Fresh anonymous human breast cancer tissues were assessed by CANscript. Samples taken from patients refractory to taxane-containing regimens and at varying disease stages were obtained from Mitra Biotech collected under institutional review board (IRB) approval from HCG Bangalore Institute of Oncology with written consent from each patient. Fresh tumor tissues were collected from breast cancer patients immediately after surgical resection at HCG Cancer Hospital, Bangalore, India. The tumor samples were transported to the laboratory at 4°C, in appropriate transport buffer within 60 min after resection, for ex vivo studies and molecular and pathological evaluation as described previously (53). Tissues were cut into thin sections and cultured in 96-well plates using optimized conditions. Tumors were treated with a taxane or doxorubicin at Cmax for 72 hours. DMSO was used as a vehicle control. After treatment, tumor cell viability was measured and immunohistochemistry was performed. Glucose uptake was evaluated 72 hours after culture.

Changes in Glut1 and CD44v6 and cleaved caspase-3 before and after drug treatment were evaluated by immunohistochemistry using specific antibodies. Initial antigen retrieval of formalin fixed paraffin embedded (FFPE) sections was done in Antigen Unmasking Solution (citrate based, Vector Laboratories) in a microwave for 30 min. Endogenous peroxidase was quenched with 3% H2O2 for 15 min. Protein blocking was carried out at room temperature for 1 hour with 10% goat serum. FFPE sections were incubated with primary antibodies at room temperature for 1 hour. Rabbit polyclonal Glut1 antibody (Abcam) was used at a 1:200 dilution, and mouse monoclonal anti-CD44v6 antibody (clone VFF-18, Abcam) was used at a dilution of 1:500. Induction of apoptosis was detected by staining for cleaved caspase-3 using a rabbit polyclonal antibody directed against cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology) at a 1:600 dilution for 1 hour at room temperature. All sections were incubated with horseradish peroxidase (HRP)–conjugated secondary antibody (SignalStain Boost IHC Detection Reagent, Cell Signaling Technology) for 1 hour at room temperature. Chromogenic signal was developed with 3,3′-diaminobenzidine (DAB Peroxidase Substrate Kit, Vector Laboratories). Tissues were counterstained with hematoxylin (Papanicolaou’s solution 1a). Scoring and calculation of drug-induced inhibition of individual tumor explants were performed as described previously (72).

Tumor cell viability was assessed by Cell Counting Kit-8 (CCK-8) (Dojindo). CCK-8 solution was added, and cells were incubated at 37°C for 3 hours in a 5% CO2 incubator under humidified conditions. The absorbance was measured at 450 nm using a multimode microplate reader (Enspire, PerkinElmer). Baseline samples (T0) were used to normalize intersample variation. The results were expressed as a percentage of viability or inhibition relative to untreated controls.

Glucose uptake assay was carried out 72 hours after culture. Culture supernatant (2 μl) was added to 200 μl of glucose reagent (Liquixx Glucose Reagent, Erbaa). All readings along with glucose standard (100 mg/dl) were performed in triplicate. The plate was incubated at room temperature for 5 min on a plate shaker at medium speed, and the absorbance was measured at 505 nm using a multimode plate reader (PerkinElmer).

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