The Prostate 30:65–68 (1997) RAPID COMMUNICATION Monoclonal Antibody 7E11.C5 Staining of Viable LNCaP Cells Robert J. Barren III,1 Eric H. Holmes,2* Alton L. Boynton,3 S. Leslie Misrock,4 and Gerald P. Murphy1 1 Cancer Research Division, Pacific Northwest Cancer Foundation, Northwest Hospital, Seattle, Washington 2 Division of Cell Surface Biochemistry, Pacific Northwest Cancer Foundation, Northwest Hospital, Seattle, Washington 3 Department of Molecular Medicine, Northwest Hospital, Seattle, Washington 4 Pennie and Edmonds, New York, New York Background: Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein defined by the monoclonal antibody 7E11.C5. The 7E11.C5 antibody forms the basis of an in vivo diagnostic imaging agent (ProstaScint, Cyt-356) for identification of metastatic prostate cancer. The epitope on PSMA recognized by 7E11.C5 has been determined to be the first 6 amino acids from the N-terminal, expressed on the cytoplasmic side of the plasma membrane. Thus, the basis for 7E11.C5 specificity in imaging studies remains unclear. Methods: Fluorescence-activated cell sorter (FACS) analysis of fixed and viable cultured cells was used to determine the staining intensity with FITC-labeled antibodies. Results: The results indicate that FITC-labeled 7E11.C5 antibody is taken up and specifically labels viable LNCaP cells in vitro. Labeling intensity of viable cells after 2 hr of antibody incubation was similar to that of fixed cells. No labeling of cells that do not express PSMA was observed, nor was labeling observed with LNCaP cells treated with an isotype-matched irrelevant antibody. Conclusions: Uptake and labeling of PSMA by FITC-labeled 7E11.C5 in viable cells in vitro strongly suggest that this is a major basis for effectiveness of the 7E11.C5 antibody during in vivo imaging applications with 111In-labeled antibody (ProstaScint, Cyt-356). © 1997 Wiley-Liss, Inc. KEY WORDS: LNCaP, prostate-specific membrane antigen, 7E11.C5, flow cytometry INTRODUCTION 7E11.C5 is a mouse monoclonal IgG1 antibody raised against a membrane fraction of LNCaP cells. Immunohistochemical studies demonstrated 7E11.C5 specificity for prostate epithelial cells . In addition, a weak expression has been shown in tissue extracts of salivary glands, smooth muscle, and lower intestine . The resulting target of the antibody has been termed prostate-specific membrane antigen (PSMA). The coding sequence for this protein has been cloned . PSMA is expressed as a 120-kD transmembrane glycoprotein whose physiological function in prostatic cells is unclear. Portions of the extracellular domain have homology with transferrin receptor . More re© 1997 Wiley-Liss, Inc. cently, PSMA was shown to have enzymatic activity as a peptidase (N-acetylated a-linked acidic dipeptidase) capable of cleaving the bioactive peptide Nacetylaspartyl-glutamate  and as a novel folate hydrolase enzyme . The 7E11.C5 antibody has been used in a competitive enzyme-linked immunosorbent assay (ELISA) and in a Western blot assay to demonstrate elevated Sponsored by the CaPCURE Foundation; the Phi Beta Psi sorority. *Correspondence to: Eric H. Holmes, Ph.D., Pacific Northwest Cancer Foundation, Division of Cell Surface Biochemistry, 120 Northgate Plaza, Suite 218, Seattle, WA 98125. Received 1 November 1996; Accepted 7 November 1996 66 Barren et al. PSMA levels in prostate cancer patients [6–9]. In addition, 7E11.C5 labeled with 111Indium has been used as a radioimmunoscintigraphy agent in the detection of distant metastatic tumors in prostate cancer patients (ProstaScint, Cyt-356) . Recent studies of the epitope specificity for the 7E11.C5 antibody indicated that the antibody bound to the first 6 amino acids from the N-terminal, a portion of the protein within the cytoplasmic domain of the protein . These results were consistent with earlier observations from electron microscopic studies with gold-labeled 7E11.C5 antibody, which showed antibody binding to the cytoplasmic side of the plasma membrane of LNCaP cells . Taken together, these results have led to questions concerning the basis for the use of the 7E11.C5 antibody as an effective in vivo imaging agent . That is, does the antibody bind to accessible epitopes of nonviable cells or debris, or does it have some ability to enter cells in vivo and bind to the intracellular epitope of viable cells? The results presented in this paper indicate that viable LNCaP cells are able to uptake fluorescently labeled 7E11.C5 antibody, which binds to the intracellular epitope of PSMA. Thus, it is reasonable to conclude that the 7E11.C5 antibody most probably also has the ability to bind to the intracellular epitope of PSMA expressed in viable prostatic tumor cells during in vivo imaging applications. MATERIALS AND METHODS Cell Lines and Reagents Prostatic cancer cell lines DU-145, PC-3, and LNCaP were obtained from the American Type Culture Collection (Rockville, MD). Both