Tryple reagent

Different cancer cell lines were grown and maintained as described in the cell culture section, and used for spheroid generation within 2–5 passages. A total of 96-well plates were prepared with Rat tail collagen I (Gibco) to form the surface for spheroid culture. On the day of cell seeding for spheroid generation, the cells were dissociated with TrypLE reagent (Gibco), neutralized the TrypLE reagent with fresh complete media, spun down, and resuspended in fresh complete media. After making a single-cell suspension, cells were counted using a Countess II automated cell counter and 1,000 cells in 100 μL volume were plated on the surface of the collagen matrix. The cells were observed daily for spheroid formation. After 5–7 days, when the spheroids reached the desired size, they were treated with selinexor and then infected with the vMyx-GFP-TdTomato virus.

The study was conducted using the MDA-MB-231 cell line. This is an epithelial human breast cancer cell line, established from a pleural effusion of a 51-year-old Caucasian woman with metastatic breast adenocarcinoma. The MDA-MB-231 breast cancer cell line was purchased from the American Type Culture Collection (ATCC) and authenticated by DNA profiling using short tandem repeat (STR) (GenePrint® 10 System, Promega) at Genomics Core Facility, Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM. Viral, bacterial and parasitic pathogen analysis by RT-PCR/PCR was performed at Dynamimed Research Company: no genetic material was detected. Cells were maintained at 37 °C in a 5% CO₂ humidified incubator (AutoFlow UN-5510, Nuaire), in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biowest) and antibiotics (penicillin, streptomycin) (Gibco®-Invitrogen). Cells were trypsinized/passaged every 2–3 days using TrypLE reagent (ThermoFisher Scientific). Cells were automatically counted using Countess cell counting chamber slides (Invitrogen) and an Eve Automatic cell counter (NanoEntek), excluding dead cells by trypan blue (Invitrogen) staining.

To evaluate whether the examined treatments increase the expression of DR4 and DR5 proteins, we performed flow cytometric analysis detecting the expression of these cell surface-bound receptors. The cells (10⁵ cells/mL) were seeded in a 12-well plate. After overnight culturing and then 72 h treatment with the combinations and single compounds, the cells were harvested with TrypLE reagent (Thermo Fisher Scientific, Waltham, MA, USA) and then washed two times with PBS (0.05 M phosphate buffer saline, pH = 7.4). The supernatant was decanted, and the cells were then resuspended in 250 μL PBS. The cells were stained with 3 μL phycoerythrin (PE) conjugated isotype control (Sony Biotechnology, Weybridge, UK), anti-DR4 antibody (Sony Biotechnology, Weybridge, UK) or anti-DR5 antibody (Sony Biotechnology, Weybridge, UK). After 20 min of incubation at room temperature in the dark, the samples were centrifuged (5 min, 1200 rpm). The cells were resuspended in 300 μL PBS and were analyzed by flow cytometry (BD FACSCalibur, Becton–Dickinson, San Jose, CA, USA). The data management was performed in CellQuest Pro (Becton–Dickinson, San Jose, CA, USA) and Flowing 2.5.1. software (Turku Centre of Biotechnology, Turku, Finland). The experiment was performed in duplicates, and the results were normalized to the untreated medium control and represented as mean ± SD.