Ether lipids influence cancer cell fate by modulating iron uptake

Abstract

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Keywords: CD44; Ether lipids; endocytosis; ferroptosis; iron; membrane tension; metastasis.

Extended Data Fig.1

a. Schematic of peroxisomal-ether lipid biosynthetic pathway. b. Cell viability following treatment with the GPX4 inhibitor RSL3 for 72 h. PyMT-1099 WT or AGPS KO cells were pretreated with TGF-β (2 ng/ml) for 10 d prior to assay. Graph is representative of two independent biological replicates. c. Amount in pmol of oxidized phosphatidylethanolamine (Oxi. PE) ether and ester phospholipids in pB3 cells treated with RSL3 for 24 hours. Five biological replicates per condition. d. Immunoblot analysis for AGPS expression in mesenchymal-enriched pB3 WT, AGPS KO, and AGPS addback cells. pB2 cells served as a control for expression of epithelial-like markers.

Extended Data Fig. 2

a. Inductively coupled plasma-mass spectrometry (ICP-MS) of cellular iron in the mesenchymal-enriched 687g WT and AGPS KO murine breast cancer cell line. b. Immunoblot analysis of OVCAR8 AGPS KO, FAR1 KO or nontargeting sg (control) cells. Unless stated otherwise, all samples were analyzed in technical triplicates and shown as the mean +/− SEM. Statistical significance was calculated using unpaired, two-tailed t-test.

Extended Data Fig. 3

a. ICP-MS of cellular iron following treatment with either hyaluronan or hyaluronidase in OVCAR8 WT or CD44 KO cells. b. Endocytosis of EGFR as assessed by quantitative colocalization of internalized EGF with an early endosomal marker (EEA1) in PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 days. Cells were treated with 200 ng/ml EGF. All data shown as mean +/− SEM and statistical significance was calculated using unpaired, two-tailed t-test; Examined n=10 fields of cells per experimental sample for all endocytosis-related experiments and n=4 replicates for ICP-MS.

Extended Data Fig. 4

a. Bright-field (top) and fluorescence images (bottom) showing reduced mesenteric metastases from athymic nude mice injected with tdTomato-labeled OVCAR8 NT sg, AGPS KO and FAR1 KO cells via the intraperitoneal route. b. Representative IVIS images of overall metastatic burden in C57BL/6 female mice following intracardiac injection of GFP-luciferized pB3 WT (n=5) and CD44 KO (n=6) cells. Mean +/− SEM. c. Quantification of overall metastatic burden in C57BL/6 female mice following intracardiac injection of GFP-luciferized pB3 WT (n=5) and CD44 KO (n=6) cells. Mean +/− SEM. d. Table showing number of cells implanted per mice for limiting dilution assay.

Fig. 1.. Ether lipids play a key role in maintaining a ferroptosis susceptible cell state. See also Extended Data Fig.1.

a. Schematic of experimental model for lipidomic analysis. b. Immunoblot analysis for AGPS expression in PyMT-1099 WT or AGPS KO cells. Cells were treated with TGF-β (2 ng/ml) for 10 d where indicated. c. Cell viability following treatment with the GPX4 inhibitor ML210 for 72 h. 1099 WT or AGPS KO cells were pretreated with TGF-β (2 ng/ml) for 10 d prior to assay. Graph is representative of two independent biological replicates. d. Bar graph showing percent of total lipids constituted by ether lipids following AGPS KO in untreated wildtype (WT) or TGF-β-treated (2 ng/ml;10 d) PyMT-1099 cells. e. Pie chart showing the relative proportion of ether lipids with various total numbers of double bonds. f. Amount in pmol of oxidized phosphatidylethanolamine (Oxi. PE) ether and ester phospholipids in PyMT-1099 TGF-β cells treated with ML210 for 24 h. Five biological replicates per condition. g. Volcano plot showing the log₂ fold change in the relative abundance of various lipid species upon knockout of AGPS in PyMT-1099 TGF-β-treated cells. Blue indicates non-ether linked polyunsaturated phospholipids with a total of at least 3 double bonds; orange indicates all ether lipids identified in lipidomic analysis and black denotes all other lipids identified. h. Volcano plot showing the log₂ fold change in the relative abundance of various lipid species upon knockout of AGPS in pB3 cells. Blue indicates non-ether linked polyunsaturated phospholipids with a total of at least 3 double bonds; orange indicates all ether lipids identified in lipidomic analysis and black denotes all other lipids identified. i. Bar graph showing the percent of total lipids constituted by ether lipids in pB3 WT, pB3 AGPS KO and pB3 AGPS addback cells. j. Bar graph showing the effects of ether lipids on the relative abundance of selected polyunsaturated diacyl phospholipids in pB3 cells. Unless stated otherwise, all samples were analyzed in technical triplicates and shown as the mean +/− SEM. Statistical significance was calculated using unpaired, two-tailed t-test. For figures 1h–1j: pB3 WT and AGPS KO cells were transduced with the respective vector control plasmids. pB3 AGPS addback cells are derivatives of AGPS KO cells transduced with a murine AGPS expression vector.

