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                                              CONFOCAL MICROSCOPY
                                                  Barry S. Levine1, Alexander V. Lyubimov1, Seraya N. Carr1, Alan P. Brown1, Jonathan J. Art2, James A. Crowell3
      1Toxicology                Research Laboratory, University of Illinois at Chicago, Chicago, IL; 2Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL;
                                                                   3Division of Cancer Prevention, National Cancer Institute, Bethesda, MD.

                              ABSTRACT                                                                                METHODS
                                                                                                                                                                                                          Figure 2. Mean Value of the Ratio of Intensity of SNARF-1
                                                                                                                                                                                                                  Loaded In Vitro Onto Rat Prostate Tissue

The knowledge of mechanisms of pH regulation and ion exchange in prostate
cancer cells may help in the development of new drugs and/or enhance the                Approximately 150 male CD (Virus Antibody Free) rats, ~400-600 g, and 13                              2.5                  n=6

anticancer potency of current drugs due to selective delivery, increased influx,        male Beagle dogs (6-10 kg)                                                                             2

and prevention of extrusion from tumor cells. As a first step in this direction, the                                                                                                                                                                        n=13                                             g/ r

objective of this study was to develop in vitro and in vivo methods of pH
                                                                                                                                                                                                                                                                                                             r/ g

                                                                                        LOADING OF THE FLUORESCENT INDICATORS (IN VITRO)
                                                                                                                                                                                                                                                                           n=12       n=13
                                                                                                                                                                                              1.5                                                                                                            Linear (g/ r)

measurements in prostate tissue and characterize intracellular pH gradients in
                                                                                                                                                                                                                                                                                                             Linear (r/ g)

                                                                                        The microdissected (rat) or sectioned (dog) prostate tissue was loaded for 30                          1
epithelial cells of normal rat and dog prostate. Freshly dissected prostate tissues     minutes with the dual emission fluorescent indicators (Carboxy–SNARF-1,
(in vitro) or the entire prostate gland (in vivo) were loaded with fluorescent dyes     SNARF calcein, and SNAFL calcein) dissolved in loading buffer (RPMI-1640

for 30 or 50 min, respectively. After loading, tissues were viewed using a Zeiss        Medium without sodium bicarbonate and amino acids). After 30 minutes, the                              0

LSM 510 Laser Scanning Confocal Microscope. Confocal images were initially taken        tissues were rinsed twice with RPMI-1640 Medium containing 250 mM
                                                                                                                                                                                                    6.4            6.6     6.8               7               7.2               7.4         7.6         7.8

in tissues perfused with RPMI-1640 medium. Calibration in situ was performed in                                                                                                                                                                   pH
                                                                                        sulfinpyrazone and viewed on the LSM 510 confocal microscope with the 25X
the same tissue with high potassium buffers of known pH containing nigericin.           objective. For measurements in the lysosomes, the prostate tissues was loaded
Carboxy-SNARF-1 was the most useful fluorescent indicator for determining the           with the single excitation and single emission indicators (Lyso Sensor Green
distribution of hydrogen ions in epithelial cells in rat and dog prostates. SNARF-1     DND-153 and DND 189) for 30 and 60 minute incubation periods and were
was visible only in the cytoplasm of the epithelial cells. The fluorescent indicator    viewed on the confocal microscope with the 63 X objective.                                                   Figure 3. Calibration Curve of the Mean Ratio of Intensity of
Lyso Sensor Green DND-189, which is only fluorescent in internal cellular                                                                                                                                 SNARF-1 Plotted Over pH (Dog Prostate Tissue)
compartments (lysosomes) at pH < 5.2, was visible inside epithelial cells of the        IN VIVO LOADING
prostate tissue. Therefore, it was concluded that the pH of lysosomes in prostate       In Vivo loading in rats was performed using a 25 G needle to load SNARF-1                         3

tissue was < 5.2. Various procedures were established, which included rat               dissolved in DMSO which was diluted in 3.5 mL of (0.4%) Trypan blue salt                      2.5
prostate microdissection, in vivo and in vitro fluorescent probe infusion, optimal      solution (0.81% sodium chloride, 0.06% potassium phosphate, dibasic) and 5                                                                                           y = -1.5124x + 12.368
concentrations of fluorescent probes, appropriate temperature regimen, and              mL of loading buffer to a final concentration of SNARF-1 at 100 mM.        The                    2                                                                        R2 = 0.9914

incubation time of cell cultures with fluorescent probes. In addition, experimental

