Specificity of PSMA expression on prostate cancer cells by confocal microscopy.

<p>Cancer cells were plated either alone (A, B) or in the presence of healthy donor derived PBMCs (C). Cells were labeled with J591-488 antibody (green) at room temperature, then fixed and immunostained for CD45 (red) and DAPI (blue). <b>A</b>) MDA cells are positive for PSMA expression. <b>B</b>) PC3 cells are negative and therefore, lack the PSMA expression. <b>C</b>) Right subfigure shows the specific expression of PSMA by MDA cells while blood cells express CD45. PBMCs isolated from normal healthy donors were mixed with MDA cells and processed as in A, B. PSMA-positive MDA cancer cells are clearly and specifically depicted (white arrow) among CD45-expressing leucocytes (arrowheads) in the mix. <i>Green= PSMA, Red= CD45, and Blue=DAPI</i>. </p

Isolation of prostate CTCs from metastatic PCa patients using anti-CD45 immunomagnetic depletion.

<p>2.5 ml blood from three metastatic PCa patients (> 50 CTCs/ 2.5 ml blood) was processed via ficoll density centrifugation and the PBMC fraction was collected. Immunomagnetic anti-CD45 depletion was performed on the obtained PBMCs and the remaining cells were washed, cytospunned onto the slides. Slides were stained for PSMA, EpCAM, sLe^x, and CXCR4 using the protocol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085143#pone.0085143.s001" target="_blank">Figure S1</a>. MDA, PC3, and KG1 cells were simultaneously stained as a control for the following markers: PSMA= Magenta, EpCAM= Yellow, HECA-452= Green, CXCR4= Red. All prostate CTCs expressed CXCR4, while, sLe^x expression was variable. The analysis of sLe^x intensity is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085143#pone-0085143-g004" target="_blank">Figure 4</a>. </p

Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

<div><p>Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm². CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (<i>p</i>=0.016). Analysis of the expression of sialyl Lewis X (sLe^x) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLe^x expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis.</p> </div

Rolling velocity of unlabeled and anti-PSMA labeled MDA cells at different shear stress.

<p>MDA cells were labeled with mAb J591-488 for PSMA. After labeling, 10⁶ J591-488 labeled MDA cells were perfused at 0.5, 1, 3, 5, and 8 dyne/cm² shear stress. Similarly unlabeled MDA cells were also perfused through E-selectin coated microtubes. Ten videos were taken at different lengths of the microtube for each shear stress. Rolling velocity was measured for both unlabeled and anti-PSMA J591-488 labeled MDA cells. Figure shows no significant difference in the rolling velocity between unlabeled and anti-PSMA J591-488 labeled MDA cells at shear stress ranging from 0.5-1 dyn/cm². The mean rolling velocities at 0.5 dyn/cm² were 5.27<u>+</u> 1.38 and 5.23 <u>+</u> 1.85 µm/sec in unlabeled and J591-488 labeled MDA cells, respectively. At higher shear stresses, a significant difference was observed between the two categories of MDA cells. Histogram shows the results from three separate experiments combined together. UnL= unlabeled MDA cells, Lab= J591-488 labeled MDA cells. Data is represented as Mean <u>+</u> SD.</p

Box plot showing the rolling velocity of MDA cells on IL-1β-stimulated HUVECs.

<p>1X10⁶ MDA cells were perfused over HUVECs. HUVECs used in the experiment were: <i>a</i>) stimulated with 50 ng/ml IL-1β for 4 h, <i>b</i>) unstimulated, and <i>c</i>) stimulated with 50 ng/ml IL-1β for 4h plus 1 h anti-E-selectin neutralizing antibody. Box plot shows the rolling velocities of MDA cells in three different categories of HUVECs. The rolling velocities in IL-1β-stimulated HUVECs ranged from 3.72<u>+</u> 2.1 to 6.01 +2.45 µm/sec at different shear stresses. The dots represent the outliers. No box plot for the category means that no cells were found rolling at the mentioned shear stress. Box plots represent three separate experiments combined together. n= total number of cells rolling in 10 different fields at a given shear stress in three independent experiments. Notice the reduction in the number of cells (n) rolling in StNeu and Unst versus St category. St= IL-1β-stimulated HUVECs expressing E-selectin. StNeu= IL-1β-stimulated HUVECs incubated with anti-E-selectin neutralizing antibody. Unst= E4ORF1 HUVECs. <i>p</i>< 0.05 was considered significant.</p

E-selectin expression and its functional assay in primary and E4ORF1 HUVECs.

<p><b>A</b>) Western blot showing E-selectin protein expression in primary (1°) and E4ORF1 HUVECs. HUVECs were either stimulated with 50 ng/ml IL-1β for 4 h or left untreated. E-selectin= 95-115 kDa, based on post-translational modifications. The blot underneath E-selectin shows actin, used as a loading control. Western blot is a representative of three independent experiments. <b>B</b>) Immunofluorescence showing E-selectin expression in IL-1β-stimulated 1° HUVECs (passage=2) and E4ORF1 HUVECs (passage 10). E-selectin= Green, DAPI= Blue. <b>C</b>) Rolling velocity of 10⁶ MDA cells on 50 ng/ml IL-1β-stimulated 1°- and E4ORF1- HUVECs. HUVECs were stimulated with IL-1β for 4 h. The mean rolling velocity of MDA cells at 1 dyn/cm² was 5.94 <u>+</u> 3.43 µm/sec and 6.35 <u>+</u> 3.92 µm/sec on E4-ORF1 and primary HUVECs, respectively. No significant difference was seen in the rolling velocities of MDA cells on either 1° or E4ORF1- HUVECs. Graph depicts Mean <u>+</u> SD.</p