Fig. 4: Co-culture of colon cancer cell lines with healthy PBMCs.
From: Molecular subtype-specific responses of colon cancer cells to the SMAC mimetic Birinapant
A Overview of the workflow. PBMCs were isolated using Ficoll-Hypaque density centrifugation, transferred in a cell culture transwell insert (0.4-µm pore size) and incubated for 5 days with the respective CRC cells. After 5 days, the insert with the PBMCs was transferred into a new well with newly seeded CRC cells. Following one day of co-culture, the cells were treated with 2 µM oxaliplatin/10 µM 5-FU/1 µM Birinapant for 2 days and analysed using an Annexin-V/PI assay. B–E Amount of apoptotic and necrotic cells after 48 h treatment in the absence (black) or presence (colored bar) of immune cells. The subtypes are represented by the colored bar with the CMS1 cell line HCT116 represented in orange (B), the CMS2 cell line GP5D in blue (C), CMS3 in pink (D, LS513), and the “CMS4” cell line MDST8 in green (E). Additionally, HCT116 (F) and GP5D (G) were co-incubated with TNFα-neutralizing antibodies during the treatments in co-culture. Significance was determined by multiple t tests using the Holm–Sidak method for multiple comparisons. N = 3; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
