Functional Characterization of Circulating Tumor Cells with a Prostate-Cancer-Specific Microfluidic Device

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents

Role of gap junctions in breast cancer

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyThu Annelise NguyenGap junctional intercellular channels allow the cells to communicate with each other. A breach in gap junctional intercellular communication (GJIC) affects cell growth and proliferation. In addition, many neoplastic cells exhibit a decrease in GJIC. Many factors that decrease GJIC have been shown to potentiate cancer formation. 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), an environmental pollutant, is a carcinogen; however, its mechanism of carcinogenicity is unclear. Therefore, we examined the effect of TCDD on GJIC in MCF-7, a human breast cell line and normal mammary epithelial cells (HMEC). TCDD showed a decrease in GJIC in MCF-7 cells caused by increased phosphorylation of gap junctional protein, Cx43. PKCα-mediated phosphorylation of Cx43 was confirmed by inhibitor studies using calphostin C. Interestingly, TCDD affected GJIC in HMEC through a novel pathway involving redistribution of Cx43 to the perinuclear membrane. Our studies suggest that TCDD causes decrease in GJIC which could potentially lead to cancer. This also indicates that if GJIC is restored it could decrease cell growth and proliferation. Therefore, we investigated the role of substituted quinolines (PQ1), shown to bind with gap junctional proteins by computational docking. The results showed that indeed PQ1 significantly increases GJIC and exerts anti-tumor effect in human breast cancer cells compared to control without treatment or HMEC. We found an increase in GJIC, growth attenutation and increased apoptosis in T47D human breast cancer cell line. Our studies suggest that PQ1 is a novel gap junctional activator causing a decrease in tumor growth. Since PQ1 alone is effective in decreasing tumor growth in breast tumors, we proposed to test its efficacy with the current drug of choice for breast cancer, tamoxifen. The combinational treatment of tamoxifen and PQ1 showed a significant decrease in cell viability, increase in BAX (Bcl2-associated X), and, increase in caspase 3 activation compared to individual treatments. Hence, combinational treatment of PQ1 and tamoxifen can potentiate decrease in tumor growth. In conclusion, downregulation of gap junctions can potentiate tumor growth while restoration of GJIC can induce apoptosis and decrease tumor growth

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

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

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