title: Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma.
abstract: BACKGROUND
  Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro.
  
  
  METHODS
  The expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting.
  
  
  RESULTS
  We observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others.
  
  
  CONCLUSIONS
  Overall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models.
authors.affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, 1665 Aurora Court Suite 1032, Aurora, CO 80045 USA.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Bhatia
authors.affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, 1665 Aurora Court Suite 1032, Aurora, CO 80045 USA.
authors.forename: Kellen
authors.initials: K
authors.lastname: Hirsch
authors.affiliation: School of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 USA.
authors.forename: Sanjana
authors.initials: S
authors.lastname: Bukkapatnam
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057 USA.
authors.forename: Nimrah A
authors.initials: NA
authors.lastname: Baig
authors.affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, 1665 Aurora Court Suite 1032, Aurora, CO 80045 USA.
authors.forename: Ayman
authors.initials: A
authors.lastname: Oweida
authors.affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, 1665 Aurora Court Suite 1032, Aurora, CO 80045 USA.
authors.forename: Anastacia
authors.initials: A
authors.lastname: Griego
authors.affiliation: School of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 USA.
authors.forename: Dylan
authors.initials: D
authors.lastname: Calame
authors.affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, 1665 Aurora Court Suite 1032, Aurora, CO 80045 USA.
authors.forename: Jaspreet
authors.initials: J
authors.lastname: Sharma
authors.affiliation: Department of Pediatrics and Section of Pediatric Hematology/Oncology/BMT, Children's Hospital Colorado and University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Donson
authors.affiliation: Department of Pediatrics and Section of Pediatric Hematology/Oncology/BMT, Children's Hospital Colorado and University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Foreman
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057 USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Albanese
authors.affiliation: Department of Pediatrics and Section of Pediatric Hematology/Oncology/BMT, Children's Hospital Colorado and University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 USA.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Venkataraman
authors.affiliation: Department of Pediatrics and Section of Pediatric Hematology/Oncology/BMT, Children's Hospital Colorado and University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045 USA.
authors.forename: Rajeev
authors.initials: R
authors.lastname: Vibhakar
authors.affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, 1665 Aurora Court Suite 1032, Aurora, CO 80045 USA.
authors.forename: Sana D
authors.initials: SD
authors.lastname: Karam
keywords: EphB2; Invasion; Medulloblastoma; Radiosensitization