Fig 1.
Schematic illustration of PpIX-metabolizing pathway and mechanism of PDD.
5-ALA is a metabolic precursor of heme in the heme synthesis pathway and is further metabolized into PpIX. PpIX emits red fluorescence (600–740 nm) when excited with blue-violet light (375–445 nm).
Fig 2.
PpIX accumulation in GIST-T1 cells in vitro.
GIST-T1 cells were loaded with PpIX for different durations. Accumulation of PpIX within cells was analyzed using FACS with excitation at 405 nm and emission at 680 nm. (A) Histogram data from FACS. The abscissa (X-axis) indicates intensity of emission and the ordinate (Y-axis) represents number of cells. The green and red histograms represent cell samples at 0 min (as a control) and cell samples at each indicated time point, respectively. (B) Temporal changes in mean fluorescence intensity (M.F.I.). The abscissa (X-axis) indicates time and the ordinate (Y-axis) of the graph represents M.F.I.
Fig 3.
Subcellular localization of PpIX.
GIST-T1 cells were loaded with 5-ALA (8 mmol/L) for 2 h and stained with MitoTracker Green, LysoTracker Green, NBD-C6 ceramide Green, or ER-Tracker Green. Intracellular localization was visualized by confocal microscopy (original magnification, × 350; scale bar, 5 μm). (A) Each row represents the fluorescence of the organelle-specific probes and PpIX, respectively. (B) The fluorescence intensity profiles of PpIX (red lines) and the organelle probes (green lines) were examined along the arrows in the confocal images. (C) Quantitative analysis of subcellular localization of PpIX. Statistical significance was determined using Tukey’s multiple comparison test and was set at **P < 0.01. The rate of concordance of PpIX levels to each organelle probe was significantly higher in lysosomes (P < 0.01).
Fig 4.
Fluorescent imaging of GIST xenograft models.
Images were captured using a high-resolution camera equipped with an optical filter. Each image was photographed under white light (left side) and LED illumination (right side). (A) Xenograft models of flank tumors. Strong fluorescence was observed in mice receiving 300 mg/kg 5-ALA for 4 h. (B) Xenograft models of peritoneal seeding tumors. Peritoneal dissemination could not be identified through skin via LED light, but strong fluorescence was observed in extracted tumors.
Fig 5.
5-ALA accumulation in GIST in vivo.
Flank tumor models were administered with 300 mg/kg 5-ALA via the tail vein. Four hours after 5-ALA administration, the spectral waveforms of normal skin tissue (blue line) and GIST (red line) were examined using a VLD-M1 spectrometer. A peak of fluorescence emission was observed at 635 nm, corresponding to PpIX.
Table 1.
Autofluorescence in normal skin tissues and fluorescence intensity using 5-ALA in GIST.