Animal studies

All in vivo mouse experiments were approved by the Ethics Committee of Jining Medical University. NOD/SCID female mice at 6-8 weeks of age were allocated randomly to each group (n = 8 or 10). Cells were subcutaneously injected into each mouse. Tumor size (mm³) was measured with calipers and calculated by the following formula: volume (mm³) = (width² × length)/ 2. The individual measuring the tumor sizes was blinded to the treatments. Primary tumor tissues were formalin fixed, paraffin embedded, and sectioned for further analysis.

Subcutaneous tumors formed by 1 × 10⁶ MDA-MB-231 cells expressing shCtrl, shCCL16, shCCL16-OECtrl, shCCL16-CCL16R, OECtrl, OECCL16, OECCL16-shCtrl, or OECCL16-shCCR2 were dissected at 48 days after implantation.

For limited dilution transplantation, 5 × 10⁵, 1 × 10⁵, 5 × 10⁴ or 1 × 10⁴, 5 × 10⁴, 1 × 10⁵ cells were subcutaneously injected into NOD/SCID male mice. 28 days after implantation, tumor tissues were harvested.

For inhibitors treatment, NOD/SCID female mice were injected with 1 × 10⁶ MDA-MB-231 or MDA-MB-231-luci cells. 12 days after cell injection, mice were treated with XAV939 in DMSO (20 mg/kg) every 4 days or Stattic (20 mg/kg) every 3 days by intraperitoneal injection or with human IL10 (0.8 × 10^-6 mg/mL) every 3 days by intratumor injection. Tumor volumes were calculated and bioluminescence images were captured.