Ferroptosis and Cuproptosis in Cancer and Neurodegeneration: A Comprehensive Review of Modulation by Iron and Copper Chelators and Related Agents

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an ambitious review addressing the emerging interplay between ferroptosis and cuproptosis in oncology and neurodegeneration. The topic is highly relevant, given the growing interest in regulated cell death pathways beyond apoptosis, and the authors demonstrate a strong command of the molecular mechanisms governing iron and copper homeostasis. The manuscript successfully integrates data from molecular biology, pharmacology, and nanomedicine, offering a broad and up-to-date perspective

Several points would benefit from clarification to improve readability:

- Although numerous preclinical strategies are discussed, the translational feasibility and current clinical status of these interventions are not always clear. A short subsection summarizing ongoing or completed clinical trials, as well as major translational barriers (toxicity, delivery, off-target effects), would add significant value.

- Some abbreviations should be consistently defined at first mention.

Author Response

Reviewer 1

This manuscript presents an ambitious review addressing the emerging interplay between ferroptosis and cuproptosis in oncology and neurodegeneration. The topic is highly relevant, given the growing interest in regulated cell death pathways beyond apoptosis, and the authors demonstrate a strong command of the molecular mechanisms governing iron and copper homeostasis. The manuscript successfully integrates data from molecular biology, pharmacology, and nanomedicine, offering a broad and up-to-date perspective

Several points would benefit from clarification to improve readability:

- Although numerous preclinical strategies are discussed, the translational feasibility and current clinical status of these interventions are not always clear. A short subsection summarizing ongoing or completed clinical trials, as well as major translational barriers (toxicity, delivery, off-target effects), would add significant value.

Response: We thank the reviewer for this constructive feedback. In response, we have added a dedicated section, “5. Translational perspectives and clinical landscape”, which summarizes the current clinical status of metal-modulated interventions, including specific analysis of trials such as FAIRPARK-II and those involving Elesclomol. This section explicitly addresses major translational barriers, including off-target toxicity, the biological constraints of delivery across the BBB and TME, and the industrial challenges of GMP scale-up for complex nanomedicines. Additionally, we have revised the final paragraph of the Conclusions to specifically acknowledge these hurdles, ensuring that the promising preclinical results are balanced against the narrow therapeutic window and the necessity for biomarker-driven patient selection in human pathology.

- Some abbreviations should be consistently defined at first mention.

Response: We have carefully audited the manuscript to ensure all abbreviations are consistently defined at their first mention. Specifically, terms ACSL4, ALOX, DHODH, FSP1, LDH, LH, LPCAT3, NCOA4, PE have been formally introduced upon its initial appearance to ensure clarity for a broad readership.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides an extensive and timely overview of iron and copper dysregulation in cancer, with a particular focus on the emerging concepts of cuproplasia and cuproptosis. The authors successfully compile a large body of recent literature and present a coherent narrative linking copper metabolism, mitochondrial function, and tumor biology. The topic is of high interest to the fields of cancer metabolism and metal-dependent cell death. However, from a biochemical perspective, the manuscript remains largely descriptive, and several mechanistic claims would benefit from deeper critical evaluation and clearer biochemical delineation. In its current form, the review summarizes what is known but does not sufficiently interrogate how or why specific copper-dependent processes predominate in particular tumor contexts. Addressing the points below would substantially improve the manuscript’s rigor and impact. Therefore, major revision of the manuscript is necessary prior to publication.

 

Detailed comments.

1. The manuscript provides a generally accurate description of ferroptosis as an iron-dependent, lipid peroxidation–driven form of regulated cell death. However, the biochemical identity of ferroptosis is not sufficiently distinguished from other ROS-associated death pathways.

 

2. GPX4 inactivation and lipid peroxidation are repeatedly cited, yet ACSL4, LPCAT3, and PUFA-PL composition, which define ferroptosis specificity, are not discussed in depth. In addition, this review would benefit from clarifying that not all lipid peroxidation equals ferroptosis, and that ferroptotic death requires a specific membrane lipid context.

 

3. The authors repeatedly emphasize GSH depletion as a central convergence point between ferroptosis and cuproptosis. While this is conceptually attractive, it risks oversimplifying ferroptosis biology. GSH depletion alone is not sufficient to induce ferroptosis if GPX4-independent lipid repair mechanisms remain intact. Alternative ferroptosis defense systems (e.g., FSP1–CoQ10 axis, DHODH in mitochondria) are absent from the discussion. How does copper-mediated GSH depletion overcome GPX4-independent ferroptosis defense pathways?

