Fig. 1: Direct inoculation of non-metastatic tumour cells into the circulation makes them metastatic.

In wild-type BALB/c mice, 67NR breast cancer cells are unable to metastasise to the lung from the mammary fat pad, while 4T1 completes the entire cascade (a–h). a A cartoon showing the mammary fat pad injection (top). Representative whole-body bioluminescence image of wild-type BALB/c mice harbouring 67NR or 4T1 tumours at the orthotopic mammary fat-pad (middle) and excised tumours (bottom) (n = 5). b The mean tumour volume of the tumours excised from these mice (n = 5). c Representative H&E staining of 67NR and 4T1 tumours from BALB/c mice (n = 5). Scale bar: 200 µm. d Representative bioluminescence images of lungs excised from BALB/c mice harbouring 67NR or 4T1 tumours at the orthotopic mammary fat-pad (top) and e the mean total flux from these lungs (bottom) (n = 5). f The number of visible lung nodules in 67NR and 4T1 tumour-bearing BALB/c mice (n = 5). g Representative haematoxylin and eosin (H&E)-stained lung tissue from 67NR and 4T1 tumour-bearing BALB/c mice. Scale bar: 300 µm. h The number of CTCs isolated from BALB/c mice harbouring 67NR and 4T1 tumours at the mammary fat pad (n = 5). Non-metastatic 67NR cancer cells metastasise to the lung similar to metastatic 4T1 cells when introduced via tail vein in wildtype BALB/c mice (i–n). i A cartoon showing the tail vein injection (top). Representative whole-body bioluminescence images of BALB/c mice injected with 67NR and 4T1 via the tail vein (bottom). j Representative bioluminescence images of lungs excised from BALB/c injected with 67NR and 4T1 via tail vein (top). k The mean total flux from these lungs (bottom) (n = 5 per group). l The number of visible lung nodules of BALB/c mice injected with 67NR and 4T1 via tail vein (n = 5). m H&E staining of representative lungs from BALB/c mice injected with 67NR and 4T1 via the tail vein. Scale bar: 5 mm and 500 µm. n The number of CTCs isolated from BALB/c mice injected with 67NR and 4T1 via tail vein (n = 5). Quantitative data are mean ± SEM. Statistical analyses were performed by unpaired two-tailed Student’s t test; ns indicates not significant, ^∗P ≤ 0.05, ^∗∗P ≤ 0.01, ^∗∗∗P ≤ 0.001.