Cell cultures

Freshly resected tumor tissues were minced and incubated with Dispase II (5 mg/ml) for 18 hours at 37 °C on a rotary shaker according to manufacturer’s instruction (Cat no: 04942078001; Roche). Briefly, the supernatants were discarded after centrifugation and pelleted cells were collected and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic. Once cells reached 90% confluency, cells were dissociated in 0.25% Trypsin/EDTA (cat no: 25–200–114; Gibco, Canada) and re-cultured at required numbers of cells (50,000, 75000, 100000 and 150,000 cells). After 48-hours, culture medium was replaced with serum free medium. After 48 hours, collected serum free media were subjected to ELISA assay. MDA-MB-231, MCF-7 and MCF10A cells were obtained from American Type Cell Cultures (ATCC) and cultured as recommended. As above, the required numbers of cells were plated and after 48 hours the medium was replaced with serum free medium and incubated for another 48 hours, and finally cell supernatants were collected and subjected to ELISA assay.