                                                                                        SNARF-1 mixture was loaded underneath the capsule of the rat prostate.                                                                                                                                               g/r

conditions for the confocal microscope to study fluorescent probes were
                                                                                        Trypan blue was used for visual confirmation of the dye uptake.
                                                                                                                                                                                                                                                                                                             Linear (g/r)
                                                                                                                                                                                                                                                                                                             Linear (r/g)

optimized. Based upon several experiments with loading carboxy-SNARF-1 either           In Vivo loading of the Beagle dog prostate was performed using a final                            1
                                                                                                                                                                                                                                  y = 0.4443x - 2.508
in vitro or in vivo for rat and dog prostates, the intracellular pH of the epithelial   concentration of 100 mM of SNARF-1 dissolved in DMSO and diluted in 1.8 mL                                                                    R2 = 0.9733

cells in both species was ~ 7.0. Besides the measurement of the pH in rat and dog
                                                                                        Trypan Blue Salt solution along with 7 mL of loading buffer. One 10 mL syringe
tissues, a method of pH measurement in prostate tissue (rather than in cell             was used to load a total volume of 8.8 mL of the dye into the Beagle dog                          0
culture) was developed with fluorescent dye infusion both in vitro and in vivo.         prostate. A clamp was placed between the base of the bladder and the prostate
                                                                                                                                                                                              6.2            6.4         6.6           6.8              7                7.2         7.4         7.6

This method may now be used for the measurement of hydrogen ion                         to prevent the dye from being lost through the urethra. SNARF-1 was loaded                                                                               pH
concentrations and other ion measurements (cellular ion metabolism) either in           for 50 minute into the prostate.
freshly excised tumor tissue or maintained in tissue culture prostate carcinoma.
This study was supported by NCI Contract No. N01-CN-95055.                              CALIBRATION
                                                                                        The probes were calibrated using the high K+/nigericin in situ approach
                                                                                        (Thomas et al., 1979). Nigericin is an K+/H+ ionophore which cause equilibrium                                                                                                                                                                          SUMMARY
                                                                                        between intracellular and extracellular pH. Each calibration buffer was adjusted
                                                                                        to the appropriate pH (6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, and 7.6) using either 1 M
                                                                                                                                                                                                                                                                                                                    • Carboxy-SNARF-1 was the most useful fluorescent indicator for pH
                                                                                        KOH or 1 M NaOH.
                                                                                                                                                                                                                                                                                                                      measurements and was visible only in the cytoplasm of the epithelial cells
                                                                                                                                                                                                                                                                                                                      collected from rat and dog prostates. However, the Lyso Sensor Green DND-
                                                                                        LASER CONFOCAL SCANNING MICROSCOPY
                                                                                                                                                                                                                                                                                                                      189 which is only fluorescent in internal cellular compartments (lysosomes)
                                                                                        The dual emission dyes were excited with Argon/Krypton (Ar/Kr) laser light at
                                                                                                                                                                                                                                                                                                                      at pKa < 5.2 was visible in cells of the prostate tissue. Therefore, it was
                                                                                        488 nm and 568 nm. The fluorescence was measured simultaneously at 585 –
                                                                                                                                                                                                                                                                                                                      concluded that the pH of lysosomes in prostate tissue was < 5.2.
                                                                                        615 nm (assigned to the green channel in all figures) and > 650 nm (assigned
                                                                                        to the red channel in all figures). For the DND-153, DND-189 and BCESF
                                                                                                                                                                                                                                                                                                                    • In vivo loading of the prostate tissue in rats and dogs was successfully
                        INTRODUCTION                                                    fluorescent dyes an absorption/emission spectrum of 488/505-550 nm was
                                                                                                                                                                                                                                                                                                                      achieved for the first time.

