Pexidartinib

Macrophage proliferation was analyzed using Cell Counting Kit-8 (Dojindo, Japan). Cultured cells (RAW264.7) were dispensed into a 96-well plate at a density of 1000 cells/well, with or without a different level of IL-34 protein (Sino Biological, China) or conditioned medium (CM) from TNBC cells. To assess the IL-34-induced p38 and mTOR signaling pathways, the cells were treated with the CSF-1R inhibitor pexidartinib (AbMole, USA), the p38 inhibitor SB203580 (MedChemExpress, USA), or the mTOR inhibitor AZD8055 (MedChemExpress). At the indicated time points, the absorbance at 450 nm was measured to determine the number of viable cells in each well.

For the xenograft tumor growth model, 4T1 or E0771 cells were orthotopically implanted into the mammary fat pad of BALB/c or C57BL/6 mice (1 × 10 5 cells/mouse). Tumor growth was monitored every 3 days, and tumor volume was calculated using the following formula: volume = (width) 2 × length ÷ 2.
Tumor growth was visualized with a bioluminescence-based IVIS imaging system (Caliper Life Sciences, USA). Mice were sacri ced after 4 weeks, and tumor weight was then assessed and xed in 4% paraformaldehyde solution for hematoxylin-eosin (H&E) and IHC staining.
For the metastatic model, 4T1 or E0771 cells were injected into the tail veins of BALB/c or C57BL/6 mice (5 × 10 4 cells/mouse). Four weeks later, the mice were sacri ced and the lungs were xed in 4% paraformaldehyde solution for H&E staining. The number and the largest size of lung metastatic nodules were evaluated under a microscope (Leica).
For drug treatment, mice were randomly divided into four groups: immunoglobulin G (IgG) (Bio X Cell, USA) group (control), pexidartinib group, anti-PD-L1 group, and pexidartinib plus anti-PD-L1 group. pexidartinib (40 mg/kg, AbMole) was given orally for 5 days every week, and anti-PD-L1 antibody (200µg/mouse, Bio X Cell) was injected intraperitoneally every 3 days.