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Published on: November 17, 2023
Isolation of human prostatic epithelial plasma membranes for proteomics using mirror image tissue banking of radical
Kanishka Sircar1, Louis Gaboury, Lydia Ouadi
1Department of Pathology, McGill University Health Center, Canada.
Purpose:
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.
Experimental Design:
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.