 

4. This manuscript excels in describing iron and copper chemistry but treats lipid metabolism largely as a downstream consequence. The biochemical origin of ferroptotic lipid peroxides (PUFA-PEs vs. other phospholipids) is not sufficiently addressed.

 

5. Enzymatic vs. non-enzymatic lipid peroxidation (ALOX-mediated vs. Fenton-driven) is not clearly distinguished.

 

6. The proposed synergy between ferroptosis and cuproptosis is intriguing, particularly via ATP depletion and inhibition of copper efflux transporters. However, from a biochemical standpoint, the causal hierarchy remains unclear. Is ferroptosis an initiator of metabolic collapse that secondarily enables cuproptosis? Or is ferroptosis merely a parallel oxidative consequence of copper-induced mitochondrial failure? The discussion would benefit from explicitly distinguishing sequential dependency from mere co-occurrence. Moreover, it is unclear whether copper acts as a primary trigger or merely an amplifier of ferroptotic processes.

 

7. The discussion of ferroptosis in neurodegeneration is well supported, but the manuscript underplays competing or complementary mechanisms. Mitochondrial dysfunction, excitotoxicity, and impaired mitophagy are mentioned only marginally. Ferroptosis is sometimes presented as the dominant mechanism, rather than one of several intersecting pathways. A cautious biochemical framing would improve credibility.

 

8. Terminology Precision: ‘Ferroptotic ROS’ Is Biochemically Vague. The authors frequently use terms such as “ferroptotic ROS” or “ferroptosis-associated oxidative stress.” Ferroptosis is driven primarily by lipid peroxyl radicals (LOO•), not generic ROS such as H₂O₂ or O₂•⁻. Clarifying this distinction would significantly improve mechanistic rigor.

 

9. The manuscript introduces cuproplasia as a copper-driven tumor-promoting process and contrasts it with cuproptosis as a copper-dependent cell death mechanism. While conceptually appealing, the biochemical boundary between these two states remains insufficiently defined. Is cuproplasia simply a physiological extension of copper-dependent enzymatic activity, or does it represent a distinct metabolic state with identifiable molecular markers? At what quantitative or qualitative threshold does copper signaling shift from supporting mitochondrial metabolism to inducing proteotoxic stress? The authors are encouraged to propose biochemical criteria (e.g., mitochondrial copper pools, lipoylation status, Fe–S cluster integrity, redox buffering capacity) that may distinguish cuproplasia from cuproptosis.

 

10. While the focus on mitochondria is justified given the central role of lipoylated TCA cycle proteins in cuproptosis, the authors underrepresent cytosolic copper buffering systems, including metallothioneins, glutathione, and copper chaperones. It is unclear whether cuproptosis reflects: a failure of cytosolic copper sequestration, excessive mitochondrial import, or altered copper redox cycling. A more balanced discussion of compartment-specific copper chemistry would strengthen the mechanistic framework.

 

11. FDX1 is repeatedly described as a central regulator of cuproptosis. However, several biochemical questions remain unresolved and should be acknowledged more explicitly: Is FDX1’s role primarily catalytic (Cu²⁺ → Cu⁺ reduction), structural (protein–protein interactions), or indirect via lipoylation control? How does FDX1-dependent copper reduction differ from other mitochondrial reductive pathways? Are there tumor types in which cuproptosis occurs independently of FDX1? A clearer distinction between established biochemical evidence and inferred regulatory roles is required.

 

12. The authors frequently reference Cu⁺ and Cu²⁺ but does not sufficiently discuss the redox chemistry of copper in biological systems. From a biochemical perspective, copper toxicity is inseparable from its redox activity. How does copper redox cycling contribute to Fe–S cluster destabilization independently of lipoylated protein aggregation? To what extent is cuproptosis driven by redox imbalance versus direct metal–protein coordination chemistry? A short subsection or few paragraphs explicitly addressing copper redox biochemistry would significantly enhance mechanistic clarity.

 

13. Figures are informative but would benefit from clearer labeling distinguishing physiological copper signaling from pathological copper overload.

Author Response

Reviewer 2

This manuscript provides an extensive and timely overview of iron and copper dysregulation in cancer, with a particular focus on the emerging concepts of cuproplasia and cuproptosis. The authors successfully compile a large body of recent literature and present a coherent narrative linking copper metabolism, mitochondrial function, and tumor biology. The topic is of high interest to the fields of cancer metabolism and metal-dependent cell death. However, from a biochemical perspective, the manuscript remains largely descriptive, and several mechanistic claims would benefit from deeper critical evaluation and clearer biochemical delineation. In its current form, the review summarizes what is known but does not sufficiently interrogate how or why specific copper-dependent processes predominate in particular tumor contexts. Addressing the points below would substantially improve the manuscript’s rigor and impact. Therefore, major revision of the manuscript is necessary prior to publication.

