Iron-Mediated Ferroptosis Impairs CAR-T Cell Function and Antitumor Efficacy in Multiple Myeloma
核心洞察
Single-cell RNA sequencing of CAR-T cells from multiple myeloma (搜索) patients reveals upregulation of iron-homeostasis genes during the diminution phase, correlating with lipid ROS accumulation and ferroptosis.
Elevated iron levels, whether from physiological serum concentrations or ferric citrate supplementation, drive CAR-T cell ferroptosis, mitochondrial damage, and impaired antitumor cytotoxicity.
The ferroptosis inhibitor ferrostatin-1 (搜索) rescues CAR-T cell viability, reduces lipid ROS, restores mitochondrial morphology, and preserves T cell memory subsets without compromising tumor control.
A new study published in Nature Cancer reveals that iron-dependent ferroptosis critically undermines the function and persistence of chimeric antigen receptor (CAR)-T cells in patients with multiple myeloma (搜索), identifying a previously unrecognized barrier to durable therapeutic responses.
Investigators performed single-cell RNA sequencing (scRNA-seq) on peripheral blood samples from four multiple myeloma (搜索) participants receiving CAR-T therapy, capturing cells at both the expansion and diminution phases of treatment. The analysis demonstrated that CAR-T cells during the diminution phase exhibited significant upregulation of iron-homeostasis genes, accompanied by elevated lipid reactive oxygen species (lipid ROS) — a hallmark of ferroptosis. Flow cytometric analysis confirmed that lipid ROS levels were significantly higher in CAR-T cells compared to matched non-CAR-T cells from both multiple myeloma (n=8) and acute lymphoblastic leukemia (搜索) (n=7) patient samples at the diminution phase.
“These findings suggest that the very success of CAR-T expansion may sow the seeds of subsequent functional decline through iron-mediated ferroptosis,” the researchers noted, as differential serum metabolite analysis between expansion and diminution phases (threshold: log2FC > 1, adjusted P < 0.1) identified significant shifts in iron-related metabolic pathways across 47 participants.
Iron Exposure Drives Ferroptotic Cell Death
To dissect the mechanistic basis of iron-induced CAR-T dysfunction, the team exposed CAR-T cells to increasing concentrations of ferric citrate (FC). Intracellular iron levels rose in a dose-dependent manner, paralleled by progressive accumulation of lipid ROS and a corresponding decline in cell viability measured by SYTOX Green-based live-cell imaging. At serum-comparable iron concentrations, CAR-T cell death was selectively rescued by the ferroptosis inhibitor ferrostatin-1 (搜索) (Fer-1), but not by inhibitors of apoptosis (Z-VAD), necroptosis (NSA), pyroptosis (VX765), or autophagy (3-MA), confirming ferroptosis as the predominant cell death modality.
Fer-1 treatment reduced lipid ROS and malondialdehyde (MDA) levels, restored redox balance, and rescued mitochondrial morphology. Transmission electron microscopy quantification revealed that Fer-1 preserved both mitochondrial area and mitochondrial length (Feret's diameter), with six independent electron micrographs analyzed per group and 25 mitochondria quantified per group. Statistical analysis using one-way ANOVA with Tukey's multiple-comparisons test confirmed the significance of these ultrastructural improvements.
Functional Consequences and Therapeutic Countermeasures
The functional impact of iron-induced ferroptosis extended to CAR-T cell differentiation and exhaustion. Treatment with ferroptosis inhibitors — Fer-1, liproxstatin-1 (搜索) (Lip-1), and UAMC-3203 (搜索) — preserved the central memory T cell (Tcm: CD45RO+, CD62L+) compartment while reducing effector memory T cell (Tem: CD45RO+, CD62L−) proportions. Expression of exhaustion markers PD-1, TIM3, and LAG3 was attenuated in a dose-dependent manner across all three inhibitors tested at escalating concentrations (group A: 1 μM/100 nM/5 nM; group B: 2 μM/200 nM/10 nM; group C: 4 μM/400 nM/20 nM for Fer-1, Lip-1, and UAMC-3203, respectively).
Critically, in vivo experiments demonstrated that ferroptosis inhibitors did not compromise tumor control when administered as monotherapy to Nalm6 tumor-bearing mice (n=5 per group), as assessed by bioluminescence kinetics and Kaplan-Meier survival analysis with log-rank (Mantel-Cox) testing.
Mitochondrial Dysfunction and ROS-Mediated Damage
Further scRNA-seq analysis comparing CAR-T cells treated with FC versus untreated controls revealed enrichment of pathways associated with mitochondrial damage and dysregulation of ROS and glutathione metabolism. Flow cytometric assessment using MitoTracker and TMRE dyes in CAR-T cells from multiple myeloma (搜索) participants (n=3) showed progressive loss of mitochondrial membrane potential over the course of therapy.
Functional assays demonstrated that FC treatment impaired total mitochondrial respiration, spare respiratory capacity, and ATP production while increasing proton leak, as measured by oxygen consumption rate (OCR). Pretreatment with ROS scavengers glutathione (GSH) and N-acetylcysteine (NAC) rescued iron-induced ROS accumulation, reduced exhaustion marker expression, and partially restored cytotoxicity.
Genetic Engineering of Ferroptosis Resistance
In a pivotal genetic experiment, ACSL4 (搜索) knockout in CAR-T cells reduced the accumulation of PUFA-derived lipid peroxidation products — including HETEs, HODEs, and HDoHEs — as determined by targeted metabolomics (n=6 independently prepared samples per condition). ACSL4-deficient CAR-T cells showed attenuated expression of exhaustion markers and preserved activation markers CD25 and CD69 even under iron-rich conditions.
In a cytokine release syndrome (CRS) mouse model, ACSL4 (搜索)-knockout CAR-T cells elicited comparable serum cytokine profiles to control CAR-T cells (n=10 mice per group), suggesting that ferroptosis resistance does not exacerbate CRS risk.
Translational Implications
The study establishes ferroptosis as a therapeutically targetable mechanism of CAR-T cell failure. Iron-modulating dietary interventions in tumor-bearing mice (n=5 per group) altered serum and organ iron levels after 10 weeks, confirming the feasibility of systemic iron modulation. These findings open avenues for combining ferroptosis inhibitors with CAR-T therapy or engineering ferroptosis-resistant CAR-T products through genetic disruption of key lipid peroxidation pathways.