P3932

A single cell suspension was prepared using enzymatic (1× Trypsin-EDTA, T3924, Sigma Aldrich), and manual disaggregation (25 gauge needle) [27 (link)]. Cells were plated at a density of 5000 cells/well in tumorsphere media (DMEM-F12/B27/EGF (20ng/ml)/Pen-Strep) in non-adherent conditions, in 6-well plates coated with (2-hydroxyethylmethacrylate) (poly-HEMA, P3932, Sigma) in the presence of mDIVI1 or vehicle. After 5 days of culture in a humidified incubator at 37° C, tumorspheres bigger than 50 μm were counted using an eye piece graticule.

The human colorectal carcinoma (CRC) cell line SW620 was obtained from the cell bank of the IRCCS Ospedale Policlinico San Martino, Genoa (kind gift of Dr. Alessandro Poggi MD, Head of the Molecular Oncology and Angiogenesis Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy). SW620 cell line was cultured in RPMI 1640 (Corning, New York, NY, USA) medium supplemented with 10% fetal bovine serum (FBS, Gibco™, Thermo Fisher Scientific Italy, Monza, Italy), penicillin/streptomycin and l-glutamine (Corning, New York, NY, USA) in a humidified incubator at 37 °C with 5% CO₂.
For the generation of tumor spheroids, SW620 adherent cells were detached with Trypsin/EDTA (Corning, New York, NY, USA) and counted using a standard hemocytometer.
Then, SW620 cell suspension was seeded at the concentration of 1.8 × 10⁴ cells per well in flat-bottom 96-well plate (Corning^®Costar^®, New York, NY, USA) pre-treated with Poly-HEMA 3% in EtOH 96% (P3932, Sigma Aldrich, Merck, Milano, Italy) to prevent cell adhesion, in serum-free DMEM-F12 medium (Euroclone, Milan, Italy), supplemented with epithelial growth factor (EGF) (Peprotech Europe, London, UK) at 10 ng/mL final concentration (≥1 × 10⁶ U/mg). Experiments were performed at proper spheroids compaction, after approximately 1 week of culture.

Tissue culture plates were coated with 1% poly (2-hydroxyethyl methacrylate) (polyHEMA; Sigma P3932) solution in 95% ethanol, and allowed to completely dry at room temperature. Cells (1 × 10⁵ cells/mL) were cultured for 24 hours on 1% polyHEMA plates to prevent adhesion, collected, washed with PBS and 2 mM EDTA and replated into 6-well plates (100 - 1000 cells/well). For some experiments ERK signaling was inhibited by pre-treating NSCLC cell lines with 60 μM PD98059 for 45 minutes prior to plating. In some cases cells were maintained in PD98059 for the duration of the clonogenic assay. Colonies were stained with crystal violet (0.5% crystal violet, 6% glutaraldehyde), and quantified using ImageJ [20 (link), 51 (link)].

Ishikawa cells in a 24-well plate were stimulated as indicated. 80%-90% confluence of BeWo cells were digested to the cell suspension and then placed in a 60 mm² dish coated with the anti-adhesive polymer, poly-2-hydroxyethyl methacrylate (P3932, Sigma), to inform 150–200 μm BeWo spheroids after 36 h culture. The spheroids were transferred to confluent Ishikawa cell monolayers. After incubation at 37 °C for 40 min, the unattached spheroids were removed by washing with PBS. The attachment rate was valued as the number of attached spheroids divided by the total number of spheroids added to the Ishikawa cells.