Article Figures & Data
Figures
- Fig. 1.
A, serial flow cytometry of SHIN3 cancer cells instilled with GmSA-1ROX or GmSA-20ROX. GmSA-20ROX showed a significant rightward shift (>1 log shift) compared with SHIN3 control cells ≥3 h after incubation (top right), whereas GmSA-1ROX failed to show a significant rightward shift (top left). The MFI progressively increased in both GmSA-1ROX and GmSA-20ROX. The slopes of regression lines, calculated from MFI and incubation time, were 0.485 a.u./h for GmSA-1ROX and 5.574 a.u./h for GmSA-20ROX, indicating greater fluorescence amplification with GmSA-20ROX compared with GmSA-1ROX. B, serial fluorescence microscopy images of SHIN3 ovarian cancer cells. Fluorescent microscopy as well as differential interference contrast imaging were done 10 min, 30 min, and 3 h after incubation with 1 μg/mL of GmSA-1ROX or GmSA-20ROX. The fluorescent dots produced by GmSA-20ROX were initially very small and minimally fluorescent (∼30 min) but became much more apparent at 3 h after incubation. Unlike the temporal changes observed in GmSA-20ROX, GmSA-1ROX showed a minimal and gradual change in the size and intensity of intracellular fluorescent dots. Original magnification, × 200. Photographic exposure time: GmSA-1ROX, 1 s; GmSA-20ROX at 10 and 30 min, 1 s; and GmSA-20ROX at 3 h, 100 μs.
- Fig. 2.
A, optical characteristics of GmSA-1ROX and GmSA-20ROX under acidic conditions. GmSA-1ROX (5 μg) and GmSA-20ROX (5 μg) in 390 μL phosphate buffer (pH 2.3, 3.3, 5.2, 6.4, and 7.4) were placed in a nonfluorescent 96-well plate (top left) and spectral fluorescence images were obtained. Emission spectra of GmSA-1ROX (middle) and GmSA-20ROX (bottom) showed the same emission peak at a wavelength of 610 nm when pH was 7.4, but the emission peak was shifted to a slightly longer wavelength under acidic conditions with both GmSA-1ROX and GmSA-20ROX. Top right, slopes of regression lines as a function of pH values in GmSA-1ROX and RmSA-20ROX were 0.023 and 0.105 (a.u.), respectively. B, certain proteases minimally activate the fluorescence of both GmSA-1ROX and GmSA-20ROX in vitro. The relative fluorescence signal intensity (SI) of GmSA-1ROX at 1, 3, and 6 h after incubation was 0.502 ± 0.219, 0.565 ± 0.174, and 0.491 ± 0.172 for trypsin, 0.608 ± 0.165, 0.723 ± 0.197, and 0.392 ± 0.098 for cathepsin C, 0.848 ± 0.265, 0.655 ± 0.200, and 0.539 ± 0.246 for cathepsin D, and 1.198 ± 0.368, 1.455 ± 0.440, and 1.927 ± 1.028 for MMP-2, respectively. Points, mean; bars, SD. The relative signal intensity of GmSA-20ROX at 1, 3, and 6 h after incubation was 1.448 ± 0.484, 1.483 ± 0.667, and 1.600 ± 0.802 for trypsin, 1.025 ± 0.334, 0.982 ± 0.295, and 0.714 ± 0.256 for cathepsin C, 2.918 ± 0.525, 2.121 ± 0.469, and 3.492 ± 1.322 for cathepsin D, and 1.055 ± 0.443, 1.406 ± 0.589, and 1.036 ± 0.404 for MMP-2, respectively. Slopes of GmSA-1ROX were −0.060, −0.080, −0.074, and 0.151 (a.u./h) for trypsin, cathepsin C, cathepsin D, and MMP-2, respectively. Slopes of GmSA-20ROX were 0.079, −0.050, 0.305, and 0.012 (a.u./h) for trypsin, cathepsin C, cathepsin D, and MMP-2, respectively. C, activation of GmSA-20ROX by SDS was 16-fold greater than that of GmSA-1ROX. The fluorescence signal of GmSA-20ROX (left column) was activated 30-fold [from 21 ± 2 (a.u.) to 624 ± 25 (a.u.)] by the addition of 5% SDS. In contrast, GmSA-1ROX (right column) was activated only 1.8-fold [from 29 ± 1 (a.u.) to 52 ± 4 (a.u.)] by the addition of 5% SDS.
