Isolation and Expansion of Patient-Derived Microtumors

Fresh dissected breast tumor tissues were transported within DMEM/F12 culture media (Gibco) and subsequently processed as previously described [23 (link)]. The isolation of patient-derived microtumors was adapted from Kondo et al. [24 (link)]. Briefly, tumors were washed in HBSS (Gibco), fragmented with forceps, and digested with Liberase DH (Roche) for 2 h at 37 °C. The digested tissue was filtered through a 500 µm stainless steel mesh (VWR) followed by a 40 µm cell strainer (Corning). Tumor fragments retained by the cell strainer were washed in HBSS and cultured in suspension in StemPro® hESC SFM (Gibco) supplemented with 8 ng/ml FGF-basic (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1.8% BSA (Gibco) and 100 µg/ml Primocin (Invivogen) in a cell-repellent culture dish (60 × 15 mm) (Corning). The single-cell filtrate was used for the expansion of tumor-infiltrating lymphocytes in Advanced RPMI 1640 (GIBCO) supplemented with 2 mM glutamine (Gibco), 1% MEM vitamins (Gibco), 5% human serum (SigmaAldrich) and 100 µg/ml Primocin (Invivogen). IL-2 (100 U/ml), IL-7 (10 U/ml) and IL-15 (23.8 U/ml) (Peprotech) were freshly added to the culture media. CD3/CD28 Dynabeads (Milteny Biotech) were added for expansion.