Establishment and Characterization of PDTO

Tumor pieces will be dissociated enzymatically and mechanically to obtain isolated cells or small cell clusters (Fig. 2). Cells will be embedded in an extracellular matrix (growth factor-reduced Matrigel or BME II) and cultured in a medium supplemented with growth factors and signal pathway inhibitors [Advanced DMEM (Gibco) supplemented with 100 UI/mL of penicillin and streptomycin (Gibco), 1% GlutaMAX (Gibco), 1X B27 (Gibco), 1.25 mM NAC (Sigma-Aldrich), 50 ng/mL EGF (PeproTech), 10 ng/mL FGF-10 (PeproTech), 5 ng/mL FGF-b (PeproTech), 500 nM A-83-01 (PeproTech), 10 μM Y27632 (Interchim), 10 mM Nicotinamide (Sigma-Aldrich), 1 μM PGE2 (PeproTech), 1 μM Forskolin (Peprotech), 0.3 μM CHIR99021 (Biogems), 100 μg/mL Primocin (InvivoGen), 50% Wnt3a, RSPO3, Noggin-conditioned media (L-WRN, ATCC), and 10% RSPO1-conditioned media (Cultrex HA-R-Spondin-1-Fc 293 T, Amsbio)]. Culture medium will be changed twice a week. Once formed, PDTO will be dissociated and reseeded to amplify them for experimental purposes. Cryovials will be prepared at regular intervals by dissociating and resuspending PDTO in Recovery Cell Culture Freezing Medium (Gibco) prior to be biobanked in liquid nitrogen. It should be noted that PDTO line will be considered as established when it will be maintained for more than 3 passages. For each PDTO line, samples will be kept frozen for DNA/RNA/protein analysis and others will be embedded in paraffin for histopathological analysis. This will allow comparisons between the characteristics of the PDTO and the tumor from which they are derived in order to validate their correspondence.Fig. 2

Establishment and characterization of PDTO derived from HNSCC and evaluation of response to treatments to assess its predictive value