Figure 1 Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (A) Confocal micrographs of the representative field of the LN section. Scale bar: 10 μm. Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.  Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (B) Histograms illustrated that the dynamic changes of GFP+ 4T1 cells metastasized to the inguinal LN. Cells were counted in whole sections (2 non-consecutive sections per LN per mouse). Each group was composed of at least two mice.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.  (C-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays: (C) Soft-agar colony forming activity was examined in 4T1PT and 4T1LN cells (5x102 cells/well, n = 4 wells per group). Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (C), **P = 0.000088 and **P = 0.000004 for the 4T1LN cells at week 2 and week 3, respectively. (C-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays:(D and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. (D and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (E), **P = 0.000006. In (G) **P = 0.000734. Level of significance was determined using two-tailed unpaired t-test. (F and G) BALB/c mice (n = 3 mice per group) were injected with 2x105 4T1PT or 4T1LN cells via tail-vein. Lung colonization was examined by lung morphology (F) and the numbers of tumor nodule (G) on day 21. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. (F and G) BALB/c mice (n = 3 mice per group) were injected with 2x105 4T1PT or 4T1LN cells via tail-vein. Lung colonization was examined by lung morphology (F) and the numbers of tumor nodule (G) on day 21. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. (H and I) NSG mice (n = 5 mice per group) were injected with 4T1PT (5x102), 4T1LN (5x102), EMT6PT (1x102), or EMT6LN (1x102) cells. Distant organ metastasis was examined by bioluminscent images on day 28. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. (H and I) NSG mice (n = 5 mice per group) were injected with 4T1PT (5x102), 4T1LN (5x102), EMT6PT (1x102), or EMT6LN (1x102) cells. Distant organ metastasis was examined by bioluminscent images on day 28. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.