FGFR3 levels predict sensitivity to BGJ-398-induced cell cycle arrest.

<p>A. Effects of BGJ-398 on cell proliferation in the drug-sensitive cells. In the left panel, cells were incubated for 48 h in the presence of the indicated concentrations of BGJ-398 and cell growth was measured by MTT reduction. Mean ± SEM, n = 6. In the center and right panels, UM-UC14 or RT4 cells were incubated with the indicated concentrations of BGJ-398 and the percentages of cells within each cell cycle quadrant were quantified by propidium iodide staining and FACS analysis. Mean ± SEM, n = 3. B. Sensitivity to the anti-proliferative effects of BGJ-398 correlates with FGFR3 expression but not with the presence of activating FGFR3 mutations. The level of growth inhibition observed after 48 h exposure to 1 µM BGJ-398 (as measured in MTT assays) was correlated with the relative level of FGFR3 (left panel) or FGFR1 (right panel) mRNA expression in a panel of 17 human BC cell lines.. C. Effects of FGFR3 knockdown on cell proliferation. Left panel: UM-UC14 or RT4 cells were transiently transfected with either non-targeting (NT) or FGFR3-specific siRNAs and cell growth was measured at 48 h by MTT reduction. Mean ± SEM, n = 6. Center and right panels: UM-UC14 or RT4 cells were transiently transfected with either non-targeting (NT) or FGFR3-specific siRNAs and percentages of cells within each phase of the cell cycle were quantified by propidium iodide staining and FACS analysis. Mean ± SEM, n = 3. Lower panel: the efficiency of FGFR3 silencing was measured by quantitative RT-PCR and immunoblotting.</p

Relationship between FGFR/bFGF expression and EMT.

<p>A. Expression of FGFRs 1–4 and bFGF in relationship to E-cadherin expression. The relative mRNA levels were measured by quantitative real-time RT-PCR. The cell lines in each panel are organized by relative E-cadherin expression (low to high, from left to right; see Fig. 1B). B. Scatterplots depicting the relationships between FGFR1, bFGF, FGFR3, and EMT marker expression. Nonparametric correlation analyses were used to evaluate the relationships between FGFR3 and E-cadherin (CDH1) expression, FGFR1 and ZEB1 expression, bFGF and ZEB1 expression, and bFGF and FGFR1 expression. Correlation coefficients and p values are indicated on the figure.</p

FGFR1 selectively regulates invasion in “mesenchymal” bladder cancer cells.

<p>A. Left panel: effects of BGJ-398 on cell growth in two “mesenchymal’ (UM-UC3, UM-UC13) and two “epithelial” (UM-UC6, UM-UC9) cell lines that were found to be resistant to the anti-proliferative effects of the drug. Growth inhibition was measured at 48 h by MTT reduction. Mean ± SEM, n = 6. Center panel: concentration-dependent effects of BGJ-398 on invasion in the UM-UC3 and UM-UC13 cells. Invasion was measured using modified Boyden chambers and standard light microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057284#s2" target="_blank">Materials and Methods</a>. Mean ± SEM, n = 3. Right panel: effects of BGJ-398 on invasion in the UM-UC6 and UM-UC9 cells. Note that the drug had no effect on invasion in either cell line. B. Stable knockdown of FGFR1 or bFGF in cells transduced with lentiviral shRNAs. Relative mRNA levels were measured by quantititative real-time RT-PCR and protein levels were measured by immunoblotting. C. Effects of FGFR1 or bFGF knockdown on invasion. Left panels: percentages of cells that invaded through Matrigel in modified Boyden chambers were quantified by propidium iodide staining and confocal microscopy. The right panels display representative confocal images where the nuclei of the cells that invaded are pseudo-colored blue and the cells that did not invade are depicted in red.</p

Effects of BGJ-398 on UM-UC3 primary tumor growth and metastasis.

<p>A. Effects on primary tumor growth. Luciferase-labelled, orthotopically recycled UM-UC3 cells were implanted into the bladders of nude mice, and tumors were allowed to grow for 8 days prior to initiating therapy with BGJ-398 (daily via oral gavage). Tumor growth was measured biweekly by luciferase imaging. Mean ± SEM from 6 (control) or 7 (treated) mice per group. B. Effects on metastasis. Whole animal metastatic burdens were determined non-invasively by luciferase imaging. Mean ± SEM, n = 6 (control mice) or 7 (treated mice). C. Representative whole body luciferase images taken just prior to the initiation of therapy and at the conclusion of the experiment. D. Effects of BGJ-398 on CTC production. CTC numbers were estimated by measuring human HLA levels in isolated whole blood by quantitative PCR; cell numbers were determined using a UM-UC3 standard curve. The scatterplot displays the results obtained from each animal; the lines denote the mean values for each group.</p

Expression of FGFR1, FGFR3, and bFGF in distinct subsets of human BC cell lines.

<p>A. Correlation of FGFR1 and FGFR3 with canonical EMT markers. mRNA levels were measured using whole genome mRNA expression profiling (Illumina platform). The heat map depicts the expression of FGFR1, FGFR3, FGF2 (bFGF), p63 (TP63), E-cadherin (CDH1), Slug (SNAI2), and vimentin. B. Quantitative analysis of EMT marker expression. Relative levels of the “epithelial” markers E-cadherin (CDH1) and p63, and the “mesenchymal’ markers ZEB1 and vimentin were measured by quantitative real-time RT-PCR.</p

Morphological Differences between Circulating Tumor Cells from Prostate Cancer Patients and Cultured Prostate Cancer Cells

<div><p>Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.</p></div

Changes in cell and nucleus after two days of storage in the CellSave tubes.

<p>A: The diameter of cultured prostate cancer cells decreased ∼6% on average. B: The nuclear diameter of cultured prostate cancer cells decreased ∼10% on average.</p

Example images of cultured prostate cancer cell (A–D) and CTCs from prostate cancer patients (E–L) captured using the CellSearch® system.

<p>CTCs were noticeably smaller than cultured cancer cells (A–D). Cultured cancer cells were mostly round with regular cell and nuclear shapes. The nucleus was typically centered and surrounded by cytokeratin (E–L). CTCs exhibited highly variable shapes, including round (E), oval (F), elongated (G–J), and clusters (L). Non-round and multi-nucleate cells were sometimes observed (G–K). The yellow scale bar is 5 µm in length.</p

Diameters of CTCs from prostate cancer patients (pre-treatment) and cultured prostate cancer cells.

<p>The average diameter of CTCs (7.97 µm) was significantly smaller than cultured cancer cells (13.38 µm) (p<0.001).</p

Image data processing using LabView software.

<p>Labview software performs the series of operations on large varying mages datasets provided by the CellSearch® system. Two parallel filtering and measurements, such as calculating area in pixels (A) and estimating the best-fit ellipse (B) are performed for optimal performance and results.</p