Extended Data Fig. 10: CXCR4 expression confers DTCs with a proliferative advantage but is not required for outgrowth.
From: Hepatic stellate cells suppress NK cell-sustained breast cancer dormancy
a, Experimental design for testing the influence of aHSC-secreted CXCL12 on cancer cell proliferation. Co-cultures of hepatocytes and sparsely seeded cancer cells were exposed to recombinant CXCL12 protein or conditioned medium (CM) from aHSCs alone or in combination with anti-CXCL12, anti-CXCR4, control IgG or a CXCR4 inhibitor, and the number of cancer cells was analysed by flow cytometry. b, Quantification of the number of cancer cells in different liver-like milieus shows that CXCL12–CXCR4 signalling induces cancer cell proliferation (for each cell line, n = 5 independent experiments). c, Scheme of Cxcr4 sites targeted by single-guide RNAs to generate 4T1 Cxcr4-KO cells. d, Genotyping of clonally derived cells obtained through CRISPR–Cas9 targeting of Cxcr4. Coloured lanes represent clones selected and pooled as 4T1 Cxcr4 wild type (Cxcr4-WT) and 4T1 Cxcr4-KO lines (n = 1 PCR per clone; selected clones were also confirmed by sequencing). bp, base pair. e, Experimental design for assessing the requirement of CXCR4 for liver metastasis. f, Representative H&E-stained livers from 4T1 Cxcr4-WT and 4T1 Cxcr4-KO lines injected in BALB/c immunocompetent mice. Arrowheads and coloured lines indicate metastases. Scale bars, 2 mm. g, Quantification of liver metastatic foci in livers of oil- and CCl4-treated mice normalized to the liver lobe area analysed (n = 6 WT oil, n = 9 WT CCl4, n = 8 KO oil, n = 11 KO CCl4). h, Quantification of metastatic area in livers of oil- and CCl4-treated mice, normalized to the liver lobe area analysed (n = 6 WT oil, n = 9 WT CCl4, n = 8 KO oil, n = 11 KO CCl4). In b, g, h, mean ± s.d.; two-tailed nonparametric Mann–Whitney U test.
