Fig. 5

CXCR4-expressing TNBC cells fail to initiate metastatic events in cxcl12a- and cxcl12b-null zebrafish mutants. (A) No differences in tumor cell invasion were found in the medusa (cxcl12a^-/-) mutants, compared to wild-type (wt) siblings. (B,C) Breast cancer cells failed to form micrometastases in 4-dpi zebrafish embryos deficient for both cxcl12a and cxcl12b ligands, whereas tumor invasion (B) and tumor burden (C) occurred in the cxcl12a^-/-/cxcl12b^+/+ and cxcl12a^-/-/cxcl12b^+/- siblings. Unpaired t-test: (A) ns, P>0.05 (wt: n=73; medusa: n=66), (B) **P=0.0012 and (C) **P=0.0033 (n=11 in each group). Graphs in A-C are cumulative of two independent experiments. (D,E) Top panels: MDA-MB-231-B breast cancer cells were highly invasive in the siblings, whereas few tumor cells remained in the metastatic region in zebrafish that were mutant for both ligands. Images were acquired using a Leica MZ16FA fluorescent microscope coupled to a DFC420C camera. Bottom panels: vessel connections are compared to distinguish siblings and double mutants: the hypobranchial arteries (HA), indicated by asterisks, failed to connect to the mandibular arch (AA1) in the cxcl12a^-/-/cxcl12b^-/- larvae. Images were acquired using a Leica TCS SPE confocal microscope with an HC PL FLUOTAR 10× DRY objective (0.30 N.A.). Scale bars: 50µm. (F) CXCL12 expression is lower in the MDA-MB-231-B compared to MCF-7 breast cancer cell line. Unpaired t-test, with Welch′s correction. **P=0.006. qPCR was performed on two biological replicates.