L glutammine

Human α-synuclein-stable transfected SK-N-SH (SK-N-SH Syn⁺) cells were routinely maintained in Dulbecco’s modified Eagle Medium Low Glucose (Sigma-Aldrich) in the presence of 1% L-Glutammine, 1% Non-Essential Amino Acids (Sigma-Aldrich), 1% penicillin/streptomycin, and 10% Fetal Bovine Serum (Euroclone). SK-N-SH Syn⁺ were plated in the presence of 50 µg/mL zeocin selection (Thermo Fisher Scientific). To induce differentiation, cells were seeded on 0.1 mg/mL Poli-D-Lysine-coated glass coverslips, treated with 3 µg/mL Retinoic Acid (Sigma-Aldrich) alone for 3 days and together with 80 nM TPA (Sigma-Aldrich) for an additional 3 days [69 (link)]. After 6 days of differentiation, cells were treated with DMSO (vehicle, Euroclone) and 6 µM or 12 µM Tubacin (Sigma-Aldrich) for 2 h at 37 °C, gently washed twice with Phosphate-Buffered Saline (PBS, Biosigma, Cona, Italy), and processed for analyses.

For intracellular cytokine detection, 2 × 10⁶ PBMCs from PCa-ADK patients or HC were cultured, overnight, in RPMI 1640 (EuroClone) supplemented with 10% FBS (Life Technologies,) 1% (v/v) l-Glutammine (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin (Euroclone) and IL-2 (100 U/ml; R&D Systems) at 37°C and 5% CO₂. For intracellular staining, the third day of polarization, cells were stimulated for 6 h with PMA (10 ng/ml) and ionomycin (500 ng/ml) (both from Sigma), in the presence of GolgiStop plus GolgiPlug (both from BD Biosciences). Cells were collected and stained for NK cell surface markers, as previously described, washed with PBS and treated with Cytofix and Cytoperm fixation and permeabilization kit (BD) for 10 min at 4°C. Cells were then washed in PBS and stained with PE-conjugated anti human CXCL8, CXCL12 (R&D System), IFNγ, TNFα, GranzymeB (Myltenyi Biotec) for 30 min. For indirect staining, cells were incubated for 1 h at 4°C with primary unlabelled antibodies anti-human Angiopoietin 1, anti-human Angiogenin, (all purchased from Abcam), washed and then stained with secondary PE-conjugated antibody anti-mouse IgG, for 30 min, at 4°C. Cytokines production was detected by flow cytometry, using a BD FACS CantoII analyzer. Isotype control and the secondary antibody alone were used as staining controls. For details on antibodies used, see Supplementary Table 2.

Neuroblastoma cell lines enriched to the neural portion (SH-SY5Y-N) were grown in Dulbecco’s Modified Eagle’s Medium High glucose, supplemented with fetal bovine serum (10%, Sigma Aldrich-Saint Louis, United States), penicillin/streptomycin (100 U/ml, Sigma-Aldrich-Saint Louis, United States), L-glutammine (2 mM, Sigma-Aldrich-Saint Louis, United States), and gentamycin (40 μg/ml, Sigma Aldrich-Saint Louis, MO, United States) at 37°C with 5% CO₂ in a humidified incubator. Cells grow adherent in 25 cm² flasks. Medium was changed twice a week. When 70% confluent, cells were enzymatically detached with trypsin-EDTA (Sigma-Aldrich-Saint Louis, United States).

Established human NSCLC cell NCI-H460 was obtained from the American Type Culture Collection (ATCC, Manassasn, VA, USA) and was cultured according to the manufacturer’s instructions. BBIRE T-238 and BBIRE T-248 primary cultures were isolates from Malignant Pleural Effusion (MPEs) of patients with LUAD, as previously described [11 (link),13 (link)]. The study was approved by the Ethics Committee (protocol number 1032/17). Cells in adherent condition were cultured in RPMI-1640 supplemented with 10% FBS, 1% L–Glutammine and 1% Penicillin/Streptomycin (Sigma, St. Louis, MO, USA), while 3D cultures in suspension were obtained as previously described [11 (link),13 (link),18 (link)]. Briefly, 20,000 cells/mL were resuspended in an appropriate amount of Spheroid Medium and seeded onto Ultralow Attachment plates (Corning Costar, MA, USA) to form 3D structures. After 4 days, the number of 3D spheres in single wells of a 96 well low attachment culture plate were counted, and their volume was estimated using Image J Software v1.50i. Cells were routinely checked for mycoplasma contamination by PCR.

For DM and DM/n-HA nanostructures synthesis, iron (II) chloride tetrahydrate (FeCl₂·4H₂O, ≥99%), iron (III) chloride hexahydrate (FeCl₃·6H₂O, ≥97%) and Dextran from LeuconostocMesenteroids (Mw 6000 Da) were purchased by Alfa Aesar. Sodium hydroxide anhydrous pellets (NaCl, ≥99%), hydrochloric acid (≥37% wt. in water), phosphoric acid (≥85% wt. in water), ammonium hydroxide ((NH₄)OH, ≥30% wt. in water), and calcium acetate hydrate (Ca(CH₃COO)₂·XH₂O, ≥99%) were purchased from Sigma–Aldrich. Ultrapure water (18.2 MΩ/cm, obtained by a Milli-Q^® Direct Water Purification System, Merck Millipore, Darmstadt, Germany) has been used in all the experiments.
For the cell-based experiments, Eagle’s Minimum Essential Medium (E-MEM), fetal bovine serum (FBS), L-glutammine, penicillin/streptomycin antibiotic mix, Dulbecco’s phosphate buffer saline (D-PBS), trypsin-EGTA, paraformaldehyde (PFA), Triton X-100, as well as the fluorescent dye phalloidin-FITC were all purchased from Sigma–Aldrich (Milan, Italy). The Vectashield Anti-fade Mounting Medium with 4′,6-diamidino-2-phenylindole DAPI (Vector Laboratories, Peterborough, UK) was used for nuclear staining. All reagents, media supplements, and plastic-ware were supplied as cell-culture tested.

Murine endothelial brain cells (bEnd.3) were grown in Dulbecco’s Modified Eagle’s Medium High glucose, supplemented with fetal bovine serum (10%, Sigma-Aldrich-Saint Louis, United States), penicillin/streptomycin (100 U/ml, Sigma-Aldrich-Saint Louis, United States), L-glutammine (2 mM, Sigma-Aldrich-Saint Louis, United States), and gentamycin (40 μg/ml, Sigma-Aldrich-Saint Louis, MO, United States) at 37°C, 5% CO₂ in a humidified incubator. Cells grow adherent in 25 cm² flasks. The medium was changed twice a week. When 70% confluent, cells were enzymatically detached with trypsin-EDTA (Sigma-Aldrich-Saint Louis, United States).