Immediately after intravenous injection of ICG solution, SHIN3 disseminated tumor nodules and parietal peritoneal vessels were optically enhanced, as shown in and supplementary video 1
. However, this tumor enhancement gradually decreased with time, whereas background signal increased, resulting in unclear tumor depictions on fluorescence images at 1 h after injection ( and supplementary video 2
). After another 5 h (6 h after injection), background signal was diminished and then tumor nodules were clearly depicted with high tumor-to-background ratios ( and supplementary video 3
). At 24 h after injection, tumor nodules were still clearly depicted with low background signals, but the camera gain had to be increased 45% due to lower overalls signal (). Similar findings were present in OVCAR 5 tumor-bearing mice, although tumor enhancement was slightly weaker than that in SHIN3 tumors ( and supplementary video 4
ICG clearly depicts disseminated peritoneal ovarian cancer nodules
In contrast to these findings with ICG, SHIN3 mice receiving HSA-IR800 was less effective. Within the first 5 minutes post injection, optical enhancement of the tumor and peritoneal vessels were observed as with ICG, however, unlike ICG, these vascular enhancements persisted even 1 h after injection due to slow clearance of HSA-IR800 (). Furthermore, in contrast to ICG, HSA-IR800 showed very high background signal even at 6 h and 24 h after injection, thus hampering efforts to visualize disseminated tumor nodules ()
These subjective results were supported by semi-quantitative analysis of fluorescence endoscopic images. A total of 542 disseminated nodules were evaluated (SHIN3-ICG group: 41 at 1 h, 55 at 6 h, and 49 at 24 h. OVCAR5-ICG group: 58 at 1 h, 85 at 6 h, and 80 at 24 h. SHIN3-HSA-IR800 group: 49 at 1 h, 71 at 6 h, and 54 at 24 h). Mean and standard deviation (SD) of each group were summarized in . In the SHIN3-ICG group, TBRs at 6 h (mean ± SD: 1.71 ± 0.20) and at 24 h (1.73 ± 0.23) were statistically higher than that at 1 h (1.17 ± 0.17). TBRs between 6 h and 24 h did not statistically differ. Furthermore, in the OVCAR5-ICG group TBRs at 6 h (1.43 ± 0.17) and 24 h (1.47 ±0.20) were also statistically higher than those at 1 h (1.24 ± 0.17). In contrast to the ICG group, the TBRs in SHIN3-HSA-IR800 group did not show statistical differences among the time points (1.19 ± 0.19 at 1 h, 1.20 ± 0.17 at 6 h, and 1.22 ± 0.14 at 24 h, respectively).
Tumor-to-background ratio improves after ICG injection
Whole body spectral fluorescence imaging clearly demonstrated differences in pharmacokinetics between ICG and HSA-IR800 (). At 1 h after injection, ICG accumulated in the liver and was excreted into the small intestine through biliary clearance. This liver uptake decreased with time, while bowel (feces) uptake increased at 6 h. Twenty-four h later, ICG containing feces were cleared from the bowel, and the tumor nodules were clearly depicted with low background signal. In contrast to ICG, the mice treated with HSA-IR800 showed high body background at all time points due to slow clearance from circulation.
ICG is retained in tumor and cleared from the background into the bile
All 32 and 29 fluorescent nodules of 1–2 mm in size sampled from the mouse peritoneum of ICG-injected, SHIN3 or OVCAR5 peritoneal implant bearing mice, respectively, were confirmed as cancer nodules by histological analysis with hematoxylin-eosin staining (). However, 40 non-fluorescent adjacent tissue samples (20 samples each from ICG-injected SHIN3 or OVCAR5 peritoneal implant bearing mice) did not contain cancer tissue. Therefore, fluorescent nodules of 1–2 mm in size, which had similar sizes to tumors observed under in vivo fluorescence endoscopy, were visualized with ICG injection of 100% sensitivity and 100% specificity.
All fluorescent nodules were validated as cancer metastasis