Figure 3.

CXCL1/2/8 promote angiogenesis through lung fibroblasts in TNBC lung metastases

(A) Representative images of blood vessels stained with CD31 (red) in mouse lung metastases of Figure 2B. The blood vessel density was quantified by ImageJ software (n = 10 view fields; two view fields per mouse, 5 mice were examined). Magnification, 400×. (B) migration assays of isolated mouse lung endothelial cells. 2 × 10⁵ freshly isolated mouse lung endothelial cells were suspended in Vasculife medium and placed into ECM-coated chambers. Chemokines and/or other different cells were added to the bottom chamber. The migrated cells were stained after 16 h of incubation, and the dye was extracted and measured at OD₅₆₀ using a luminometer. Recombinant human CXCL1 (8 nM)/2 (2 nM)/8 (20 nM) proteins or different cell types were added to the bottom chamber of the assay system. (C) Tube formation assays of isolated mouse lung endothelial cells. Recombinant human CXCL1 (8 nM)/2 (2 nM)/8 (20 nM) proteins or the supernatant of single or co-culture of different cell types were added to the medium of endothelial cells cultured on the surface of Matrigel. (D) Migration assays of isolated mouse lung endothelial cells. Recombinant human CXCL1 (8 nM)/2 (2 nM)/8 (20 nM) proteins, antibodies, or different cell types were added to the bottom chamber. (E) Tube formation assays of the isolated mouse lung endothelial cells. Recombinant human CXCL1 (8 nM)/2 (2 nM)/8 (20 nM) proteins, neutralizing antibodies, or the supernatant from single- or co-culture of different cell types were added to the medium of endothelial cells cultured on the surface of Matrigel. The bar graph indicates mean ± SD. ∗p < 0.05, ∗∗p < 0.01.