Mammocult

The reagents were procured from following sources. DMEM/F12 and hydrocortisone were obtained from Corning/Cellgro (Manassas, VA). Mammocult was from StemCell Technologies (Cambridge, MA). Cholera toxin and collagenase were obtained from EMD‐Millipore (Billerica, MA) and Gibco/Thermo Fisher, respectively. Insulin, hyaluronidase, IGF1, TNFα, and all other reagents were purchased from Sigma–Aldrich unless otherwise indicated.

Cells were transfected with miR‐206 mimics or negative control, incubated (24 hr), and diluted in MammoCult human medium, supplemented with MammoCult proliferation supplements (Stemcell Technologies, BC, Canada) in a 1:10 ratio, 4 μg/ml (0.0004%) heparin solution, 0.48 μg/ml hydrocortisone, and 1% PEST. Cells were seeded in low‐adherent six‐well plates (10,000 cells/well) with 2 ml of final volume. Cells were incubated (5% CO₂; 37°C) for 7 days to allow primary mammospheres to form. Plates were scanned in 2400 dpi resolution using GelCount, version 1.1.2.0 (Oxford Optronix Ltd, Milton, UK). For secondary cultures, cells from the primary culture were rinsed twice with PBS, brought to single‐cell suspension using Trypsin–EDTA treatment, and mammosphere assays conducted following the miR‐206 mimics or control transfection as described above, but with 5000 cells/well. Plates were scanned and images of the mammospheres were analyzed as above. For primary mammosphere assays, three experiment were performed using HC11 cells of three different passages, each in technical triplicates. For secondary mammosphere assays, three experiment were performed using HC11 cells of three different passages, one in technical triplicates and two without technical replicates.

The mammosphere assay was performed using Mammocult (Stem Cell Technologies, Vancouver, Canada) as previously described^{16 (link)}. Briefly, 20,000 cells were cultured in ultra-low adherence 6 well plates in triplicate. The culture was allowed to grow for 7 days before the number of spheres greater than 60 µM in diameter were assessed. Thioridazine treatment was applied once immediately upon plating. Quinpirole and SKF83959 was applied daily.

Separation-sorted human breast cancer cell concentrations were determined using the cell counter and then seeded at densities of 5 × 10⁵ cells/mL in low adherence 6-well plates. The cultures were maintained in Human EpiCult-C (StemCell Technologies, Catalog #05630) supplemented with 5% FBS (StemCell Technologies, Catalog #07904) for 24 hours, and then the medium was replaced with serum-free conditions and maintained for an additional 10 days. At the end of the assays, the colonies were enumerated under a microscope. After 10 days, the medium was removed and the plates gently rinsed with PBS. The culture cells were then counted. The procedure was repeated three times. Further, the sorted cells were seeded and cultured in the presence MammoCult™ (StemCell Technologies, Catalog #05620) supplemented with 0.48 μg/mL freshly dissolved hydrocortisone (StemCell Technologies, Catalog #07904) and 4 μg/mL heparin (StemCell Technologies, Catalog #07980) and IL-6 (Sigma, Catalog # SRP3096) to induce greater numbers of mammospheres and tumorspheres, and the cultures were maintained for an additional 7 to 10 days. At the end of the culture period, the colonies were enumerated under a microscope.
At the end of the assay, the cells were assessed by flow cytometry and image processing of each well with Cytation™ 3.

3D CTC tumorspheres were dissociated as single CTC units or pairlets and then scored using hemocytometer and confirmed for cell viability using 1:1 Trypan Blue (Gibco Life Technologies, Inc.). Twenty-four well flat-bottom plates were coated with 1% soft agar and approximately 10-35 trypsinized CTC units/subset were suspended in 100 μl of Mammocult^™ (StemCell Technologies, Inc.) media were added in each well in multiples. The tissue culture plate was then incubated at 37 °C to analyze the spatial-temporal kinetics of 3D CTC tumorsphere formation with a 10 week period. CTC growth rate was observed under 10X magnification and images were captured and analyzed every week under 40X magnification using phase-contrast microscopy (Zeiss, Inc.).