Characterization of 3D Spheroid Cultures

Indirect immunofluorescence was performed as described previously [32 (link)]. Spheroids in suspension were stained applying the same protocol but using centrifugation between every step. Primary antibodies: rabbit-anti-human EpCAM (EGP40/1556R; 1:100; Novus Biologicals, Centennial, CO, United States), rabbit-anti-human cytokeratin 14 (1:200, Thermo Fisher Scientific, Waltham, MA, United States), rabbit-anti-human cytokeratin 19 (1:75; Novus Biologicals), mouse-anti-human Vimentin (V9; 1:100; Santa Cruz Biotechnology Inc., Dallas, TX, United States), mouse-anti-human α-smooth muscle actin (1A4; 1:200; Sigma-Aldrich), mouse-anti-human Thy-1 (AS02; 1:100; Dianova, Hamburg, Germany), rabbit-anti-human ALDH1A1 (20H2L4, 1:100; Thermo Fisher Scientific), and rabbit-anti-human CDKN2A/p16INK4a (EPR1473; 1:200; Abcam, Cambridge, UK). Secondary antibodies were goat-anti-mouse-IgG-Alexa Fluor 555 and donkey-anti-rabbit-IgG-Alexa Fluor 488 (both Thermo Fisher Scientific). DNA was stained with Hoechst 33,342 and the actin cytoskeleton was visualized using phalliodin-PF647 (Promokine, Heidelberg, Germany). Imaging was conducted using a confocal laser scanning microscope CLSM 780 (Carl Zeiss, Oberkochen, Germany) and ZEN software (Carl Zeiss).