Cancer cell isolation kit

All primary tumors were diagnosed at the Cross Cancer Institute (Edmonton, AB, Canada). The use of these tissues has been approved by our Institutional Ethics Committee. All archival tissues were formalin-fixed and paraffin-embedded. All fresh tumor samples were processed immediately after surgery to isolate primary BC cells using a protocol described previously [9 (link)]. Briefly, tumors were cut into small pieces of 2–4 mm in the greatest dimension, which were then incubated with RPMI supplemented with Enzyme H, Enzyme R and Enzyme A (Macs Miltenyi Biotec, Auburn, CA, USA) in MACS C-tubes. Tissue shearing was performed by providing three pulses every 30 min at 37 °C. Cells were collected after centrifugation at 300× g for 7 min. The subsequent steps of BC isolation were based on the manufacturer’s instructions (Cancer Cell Isolation kit, Panomics, Redwood, CA, USA). After culturing for 1–2 days, cells were infected with lentivirus containing either mCMV or the SRR2 reporter. Infection was repeated twice (24 h apart) and cells were sorted into RU or RR cells approximately 48 h later, based on the green fluorescence protein (GFP) expression [9 (link)].

Tissue was obtained from breast ductal carcinoma in situ (DCIS) of a 47 year-old female of Chinese origin. The Cancer Cell Isolation Kit (Panomics) was used to obtain primary cells from the tissue. Fibroblasts were removed using the MAC Separator (Miltenyi Biotech). Primary cells obtained were labeled as ETCC001 and were grown in M171 media (Life Technologies) with mammary epithelial growth supplement (MEGS). Cell lines arising from ETCC001 after hTERT transformation were grown in RPMI supplemented with 10% fetal bovine serum (FBS), L-glutamine and Penicillin/Streptomycin (Life Technologies). Cell cultures were maintained in a humidified incubator at 37°C with 5% atmospheric CO₂.

NSCLC cells were isolated from surgical tumor tissues using Cancer Cell Isolation Kit (Panomics). According to the manufacture’s instructions, cells were cultured for 4 days at 37 °C under 5 % CO₂ in complete RPMI 1640 medium (GIBCO, containing 10 % heat-inactivated fetal bovine serum supplemented with 2 mM glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate), and used for experimental research. MiR-125b mimics and miR-125b inhibitor were from Ribobio (Guangzhou, China). Human TP53INP1 expression plasmid was purchased from Origen. Human TP53INP1 siRNA was from Santa Cruz. The nucleofector transfection kit was purchased from Amaxa.