title: Isolation of human prostatic epithelial plasma membranes for proteomics using mirror image tissue banking of radical prostatectomy specimens.
abstract: OBJECTIVE
  To isolate human prostatic epithelial plasma membranes for the identification of cell surface proteins in the therapeutic targeting of cancer cells while permitting the retrieval of banked samples for clinical purposes.
  
  
  METHODS
  Radical prostatectomies from 84 patients (median, 61 years; prostate-specific antigen, 5.9; 66% nonpalpable) were processed with alternate, mirror image slices submitted for histology and tissue banking. Benign and malignant foci were macrodissected from the banked sections using the pathologically mapped, mirror image histology sections as a guide. Epithelial plasma membranes were isolated using novel immunomagnetic purification and their purity was assessed. Tissue homogenates were probed by Western blot for malignant (AMACR) and benign (p63) markers to test the accuracy of this protocol. Selected banked tissue slices were retrieved, thawed, and compared pathologically to their corresponding routinely processed alternate slices.
  
  
  RESULTS
  Plasma membrane preparations showed the enrichment of epithelial plasma membrane markers (prostate-specific membrane antigen and epithelial-specific antigen) with minimal marker expression from nonepithelial cells or intracellular organelles. Cancer homogenates showed up-regulated AMACR and down-regulated p63, whereas benign homogenates showed up-regulated p63 and down-regulated AMACR. There was 30% benign (p63+) contamination in cancer slices and <6% cancer (AMACR+) contamination in benign slices. Retrieved tissues showed the retention of immunoreactivity while their histology was always adequate for diagnosis.
  
  
  CONCLUSIONS
  We have successfully isolated purified epithelial plasma membranes from benign and malignant human prostates and provided validation data for the accuracy of our protocol in a prostate-specific antigen-screened cohort. Our method also enabled the retrieval of banked tissues for clinical purposes with the retention of good histologic and immunohistochemical quality.
authors.affiliation: Department of Pathology, McGill University Health Center, Canada.
authors.forename: Kanishka
authors.initials: K
authors.lastname: Sircar
authors.forename: Louis
authors.initials: L
authors.lastname: Gaboury
authors.forename: Lydia
authors.initials: L
authors.lastname: Ouadi
authors.forename: Melanie
authors.initials: M
authors.lastname: Mecteau
authors.forename: Eleonora
authors.initials: E
authors.lastname: Scarlata
authors.forename: Fred
authors.initials: F
authors.lastname: Saad
authors.forename: Armen
authors.initials: A
authors.lastname: Aprikian
authors.forename: Simon
authors.initials: S
authors.lastname: Tanguay
authors.forename: Steven
authors.initials: S
authors.lastname: Lapointe
authors.forename: Christian
authors.initials: C
authors.lastname: Lussier
authors.forename: Tina
authors.initials: T
authors.lastname: Miletti
authors.forename: Joel
authors.initials: J
authors.lastname: Lanoix
mesh_terms: D000368:Aged; D002462:Cell Membrane / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008875:Middle Aged; D011467:Prostate / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000096:biosynthesis; D011468:Prostatectomy / Q000379:methods*; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000601:surgery*; D040901:Proteomics / Q000379:methods
chemical_list: D017430:Prostate-Specific Antigen