Enrichment of Breast Cancer Stem Cells

Mouse breast cancer 4T1 cells (CRL-2539™, ATCC), EMT6 (CRL-2755™, ATCC), and normal mouse mammary HC11 cells (CRL-3062™, ATCC) were routinely cultured in RPMI 1640 (10-040-CV, Corning) with 10% FBS (10082-147, Gibco) and 1% penicillin plus streptomycin supplement (SV30010, Citiva) at 37 °C in a high humidity, 5% CO2 and 95% air incubator. Three-dimensional cancer stem cell (CSC) spheroid culture was performed as previously described [34 (link), 35 (link)]. Briefly, 4T1 cells were cultured using ultralow attachment plates (3471, Corning) in serum free RPMI1640 supplemented with 2% SM1 supplement (5711, StemCell Technologies), 20 ng/mL EGF (78,006, StemCell Technologies), 20 ng/ml FGF (78,003, StemCell Technologies) and 4 µg/ml heparin (7980, StemCell Technologies) for 7 days. Under these conditions, tumor cells with stemness continue to proliferate and grow into spheres, while other cells in the culture are either unable to proliferate or die. At day 7, CSC spheroids, greater than 40 μm, were enriched and collected using a 40 μm cell strainer (50-196-0596, ThermoFisher). CSC spheroids smaller than 40 μm and non-proliferating non-CSCs pass through the strainer and are discarded.

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Chen Y.L., Lowery A.T., Lin S., Walker A.M, & Chen K.H. (2022). Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization. Cancer Cell International, 22, 351.

Publication 2022

CRL-2539 FBS ultralow attachment plates EGF FGF heparin cell strainer

Breast cancer Cancer stem cell Cell Cells culture Hc11 Heparin Humidity Mammary Mouse Penicillin Serum Stemcell Streptomycin Supplement Tumor

Corresponding Organization :

Other organizations : National Kaohsiung University of Science and Technology, Delta State University, University of California, Riverside

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Variable analysis

independent variables

Cell line (4T1, EMT6, HC11)

dependent variables

Cancer stem cell (CSC) spheroid formation and growth

control variables

Culture conditions (RPMI 1640 with 10% FBS and 1% penicillin plus streptomycin, 37 °C, 5% CO2, high humidity)
Ultralow attachment plates for 3D CSC spheroid culture
Serum-free RPMI 1640 supplemented with 2% SM1, 20 ng/mL EGF, 20 ng/mL FGF, and 4 µg/mL heparin for CSC spheroid culture
Cell strainer (40 μm) to enrich for CSC spheroids greater than 40 μm

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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