Fig. 2: Hirudin suppresses tumor retention and colonization by disrupting the CTC clusters.

a MCF-7-GFP cells were used to create in vitro CTC clusters. After treating with 50 μg/ml or 100 μg/ml hirudin for 24 h, the formation of CTC clusters was observed. n = 3. b The protein expression of vimentin and CEACAM6 after 24-h treatment of hirudin or PBS. n = 3. c Quantitative analysis of vimentin and CEACAM6 protein expression. d Quantitative analysis of the relative cluster area of MCF-7 cell clusters. n = 4. e CTC clusters were blown apart and the proliferation of tumor cells within the clusters was examined by Edu staining. n = 3. f Transmission electron micrographs displaying the morphological characteristics of tumor cells in different states. The arrow indicates cells in apoptosis or premature apoptosis. g A schematic illustrating the establishment of the lung metastasis model via tail vein injection of 4T1-luc single cells or cell clusters pre-treated with hirudin or PBS for 24 h. h Representative bioluminescence images (IVIS spectral system) monitoring lung metastases on days 1, 2, 3, 7 and 14 after injection. n = 7. i–k Ki67 (i, j) and pan-CK (k) immunohistochemical images of lung tissue in retention model, and quantitative analysis of Ki67 (j). n = 3. l, m Ki67 immunohistochemical images (l) and quantitative analysis (m) of lung tissue in the colonization model. n = 3. n,o Representative HE staining (n) and quantitative analysis (o) of lung metastases in colonization model mice. n = 3. Data are mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 versus clusters.