcinelli
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Gradilone
authors.affiliation: 1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Gazzaniga
keywords: Circulating tumor cells (CTCs); bevacizumab (BEV); colorectal cancer (CRC); epithelial cell adhesion molecule (EpCAM) isoform