Response: We thank the reviewer for this profound biochemical critique. We agree that a high-level review must move beyond descriptive summaries to interrogate the causal hierarchies and threshold-dependent transitions of metal-driven death. We have substantially revised the manuscript to incorporate the requested mechanistic rigor, specifically addressing lipid specificity, non-canonical defense systems, and the redox biochemistry of copper.

 

Detailed comments.

1. The manuscript provides a generally accurate description of ferroptosis as an iron-dependent, lipid peroxidation–driven form of regulated cell death. However, the biochemical identity of ferroptosis is not sufficiently distinguished from other ROS-associated death pathways.

 

Response: We appreciate this request for mechanistic rigor. We have revised the Introduction to explicitly distinguish ferroptosis from other ROS-associated modalities like apoptosis or necroptosis. We now clarify that while other death pathways may involve generic oxidative damage to DNA or proteins via species like H2O2, ferroptosis is uniquely defined by the iron-dependent propagation of lipid peroxyl radicals specifically within the phospholipid bilayer. This “biochemical fingerprint” ensures that the description moves beyond generic oxidative stress to a specific metabolic failure of lipid-oxygen homeostasis.

 

2. GPX4 inactivation and lipid peroxidation are repeatedly cited, yet ACSL4, LPCAT3, and PUFA-PL composition, which define ferroptosis specificity, are not discussed in depth. In addition, this review would benefit from clarifying that not all lipid peroxidation equals ferroptosis, and that ferroptotic death requires a specific membrane lipid context.

 

Response: The reviewer is correct that these enzymes are the gatekeepers of ferroptosis specificity. We have updated Section 1 to discuss the essential roles of ACSL4 and LPCAT3 in remodeling the membrane lipidome. Specifically, we have added text explaining how these enzymes prime the cell for ferroptosis by enriching the membrane with polyunsaturated fatty acid-containing phosphatidylethanolamines. We have also clarified that lipid peroxidation is a broad chemical event, but ferroptosis only occurs when LPO is sustained within this specific enzymatic and structural lipid context.

 

3. The authors repeatedly emphasize GSH depletion as a central convergence point between ferroptosis and cuproptosis. While this is conceptually attractive, it risks oversimplifying ferroptosis biology. GSH depletion alone is not sufficient to induce ferroptosis if GPX4-independent lipid repair mechanisms remain intact. Alternative ferroptosis defense systems (e.g., FSP1–CoQ10 axis, DHODH in mitochondria) are absent from the discussion. How does copper-mediated GSH depletion overcome GPX4-independent ferroptosis defense pathways?

 

Response: We thank the reviewer for pointing out this risk of oversimplification. While GSH depletion is a major convergence point in our metal-synergy model, we now explicitly acknowledge the GPX4-independent defense systems. We have integrated a discussion of the FSP1-CoQ10 axis and mitochondrial DHODH into Section 1. We have also addressed the reviewer's question regarding copper synergy: we propose that copper-mediated GSH depletion, when combined with the mitochondrial metabolic collapse (ATP depletion) characteristic of cuproptosis, likely creates a multimodal failure that overwhelms these parallel defense systems. This revised framing provides a more cautious and accurate biochemical perspective on how metal-driven RCD is executed.

 

4. This manuscript excels in describing iron and copper chemistry but treats lipid metabolism largely as a downstream consequence. The biochemical origin of ferroptotic lipid peroxides (PUFA-PEs vs. other phospholipids) is not sufficiently addressed.

 

Response: We thank the reviewer for this critical observation. We agree that lipid metabolism is not merely a downstream effect but is a co-determinant of ferroptosis specificity. We have revised Section 1 and the Figure 2 legend to explicitly address the biochemical origin of lipid peroxides. Specifically, we have moved the discussion of the ACSL4-LPCAT3 axis to the foreground to emphasize how these enzymes prime the cell by enriching membranes with arachidonic acid- and adrenic acid-containing PUFA-PEs. We now clarify that ferroptosis is a substrate-specific event targeting these particular phospholipids, rather than a stochastic process involving generic lipid peroxidation. This revision ensures that lipid metabolism is recognized as an essential upstream requirement for the execution of metal-dependent cell death.

