Fungizone

Resected tumor lesions were immediately transported from the department of surgery to a GMP facility at Herlev Hospital for further processing. The tumors were manually dissected into 48 fragments and cultured in two 24-well plates (Nunc, Roskilde, Denmark) with complete medium (CM) containing; RPMI (Invitrogen, Waltham, MA), 1% penicillin and streptomycin, 0.5% Fungizone (Bristol-Myers Squibb, New York, NY), 10% Human serum (Sigma-Aldrich, St. Louis, MO) and 6000 U/mL IL-2 (Aldesleukin; Novartis, Basel, Switzerland). The CM was exchanged every 2–3 days until reaching approximately 50 × 10e6 cells, when the cells were either cryopreserved for later use or further expanded in a rapid expansion protocol (REP). In the REP 20 × 10e6 cells, together with irradiated (40 Gy) allogeneic feeder cells at a 1:200 ratio, were cultured in 80/20 medium (80% CM and 20% AIM-V medium) supplemented with 30 ng/mL anti-CD3 antibody (OKT3; Miltenyi Biotech, Bergisch Gladbach, Germany) and IL-2 at a concentration of 6000 U/mL. After 7 days, the cells were moved from a static expansion to the dynamic Wave^® system (GE Healthcare, Chicago, IL). After another 7 days of culture, the cells (REP-TILs) were infused into the patient. The method is further elaborated elsewhere [40 (link)] and identical to our previous clinical trial in ovarian cancer.

Mesenchymal stem cells were isolated as previously described 15. Briefly, BM nucleated cells were plated at 50,000 cells/cm² in proliferation medium consisting in alpha‐modified Eagle's medium (αMEM; Invitrogen, Saint Aubin, France) supplemented with 2 mM l‐glutamine (Invitrogen), 100 U/ml Penicillin–Streptomycin (Invitrogen), 0.25 mg/l amphotericin B (Fungizone^®; Bristol‐Myers Squibb, New York, NY, USA), 10% foetal calf serum (FCS; Logan, UT, USA) and 1 ng/ml fibroblast growth factor‐2 (R&D Systems, Inc., Minneapolis, MN, USA) and incubated in a humidified atmosphere with 5% CO₂ at 37°C. The medium was changed twice a week. When cultures reached 70–90% confluency, cells were detached with trypsin/ethylenediaminetetraacetic acid (EDTA; Invitrogen) and replated at 1000 cells/cm² (passage 1, P1). For the present study, all cells were derived from P2 cultures.

Human AB serum (HS) was purchased from Sigma- Aldrich (Brøndby, Denmark); RPMI-1640 with GlutaMAX and AIM-V medium were obtained from Invitrogen (Nærum, Denmark). RhIL2 (Proleukin) was from Novartis (Basel, Switzerland), and OKT3 (anti-CD3) antibody was from Cilag AG (Schaffausen, Switzerland). Pulmozyme was purchased from Roche (Basel, Switzerland). Allogeneic peripheral blood mononuclear cells (PBMCs) (or feeder cells) were obtained from buffy coats from healthy donors. Solu-Cortef (hydrocortisone sodium succinate) was obtained from the local hospital pharmacy. Fetal bovine serum (FBS) was from Gibco (Nærum, Denmark). Complete medium (CM) consisted of RPMI-1640 with GlutaMAX, 25 mM HEPES pH 7.2, 100 U/ml penicillin, 100 μg/ml streptomycin and Fungizone^® (Bristol-Myers Squibb, New York, NY, USA) 1,25 lg/ml supplemented with 10% HS and 6000 IU/ml of rhIL2. Rapid expansion medium (RM) consisted of AIM-V medium and Fungizone^® 1,25 lg/ml supplemented with 6000 IU/ml rhIL2.

For VCZ (Vfend; Pfizer, USA), a stock solution of 10 mg/mL was prepared in 0.9% sterile saline and adjusted to obtain a final dose of 20 mg/kg in 0.9% sterile saline when injecting 10 μL and assuming an average weight of 300 mg per larva. For amphotericin B (AMB) (Fungizone; Bristol Myers Squibb, Canada), a stock solution of 5 mg/mL was prepared in sterile AD and adjusted to obtain a final dose of 10 mg/kg in 5% glucose when injecting 10 μL and assuming an average weight of 300 mg per larva. All treatments were freshly prepared and administered daily by intrahemocoel injection alternately in the last left and right prolegs to prevent potential injury by repeated injection in the same proleg. Infected control groups were injected with the treatment vehicle, and noninfected control groups received the treatment as controls and to assess toxicity.

After isolation, PBMCs were immediately cultured in a T75 flask for 2 h at 37 °C in a humidified air 5% CO₂ atmosphere at a concentration of 3–4 × 10⁶ cells/mL in X-VIVO 15 media (Lonza, Basel, Switzerland), supplemented with 1% human AB serum (Sigma-Aldrich), 50 μg/mL gentamicin (B/Braun Medical, Melsungen, Germany) and 2.5 μg/mL amphotericin B (fungizone; Bristol-MyersSquibb, Rueil-Malmaison, France). After 2 h of incubation, monocytes were observed as adherent cells and nonadherent cells were removed by three washes with PBS 1X. Subsequently, 1000 U/mL each of human recombinant IL-4 and GM-CSF (both from ProSpec, Rehovot, Israel) were added to monocytes (days 0 and 2) and after 5 days the immature MDDCs were obtained and washed with cold PBS 1X.