Lipidomics data, AMPK-mediated lipid droplet plasticity and dispersion dictate the differential sensitivity of melanoma cells to PUFA- and iron-induced ferroptosis

Description

Treatment-resistant cancer cells often remain sensitive to ferroptosis, a lipid peroxidation-mediated form of regulated cell death emerging as a promising interventional strategy. Here, using a panel of melanoma cell lines, we find that the ability of iron and polyunsaturated fatty acids (PUFA) to evoke ferroptosis differed substantially among the models and was time-dependent (early versus late onset of ferroptosis). This difference could largely be attributed to the sequestration of PUFA into lipid droplets (LDs) and their differential subcellular (re)distribution and association with lipid-metabolizing organelles like mitochondria. We found that starvation-dependent activation of adenosine monophosphate protein kinase (AMPK) drives the relocation of LDs toward mitochondria in the late responder group. Mitochondrial inhibitors largely rescued cells from PUFA- and iron-induced ferroptosis, corroborating the involvement of mitochondria in ferroptosis induction in these models. Collectively, our work demonstrates that nutrient availability through redispersion of LDs plays a critical regulatory role in PUFA-mediated ferroptosis induction. These findings may have important implications for the further exploitation of ferroptosis in cancer treatment.

Steps to reproduce

Lipid extraction :An amount of cells containing 10 μg of DNA was homogenized in 700 μL of water with a handheld sonicator and was mixed with 800 μl HCl(1M): CH3OH 1:8 (v/v), 900 μl CHCl3, 200 μg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma Aldrich), 3 μl of Ultimate SPLASH® LIPIDOMIX® Mass Spec Standard (#330707, Avanti Polar Lipids), 3 μl of Ceramides and 3 μl of Hexosylceramides internal Standards (#5040167 and #5040398, AB SCIEX). After vortexing and centrifugation, the lower organic fraction was collected and evaporated using a Savant Speedvac spd111v (Thermo Fisher Scientific) at room temperature, and the remaining lipid pellet was stored at -20°C under argon. Mass spectrometry:Immediately before mass spectrometry analysis. Lipid pellets were reconstituted in 100% ethanol. Lipid species were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX). Chromatographic separation was performed on a XBridge amide column (150 mm × 4.6 mm, 3.5 μm; Waters) maintained at 35  °C using mobile phase A [1 mM ammonium acetate in water-acetonitrile 5:95 (v/v)] and mobile phase B [1 mM ammonium acetate in water-acetonitrile 50:50 (v/v)] in the following gradient: (0-6 minutes: 0% B  6% B; 6-10 minutes: 6% B  25% B; 10-11 minutes: 25% B  98% B; 11-13 minutes: 98% B  100% B; 13-19 minutes: 100% B; 19-24 minutes: 0% B) at a flow rate of 0.7 mL/minutes which was increased to 1.5 mL/minutes from 13 minutes onwards. SM, CE, CER, DCER, HCER, and LCER were measured in positive ion mode with a precursor scan of 184.1, 369.4, 264.4, 266.4, 264.4, and 264.4, respectively. TAG, DAG, and MAG were measured in positive ion mode with a neutral loss scan for one of the fatty acyl moieties. PC, LPC, PE, LPE, PG, PI, and PS were measured in negative ion mode by fatty acyl fragment ions. Lipid quantification was performed by scheduled multiple reactions monitoring (MRM), the transitions based on the neutral losses or the typical product ions described above. The instrument parameters were as follows: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = 5500 V and −4,500 V; Temperature = 550  °C ; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = 60 V and −80 V; Entrance Potential = 10 V and −10 V; Collision Cell Exit Potential = 15 V and −15 V. Data Analysis: Peak integration was performed with the MultiQuantTM software version 3.0.3. Lipid species signals were corrected for isotopic contributions (calculated with Python Molmass 2019.1.1). Lipids were quantified based on internal standard signals, adhering to the guidelines of the Lipidomics Standards Initiative (LSI) (level 2 type quantification as defined by the LSI).

Institutions

• Katholieke Universiteit Leuven Departement Oncologie

Categories

Funders

• Research Foundation - Flanders

  Belgium

  Grant ID: G0C2219N
• Stichting tegen Kanker

  Belgium

  Grant ID: 2022-043 (ATE-2022/1871
• KU Leuven

  Belgium

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