Agnosphere Isolation and Propagation Protocol

Agnospheres were isolated and cultured as described previously (Verginelli et al, 2021 (link)). Briefly, tumor samples were digested with collagenase I (Gibco), and after filtration, single‐cell suspensions were resuspended in culture medium [Dulbecco's Modified Eagle's Medium: F12 (DMEM; Sigma) supplemented with N2 supplement (Life Technologies‐GIBCO), BSA 0.5% (Sigma), Heparin 4 μg/ml (Sigma), 2 mM Glutamine (Sigma), and Penicillin–Streptomycin (EuroClone)]. Ultralow‐attachment flasks (Corning, cat. CLS3814) were used in case of AS901. The same medium composition was used for further propagation of the agnospheres; when subculturing, agnospheres dissociation was mechanically achieved by pipetting and by trypsin treatment. MCF7 and HEK‐293T cells were provided by American Type Culture Collection (ATCC) and cultured in DMEM or Iscove medium (Sigma‐Aldrich), respectively, supplemented with 10% fetal bovine serum (FBS; Euroclone), 2 mM Glutamine (Sigma‐Aldrich), and Penicillin–Streptomycin (EuroClone). The cells were periodically checked for mycoplasma contamination.