Grgdsp peptide

For stability assays, SSTN_IGF1R and GRGDSP peptide (EMD Millipore) were incubated in either human plasma (obtained from the UW-Madison Hospital Blood Bank) or sterile CMF-PBS (control) for 0, 8, 24 or 72 hr and their activity (ability to block endothelial cell adhesion) measured against a standard curve using HUVECs plated on wells coated with either VN (SSTN_IGF1R) or FN (GRGDSP). For in vivo stability (steady-state accumulation) assays, female BALB/c mice (3 mice/treatment condition) were serially injected every 24 hr subcutaneously with biotinylated SSTN_IGF1R (0.365 mg/kg) for up to 6 total injections. Mice were then terminally bled under anesthesia at 0, 24, 48, 72, 96, 120 and 144 hr post-injection. Alternatively, mice were given a single bolus of SSTN_IGF1R (0.365 mg/kg) and were terminally bled at 0, 0.1, 0.3, 1, 3, 6, 12 and 24 hr post-injection. Levels of SSTN_IGF1R in serum samples spotted on nitrocellulose membranes were quantified by IR scanning using anti-biotin antibody on a LI-COR Biosciences Odyssey digital imaging system and LI-COR Image Studio v4.0 analysis software. Pharmacokinetic and pharmacodynamic variables were calculated using the WinnonLin, Phoenix 64 software package.

Detailed information about cell lines is included in Supplementary Table 6. All melanoma cell lines were grown in high glucose DMEM (Lonza) supplemented with 10% fetal bovine serum (Hyclone), 2 mM glutamine and 20 μg/mL gentamicin. Primary melanocytes were cultured in 254CF medium (Gibco) supplemented with human melanocyte growth supplement (Gibco); melan-a⁶¹ were cultured in RPMI (Gibco), supplemented with 5% fetal calf serum (Gibco) and 200 nM 12-o-tetradecanoyl phorbol-13-acetate (Sigma). Human dermal lymphatic microvascular endothelial cells HMVEC-dLyAd and human lymph node endothelial cells HLEC were cultivated in Clonetics EGM-2 MV BulletKit (Lonza) following the manufacturer’s instructions. Human lymphatic fibroblasts HLF were cultured in high glucose DMEM (Lonza) supplemented with 10% fetal bovine serum (Hyclone). All cells were grown at 37°C in a humidified 5% CO2 atmosphere.
When indicated, HLECs were treated with the following compounds: NGFR inhibitor THX-B (15 μM) and MEKi PD0325901 (1 μm) synthetized by CNIO Experimental Therapeutics Program; NF-kB inhibitor JSH-23 (5 μM, Merck), GRGDSP peptide (10 ng/mL, Merck), anti-integrin α_v blocking antibody (0.1 μg/mL, Merck), Fc soluble ICAM-1 (10 μg/mL, R&D Systems), diphtheria toxin (10 μg/Kg mouse body weight, Sigma Aldrich), human Pro-NGF (50ng/mL, Alomone) and human BDNF (5 ng/mL, Peprotech).