DU-145 and PC-3 cells do not express the PSMA antigen found in LNCaP cells . All three cell lines were grown in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS) (Gibco-BRL), and maintained at 37°C in a 5% CO2– 95% air incubator. The monoclonal antibody 7E11.C5 (CYT-356) was obtained from Cytogen Corporation (Princeton, NJ). In some experiments, LNCaP cells were exposed to Eagle’s Basal Medium (BME; GibcoBRL) containing only 0.025 mM calcium. It should be noted that neoplastic cells in general maintain viability and cellular proliferation in calcium-deficient medium . Direct Labeling of FITC to 7E11.C5 7E11.C5 was labeled with fluorescein isothiocyanate (FITC), using the FluoroTag Conjugation Kit (Sigma Chemical Company, St. Louis, MO). In brief, 0.25 mg of FITC was added to 5 mg of 7E11.C5 in 0.1 M sodium carbonate–bicarbonate buffer pH = 9.0. The conjugation reaction was allowed to take place for 2 hr at room temperature in the dark with continual mixing. The reaction mixture was then placed over a Sephadex G-25 column (9.2 ml resin bed volume) equilibrated in phosphate-buffered saline (PBS) to separate the FITC-bound antibody from the unbound, free FITC. Antibody concentration was determined by absorbance at 280 minus 35% of the absorbance at 495, to correct for the FITC absorbance. The antibody was diluted to the stock concentration of 1 mg/ml in PBS. For FACS staining, the stock antibody solution was diluted 1:100 in RPMI or BME medium containing 5% FCS. Cell Staining For Flow Cytometry Analysis Cells were grown to approximately 80% confluence on 150-mm tissue culture dish (Falcon, Franklin Lakes, NJ). The media was then replaced with either RPMI 5% fetal calf serum or under low calcium conditions in BME with 1% EDTA-treated FCS. The cells were allowed to incubate with the respective medium for 24 hr. After 24 hr, 35 mg of propidium iodide (PI) (Sigma, St. Louis, MO) in 7 ml of RPMI containing 5% FCS or BME containing 1% EDTA-treated FCS was added directly onto the cells. The cells were incubated at 37°C in the dark for 15 min. The stain was removed from the plates and the cells washed extensively with PBS (0.1 M sodium phosphate, 0.15 M NaCl pH = 7.4). The cells were then incubated with 70 mg of FITC-labeled 7E11.C5 or FITC-labeled IgG1 mouse monoclonal antibody to keyhole limpet hemocyanin (KLH) (Becton Dickinson, San Jose, CA) as a negative control. The antibodies were diluted in a solution composed of 7 ml RPMI containing 5% FCS or BME containing 1% EDTA-treated FCS and the cells treated for 15 min or 2 hr. The cells were then harvested by scraping, pelleted by centrifugation for 5 min at 400g, washed with PBS, and passed through a 35-mm strainer cap (Falcon, Franklin Lakes, NJ) to prepare a single-cell suspension. The cells were then subjected to FACS analysis. Control LNCaP and PC-3 were harvested by scraping, fixed with 2 ml of 3% paraformaldehyde for 10 min on ice, and collected by centrifugation for 5 min at 400g. The cell pellets were resuspended in 0.2 ml of PI solution and incubated for 15 min on ice. The cells were then washed with PBS, collected by centrifugation for 5 min at 400g, and 0.2 ml of either the negative control, isotype-matched FITC-labeled anti-KLH, or 7E11.C5 antibody was added for a 30-min incubation period on ice. At the end of this period, 2 ml of PBS was added to the cells and centrifuged for 5 min at 400g. The pellets were resuspended in PBS and passed through a 35-mm strainer cap. The FACSCaliber flow cytometer (Becton Dickin- LNCaP Staining by 7E11.C5 67 son, San Jose, CA) was used to collect the sample information, forward, side scatter, and fluorochrome measurements. The data were collected in list mode. The analysis was done using Cell Quest software on a Macintosh 7600. RESULTS Figure 1 shows the results as three-dimensional graphs comparing cell number with staining intensity from FITC-labeled 7E11.C5 and propidium iodide (PI) treatments. In all treatment conditions, elimination of the initial PI staining step had no effect on the uptake and intensity of staining with FITC-labeled 7E11.C5 (results not shown). The results indicate that no staining of either DU-145 or PC-3 cells was observed using FITC-labeled 7E11.C5 (Fig. 1A,B) or with LNCaP cells using the isotype-matched negative control FITClabeled anti-KLH antibody (Fig. 1C). All three had greater than 95% of the cells, which were negatively stained with the respective antibody. Positive staining with 7E11.C5 (representing about 57% of the population) was observed for both the 15-min (Fig. 1D) and the 2-hr (Fig. 1E) incubations. In addition, 63% of both populations were positively stained with PI. The largest percentage of the population of these two time points were the double positive population at 54% for 15 min and 43% for the 2-hr incubation. The positive 7E11.C5 staining, negative PI staining cells made up 5% of the population at 15 min and 