CAFs and NFs culture

Fresh specimens and adjacent normal breast tissue samples (>3–5 cm away from the tumor) were collected from 5 TNBC patients in Zhujiang Hospital. The specimens were sectioned into 1-mm³ pieces and digested with 1 ml 0.12% collagenase A in a 37°C humidified atmosphere containing 95% air and 5% CO₂ for 8 h, after which the digestion was stopped by supplementation with Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) plus 10% fetal bovine serum (FBS, Gibco, USA). Tissue debris was removed and cells were collected and cultured in a 37°C humidified atmosphere containing 95% air and 5% CO₂. Once cells reached 80% confluence, they were harvested and reseeded.

CAFs conditioned medium (CAFs-CM) and NFs conditioned medium (NFs-CM) were prepared as follows: CAFs and NBFs in logarithmic growth phase were harvested, cell density was adjusted to 1×10⁶/mL, and a total of 20 mL cells were inoculated in a 75-cm² cell culture flask. When cells reached 80–90% confluency, the supernatants were collected and centrifuged at 1200 rpm for 15 min to remove cell debris, then the suspension was stored at −20°C until use.