Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture

Abstract

Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1,2. Although other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3,4,5,6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies have suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7,8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3,4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2,9 and C′ dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme glutathione peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.

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Fig. 1: Ferroptosis exhibits propagative spatiotemporal patterns.
Fig. 2: Ferroptosis spreading requires lipid peroxidation and iron and involves cell swelling.
Fig. 3: Ferroptotic cell rupture is inhibited by osmoprotectants.
Fig. 4: Ferroptosis spreading involves calcium flux and does not require cell rupture.
Fig. 5: PEG3350 slows ferroptosis propagation.

Data availability

The statistical source data that support the findings of this study have been provided as part of this publication. All other data are available from the corresponding authors upon request. Source data are provided with this paper.

Code availability

Our source code is available via GitHub at https://github.com/AssafZaritskyLab/PropagationOfCellDeath. This repository includes all code used to measure the mean time difference between neighboring deaths and to run the random simulations, as well as a demo dataset.

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Acknowledgements

This research was supported by grant 1R01GM122923 from the NIH to S.J.D. and grant CA154649 to M.O. from NCI. A.Z. was supported by the Data Science Research Center, Ben-Gurion University of the Negev, Israel. We thank members of the Overholtzer laboratory for helpful discussions.

Author information

Affiliations

Authors

Contributions

M.R. and M.O. designed the study. M.R. designed, performed and analysed experiments. L.S., C.G., T.L., N.S. and A.Z. wrote the analysis code and performed computational analyses. M.R., A.Z. and M.O. wrote the paper. S.J.D., U.W., M.S.B. and P.N. provided key reagents and edited the manuscript.

Corresponding authors

Correspondence to Assaf Zaritsky or Michael Overholtzer.

Ethics declarations

Competing interests

Memorial Sloan-Kettering Cancer Center and three investigators involved in this study (M.S.B., U.W. and M.O.) have financial interests in Elucida Oncology. Research involving C′ dots may involve one or more US or international patent applications.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Treatment of cells with FAC and BSO induces ferroptosis.

a, Viability of HAP1 cells after treatment with FAC and BSO and either DMSO or ferroptosis inhibitors as measured by crystal violet staining. N=three independent experiments. Dunnett’s test; **p = 0.0024 for Lip-1; **p = 0.0045 for Fer-1; *p = 0.0107 for Trolox. b, Confocal images of HAP1 cells treated with FAC and BSO and stained with C11-BODIPY581/591. Non-oxidized probe is shown in red, oxidized probe is shown in green (arrow). Scale bar = 10 μm. Images are representative of three independent experiments. c, Values from the analysis of the experiment shown in panels 1c and d. Note that the experimental mean time difference between neighbors (µexpΔt) is much smaller than the mean (μperm∆t) and 95th percentile (μ95perm∆t) obtained from the randomly permuted data. (d) Spatiotemporal distribution of cell death in HAP1 cells treated with ML162 to induce ferroptosis. Each dot represents a cell from a single movie representative of five fields of view from one experiment. Colors indicate relative times of cell death as determined by SYTOX Green staining. e, Distribution of experimental time differences between neighboring deaths in blue and averaged distribution of the corresponding permuted data in orange. Data belong to the experiment shown in panel d and are representative of five fields of view from one experiment. Statistical source data can be found at Source data Extended Data Fig. 1. Source data

Supplementary information

Reporting Summary

Supplementary Video 1

B16F10 melanoma cells treated with C′ dot nanoparticles undergo ferroptotic cell death with wave-like propagation. Time-lapse images show DIC and SYTOX Green fluorescence. Times are shown as minutes (min). Scale bar, 20 μm.

Supplementary Video 2

TRAIL-induced apoptosis of MCF10A cells. Time-lapse DIC images show apoptosis induced by treatment with TRAIL; times are shown as minutes (min).

Supplementary Video 3

Ferroptosis spreading requires lipid peroxidation and iron. Time-lapse images show HAP1 cells treated with FAC and BSO to induce ferroptosis, and control vehicle (DMSO), or liproxtstatin-1 (middle panel) or deferoxamine (DFO, right panel) were added when indicated as ‘Treated’ in white text. Times are shown as minutes (min). Images show DIC and SYTOX Green fluorescence.

Supplementary Video 4

Ferroptotic cells undergo swelling prior to rupture. Time-lapse confocal images show that the cell swelling marker cPLA2-mKate (red, middle panel) translocates to the nuclear envelope prior to SYTOX Green labelling (right panel) in HeLa cells treated with FAC and BSO.

Supplementary Video 5

HAP1 cells treated with FAC and BSO in the presence of PEG1450 exhibit waves of cell rounding. Time-lapse images show DIC; times are shown as hours:minutes.

Supplementary Video 6

Calcium flux occurs prior to ferroptotic cell rupture. Time-lapse images show spreading of GCaMP fluorescence (green) prior to cell rupture marked by SYTOX Orange (red) in HAP1 cells treated with FAC and BSO. Times are shown as minutes (min).

Supplementary Video 7

Calcium fluxes spread in a wave-like manner in the absence of cell rupture. Time-lapse images show spreading of GCaMP fluorescence (green) and SYTOX Orange staining (red) in HAP1 cells treated with FAC and BSO and PEG1450. Note that PEG1450-treated cells maintain GCaMP fluorescence and do not label with SYTOX Orange, unlike control cells from Supplementary Video 6.

Supplementary Video 8

Wave-like spreading of ferroptosis in U937 cells treated with FAC and BSO, shown by DIC microscopy. Time-lapse images show waves occurring in control (left panel) and PEG3350- treated (right panel) conditions. Times are shown as minutes (min).

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

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Riegman, M., Sagie, L., Galed, C. et al. Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture. Nat Cell Biol 22, 1042–1048 (2020). https://doi.org/10.1038/s41556-020-0565-1

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