14% of the population at 2 hr. The positive control paraformaldehydefixed LNCaP cells (Fig. 1F) showed strong PI and 7E11.C5 staining with greater than 95% of the cells staining positive for both PI and 7E11.C5–FITC. This was expected, since paraformaldehyde compromises the cellular membrane, allowing efficient staining with both PI and 7E11.C5. The intensity of the 7E11.C5 staining was similar in the positive control and the 2-hr incubation, with mean channel values of 98 and 101, respectively. The 15-min incubation showed reduced staining intensity with a mean channel value of 67. LNCaP cells were next incubated in calciumdeficient medium (BME, containing 0.025 mM calcium). Previous reports indicate that this treatment results in plasma membrane permeability to calmodulin antibodies  and under this condition neoplastic cells maintain viability and proliferate in calciumdeficient medium when compared to their nonneoplastic counterparts . LNCaP cells were exposed to calcium-deficient medium for 24 hr and then stained with PI and FITC-labeled 7E11.C5 for 15 min (Fig. 1G) or 2 hr (Fig. 1H). Under conditions of low calcium, LNCaP cells positively stain for both 7E11.C5 and internalization of PI (Fig. 1G) equivalent Fig. 1. FACS analysis of cells. Three dimensional representations of cell number, FITC intensity, and PI intensity are shown on the indicated axes. Panel A) DU-145 cells labeled with PI for 15 min followed by 7E11.C5-FITC for 2 hours; Panel B) PC-3 cells labeled with PI for 15 min followed by 7E11.C5 for 2 hours; Panel C) LNCaP cells labeled with PI for 15 min followed by the isotype matched negative control Anti-KLH-FITC antibody for 2 hours; Panel D) LNCaP cells labeled with PI for 15 min followed by 7E11.C5-FITC for 15 min; Panel E) LNCaP cells labeled with PI for 15 min followed by 7E11.C5-FITC for 2 hours; Panel F) Positive control fixed LNCaP cells labeled with PI for 15 min followed by 7E11.C5 for 30 min; Panel G) LNCaP cells labeled with PI for 15 min followed by 7E11.C5-FITC (in BME) for 15 min; Panel H) LNCaP cells labeled with PI for 15 min followed by 7E11.C5-FITC (in BME) for 2 hours. The conditions of the analysis were as described under ‘‘Materials and Methods.’’ to the fixed control cells (Fig. 1F). Similar results were observed after the 2-hr incubation period, except that the binding of 7E11.C5 and PI was increased above that of the fixed control cells (Fig. 1H). It is clear that calcium-deficient medium enables PI increased access to the cellular interior and that the 7E11.C5 antibody also gains access to its cytoplasmic epitope. The observation that a rapid 15-min exposure 68 Barren et al. to the 7E11.C5 antibody stains a significant fraction of the cell population when maintained in either calcium-containing RPMI medium (Fig. 1D) or calciumdeficient BME medium (Fig. 1G), compared to the control irrelevant anti-KLH antibody (Fig. 1C) indicates membrane permeability and specific binding of the 7E11.C5 antibody to its intracellular epitope. DISCUSSION The finding that the 7E11.C5 epitope on PSMA is composed of the first 6 amino acid residues from the N-terminal of the protein, a portion of the protein found on the cytoplasmic side of the plasma membrane, has led to questions concerning the basis for targeting of the radiolabeled antibody to prostatic tumors during in vivo imaging with ProstaScint (Cyt356) . It has been postulated that the labeled antibody binds to dead cells and cellular debris present in metastatic tumor sites, as opposed to labeling viable cells within the tumor. Such a requirement would limit the value and applicability of an immunoscintigraphic scan. To address this question experimentally, we have conducted a FACS analysis of viable LNCaP cells, which are high expressors of the 7E11.C5 antigen, PSMA. The results presented clearly indicate that viable prostatic cancer cells uptake and specifically bind FITC-labeled 7E11.C5. Prostatic cancer cell lines that do not express PSMA (DU-145 and PC-3 cells) were not stained by FITC-labeled 7E11.C5. Furthermore, LNCaP cells were not stained by the isotype-matched irrelevant FITC-labeled anti-KLH antibody. The staining intensity of viable LNCaP cells with 7E11.C5 increased with time of incubation from 15 min to 2 hr to the point where the staining intensity resembled that of fixed cells. Uptake of PI was also observed throughout the cell populations despite the fact that these were viable cells initially in log phase of growth. These results suggest that the LNCaP cell membranes are highly permeable. Further, 7E11.C5 uptake was independent of the initial PI staining step, since elimination of it had no impact on 7E11.C5 uptake and labeling of the intracellular epitope of PSMA. The results in this paper indicate that antibodies such as 7E11.C5 are accessible to intracellular epitopes of prostatic cancer cells in vitro. 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