Figure 3.

Radiation-induced NETs protect tumor cells from cytotoxicity and favor metastasis. A–E, HT29 cancer cell spheroids were cultured in the presence of γ-irradiated (2 Gy) neutrophils in the presence/absence of DNAse I to allow their coating by NETs for 4 hours. Then, NET-covered or not-covered cancer cell spheroids were cocultured with activated human T cells (A–C) or IL2-activated human NK cells (D and E). A and D, Representative microphotographs of time-lapse confocal videos (Supplementary Video S2) of such cocultures showing cytotoxic immune cells in blue, tumor cells in red, and NETs (DNA) in green. A representative time point and a single z-slice are shown at the top, and the 3D reconstruction of the whole z-stack that includes the tracks of cytotoxic immune cells is shown below. B and E, Quantification of the minimum distance of cytotoxic immune cell tracks to tumor cells in the spheroids to account for cell-to-cell interactions with tumor cells. Each dot represents one individual tracked lymphocyte. Mann–Whitney U-tests were used for statistical comparisons. C and F, Percentage of viable tumor cells recovered after 48 hours from such cocultures to account for cytotoxicity. To redirect T-cell–mediated cytotoxicity, 1 μg/mL of EpCAM–CD3ε bispecific Ab (T-cell engager) was added to the cocultures. Pooled data from two to four experiments were analyzed using paired t tests. G, Balb/c mice were injected twice with mildly sonicated NETs isolated from γ-irradiated human neutrophils, and 24 hours later, 4T1 fluorescent (mCherry⁺) tumor cells were injected in the tail vein of such mice. Representative fluorescence images of the lung of the experimental groups of mice 10 days after tumor cell intravenous injection and quantification of the tumor area on the surface of such lungs. H, Experiments were performed as shown in (G) but included Ab-based depletion of NK cells (anti-Asialo GM1) and/or CD8⁺ T cells (anti-CD8β mAb). Mann–Whitney U-tests were used for statistical comparisons.