Figure 1.
ICG flow from the SiLN to the proper-ALN, observed using an in vivo fluorescence imaging system (IVIS; n = 1). B. Representative PDE images, following an ICG injection speed of 0.5 mL/h. The speed of ICG flow was calculated by dividing the distance, l, by the duration of time that had elapsed post-injection. C. Graph of the relationship between ICG flow speed and intra-SiLN injection speed (low, 0.5; medium, 1.0; high, 3.0 mL/h; n = 4 per group), revealing a low level of variation between individual experiments in the low-speed group. D. HS-FVCS image of the afferent lymphatic vessels after intra-SiLN injection of FITC-BSA solution (n = 2). (a) Area near the SiLN and proper-ALN captured by a normal digital camera. Two regions of interest were selected. (b) Bright field images obtained by HS-FVCS, without use of a fluorescence filter. A thick superficial epigastric vein (→) was observed. (c) Fluorescence images obtained by HS-FVCS, with use of an appropriate fluorescence filter (bandwidth: 510±2 nm). A new flow channel filled with FITC-BSA solution (→) appeared at a distance of about 200 µm from the vein. (d) Results of hematoxylin and eosin (H&E) staining. The flow channel was identified as the afferent lymphatic vessels by injection of Indian ink. The vein was not stained (n = 1).
Figure 2.
Establishment of the model of lymph node metastasis.
A. Representative images captured by in vivo bioluminescence imaging of a mouse with tumor cells grafted into the SiLN to promote metastasis to the ALN. (a) In vivo and (b) ex vivo bioluminescence signals in the proper-ALN and SiLN on day 14 post-inoculation, indicating that the proper-ALN is the draining lymph node. B. Graph showing the high correlation between in vivo and ex vivo bioluminescence (P = 0.0023; Spearman’s rank correlation coefficient [rs] = 0.9161; SiLN, n = 6; proper-ALN, n = 6). C. Results of histological verification. Tumor cells stained with H&E and luciferase-positive immunohistochemical signals in the proper-ALN and SiLN. MS: marginal sinus. T: tumor. D. Dissemination of KM-Luc/GFP cells (metastasis, n = 4) or PBS alone (control, n = 3) to each organ, assessed on day 14 post-injection of the SiLN. I, ipsilateral; C, contralateral; LN, lymph node. Error bars indicate the SEM values.
Figure 3.
Parameters related to the incidence of metastasis.
A. Effects of inoculation conditions on the incidence of lymph node metastasis. Metastatic incidence increased with injection of a larger number cells (a), did not vary significantly with injection duration (b), but decreased with a larger SiLN volume (c). In the values presented above the bars, the denominator represents the number of inoculated mice while the numerator represents the number of mice with metastases in the proper-ALN. NS indicates P>0.05; *P<0.05 calculated by Fisher’s exact probability test. B. Assessment of the correlation between the above 3 parameters and metastasis incidence, using a new parameter, cells mm−3 min−1. When metastasis incidence was set at 1, 100% metastasis was achieved when the cells mm−3 min−1 value exceeded 4.72×102 (n = 39). Analyses were performed with the Mann-Whitney U test.
Figure 4.
Monitoring of metastatic progression by CE-HFUS with Als.
A. Imaging of the temporal changes in angiogenic vessel density in a cross-section of a metastatic proper-ALN, visualized by CE-HFUS with ALs. Blood vessel density increased with tumor progression. Red circles indicate the proper-ALN boundary, arrows and dotted lines indicate the AL-enhanced region, and green highlighting indicates the dense area of neovasculature in the proper-ALN. B. Results of 3D quantitative analysis of the temporal changes in blood vessel volume and density in metastatic proper-ALNs. The term “cells” indicates the metastasized group (1×105 cells/min, n = 4) and “PBS” the negative control group (n = 3). Values for each group were normalized against the measurement on day 0. Error bars indicate the SEM values. * is for comparison of the temporal change within each group; # is for comparison between groups. * or #, P<0.05; ** or ##, P<0.01, calculated using two-way ANOVA followed by the Tukey-Kramer test.