Rpmi 1640

The human breast-cancer cell line T-47D (ATCC, Manassas, Virginia, United States) was routinely maintained in growth medium, composed of RPMI-1640 (Bioconcept AG, Allschwil, Switzerland), 2 mM l-Glutamine (Bioconcept AG), 10 mM HEPES (Bioconcept AG), 1 mM sodium pyruvate (Bioconcept AG), 0.02 mg ml⁻¹ bovine insulin (Sigma-Aldrich), 1x Penicillin/Streptomycin (P/S, Bioconcept AG), and 10% fetal calf serum (FCS, Bioconcept AG) at 37°C in a humidified 5% CO₂ incubator. For imaging, RPMI-1640 was replaced with RPMI-1640 without phenol-red (Bioconcept AG). T-47D constitutively expressing mKate2-labeled nuclei were generated by lenti-viral transduction with NucLightRed (Essen BioScience, Ltd., Newark, United Kingdom), followed by selection and maintenance of transduced cells in the presence of puromycin (2 μg ml⁻¹). Multicellular spheroid generation was initiated 4 days prior to the start of the in vitro PK/PD experiment by seeding 200 cells in 50 μl of growth medium into each well of a non-adherent Akura™ 96 plate (InSphero AG). The seeding density, resulting in spheroids with diameters of 200–250 μm, was based on prior cell-seeding density titrations performed with T-47D cells (data not shown). All cell culturing was performed under standard mammalian cell culturing conditions (37°C, 95% humidity, 5% CO₂).

For transfer of daughter cells, when confocal microscopy analysis of mitotic cells was performed or when cell proliferation was assessed, cells were cultured in T-cell medium (RPMI 1640 (Bioconcept), 2 mM L-Glutamine (Bioconcept), 2% penicillin-streptomycin (Sigma-Aldrich), 10% foetal bovine serum (Omnilab), 25 mM HEPES (Gibco, Life Technologies, Zug, Switzerland), 1× non-essential amino acids (Sigma-Aldrich), 50 µM β-Mercaptoethanol (Gibco), 1 mM sodium pyruvate (Gibco) supplemented with self-made human IL-2, and stimulated on plate-bound human Fc-ICAM-1 (50 µg/ml) (R&D Biosciences, Bio-Techne AG, Zug, Switzerland), α-CD3 (5 µg/ml) (145-2C11, BioLegend) and α-CD28 (5 µg/ml) (37.51, BioLegend) for 30–36 h. mTOR modulation was done by adding 20 nM of rapamycin (Santa Cruz, LabForce AG, Muttenz, Switzerland) or 1–2 µM of Akt-kinase inhibitor (Sigma-Aldrich) 12 h post stimulation. For adoptive transfer, 1 × 10⁴ purified CD45.1 P14 cells (stimulated as indicated) were intravenously injected into naive C57BL/6 CD45.2 recipient mice. For cell proliferation assays, cells were stained with CellTrace Violet^TM, CellTrace Yellow^TM or CellTrace Violet^TM (Life Technologies) following manufacturer’s instructions prior to stimulation.