Heparin

Spheroids were generated on PDMS-based concave microwells fabricated using soft lithography techniques and the meniscus of the PDMS prepolymer, as previously described^{46 (link)}. The concave micromolds consisted of 1 × 1 cm arrays of 500-μm diameter microwells, for a density of 100 wells per cm². All substrates were coated with 3% (w/v) bovine serum albumin to prevent cell attachment. Trypsin-dispersed single PD-MSCs (200,000 cells per mold, passages 8–11) were seeded on top of the concave microwells, which allowed the cells to become trapped within the wells. At 5 min post-seeding, a flow of culture medium was gently applied to remove non-docked cells. The plated cells were cultured with medium containing FGF-4 (75 ng/ml) and heparin (3 μg/ml) (Peprotech). For measuring viability, spheroids were labeled with calcein-AM and ethidium homodimer-1 (EthD-1; Molecular Probes). Confocal microscope images were used for measuring diameters of spheriods and viability, and ImageJ software was used for quantification.

Peripheral lung tissue without the trachea and bronchi was mechanically dissociated with scalpels and digested by a solution of 2 mg/ml collagenase A, 2 mg/ml trypsin, and 50 μg/ml gentamicin (Sigma) in DMEM/F-12 (Gibco) for 45 min at 37°C with shaking. The digestion was stopped by the addition of 10% (v/v) fetal bovine serum (Gibco). The cell suspension was filtrated by a 100 μm cell strainer (Falcon) to eliminate tissue debris and cell clumps and was then collected by centrifugation at 4°C for 10 min. After being washed with DMEM/F-12 and undergoing centrifugation, the cell pellet was treated with red blood cell lysis buffer (Beyotime) 3 times for 2 min each. The cells were centrifuged again and resuspended in 20 U/ml DNase I (Sigma). After a final wash with DMEM/F-12, the cell suspension was filtrated through a 40 μm cell strainer (Falcon) and then seeded into nonadherent 25 cm² flasks pretreated with poly-HEMA (Sigma) in lungosphere medium [1×B-27, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), 4 μg/ml heparin (STEMCELL), 20 ng/ml murine epidermal growth factor (mEGF; Peprotech), 10 ng/ml murine fibroblast growth factor 2 (mFGF2; Peprotech), and 10 μM Y-27632 (STEMCELL) in DMEM/F-12] (1.0×10⁶ cells/ml). The flasks were incubated in a humidified atmosphere at 37°C and 5% CO₂, and half the volume of medium was changed every 2 to 3 days.

PCa metastases were dissected from lung tissues under a fluorescent microscope. The isolated tissues were minced with scissors in complete DMEM supplemented with FBS, penicillin–streptomycin and Gentamicin. The tissue fragments were mechanically dissociated by vigorous pipetting with a 10 ml serological pipette before being washed twice in PBS. Subsequently, the fragments were digested in Accutase supplemented with DNaseI (Thermo Fisher) and ROCK inhibitor (Y27632; STEMCELL Tech) at 37 °C for 30 minutes. The cells were then resuspended in DMEM and filtered through a 100 μm mesh followed by a 70 μm mesh to select for single cells and small cell clusters. The cells were resuspended in Neurobasal medium supplemented with B27, Glutamax (NBM; Life Technologies), hEGF (20 ng/ml), FGF2 (20 ng/ml), Heparin (100 ng/ml; Peprotech), and dihydrotestosterone (DHT, 100 nM; Methods), and cultured in a low-attachment flask.
Primary cells transduced with LV underwent puromycin selection (2 μg/ml) two days after infection and were selected for 10 days. The single cells were sorted by FACSAria III Cell Sorter (BD) into 96-well plates with low-attachment, and clones were generated.

hPD-MSCs were kindly provided by Gi Jin Kim (CHA University, Pangyo, Korea). hPD-MSCs were isolated and characterized as reported previously [77 (link),78 (link)]. Briefly, hPS-MSCs were cultured in α-MEM containing 10% fetal bovine serum (FBS; Corning Inc., Corning, NY, USA), 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA), 1 mg/mL heparin, and 100 μg/mL FGF4 (PeproTech, Rocky Hill, NJ, USA) with 5% CO₂ at 37 °C.

HUVEC were obtained from Lonza (Breda, The Netherlands) and the Endothelial Cell Facility of University Medical Center Groningen, The Netherlands. Cells were maintained in endothelial cell medium, composed of, RPMI 1640 basal medium (Lonza, Verviers, Belgium), supplemented with 20% heat-inactivated foetal bovine serum (Invitrogen/GIBCO, CA, USA), 50 μg/ml endothelial cell growth factors supplement (bovine brain extract; homemade), 1% penicillin-streptomycin (Sigma-Aldrich, MA, USA), 2 mM L-glutamine (Lonza) and 5 U/ml heparin (Leo Pharma, Ballerup, Denmark). Cells were used for experiments at passage 6 and 7.
Confluent monolayers of HUVEC were stimulated for 48 h with or without 5 or 10 ng/ml citric acid activated-TGF-β1 (Peprotech, NJ, USA; #100–21 C) in RPMI 1640 basal medium, supplemented with 20% heat-inactivated foetal bovine serum, 2 mM L-glutamine, 5 U/ml heparin and 1% penicillin-streptomycin. Cells treated with endothelial cell medium were harvested as unstimulated controls, Cells were treated with pharmacological inhibitors for 48 h to inhibit desired signalling pathways.

HSJD-DIPG-007 (DIPG007) cell line was provided by Dr Angelo Montero Carcaboso (Sant Joan de Deu Hospital, Barcelona, Spain), where it was established from human DIPG tumor tissue [38 (link)]. Cells were cultured in neurospheres in DMEM-F12/Neurobasal-A (1:1, Gibco Grand Island, NY, USA) serum-deprived medium supplemented with: HEPES (10 mM, Gibco), sodium pyruvate (1 mM, Gibco), non-essential amino-acids (1%,Gibco), GlutaMax (1%,Gibco), B27-Supplement (Gibco), hEGF (20 ng·mL⁻¹, Preprotech, Cranbury, NJ, USA), human bFGF (20 ng·mL⁻¹, Preprotech), hPDGF-AA (10 ng·mL⁻¹, Preprotech), hPDGF-BB (10 ng·mL⁻¹, Preprotech), heparin (2 µg·mL⁻¹, Preprotech) and antibiotic-antimycotic mix (1%, Gibco). U87 cells were cultured in DMEM supplemented with fetal bovine serum (10%, Gibco) and antibiotic-antimycotic mix (1%, Gibco). Cells were maintained in a 37 °C, 5% CO₂ atmosphere and were periodically tested for the absence of mycoplasma contamination (MycoAlert^®, Lonza, Basel, Switzerland).