Extended Data Fig. 5: BACH1/p53R175H–mediated regulation of CEMIP expression is critical for lung metastasis.
a, Diagram of lung metastasis models by tail vein injection in nude mice. Cal-33 cells or other cancer cells were transduced with Tomato fluorescence gene lentivirus (system 1) or luciferase-Tomato lentivirus (system 2) and sorted by FACS and then 0.5~4 million cells (dependent on cell type) were tail vein injected into each mouse. For system 1, 6~8 weeks later, mice were sacrificed and lungs were isolated for imaging using KEYENCE BZ-X800 fluorescence microscope. For system 2, 3~5 weeks later, mice were i.p. injected with luciferase substrate D-luciferin and imaged on IVIS Spectrum Optical Imaging System. b, Representative fluorescence images of liver and kidney isolated from mice tail vein injected with Cal-33 Ctrl cells, related to main Fig. 5a. Scale bar, 500μm. c, Quantitative analysis of total metastatic area (μm2) in lungs, related to main Fig. 5a. n = 7 mice for each group. P values (from left to right): 0.0035; 0.0062; 0.0048; 0.9274; 0.6024. d, H&E staining of lung tissues infiltrated with Cal-33 cells, related to main Fig. 5a. Red arrows indicate the metastatic nodes; panel d is a representative image of 5 lung sections from 5 mice (each group). Bar scale=20 μm. e, Immunohistochemical staining of Ki67 of lung tissues infiltrated with Cal-33 cells, related to main Fig. 5a. Red arrows indicate the Ki67 positive cell clusters; panel e is a representative image of 5 lung sections from 5 mice (each group). Bar scale=20 μm. f, Identification of CEMIP-/- Cal-33 cells made by CRISPR method. g, In vivo luminescence imaging of mice 4 weeks after tail vein injection of 0.5 million of luciferase carrying Cal-33 Ctrl or CEMIP-/- cells. Mice were imaged on IVIS System. n = 5 mice for each group. h, i, Quantitative analysis of total counts of luminescence (h) and average counts of luminescence (i) in lungs, related to panel (g). n = 5 mice for each group. P value (h): 0.0027. P value (i): 0.0028. Two-tailed Student’s t-test were used for statistical analysis. ns, not significant, **p < 0.01; Data represent mean + S.E.M.