Fig. 2.. Ether lipids regulate cellular redox-active iron levels in cancer cells. See also Extended Data Fig. 2.

a. Relative lysosomal iron levels based on Rhodox-M fluorescence intensity normalized to the fluorescence intensity of lysotracker. Fold change is calculated relative to untreated PyMT-1099 wild-type (WT) cells. b. Relative lysosomal iron levels based on Rhodox-M fluorescence intensity normalized to the fluorescence intensity of lysotracker. Fold change is calculated relative to pB3 WT cells. c. Inductively coupled plasma-mass spectrometry (ICP-MS) of cellular iron in PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 d. d. Inductively coupled plasma-mass spectrometry (ICP-MS) of cellular iron in pB3 cell line derivatives. e. Relative lysosomal iron levels in OVCAR8 NT sg, FAR1 KO or AGPS KO cells pretreated with FAC (50 µg/ml) for 24 h. Data shown are based on Rhodox-M fluorescence intensity normalized to lysotracker fluorescence intensity. Fold change is calculated relative to NT sg. f. Inductively coupled plasma-mass spectrometry (ICP-MS) of cellular iron in OVCAR8 NT sg, FAR1 KO or AGPS KO cells pretreated with FAC (50 µg/ml) for 24 h. g. Cell viability of OVCAR8 NT sg, FAR1 KO or AGPS KO cells pretreated with FAC (50 µg/ml) for 24 h followed by ML210 treatment for 72 h. h. Cell viability in response to ML210 treatment. PyMT-1099 WT or AGPS KO cells were pretreated with TGF-β (2 ng/ml) for 10 days followed by FAC treatment (100 µg/ml) for an additional 24 h. Cells were then treated with ML210 in the presence or absence of liproxstatin-1 (0.2µM) and cell viability was assessed after 72 h. i. ICP-MS of cellular iron from primary tumors derived from pB3 WT, pB3 AGPS KO, and pB3 AGPS addback cells. Mean +/− SEM from 3 independent tumor samples per condition. Each datapoint represents the average iron measurement from 5 technical replicates per tumor sample. All samples were analyzed with 3–6 technical replicates and shown as the mean +/− SEM unless stated otherwise. Statistical significance was calculated using unpaired, two-tailed t-test. Abbreviation: NT – nontargeting.

Fig. 3.. Ether lipids facilitate CD44-mediated iron endocytosis. See also Extended Data Fig. 3.

a. Endocytic transport of fluorescently labeled transferrin as assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 cells. b. Endocytic transport of fluorescently labeled hyaluronate probe as assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 cells. c. Endocytic transport of fluorescently labeled transferrin as assessed by quantitative colocalization with an early endosomal marker (EEA1) in PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 d. d. Endocytic transport of fluorescently labeled hyaluronate probe as assessed by quantitative colocalization with an early endosomal marker (EEA1) in PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 d. e. ICP-MS of cellular iron following treatment with either hyaluronan or hyaluronidase in PyMT-1099 WT or CD44 KO cells pretreated with 2 ng/ml TGF-β for 10 d. f. Endocytic transport of dextran as assessed by quantitative colocalization with the early endosomal marker EEA1 in pB3 cells. g. Endocytic transport of dextran as assessed by quantitative colocalization with the early endosomal marker EEA1 in PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 d. h. Endocytosis of EGFR as assessed by quantitative colocalization of internalized EGF with an early endosomal marker (EEA1) in PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 days. Cells were treated with 2 ng/ml EGF. All data shown as mean +/− SEM and statistical significance was calculated using unpaired, two-tailed t-test; Examined n=10 fields of cells per experimental sample for all endocytosis-related experiments and n=4 replicates for ICP-MS.

