Xenograft tumour models

BALB/c nude mice (4-6 weeks old, female) were weighed and randomly divided into the shBCKDK and shCtrl groups (12 animals). BT-549 cells (5 × 10⁶ cells in 100 µL PBS) harbouring empty vector and stable knockdown of BCKDK were subcutaneously injected on the right flank. Body weight and tumour volumes were measured twice a week. After 22 days, all mice were executed by cervical dislocation.

Patient-derived tumour xenograft (PDX) models were generated from fresh tumour samples of patients with TNBC, which were subcutaneously implanted on the right flank (F0). After reaching the appropriate volume, the tumours were excised and divided into equal pieces for implanting the next generation. When the tumours became palpable, the tumour-bearing mice were randomised to two groups (12 animals). BT2 (MedChemExpress, HY-114855, 20 mg/kg/day) or PBS was intraperitoneally injected daily for 1 week. Tumour size was evaluated using calliper measurements every 2–3 days by the following formula:

V = 0.5 × D × W² (V, volume; D, the larger diameter; and W, width).

The mice were sacrificed after 17 days, and the tumour tissues were excised from mice, weighed, and fixed in a 10% formalin solution for further analysis.