Prostaglandin e2

The establishment and characterization of the human colon cancer organoids has been described [62 (link)]. The organoids were cultured in drops of Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco, cat no. A1413202) in DMEM/F12 medium (Thermo), supplemented with 1% (v/v) penicillin/streptomycin (Gibco), 1% Hepes buffer, pH7.5, 1% L-glutamine (Gibco), 1 μg/mL R-spondin (Sigma), 100 ng/mL Noggin (Peprotech), B27 vitamin A-free (Invitrogen, cat no. 12587010), 1 mM n-acetyl cysteine (Sigma), 10 mM nicotinamide (Sigma), 50 ng/mL EGF (BD Biosciences), 500 nM A83-01 (Tocris), 10 μM SB202190 (Sigma), 10 μM Y-27632 (Sigma) and 10 nM Prostaglandin E2 (Cayman). Medium was refreshed every four days and organoids were splitted by treatment with TryPLE Express (Gibco, cat. no. 12604013).

Arachidonic acid (AA: 5,8,11,14-eicosatetraenoic acid), dexamethasone (DEX: (11β,16α)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3), naproxen (Nap: (S)-(+)-6-methoxy-α-methyl-2-naphthaleneacetic acid), esculetin (Esc: 6,7-dihydroxycoumarin) and cAMP analogue (8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt) were purchased from Sigma-Aldrich Korea (Seoul, Korea). Prostaglandin D₂ (PGD₂: 9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid) and prostaglandin E₂ (PGE₂: 9-oxo-11α,15S-dihydroxy-prosta-5Z,13E-dien-1-oic acid) were purchased from Cayman Chemical (Ann Arbor, MI, USA). PKA inhibitor H89 dihydrochloride (N-2-3-(4-bromophenyl)-2-propenylaminoethyl-5-isoquinolinesulfonamide dihydrochloride) was purchased from Tocris (Bristol, UK). Adenylyl cyclase type V inhibitor (2-amino-7-(furanyl)-7,8-dihydro-5(6H)-quinazolinone) was purchased from Calbiochem (San Diego, CA, USA). All these chemicals were dissolved in dimethyl sulfoxide (DMSO).

The canine melanoma cell lines CMeC, LMeC, CMM-1, and CMM-2^{32 (link),33 (link)}, were cultured as described previously^{6 (link)}. The canine osteosarcoma cell lines POS^{34 (link)} and HMPOS^{35 (link)} were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 200 μg/mL streptomycin, and 200 U/mL penicillin (Thermo Fisher Scientific) at 37 °C under a 5% CO₂ atmosphere. Canine PBMCs were purified from heparinized blood obtained from healthy beagles by density gradient centrifugation on Percoll (GE Healthcare UK, Buckinghamshire, UK) and cultured as described previously^{6 (link)}. PBMCs were stimulated with 5 μg/mL Staphylococcal Enterotoxin B (SEB) (Sigma-Aldrich) and 1 μg/mL anti-canine CD28 antibody (eBioscience, San Diego, CA). In some experiments, cells were co-treated with 5 μM meloxicam (Sigma-Aldrich), 2.5 μM Prostaglandin E2 (Cayman Chemical), and/or 20 μg/mL canine chimeric anti-PD-L1 antibody c4G12^{11 (link)} as indicated. The same concentrations of DMSO (Nacalai Tesque, Kyoto, Japan) and dog IgG (Jackson ImmunoResearch, West Grove, PA) were used as negative controls.

Immediately postmortem, the thoracic cavity was opened and the pulmonary vasculature flushed of blood with PBS via the right ventricle. The right atrium of the heart was then cannulated with PE10 tubing, secured with 5-0 silk, and the lung and heart were removed intact. The pulmonary vasculature was perfused via the right atrium with DMEM media at 37 °C for 20 minutes using a peristaltic pump at 50 μL/min and the venous outflow collected from the left atrium.
Levels of the prostacyclin breakdown product 6-keto PG F1_α (6-ketoPGF_1α), the thromboxane breakdown product thromboxane B₂, prostaglandin E₂, prostaglandin F_2α, prostaglandin D2, 12-HETE, and 15-HETE (Cayman Chemical) were measured in supernatants/plasma by commercial immunoassay. In some cases, tissue/vessel segments were weighed and prostanoid levels expressed relative to tissue mass.

Methanol, acetonitrile (ACN), ethyl acetate, water, formic acid (FA) were acquired from Merck Millipore (Warsaw, Poland). Standards of Thromboxane B2, Leukotriene B4, Prostaglandin D2, Prostaglandin E2, 6-keto Prostaglandin F1α, Prostaglandin F2α, 15-deoxy-Δ12,14-Prostaglandin J2, 13,14-dihydro Prostaglandin E1, and their isotope-labeled standards were procured from Cayman Chemical Company (Ann Arbor, MI, USA).