Recombinant epidermal growth factor

Three immortalized cell lines originated from Barrett’s esophagus, BAR10T (kindly provided by Dr. Rhonda Souza), CPA and CPB (ATCC, Manassas, VA, USA), were cultured with DMEM/F12 (GIBCO, New York, NY, USA), supplemented with 5% fetal bovine serum (FBS, GIBCO), 1% penicillin/streptomycin (GIBCO), 0.4 μg/ml hydrocortisone (Sigma-Aldrich, Saint Louis, MO, USA), 20 mg/L adenine hydrochloride hydrate (Sigma-Aldrich), 140 μg/ml bovine pituitary extract (Thermo Fisher Scientific, Waltham, MA, USA), Insulin-transferrin-sodium selenite media supplement (Sigma-Aldrich) and 20 ng/ml recombinant epidermal growth factor (Sigma-Aldrich). EAC cell lines, FLO1 (ATCC), OE19 (Sigma-Aldrich), SK-GT4 (kindly provided by Dr. Xiaochun Xu at MD Anderson), ESO26 (Sigma-Aldrich) and OE33 (kindly provided by Dr. David Beer) were cultured in DMEM or RPMI 1640 medium (GIBCO), supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were grown at 37 °C in 5% CO₂. Cell lines were authenticated by Genetica DNA Laboratories using short tandem repeat profiling (Genetica DNA Laboratories, Burlington, NC, USA). Cells used were from the stocks immediately after authentication and cultured less than 6 months. Mycoplasma was tested periodically using the qRT-PCR method (Southernbiotech, Birmingham, AL, USA).

Tumorosphere formation was assessed as previously described. Briefly, cells were trypsinized, placed in mammosphere media [DMEM/F12 (Life Technologies, Burlington, ON, Canada), 2% B27 supplement (Life Technologies, Burlington, ON, Canada), 20 ng/mL recombinant epidermal growth factor (Sigma), 0.5 µg/mL hydrocortisone (Stem Cell Technologies, Vancouver, BC, Canada), 5 µg/mL insulin (Sigma)] and resuspended as single cells using a 25-gauge needle [44 (link)]. Cells were plated in ultra-low-attachment six-well plates at a density of 10,000 cells/well and treated with deB-C6.5-diab, T-DM1, or T-MMAE at the time of plating. After 10 days, all tumorospheres greater than 50 µm in diameter were counted using an inverted microscope fitted with a graticule.
To assess the tumorosphere-forming efficiency of cells pretreated with deB-C6.5-diab, T-DM1, or T-MMAE, tumor cells were seeded at 150,000 cells per well in a six-well plate and allowed to adhere for 3 h at 37 °C. DeB-C6.5-diab, T-DM1, and T-MMAE were added to the cells at the indicated concentrations and incubated for five days. Cells were trypsinized and plated in mammosphere media as described above. The tumorosphere-forming efficiency was reported as a percentage relative to the corresponding untreated control.

SILAC labelled HeLa cells were grown in 15 cm cell culture dishes containing 25 mL of medium, to 50–80% confluency. Recombinant Epidermal Growth Factor (Sigma, #E9644) was reconstituted in PBS (Gibco, #14190–094) and added to dishes at a final concentration of 20 ng/mL. Dishes were returned to 37°C for 20 min.