Xenograft tumor models and treatment regimes

All animal experiments were performed in accordance with the policies of the animal ethics committee of the Animal Research Committee of Wuhan University, and maintained in accordance with the guidelines by the Association for Assessment and Accreditation of Laboratory Animal Care International. BALB/c mice (female, 6–8 weeks old) and C57BL/6 mice (female, 6–8 weeks old) were purchased from Center for Disease Control and Prevention (Hubei, China), and GFP transgenic C57BL/6 mice were purchased from Shanghai Biomodel Organism Science & Technology Development Co., Ltd (Shanghai, China). All groups were randomly divided.

For tumor inoculations, 4T1-mCherry-luciferase cells (1 × 10⁶) or B16F10-mCherry-luciferase cells (2 × 10⁵) were injected to the right mammary fat pad of BALB/c mice or the armpit of C57BL/6 mice, respectively. HET0016 (5.0 mg/kg), DDMS (7.5 mg/kg) or vehicle (DMSO) were administrated intraperitoneally five times a week, starting on the day after tumor implantation. After the mice were killed on day 14 or day 28 peripheral blood, tumors and lungs were collected and analyzed. Lung metastasis was measured by hematoxylin/eosin and mCherry staining. Pulmonary metastatic nodules were counted in three serial sections. Lung metastasis also detected by ex vivo luciferase based noninvasive bioluminescence imaging system (In-Vivo Xtreme II, Bruker, Billerica, MA, USA). The lung images were analyzed by quantification of total photon flux of each lung using Molecular Imaging Software (Bruker).

In another experiment, bone marrow-derived cells were harvested from GFP transgenic C57BL/6 mice, and then the VEGFR1⁺GFP⁺ myeloid cells were isolated from bone marrow-derived cells by flow cytometry with purities of >95%. The B16F10-bearing mice treated with or without HET0016 (5.0 mg/kg) or DDMS (7.5 mg/kg) were injected with VEGFR1⁺GFP⁺ myeloid cells by tail vein on day 12 after tumor implantation. After 48 h, VEGFR1⁺GFP⁺ myeloid cells in the lungs were analyzed by flow cytometry.

To confirm that any beneficial effects observed reflect TAM depletion rather than off-target effects of the drug, we used two complementary methods to deplete macrophage. The Clod (100 mg/kg) liposomes were administrated intraperitoneally 24 h after 4T1 cells (1 × 10⁶) were injected to the mammary fat pad of BALB/c mice, followed by repeated injections of 50 mg/kg every fourth day. In another experiment, ZA (100 μg/kg) liposomes were injected intravenously once a week, and then HET0016 (5.0 mg/kg) was administrated intraperitoneally five times a week. Clod and ZA doses were based on the literature^{39, 40} and the efficiency of macrophage depletion was assessed by flow cytometry analysis of CD11b⁺ cells in peripheral blood and immunohistochemical analysis of tumor sections for F4/80.

In order to investigate the effects of macrophage M2 polarization regulated by CYP4A on pre-metastatic niche formation and metastasis, the mice were treated by intraperitoneal injection of the CM (300 μl) from control, M2 macrophages (M2) treated with or without HET0016 (5 μM), DDMS (10 μM) or CYP4A10^high M2 daily for 2 weeks, followed by tail vein injection of 4T1 cells (2 × 10⁵). The lungs were harvested and analyzed by flow cytometry on day 14. Lung metastasis was measured by hematoxylin/eosin staining on day 24.

For the in vivo co-injection model, 6 × 10⁵ 4T1 cells were mixed with 2 × 10⁵ wild-type RAW264.7 cells, negative control lentivirus (LV-NC) RAW264.7 cells or CYP4A10^high RAW264.7 cells at a ratio of 3:1 and orthotopically injected into BALB/c mice. On day 14 or day 28, tumors and lungs were collected and analyzed.

Cell culture, lentiviral transduction, preparation of CM, cell proliferation assay, migration assay, flow cytometry, gene expression analysis, immunoblotting assay, measurement of 20-HETE, immunohistochemisty and tissue microarray analysis, immunofluorescence, enzyme-linked immunosorbent assay and statistical analysis were described in Supplementary Materials and Methods.