3D Cultures and Spheroid’s Viability by MTT Assay

Spheroids were generated as reported (Di Donato et al., 2021). MDA-MB-231 and MDA-MB-453 cells (3 × 10⁴) were mixed in each well with 250 μl of phenol red-free growth factor-reduced Matrigel (10 mg/ml; BD Biosciences) and 50 μl of spheroid plating medium. It was made using phenol red-free DMEM/F12 medium containing 7% CSS, 100 U/ml penicillin, 100 U/ml streptomycin, GlutaMAX 100× (Gibco), 10 mM HEPES, 1 M nicotinamide (Merck), 500 mM N-acetylcysteine (Sigma-Aldrich), and 10 μM Y-27632 (Merck). After 3 days, the spheroid plating medium was replaced with a similar medium in the absence of N-acetylcysteine and Y-27632. On day 4, the spheroids were untreated or treated with the indicated compounds. Unless otherwise stated, the medium was changed every 2 days. In Figures 3D,E, the media were not changed until the 9th day. Different fields were analyzed using Leica DMIRB (Leica) microscope equipped with C-Plan ×40 objective (Leica) and phase-contrast images were acquired using a DFC 450C camera (Leica). The relative spheroid size was calculated using the Application Suite software (Leica) and expressed as a fold increase over the basal spheroid size, which was measured on the 3rd day. After 15 days, spheroid viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich). Briefly, MDA-MB-231 and MDA-MB-453 spheroids were incubated with a 2% (w/v) SDS solution to solubilize the Matrigel. After 2 h at 37°C, the MTT solution (final concentration of 500 μg/ml) was added to the spheroids at 37°C and 5% CO₂. Two hours later, DMSO (100 μl) was added and the mixture was incubated for 1 h at 37°C. The optical density (OD) from duplicate samples was measured at 562 nm using an EnSpire plate reader (PerkinElmer, Waltham, MA, United States).

Neurotrophin β-nerve growth factor (NGF) challenge increases the size of triple-negative breast cancer (TNBC) cell spheroids and the MMP-9 release. (A,B) MDA-MB-231 (A) and MDA-MB-453 (B) cells embedded in Matrigel were used. Four days later, representative images were acquired. Cells were left untreated or treated with 100 ng/ml NGF in the absence or presence of {"type":"entrez-nucleotide","attrs":{"text":"GW441756","term_id":"315858226","term_text":"GW441756"}}GW441756 (GW; 1 μM) for 15 days. Shown are phase-contrast images, representative of three different experiments, captured on the 15th day. Scale bar, 100 μm. (C) Spheroid viability was analyzed by the MTT assay after 15 days of treatment. Optical density (OD) was measured at 562 nm and the obtained results are shown in the graph. Means and SEMs are shown. n represents the number of experiments. (D,E) MDA-MB-231 (D) and MDA-MB-453 (E) cells were used to generate spheroids, as in (A,B). Spheroids were left untreated for 9 days, avoiding refreshing the medium, in the absence or presence of the anti-NGF antibody (anti-NGF Ab). Shown are phase-contrast images, representative of three different experiments, captured at day 9. Scale bar, 100 μm. (F) MDA-MB-31 cells were treated for 30 h with 100 ng/mL NGF in the absence or presence of {"type":"entrez-nucleotide","attrs":{"text":"GW441756","term_id":"315858226","term_text":"GW441756"}}GW441756 (GW; 1 μM). The release of MMP-9 in conditioned media was analyzed by zymography. *p < 0.05 for the indicated experimental points vs. the corresponding untreated controls.