Fig. 7. Analysis of lymphatic vessels in B16F10 lung metastasis model.

a Scheme of the experimental procedures. The lung image with B16F10 metastatic colonies after CUBIC procedures is shown. b The 3D whole-lung images at days 1, 4, and 10 after intravenous B16F10 injection. B16F10 cells expressing mCherry were inoculated intravenously in Prox1-GFP mice (female, 3–5 months). At 1, 4, and 10 days after inoculation, the excised lungs were subjected to CUBIC procedures. Representative 3D whole-lung images of lymphatic vessels (yellow) and cancer cells (red) are shown. The enlarged 3D images (white insets) are shown in the bottom (Z = 10 μm, digital zoom:1.25). c The 3D whole-lung images of original signals and classified signals using ilastik software from B16F10 cancer metastasis are shown. The enlarged 3D images (white insets) are shown in the bottom. d The 2D images at days 1 and 10 after intravenous B16F10 injection. Representative 2D (XY) images are shown. White arrowheads indicate cancer cells and tumor colonies. e The distances between B16F10 tumors and lymphatic vessels. The distances between B16F10 cancer cells or tumors and lymphatic vessels (GFP⁺ lymphatic endothelial cells) at days 1, 4, and 10 were calculated using the 3D images (pixel-based, n = 3 mice per group). Representative data from 2 independent experiments are shown. These boxplots show five-number summary statistics; i.e., the middle thick line, two hinges, and two whisker correspond to the median, interquartile range (IQR), and the largest and the smallest values unless “outliers”, respectively. “Outliers” are values which are larger or smaller than from 1.5 IQR, and shown in the black points. f Quantification of lymphatic vessels (volume, branching/end points, and lengths and radii of branches). All the classified signals as lymphatic vessels are counted as a volume at the whole lung level. With all the classified signals as lymphatic vessels, branching/end points (vertex), and lengths and radii of branches were calculated using TubeMap. The results from 2 independent experiments are shown (control; n = 3 mice, day 1; n = 5 mice, day 4; n = 5 mice, day 10; n = 4 mice). Data are shown with mean ± SD. One-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test were used. Source data are provided as a Source Data file. g The geometric features of lymphatic vessels in B16F10 experimental metastasis model using PH. The extracted signals of the lymphatic vessels were subjected to PH. Persistent diagrams are shown (left). The red points (dimention1) and blue triangles (dimention2) represent planar feature points (loop) and spatial feature points (void), respectively, observed by the persistent homology method. The results from 2 independent experiments are also shown in MDS (control; n = 3 mice, day 1; n = 5 mice, day 4; n = 5 mice, day 10; n = 4 mice). Source data are provided as a Source Data file.