Autophagy inhibition reduces tumor mass in combination of immunotherapy and anti-EGFR treatment in colon cancer cell lines.

Confocal microscope images of three-dimensional culture in RKO and Colo-205 cell lines.(A) RKO cells treated with 1μM anti-EGFR mAbs Cetuximab (C) or panitumumab (P), 5mM of the autophagic inhibitor 3-MA or 20μΜ of (HCQ), 0,5 μM checkpoint inhibitors nivolumab (N), Pembrolizumab (PE) or ipilimumab (IPI) for 48 hours alone or in 3-Methyladeninein combination with a constant dose of E, I and / or A. (B) Colo-205 cells treated with 1μM anti-EGFR mAbs Cetuximab (C) or panitumumab (P), 0,5μM of check point inhibitors nivolumab (NI), pembrolizumab (PE), ipilimumab (IPI), 5mM of the autophagic inhibitor 3-MA or 20μΜ of HCQ and 1μΜ MEK inhibitor PD-0325901 for 48 hours. Colo-205 were treated with [3-Methyladeninee (3-MA)] and in combination with a constant dose of E, I, PD, and / or A respectively for 48 hours. (C) HT29 cells treated 1μM anti-EGFR mAbs Cetuximab (C) or panitumumab (P), 0,5μM of check point inhibitors nivolumab (NI), pembrolizumab (PE), ipilimumab (IPI), 20μΜ of the autophagy inhibitor HCQ and 1μΜ MEK inhibitor PD-0325901 for 48 hours alone or in combination with a constant dose of E, I, PD and /or A for 48 hours. Nuclei were detected with DAPI (blue) and cleaved caspase-3 with the specific antibody (red). High concentration of cleaved caspase-3 and apoptotic nuclei are shown with yellow arrows.