Fig. 2: Formation of osteoclastic pre-metastatic niche promotes bone metastasis.

A Experimental design for the injection of tumor cells after the formation of a pre-metastatic microenvironment. B Representative radiographs of long bones bearing tumors from mice treated with vehicle or CM. The yellow arrows indicate sites of bone destruction. C Ratio of bone fractures on day 21 after injection of cancer cells between the vehicle and CM treatment groups. n = 6 per group. D Micro-CT images showing trabecular and cortical bone destruction in the proximal tibia. E Morphometric quantification of cortical bone micro-CT images with analysis of bone volume fraction (BV/TV) from mice treated with vehicle or CM. n = 6 per group. F Micro-CT images showing bone destruction on the surface of the vertebral bodies. The yellow arrows indicate sites of bone destruction. G–L Representative images of immunofluorescence staining of CTSK (red) and Netrin-1 (green) in the femur, tibia, and spine (right, from top to bottom) and quantitative analysis (left). Scale bars, 1000 µm. n = 6 per group. M–R Representative immunofluorescent images (M, O, Q) and quantification (N, P, R) of proliferative tumor cells in the femur, tibia, and spine (n = 6 mice), as determined by the presence of Ki-67⁺. Scale bars, 1000 µm. Data are presented as mean ± SD. Significance levels are indicated on top of each comparison. Comparisons between the two groups were performed using an unpaired two-sided Student’s t-test.