L lysine

Whole intact tumours were harvested on days 10–13 post-engraftment and immediately immersed in fixative solution (1% PFA, 75 mM l-lysine [Sigma, Cat. L5501], 10 mM sodium m-periodate [Thermo Scientific Pierce, Cat. 20504], diluted in 0.2 M PBS adjusted to pH 7.4) for 16–20 h at 4 °C under gentle agitation. Fixative solution was discarded, and tumours were washed using 1X PBS for 1 h at 4 °C under gentle agitation to remove remaining PFA. Tumours were then resuspended in 30% sucrose (w/v, diluted in pH 7.4 PBS) for 24–36 h at 4 °C without agitation, until tissue was no longer floating. Tumours were cryogenically frozen in OCT compound (Thermo Fisher, Cat. 15212776) using methanol and dry ice bath, and stored at −80 °C until cryosectioning. Frozen tumour blocks were cryosectioned at 10 μm thickness using a Leica CM1900UV and mounted onto glass slides (VWR, Cat. 631-0108). Cryosections were stored at −80 °C until staining. For staining, sections were washed with PBS to remove OCT compound on the slides, and blocked with solutions containing imaging buffer (2% FCS, 0.1% Triton X-100 (Sigma, Cat. X100), 0.01% sodium azide), FcBlock, and species-specific serum depending on the fluorochrome-conjugated antibodies used in each staining panel. Sections were blocked for a minimum of 3 h at room temperature, before incubation with fluorescently conjugated antibodies diluted in blocking solution overnight at 4 °C. A final wash was performed twice using imaging buffer before the sections were mounted using Fluoromount G (Southern Biotech, Cat. 0100-01) and glass coverslips were placed on top of the sections. Images were collected using Zeiss Axioscan 7 Slide Scanner or Zeiss LSM 980 confocal microscope, and analysed using Imaris software (Bitplane, V10.0).

All chemicals and reagents utilized in this study were procured from reputable commercial suppliers. Ethanol and glycerol were sourced from ZORKA Pharma (Šabac, Serbia). The free radical 2,2-iphenyl-1-picrylhydrazyl (DPPH) was supplied by Fluka (Buchs, Switzerland). Phosphate buffer components (disodium phosphate and monosodium phosphate), as well as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), protocatechuic acid (PCA), choline chloride, 1,2-propanediol, lactic acid, betaine, urea, and L-lysine, were purchased from Sigma-Aldrich (Darmstadt, Germany). Thermo Scientific (Waltham, MA, USA) sourced D-glucose, D-fructose, xylitol and citric acid. Ascorbic acid was obtained from Betahem (Belgrade, Serbia). In contrast, the Folin–Ciocalteu (FC) reagent, potassium persulfate, sodium carbonate, glycerol, 3,4-dihydroxy-L-phenylalanine (L-DOPA) and 3,5-dinitro salicylic acid (DNSA) were sourced from Merck (Darmstadt, Germany). Tripton LP0042 and yeast extract LP0021 were supplied by Oxoid LTD (Basingstoke, UK) and nutrient agar was provided by Lab M (Bury, UK). Phosphate-buffered saline (PBS, 1X, pH 7.4) was sourced from UFC Biotechnology (Amherst, NY, USA). Several other reagents, including streptomycin, sodium chloride, disodium phosphate, monosodium phosphate, mushroom-derived tyrosinase enzyme, kojic acid, α-amylase from porcine pancreas, and acarbose were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).