Bt474

The breast cancer (BCa) cell lines BT474, SKBR-3, and MDA-MD-361 were obtained from Banca Biologica and Cell Factory in IRCCS Ospedale Policlinico San Martino (Genova, Italy) affiliated to the European Culture Collection’s Organization. Culture media for routine cell expansion was Dulbecco’s modified Eagle medium (DMEM) high glucose supplemented with 1% glutamine, penicillin, and streptomycin, and 10% heat-inactivated fetal bovine serum for BT474 and SKBR-3 or 20% for MDA-MD-361 (Euroclone S.p.A, Pero, Italy). EVs production medium was prepared as reported by Thery et al. [10 (link)]. Cultures were performed at 37 °C in humidified 5% CO₂ atmosphere. Normal human immunoglobulins G IgGs (CLS Behring, King of Prussia, PA, USA) and Tz (Genentech-Roche, South San Francisco, CA, USA) were dissolved with saline solution with 0.9% NaCl in a stock concentration of 21 mg/mL, donated by the Unità Farmaci Antiblastici of the IRCCS Ospedale Policlinico San Martino. Tz was used at a concentration of 10 µg/mL. Control cells were cultured with normal human IgGs at the same concentrations used for Tz.

For in vitro studies, we used three categories of breast cell lines. For Luminal A (LumA) phenotype we used T47D (from ICLC cell biobank, Genova, It), for luminal B (LumB) we used the BT474 (kindly provided by Dr. Daniela Gaglio, IBFM-CNR), for HER2+ phenotype we used SKBR3 (ATCC, Manassas, Virginia, USA), and lastly, for TNBC phenotype we used MDA-MB-231 (ICLC). These cell lines have been selected as representative of the BC subtypes, as reported in [31 (link)]. MCF-10 (ATCC) cell line was used as control, being representative of a non-tumorigenic epithelial cell line. T47D cells were cultured in High Glucose DMEM (Gibco, Life Technologies, Carlsbad, California, Stati Uniti); SKBR3 and BT-474 in RPMI (Euroclone, Italy) and MDA-MB-231 and MCF-10 in Advanced DMEM (Euroclone, Italy). All of the media were supplemented with 10% heat-inactivated foetal bovine serum, penicillin and streptomycin (50 IU/mL), and 2 mM glutamine (all Euroclone, Italy). The cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

The BC cell lines BT474, MDA-MD-361 and SKBR-3 were obtained from Banca Biologica and Cell Factory in IRCCS Ospedale Policlinico San Martino affiliated to the European Culture Collection’s Organization. Culture media for routine cell expansion was DMEM high glucose supplemented with 1% glutamine, penicillin, streptomycin, and 10% heat-inactivated fetal bovine serum for BT474 and SKBR-3 or 20% for MDA-MD-361 (Euroclone S.p.A, Pero, Italy). To obtain Tz-R cells, each cell line was cultured and expanded in medium containing Tz at the initial concentration of 10 µg/mL that was progressively increased over about 6 months, to the final concentration of 160, 250, and 200 µg/mL for BT474, SKBR-3, and MDA-MD-361, respectively. Cells were routinely cultured in the Tz-containing medium for over a year before analysis. Wt and Tz-Res cells were cultured in parallel to minimize differences in passage number. Tz was kindly provided by the pharmacy (UFA-Unità Farmaci Antiblastici) of the IRCCS Ospedale Policlinico San Martino.