Fig. 4.. Ether lipid deficiency impairs membrane biophysical properties.

a. Schematic of membrane tether pulling assay and fluorescence image showing a tether pulled from the plasma membrane of a pB3 cell using an optically trapped 4 µm anti-Digoxigenin coated polystyrene bead. b. Graph showing tether radius (R) and tether force measurements (f) in pB3 WT, AGPS KO, and AGPS addback cells. All data shown as mean +/− SD. c. Membrane tension measurements in pB3 WT, AGPS KO pretreated with 20µM of the indicated ether phospholipid liposomes, and AGPS addback cells. All data shown as mean +/− SEM. d. Endocytic transport of fluorescently labeled hyaluronate probe as assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 WT or AGPS KO cells pretreated with 20µM of the indicated ether phospholipid liposomes. All data shown as mean +/− SEM. e. Endocytic transport of fluorescently labeled transferrin as assessed by quantitative colocalization with an early endosomal marker (EEA1) in pB3 WT or AGPS KO cells pretreated with 20µM of the indicated ether phospholipid liposomes. All data shown as mean +/− SEM. f. GP values of C-Laurdan-labeled plasma membranes from pB3 WT, AGPS KO and AGPS addback cells. Data is shown as mean GP +/− SD. g. GP values of C-Laurdan-labeled intracellular membranes from pB3 WT, AGPS KO and AGPS addback cells. Data is shown as mean GP +/− SD. h. GP maps of C-Laurdan-labeled intracellular membranes from PyMT-1099 WT or AGPS KO cells treated with 2 ng/ml TGF-β for 10 d. Data shown as mean GP +/− SD. i. Representative curves showing a leftward shift of the phase separation curve in GPMVs from pB3 AGPS KO cells in comparison to wildtype pB3 control cells. This is indicative of less stable phase separation upon loss of AGPS. Curves were generated by counting >/= 20 vesicles/temperature/condition at >4 temperature. The data was fit to a sigmoidal curve to determine the temperature at which 50% of the vesicles were phase-separated (T_misc). Data shown as the average fits of 3 independent experiments. Inset showing a decrease in miscibility transition temperatures (T_misc) upon loss of AGPS in pB3 cells. Graph shows the mean +/− SEM of 3 independent experiments. Statistical significance was calculated using unpaired, two-tailed t-test.

Fig. 5.. Loss of ether lipids decreases metastasis and cancer cell stemness. See also Extended Data Fig. S4.

a. Representative confocal images of extravasated tdTomato-labeled pB3 WT and AGPS KO cells from an in vitro microvascular network established using HUVEC (green) and normal human lung fibroblasts (unlabeled), over a time period of 24 h. b. Quantification of extravasated tdTomato-labeled pB3 WT and AGPS KO cells from an in vitro microvascular network established using HUVEC (green) and normal human lung fibroblasts (unlabeled), over a time period of 24 h. Each datapoint represents number of extravasated cells per device. Data is representative of two independent biological replicates. Graph shows the mean +/− SEM and statistical significance was calculated using unpaired, two-tailed t-test. c. Quantification of extravasated tdTomato-labeled PyMT-1099 cell line derivatives from an in vitro microvascular network established using HUVEC (green) and normal human lung fibroblasts (unlabeled), over a time period of 24 h. Each datapoint represents number of extravasated cells per device. Data is representative of two independent biological replicates. Graph shows the mean +/− SEM and statistical significance was calculated using unpaired, two-tailed t-test. d. Representative IVIS images of overall metastatic burden in C57BL/6 female mice following intracardiac injection of GFP-luciferized pB3 WT (n=5) and AGPS KO (n=5) cells. Mean +/− SEM. e. Quantification of overall metastatic burden in C57BL/6 female mice following intracardiac injection of GFP-luciferized pB3 WT (n=5) and AGPS KO (n=5) cells. Mean +/− SEM. f. Representative images of H&E-stained sections of harvested kidneys from C57BL/6 female mice following intracardiac injection of pB3 WT or pB3 AGPS KO cells. g. Gross images of primary tumors derived from pB3 WT control cells and pB3 AGPS KO cells. h. Tumor growth kinetics of primary tumors derived from pB3 WT control cells and pB3 AGPS KO cells. (n=5 mice per group). i. Bar graph showing the average weight from primary tumors derived from pB3 WT control cells and pB3 AGPS KO cells. Data shows the mean +/− SEM. j. Estimated number of cancer stem cells (CSCs) per 10,000 cells as calculated by extreme limiting dilution analysis (ELDA) software. Tumor-initiating capacity was assessed following implantation of indicated amounts of pB3 WT or pB3 AGPS KO cells into the mammary fat pad of C57BL/6 mice. P values, χ² pairwise test. k. Table showing the number of mice with palpable primary tumors at 121 d post orthotopic implantation of PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 d into NSG female mice. l. Quantification of lung metastases for aforementioned experiment. Data shows the mean number of lung metastases +/− SEM. m. Representative images of H&E-stained lungs harvested from C57BL/6 female mice following orthotopic injection of PyMT-1099 WT or AGPS KO cells pretreated with 2 ng/ml TGF-β for 10 d. Lungs were harvested after 121 d post-injection.