- Fig. 3.
Serial side-by-side in vivo spectral fluorescence images of peritoneally disseminated cancer model after injection with 50 μg GmSA-1ROX or 50 μg GmSA-20ROX. A, white light and composite image of the peritoneal cavity and the peritoneal membrane. Immediately after injection, aggregated tumor foci (abdominal cavity) and disseminated cancer implants (peritoneal membrane) were undetectable due to high background signals (blue and yellow arrowheads, respectively). At 1 h after injection, GmSA-20ROX clearly depicted the aggregated tumor foci (red arrow) and the submillimeter peritoneal implants (pink arrow), whereas GmSA-1ROX minimally visualized the cancer foci (yellow and blue arrows) due to the insufficient fluorescence intensity from the cancer cells. At 3 h after injection, aggregated tumor foci as well as submillimeter peritoneal implants were more clearly visualized by GmSA-20ROX, whereas GmSA-1ROX failed to show the cancer foci due to insufficient fluorescence, although small cancer foci were detected by white light images. B, semiquantitative assessment of in vivo fluorescence intensities of GmSA-1ROX and GmSA-20ROX. ROI was drawn inside the intestine on the unmixed GmSA-ROX fluorescence image using both the white light (top) and unmixed rhodamineX image (middle). Histogram of fluorescence intensity of a ROI drawn on each of the peritoneal membranes instilled with GmSA-1ROX and GmSA-20ROX. The dynamic range of the fluorescence intensity was split into equal-sized 256 bins (1–256). Then, for each bin (X axis), the number of pixels from the data set that fall into each bin (Y axis) is counted. The regression lines were calculated from the data sets (fluorescence threshold values 31–241, total number of pixels within the threshold range 1–100,000 in common logarithm). Bottom, the slopes of GmSA-1ROX and GmSA-20ROX were −0.009 and −0.012 for immediate, −0.017 and −0.005 for 1 h, and −0.074 and −0.007 for 3 h after injection, respectively.
- Fig. 4.
Sensitivity of GmSA-20ROX spectral fluorescence imaging to detect peritoneal cancer foci was higher than that of GmSA-1ROX. In vivo spectral fluorescence imaging of RFP-transfected SHIN3 ovarian cancer–bearing peritoneal membrane was done 3 h after injection with 50 μg GmSA-1ROX or 50 μg GmSA-20ROX. The spectral fluorescence image was unmixed based on the spectral patterns (top middle) of GmSA-1ROX and GmSA20ROX, as well as the autofluorescence, and then, composite images consisting of GmSA-ROX (green), RFP (red), and autofluorescence (black and white) were made. Most foci detected by unmixed GmSA-1ROX images or GmSA-20ROX images were colocalized with unmixed RFP images. Two-color in vivo fluorescence intensity plots of the foci detected by unmixed GmSA-ROX images, unmixed RFP images, or both and nontumorous areas (bottom left, GmSA-1ROX; bottom right, GmSA-20ROX). All foci with signal intensities ≥30 (a.u.) on spectral unmixed RFP images and diameters ≥0.8 mm were defined as cancer foci (n = 207 for GmSA-1ROX and n = 190 for GmSA-20ROX). For comparison, ROIs were drawn in the surrounding nontumorous areas on the unmixed RFP images. When the foci positive for GmSA-ROX were defined as those whose fluorescence intensities ≥10 (a.u.) on spectral unmixed GmSA-1ROX images or GmSA-20ROX images, sensitivity and specificity were 24% (49 of 207) and 100% (181 of 181) for GmSA-1ROX and 99% (189 of 190) and 99% (144 of 146) for GmSA-20ROX, respectively.
Additional Files
Supplementary Data, Hama, et al.
Supplementary Figure S1; Supplementary Movie S1.
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Volume 13, Issue 21
