100 mm plates

Anoikis resistance was determined by incubating cancer cells on low attachment, hydrophobic 100 mm-plates (#82.1472.001, Sarstedt) either in a static or dynamic mode, the latter being obtained by slight shaking of the plate within the incubator. Cells under suspension after 24 h incubation were collected by centrifugation and adherent cells were detached with trypsin (# 25300-054, Life Technologies) for cell counting (Cellometer, Auto T4 Bioscience).

1C11 cells were cultured according to standard protocol.¹⁵, ¹⁶ Briefly, 1C11 cells were kept on 100 mm plates (Sarstedt) in DMEM Glutamax with 10% fetal bovine serum, 1% non‐essential amino acids, 1% penicillin/streptomycin, and 1% L‐glutamine (all Life Technologies) at 37°C and 5% CO₂. For differentiation to serotonergic 1C11 cells (1C11^5‐HT), 40,000 cells were transferred to a 3.5 cm plate (Sarstedt) containing 3 coverslips or slide chambers (Ibidi) for live cell imaging. Then, culture medium was supplemented with 1 mM dibutyryl cyclic adenosine monophosphate (cAMP) and 0.05% cyclohexanecarboxylic acid for 4 days. On day 4, 1C11^5‐HT were shown to display a complete 5‐HT metabolism.¹⁶ Fetal bovine serum in the medium, which contains 5‐HT, does not impact differentiation to a complete 5‐HT phenotype, but reduces 5‐HT synthesis, content and uptake by activation of 5‐HT_2B and 5‐HT_1B/D autoreceptors.¹⁶ As we did not quantify 5‐HT synthesis, content or uptake, this did not impact on our experiments. Additionally, medium was removed, and cells were washed three times in 1x phosphate‐buffered saline (PBS) prior to all experiments to remove medium residues and avoid any bias caused by 5‐HT in the medium. For each treatment, a randomized three‐digit code was applied before image acquisition. Image acquisition and analysis were performed under blinded conditions.

Two breast cancer cell lines (MCF-7 and MDA-MB-231) and fibroblasts as normal cell lines, which were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were used to investigate the effects of the novel synthesized compounds. The cells were cultured in DMEM (Corning, Kennebunk, ME, USA), supplemented with FetalBovine Serum (Eurx, Gdansk, Poland) at concentration 10% and penicillin/streptomycin at 1%. Cells were cultured on 100 mm plates (Sarstedt, Newton, NC, USA) and then kept in an incubator that provided optimal growth conditions (5% CO₂, temperature: 37 °C, humidity at 90–95% level). After reaching 80% confluence, cells were detached from the bottom of the plate using 0.05% trypsin supplemented with 0.2% EDTA (Gibco, San Diego, CA, USA). Then, using a hemocytometer, the number of cells was quantified and seeded at density 5 × 105 cells per well in six-well plates (Sarstedt, Newton, NC, USA) in 2 mL of DMEM. In this research, cells that obtained 80% of confluency were used.

Human colorectal adenocarcinoma cell line DLD-1 (CCL-221) and HT-29 (HTB-38) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The DLD-1 line histologically is the most similar to a primary tumour. Line HT-29, in turn, is used to assess multidrug resistance, absorption of nutrients, and chemically induced differentiation of enterocytes. Cells (passage number range of 6–8) were cultured in RPMI 1640 medium (Sigma, St. Louis, MO) and McCoy’s 5a medium (ATCC), respectively, complemented with 10% of foetal bovine serum (FBS) and 1% of antibiotics: penicillin/streptomycin. The cells were maintained in an incubator, which provides optimal growth conditions for cell culture: 5% CO₂, 37 °C, and humidity in a range of 90–95%. The cells were cultured in 100 mm plates (Sarstedt, Newton, NC). Subsequently, after obtaining a subconfluent cell culture, the cells were detached with 0.05% trypsin and 0.02% EDTA phosphate buffered saline without calcium and magnesium (Corning, Corning, NY). Then, utilising a haemocytometer, the number of cells was quantified and seeded at a density of 5 × 10⁵ cells per well in six-well plates (“Nunc”) in 2 mL of the growth medium (RPMI 1640 and McCoy’s 5a). In the present study, cells that obtained 80% of confluency were used.

Human breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human breast epithelial cells (MCF-10A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 and MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, San Diego, CA, USA), MCF-10A cells were cultured in Mammary Epithelial Cell Growth Medium with supplements: BPE, hEGF, insulin, hydrocortisone, GA-1000 (Lonza, Basel, Switzerland). All media were complemented by 10% of fetal bovine serum (FBS) and 1% of antibiotics: penicillin and streptomycin (both Gibco, San Diego, CA, USA). The cells were maintained in an incubator that provides the optimal growth conditions for the cell culture: 5% CO₂, 37 °C, and humidity in a range of 90–95%. The cells were cultured in 100 mm plates (Sarstedt, Newton, NC, USA). Subsequently after obtaining a subconfluent cell culture, the cells were detached with 0.05% trypsin with 0.02% EDTA (Gibco, San Diego, CA, USA). Then, utilizing a Scepter 3.0 handheld automated cell counter (Milipore, Burlington, MA, USA), the number of cells was quantified and seeded at a density of 5 × 10⁵ cells per well in six-well plates (“Nunc”) in 2 mL of the growth medium (Dulbecco’s Modified Eagle Medium or Mammary Epithelial Cell Growth Medium, respectively). In the present study, cells that obtained 80% of confluence were used.