Figure 2.

Targeting CX3CR1 with a neutralizing antibody or transcriptional silencing by CRISPRi impairs the homing of breast cancer cells to the bone. A, The neutralizing antibody blocked ERK phosphorylation in SKBR3 human breast cancer cells induced by soluble FKN and assessed by Western blotting. B, Pre-incubation of cells with the same antibody prior to their IC inoculation in mice dramatically reduced the number of fluorescent MDA-231 cells detected in the knee joints 24 hours post-inoculation, as compared to animals that received cells pre-incubated with either vehicle or an irrelevant immunoglobulin (Vehicle: n=6; IgG: n=7; CX3CR1-Ab: n=6. **p<0.01). C, Representative images of fluorescent MDA-231 cells detected in the lung parenchyma of inoculated animals. Magnification 200x. D, Preventing the activation of CX3CR1 also significantly reduced the number of DTCs seeding the lungs from the systemic blood circulation, as compared to controls (IgG: n=6; CX3CR1-Ab: n=6. **p<0.01). E, The effective silencing of CX3CR1 in MDA-231 cells by CRISPRi was confirmed by Western blotting. F, when CX3CR1-silenced cells were inoculated in mice, the number of DTCs detected in bone was significantly reduced (gRNA: n=3; dCas9: n=3; g(CX3CR1)dCas9: n=5. *** p<0.001).