Purecol collagen

WM239A cells were trypsinized and counted with a hemocytometer. WM239A cells were pelleted and re-suspended in monomer solution at a concentration of 300,000 cells per mL for single cell migration studies and 400,000 cells per mL for cluster growth studies. A hydrogel disk loaded with WM239A cells was formed by pipetting 30 μL of 6 mM/6 mM –ene/thiol monomer solution into a 1 mL syringe with the tip cut off. The monomer solution containing single cells or the PDX tumor was exposed to 365 nm light (XX-40 light source, UVP) at 10 mW/cm² for 2 minutes. Hydrogels were then transferred to a 24-well plate containing RPMI medium and maintained at 37 ^oC at 5% CO₂. For cluster studies, cell aggregates were grown from a single cell suspension for 7 days. After 7 days, human dermal fibroblasts were pelleted and resuspended in a 3 mg/mL PureCol® collagen (Advanced BioMatrix) solution at a cell density of 500,000 cells per mL. One half mL of the resulting collagen solution was placed in the bottom of the 24-well plate containing the hydrogel, thus surrounding the hydrogel. For control conditions, collagen without human dermal fibroblasts was pipetted into the bottom of the 24-well plate to surround the PEG hydrogel disk containing WM239A clusters. The cultures were maintained at 37 ^oC at 5% CO₂.

The protocols for the differentiation of pNECs at ALI are adapted versions based on the protocol from Muller et al.^{10 (link)}. Detailed protocols of the described procedures are attached as Supplementary Files (Supplement File 1). Nasal brush biopsies were performed to obtain pNECs. A moistened cytobrush (Cooper Surgical, Trumbull, Connecticut, USA) was inserted into the nostril and rotated gently several times. A new cytobrush was used for each nostril. Next, both brushes were transferred into a 15 ml conical tube, containing 8 ml RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 1% Antibiotic-Antimycotic (100X, Gibco, Thermo Fisher Scientific) and 0.1% Gentamicin (50 mg/ml, Gibco, Thermo Fisher Scientific) and mucus and cells were detached by gently swirling from the brushes. The cell suspension was centrifuged, resuspended in Pneumacult-Ex Plus medium (Ex-P; Stemcell Technologies, Vancouver, CAN) and treated for 20 min with DNase I (1.5 mg/ml; Sigma Aldrich, St. Louis, Missouri, USA). Afterwards, the cell suspension was centrifuged, resuspended in Ex-P and seeded onto coated 12-well plates (coating buffer containing fibronectin (1 mg/ml, Gibco, Thermo Fisher), bovine serum albumin fraction V (BSA; 1 mg/ml; Sigma), PureCol collagen (1:100, AdvancedBioMatrix, San Diego, CA) in PBS without Ca²⁺/Mg²⁺)^{21 (link)}.

Human UVEC spheroids were generated by seeding primary HUVEC at passage 3 to 5 in each well of 384-well hanging-drop plates (3D Biomatrix, Ann Arbor, MI, USA) in complete EBM-2 medium containing 0.25% methyl cellulose (750 cells in 30 μL/well). After 18 hours, HUVEC spheroids were collected, resuspended in serum-free EBM-2 medium, and mixed with collagen solution (PureCol collagen; Advanced BioMatrix, San Diego, CA, USA; 2.2 mg/mL in M199 medium, pH adjusted to 7.4 with NaHCO₃ and NaOH). To determine the effect of the inhibitor on VEGF-A–induced sprouting, aliquots of the HUVEC spheroid/collagen mixture (300 μL/well of 48-well plate) were pretreated with various concentrations of 33DFTG (0.01–10 μM) for 6 hours. After the 6-hour incubation period, 300 μL VEGF-A (100 ng/mL in serum-free EBM-2 medium; PeproTech, Rocky Hill, NJ, USA) was combined with 300 μL collagen mixture in the presence or absence of corresponding doses of 33DFTG. Plates were incubated for 24 hours at 37°C; cumulative sprout length of each sprout was calculated, and the results were expressed as fold change compared to VEGF-A–treated group. Because 33DFTG stock was dissolved in DMSO, all treatments contained 0.05% DMSO. Of note, sprout lengths vary from passage to passage (cells with higher passage number showed fewer sprouting length/numbers in response to the same concentration of VEGF-A).

The cell culture medium LHC-9 and DMEM:F12, and Trypsin-EDTA, were bought from Gibco, Thermo Fisher Scientific, Waltham, MA USA. The cell culture flasks were obtained from Nunc A/S, Roskilde, Denmark, while the cell culture plates were from Corning, NY 14,831 USA. PureCol^™ collagen was from Advanced BioMatrix, Inc, CA, USA.The ELISA cytoset for CXCL8 was purchased from Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, while IL-1α and IL-1β DuoSet were from R&D Systems, Inc, Minneapolis, MN, USA. Cytotoxicity Detection Kit (lactate dehydrogenase; LDH) was obtained from Merck KGaA, Darmstadt, Germany. The RNA isolating kit; NukleoSpin RNA plus, was bought from MACHEREY-NAGEL, Duren, Germany. CH22319 and N-acetyl cystein (NAC) were bought from Merck KGaA, Darmstadt, Germany. High Capacity cDNA Archive Kit, TaqMan Universal PCR Mastermix, TaqMan Gene Expression Assays (CXCL8, Hs00174103_m1, IL-1α; Hs00174092_m1, IL-1β; Hs01555410_m1, TNF-α; Hs01113624_g1, HO-1; Hs01110250_m1, CYP1A1; Hs01054797_g1, CYP1B1; Hs02382916_s1, PAI-2 ; Hs01010736_m1, COX2; Hs00153133_m1 and GAPDH; Hs02758991_g1) were bought from Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA. Other chemicals were purchased from commercial sources at the highest purity available.