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. 2022 Jun 15;42(8):768–783. doi: 10.1002/cac2.12321

FIGURE 6.

FIGURE 6

Involvement of ferroptosis in p‐PSMA‐CAR‐NK92MI cell‐mediated killing. (A) C4‐2 cell morphology was observed by transmission electron microscopy after 2 hours of culture alone or coculture with p‐PSMA‐CAR‐NK92MI cells. The cells that died by ferroptosis primarily exhibited shrunken and damaged mitochondria. Scale bar: 200 nm. (B) Flow cytometry revealed a significant difference in the relative lipid ROS levels in C4‐2 cells after coculturing with p‐PSMA‐CAR‐NK92MI cells (E:T ratio of 0.5:1) for 24 hours. (C) Our biochemical analyses revealed that C4‐2 cells treated with CAR‐NK92MI cells for 4 hours contained higher intracellular Fe2+ levels than untreated C4‐2 cells. (D) C4‐2 cells cocultured with p‐PSMA‐CAR‐NK92MI cells (E:T ratio of 0.5:1) for 24 hours were treated with RSL3 (2 μmol/L) or ferrostatin‐1 (10 μmol/L). p‐PSMA‐CAR‐NK92MI cells increased the lipid ROS levels in C4‐2 cells, and RSL3 increased the lipid ROS levels, these increases were inhibited by ferrostatin‐1. (E) C4‐2 cells cocultured with p‐PSMA‐CAR‐NK92MI cells (E:T ratio of 0.5:1) for 24 hours were treated with RSL3 (2 μmol/L) or ferrostatin‐1 (10 μmol/L). And augmented RSL3‐induced cell death in C4‐2 cells, and these effects were reversed by ferrostatin‐1. (F) The concentration of lipid ROS in C4‐2 cells after p‐PSMA‐CAR‐NK92MI cell therapy increased over time. (G) ELISA revealed evident increases in IFN‐γ production over time after p‐PSMA‐CAR‐NK92MI cell introduction. (H) The increase in lipid ROS in C4‐2 cells induced by the p‐PSMA‐CAR‐NK92MI cell supernatant could be reversed by anti‐IFN‐γ antibodies but not by anti‐TNF antibodies. (I) Immunohistochemistry revealed a significant reduction in SLC7A11 and SLC3A2 expression in tumor sections from the p‐PSMA‐CAR‐NK92MI cell treated mice. Scale bar: 100 μm. (G) The intracellular GSH levels were significantly higher in C4‐2 cells treated with CAR‐NK92MI cells (E:T ratio of 1:1) for 2 hours than in untreated C4‐2 cells. The data are presented as the means ± SEMs from three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001; ns, not significant. Abbreviations: ROS, reactive oxygen species; E:T, effector‐to‐target; ELISA, enzyme linked immunosorbent assay; IFN‐γ, interferon gamma; TNF, tumor necrosis factor; SLC7A11, solute carrier family 7 member 11; SLC3A2, solute carrier family 3 member 2; GSH, glutathione; SEM, standard error of the mean.