Many tumors contain hypoxic areas due to decreased vascular supply which may                                                                                                                                                                                                                                        • Based upon several experiments with loading carboxy-SNARF-1 either in
                                                                                        PERFUSION AND IMAGING OF PROSTATE TISSUE
result in increased glycolysis and lactic acid production, resulting in decreased                                                                                                                                                                                                                                     vitro or in vivo for rat and dog prostates, the intracellular pH of the
                                                                                        A Lambda Double Perfusion tube set was used with the Lambda Perfusion Pump
local pH (Wike-Hooley et al., 1985). Recent studies have demonstrated that the                                                                                                                                                                                                                                        epithelial cells was measured at ~ 7.0. Other cells located in the prostate
                                                                                        in order to simultaneously irrigate fresh media into the confocal dish and to
extracellular pH of solid tumors is more acidic than the intracellular pH of tumor                                                                                                                                                                                                                                    tissue such as the stroma cells were not stained by any fluorescent
                                                                                        aspirate media out of the dish (approximately 35-45 mL/hr). The Bioptechs
cells (McCoy et al., 1995; Raghunand et al., 1999). The pH gradients in solid                                                                                                                                                                                                                                         indicators including the acetoxymethyl (AM) ester form of SNARF-1 dye
                                                                                        Objective Temperature Control System was used to provide a temperature
tumors may provide a means for more selectively targeting neoplastic cells with                                                                                                                                                                                                                                       which was easily and selectively up taken only by epithelial cells. This fact
                                                                                        controlled environment (Figure 1). RPMI-1640 medium was perfused onto the
cytotoxic or anticancer agents.                                                                                                                                                                                                                                                                                       is opening a theoretical possibility of the synthesis of the AM forms of new
                                                                                        microdissected (rat) or sectioned (dog) tissue while it was in a confocal dish on
                                                                                                                                                                                                                                                                                                                      constructed therapeutic compounds and its selective delivery to the
                                                                                        the stage of the LSM 510 confocal microscope. Images were obtained every 2
                                                                                                                                                                                                                                                                                                                      prostate epithelial cells based on the intercellular esterase cleavage.
The objective of this research program was to investigate the hypothesis that           minutes until the intensity reached a plateau. The tissue was then perfused
prostate cells contain a relatively acidic intracellular environment and to             with the calibration buffers at pHs 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, and 7.6.
                                                                                                                                                                                                                                                                                                                    • A method of pH measurement in the prostate tissue (rather than in
characterize the organelles that demonstrate low intracellular pH. This work was        Images were obtained every 3, 5, 7, and 9 minutes for each buffer.
                                                                                                                                                                                                                                                                                                                      individual cells) was developed with fluorescent infusion both in vitro and in
of considerable importance because the characterization of intracellular pH of
                                                                                                                                                                                                                                                                                                                      vivo. This method may now be used for the measurements of hydrogen ion
various cells within the prostate of an animal model reflective of the human            DATA ANALYSIS
                                                                                                                                                                                                                                                                                                                      concentrations and other ion measurements (cellular ion metabolism) in
prostate may have implications for drug development based upon pH-dependent             The intensity of the dye was obtained by subtracting each density level from
                                                                                                                                                                                                                                                                                                                      prostate cancer tissues.
drug delivery and activity. The present study was the first attempt to evaluate         the maximum possible level of density (density of absolute black = 255). The
the hydrogen ion concentration of the epithelial cells from the prostate of rats        mean intensity and standard deviation of five measurements were calculated.
                                                                                                                                                                                                                                                                                                                    • A method of viability assessment of the cells in tissue sections was
and dogs. This study created a model for studying the spatial distribution of pH        Standard curves of the ratios of intensity (red/green and green/red) of the
                                                                                                                                                                                                                                                                                                                      developed by loading with the second dye (BCECF AM) after the completion
within normal and tumor epithelial cells in prostate tissues.                           fluorescent indicator versus pH were plotted. Intracellular pH levels of the
                                                                                                                                                                                                                                                                                                                      of pH measurements with the primarily fluorescent indicator (SNARF-1).
                                                                                        prostate cell were determined from linear regression of the calibration data.
                                                                                                                                                                                                                                                                                                                      Both dyes are fluorescent only in live cells.

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