Methocel

Fertilized chicken eggs were incubated in a hatching incubator (relative humidity 65%, 37 °C), as previously described63 (link). On embryo development day (EDD) 8, A2780 or A2780cisR cells were prepared in 25 μL hanging spheroids containing 10⁶ cells in 20% Methocel (Sigma-Aldrich) and 80% serum free RPMI-1640 medium. The spheroids were then incubated for 3 h and transplanted on the surface of the CAM. Vascularized three-dimensional tumors were visible 3 days after tumor cell implantation and randomized on EDD 11, when treatment was initiated. Tumors were treated on EDD 11 and 12 (referred to as treatment day 1 and 2) by 20 μl intravenous injection into a main blood vessel of the CAM. Erlotinib and RAPTA-C were freshly prepared and pre-mixed in 0.9% NaCl. The injected doses (10–20 μg/kg for erlotinib and 21.6–216 μg/kg for RAPTA-C) were adjusted to the normalized embryo weight at EDD 11/12 (4.27 g). Control tumors were treated with vehicle (0.14% DMSO in 0.9% NaCl). Tumors were monitored daily for 8 days and tumor size was calculated with the formula: volume = [large diameter] x [perpendicular diameter] 2 × 0.52. At EDD 18: (i) the embryos were sacrificed and weighed and (ii) the tumors were resected, weighed and fixed in zinc-fixative for additional analysis.

Spheroids formation was performed following Wang et al. [29 (link)]. In brief, cells were seeded in 35 mm diameter dishes high Bioinert (Ibidi, Germany) at 0.1 × 10⁶ cells/dish density in complete medium supplemented with 25% of Methocel (Sigma-Aldrich) stock solution (0.5 g in 500 ml of complete medium). Spheroids size (µm) was determined by measuring feret’s diameter using standard ImageJ software tools (ImageJ, National Institutes of Health). Intracellular oxygen level was measured using BTP (bis(2-(20-benzothienyl)-pyridinato-N,C30) iridium (acetylacetonate) (Sigma-Aldrich), a fluorescent dye quenched by molecular oxygen [30 (link)]. Spheroids were stained with 5 µM BTP in complete medium for 4 h. After this time, spheroids were carefully washed with phenol-red free medium. Images of at least ten spheroids for condition were acquired using a Nikon C1si confocal microscope (Nikon). Fluorescence intensity was analyzed using standard ImageJ software tools.

Artificial seawater (pH 8.2, osmolality 1300 mmol/L, specially weight 1.05, NaCl 26.518 g/L, MgSO4 3.305 g/L, MgCl₂ 2.447 g/L, CaCl₂ 1.141 g/L, KCl 0.725 g/L, NaHCO₃ 0.202 g/L, and NaBr 0.083 g/L) was prepared according to the main components of seawater in the East China Sea provided by the China Oceanic Administration [23 (link)]. Dimethyl sulfoxide (DMSO) and dihydroethidium (DHE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Biosharp Life Sciences (Biosharp, China). DMF (Cat# HY-17363), ML385 (Cat# HY-100523) and Ferrostatin-1 (Fer-1, Cat# HY-100579) were obtained from Med Chem Express (MCE, USA). Primary antibodies for anti-GAPDH (Cat# ab181602) and anti-Nrf2 (Cat# ab62352) were obtained from Abcam (Abcam, USA).
DMF was suspended in 0.9% NaCl and 0.5% Methocel (Sigma) to a final concentration was 4 mg/ml. Fer-1 was dissolved in dimethyl sulphoxide and then diluted in saline to a final concentration of 0.5 mg/ml.

HUVEC and BP were mixed 1:1 in Endopan 3 containing 20% Methocel (Sigma). Twenty-five microliters cell suspension drops were pipetted on non-adherent plastic plates37 (link). Subsequently, plates were turned upside-down to form the so-called hanging drops in which spheroids are contained. Plates were incubated for 24 h at 37 °C and spheroids were collected by washing the plates with 10% FCS/PBS. Spheroids were centrifuged at 200g for 5 min and resuspended in 20% FCS and 80% Methocel. The collagen mix was prepared on ice using Collagen type I (isolated from rat tail tendons), Medium 199 and NaOH (1 M) in an 8:1:1 ratio. Additionally, 1 × HEPES buffer was added to the mix to adjust the pH. The collagen solution was mixed with the spheroid solution in a 1:1 ratio and transferred to a 24-well plate. For polymerization, gels were incubated for 30 min at 37 °C and then stimulated with 50 ng ml⁻¹ hVEGF (R&D) in basal EC medium. The assay was stopped after 24 h of incubation at 37 °C by adding 1 ml 10% PFA per well. Pictures of ten spheroids per gel were taken on an Olympus TH4–200 microscope and average sprout length was measured with Fiji.

For local treatment of ocular lesions, disulfiram (Tetraethylthiuram disulfide, Sigma-Aldrich, Munich, Germany) was dissolved to a concentration of 300 μM in 2% (w/v) methocel ([Hydroxypropyl-methylcellulose], Sigma-Aldrich, Munich, Germany) and administered to the diseased C57BL/6J mice (n=8) daily from Day 8 to Day 19 as eye drops. The control group (n=8) received methocel only. In addition, C57BL/6J mice (n=8/group) were treated systemically with p.o. 200 mg/kg disulfiram once daily or olive oil (ALDI, Essen, Germany) over a period of 12 days.