Fig. 3.
Irradiated HNSCC cells are better taken up by M1 macrophages compared to mock-treated ones. a Timeline shows the simultaneous treatment of the HNSCC cells and the monocytes/macrophages. On day 6 the tumor cells were first stained with the cytoplasmic membrane dye CellBrite and subsequently added to the differentiated and pre-polarized macrophages at a ratio of 3:1 (6 × 105 tumor cells per 2 × 105 macrophages) and incubated at 37 °C in the corresponding supernatants (SN) in which the tumor cells were treated for the previous 3 days. After 2 h, all cells were harvested and stained with the viability stain Zombie NIR and CD11b to distinguish between the two cell types. As a control group, macrophages and tumor cells were incubated at 4 °C. The gating strategy of the phagocytosis analysis is shown in b. After pre-gating on singlets (FSC-A/FSC-H) and viable cells based on morphological properties (FSC-A/SSC-A) and live/dead stain (Zombie NIR), macrophages were identified by CD11b expression. The phagocytosis rate was determined by the percentage of CellBrite-positive macrophages. Data for the uptake of the 2 × 5Gy-irradiated and mock-treated (0 Gy) HPV-negative HNSCC cell line cal33 (c) and the HPV-positive HNSCC cell line UD2 (d) are presented as median with interquartile range. Statistical analysis was performed with the Kruskal–Wallis test and Dunn’s correction for multiple testing comparing all “0 Gy” and all “2 × 5 Gy” of all four phenotypes with each other, and with the Mann–Whitney U test comparing “0 Gy” with “2 × 5 Gy” within each phenotype. Additionally, the Mann–Whitney U test was used to compare the influence of the HPV status on the uptake of tumor cells through the respective macrophage subtypes and treatment modalities (# p < 0.01). Significance is indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001