Fig. 3

Differential metastasis and splenomegaly in 4T1 + RAW264.7 versus 4T1 intraductally inoculated mice. a Representative image of the ex vivo bioluminescence imaging of 4T1 metastases in isolated axillary lymph nodes and lungs at 5 w p.i. b Quantification of 4T1-derived bioluminescence signals (in p/s/cm²) in the axillary lymph nodes and lungs of 4T1 + RAW264.7 and 4T1 inoculated mice (4T1 + RAW264.7 inoculation group: n = 7 axillary lymph nodes, n = 8 lungs; 4T1 inoculation group: n = 8 axillary lymph nodes, n = 7 lungs). c H&E histology and Ki67 immunohistochemistry of axillary lymph node and lung metastases in 4T1 + RAW264.7 and 4T1 inoculated mice at 5 w p.i. White dashed lines and black arrows indicate tumor-rich regions. The detailed image of Ki67 staining in the axillary lymph nodes identifies the border between normal lymph node tissue (N) and tumor-rich tissue (T). The bar graphs show the percentage of Ki67-positive staining in the axillary lymph node and lung metastases from 4T1 + RAW264.7 and 4T1 inoculated mice (n = 5 axillary lymph nodes and n = 3 lungs for each inoculation group). Black scale bars = 200 μm, green scale bars = 100 μm, red scale bars = 50 μm. d Weight measurement of spleens isolated at 3 and 5 w p.i. from 4T1 + RAW264.7 and 4T1 inoculated mice (number of spleens: 4T1 + RAW264.7 inoculation group: n = 5 at 3 w p.i., n = 7 at 5 w p.i.; 4T1 inoculation group: n = 5 at 3 w p.i., n = 6 at 5 w p.i.). e Representative images of isolated spleens at 5 w p.i. from 4T1 + RAW264.7 and 4T1 inoculated mice. The image of an isolated spleen from a non-inoculated mouse is also shown for comparative purposes. Data in panel (b), (c) and (d) are presented as the means +/− SEM. NS: not significant, *: P < 0.05