Fig. 2

cxcr4b is highly expressed in neutrophils and loss of function results in reduced triple negative breast cancer burden. (A) cxcr4a and cxcr4b expression levels were quantified in neutrophils and compared to the GFP negative cell population. Data are read counts from RNA sequencing performed on three biological replicates. FACS-sorted neutrophils were obtained from 5 dpf Tg(mpx:GFP)ⁱ¹¹⁴ larvae. cxcr4a and cxcr4b gene expression was enriched in neutrophils compared to GFP negative cells in zebrafish larvae (~4-fold and ~10-fold, respectively). cxcr4b was highly expressed in neutrophils compared to cxcr4a(~100-fold increased gene expression). (B) Relative metastatic tumor burden of MDA-MB-231-B-DsRed cells was quantified in ody and wt siblings at 2 dpi. Data are mean ± SEM of two independent experiments (wt: n = 64, ody: n = 57). Un-paired t-test ****p < 0.0001. (C) MDA-MB-231-B tumor cells established a secondary tumor mass, with initiation of single cell extravasation, in wt larvae, whereas a phenotype inhibition was found in ody mutants at 2 dpi (22.5% reduction). (D) MDA-MB-231-B tumor burden was measured in wt and cxcr4b null mutants at 4 dpi, at the metastatic site where secondary growth began at 2 dpi. A 40.5% reduction in tumor burden was observed. Data are mean ± SEM of two independent experiments (wt: n = 59, ody: n = 43). Un-paired t-test, with Welch’s correction ****p < 0.0001. (E) Highly invasive cancer cells displayed aggressive and metastatic features in wt siblings, whereas few cells remained in the CHT region of 4 dpi odylarvae. Scale bars: 50 µm. Micrographs are acquired using a Leica MZ16FA fluorescent microscope coupled to a DFC420C camera.