Article Figures & Data
Figures
- Figure 1.
Ex vivo whole bladder organ culture. A, female rats are catheterized with a 20-gauge catheter, and the bladder is drained. The ureters are ligated, and the bladder is conditioned by removal of the glycosaminoglycan layer with brief acid treatment (0.1 N hydrochloric acid), following by neutralization with 0.1 N sodium hydroxide and washing with PBS. Qdot-labeled tumor cells suspended in culture medium are instilled into the bladder lumen. The urethra is ligated with the catheter in place, and the bladder and urethra are excised en bloc and transferred to a sterile 50-mL tube containing culture medium (B). Bladders are cultured ex vivo for up to 20 days at 37°C, 5% CO2. C, bladders instilled with either 253J-LacZ cells (i) or 253J-BV-LacZ (ii) cells were harvested at 7 days after instillation of cells. Tissue was incubated for 2 hours with X-gal substrate solution to reveal β-galactosidase activity. To confirm the presence of LacZ-expressing 253J-BV cells within the bladder wall, tissue was paraffin-embedded, sectioned, and counterstained with Nuclear Fast Red (iii). D, i, fluorescence image showing TCCSUP cells labeled with 15 nmol/L Qdots in vitro and the subcellular distribution of the nanocrystals. More than 90% of the cells are labeled with Qdots after 1 hour. Nuclei are stained with 4′,6-diamidino-2-phenylindole. Bar, 50 μm. iii, two-photon image of Qdot-labeled TCCSUP/GFP cells within the bladder wall. Original magnification, ×400. iv, high-magnification two-photon image of Qdot-labeled TCC/GFP cells within the bladder wall. Original magnification, ×1,000. v, differential interference contrast/epifluorescence cross-section of GFP-expressing, Qdot-labeled TCC cells within the bladder wall, showing GFP (green), Qdot (red), and Hoechst (blue) signals. Original magnification, ×400.
- Figure 2.
Optical imaging system. A, block diagram showing the major system components and the configuration for imaging specimens (adapted from ref. 24). B, transmission curves for excitation and emission filters, overlaid with the absorption and emission characteristics of the Qdot655 nanocrystals.
- Figure 3.
Epifluorescence imaging of Qdot-labeled tumor cells in the whole bladder organ culture model. A, steps in measurement of tumor area with Image J software: (i) representative epifluorescence image of tumor involvement within the bladder wall; (ii) inverted image of (i); (iii) overlay of red pseudocolor for determination of area; (iv) outline view of area measured using “Analyze Particles” command. Arrowheads, region of interest. B, images captured on two aspects (180 degrees and 270 degrees) of a representative bladder specimen incubated for 7, 14, or 18 days; 200 μmol/L thiotepa was added on day 14 for the last 4 days. C, fluorescent areas captured in (B) were measured using Image J and graphed. In each case, the area comprising tumor was expressed as a percentage of the total bladder surface area to control for differences in the area of individual organs. The surface area of bladders cultured ex vivo ranged from 18.2 to 21.4 mm2, with tumor areas ranging from 0.11 mm2 at 7 days to 2.11 mm2 by 18 days. *, P < 0.05, compared with 18-day control bladders (two-tailed t test).
- Figure 4.
Imaging of tumor invasion into the bladder wall using TPLSM. A, representative focal planes of a z-series through a Qdot-labeled TCCSUP cell mass of 49-μm height. The tumor mass invaded to a depth of only 8 μm. The bladder surface (WGA positive, green; Qdot negative, absence of red) visible in the bottom middle and right (white arrowheads) was not evident until 56 μm into the stack. B, representative z-series of a Qdot-labeled 253J-BV cell mass of 58-μm height. The tumor mass invaded to a depth of 63 μm. In contrast to (A), the bladder surface visible in the top left (white arrowhead) represented the start of the z-stack. C, graphical representation of mean tumor height and mean depth of invasion of TCCSUP and 253J-BV tumor foci into the bladder wall based on quantitative measurement of z-stacks. The average vertical dimension of the tumors is similar but the depth of invasion is significantly different between the two TCC cell lines (P = 0.00001, t test). D, table showing relative vertical dimensions and depths of invasion for three TCC cell lines. E, schematic representation of cell lines with low (Phenotype A) or high (Phenotype B) invasive potential represented by TCCSUP and 253J-BV, respectively.
- Figure 5.
Measurement of tumor volume using Volocity software. A representative focus of TCCSUP cells within the bladder wall was imaged by TPLSM. Fluorescence emitted by (A) Qdots655 (red), (B) Hoechst 33342 (blue), and (C) lectin WGA (green) was captured as described in Materials and Methods. Merged image (D). A z-stack characterizing the tumor focus shown in (A) to (D) was exported to Volocity and the volume measured as 59,652 μm3 using the visualization and classification software.
Volume 66, Issue 6