 

5. Enzymatic vs. non-enzymatic lipid peroxidation (ALOX-mediated vs. Fenton-driven) is not clearly distinguished.

 

Response: We have clarified the distinction between these two processes in Introduction. We now describe enzymatic peroxidation (via 12/15-ALOX) as the site-specific initiator and iron-catalyzed Fenton chemistry as the propagator. This dual-layered model explains how the biochemical specificity of ferroptosis is established and then amplified into a catastrophic, non-enzymatic chain reaction.

 

 

6. The proposed synergy between ferroptosis and cuproptosis is intriguing, particularly via ATP depletion and inhibition of copper efflux transporters. However, from a biochemical standpoint, the causal hierarchy remains unclear. Is ferroptosis an initiator of metabolic collapse that secondarily enables cuproptosis? Or is ferroptosis merely a parallel oxidative consequence of copper-induced mitochondrial failure? The discussion would benefit from explicitly distinguishing sequential dependency from mere co-occurrence. Moreover, it is unclear whether copper acts as a primary trigger or merely an amplifier of ferroptotic processes.

 

Response: We thank the reviewer for this profound critique regarding the causal hierarchy of the iron-copper nexus. We agree that a high-level review must move beyond descriptive co-occurrence to interrogate sequential dependency. Accordingly, we have added a dedicated analysis to Section 3.2a explaining that the observed synergy is a contingent cascade: iron-driven lipid peroxidation leads to mitochondrial failure and ATP depletion, which subsequently locks the ATP-dependent copper exporters (ATP7A/B), transforming a sublethal copper load into a cuproptotic trigger. To support this, we have comprehensively revised the captions for Figures 4 and 7 to provide a stepwise, stage-by-stage narrative of this metabolic “power outage”. These revisions clarify that ferroptosis often acts as the metabolic initiator that disables cellular clearance mechanisms, while copper acts as a preemptive amplifier by competing for the glutathione pool, thereby creating a mutually reinforcing feedback loop with a clear biochemical roadmap.

 

 

7. The discussion of ferroptosis in neurodegeneration is well supported, but the manuscript underplays competing or complementary mechanisms. Mitochondrial dysfunction, excitotoxicity, and impaired mitophagy are mentioned only marginally. Ferroptosis is sometimes presented as the dominant mechanism, rather than one of several intersecting pathways. A cautious biochemical framing would improve credibility.

 

Response: We thank the reviewer for this insightful comment regarding the multi-faceted nature of neurodegeneration. We agree that a cautious biochemical framing is essential for credibility. To address this, we have implemented a two-fold revision. First, in Section 2, we added a dedicated paragraph on cell-type specific regulatory mechanisms, explicitly mentioning localized mitophagy and neuroinflammatory control as intersecting factors in neuronal and oligodendrocyte vulnerability. Second, in Section 4.1, we have added text to clarify that ferroptosis is a convergent terminal pathway that exists alongside, and is often triggered by, mitochondrial dysfunction, excitotoxicity, and impaired mitophagy. By framing iron-dependent lipid peroxidation as the biochemical “tipping point” within a broader landscape of neurodegenerative stress, we believe the manuscript now offers a more balanced and integrated perspective on regulated cell death in the CNS.

 

8. Terminology Precision: ‘Ferroptotic ROS’ Is Biochemically Vague. The authors frequently use terms such as “ferroptotic ROS” or “ferroptosis-associated oxidative stress.” Ferroptosis is driven primarily by lipid peroxyl radicals (LOO•), not generic ROS such as H₂O₂ or O₂•⁻. Clarifying this distinction would significantly improve mechanistic rigor.

 

Response: We thank the reviewer for this technical correction. We agree that the term “ferroptotic ROS” lacks the precision required for high-impact redox biology. We have performed a comprehensive linguistic revision of the manuscript to distinguish generic reactive oxygen species (the initiators) from the specific executioners of ferroptosis. Specifically, we have added a clarifying statement in Section 1 to emphasize that while species like H₂O₂ may act as upstream triggers, the execution of ferroptosis is uniquely driven by the iron-dependent propagation of lipid peroxyl radicals (LOO•) and subsequent lipid hydroperoxides (LOOH) within the membrane context. Throughout the text and Table 4, we have replaced vague terminology with precise descriptors, favoring “lipid peroxyl radicals” when discussing chain propagation and RTA activity, to ensure mechanistic rigor and full alignment with current biochemical standards in the field.

 

 

9. The manuscript introduces cuproplasia as a copper-driven tumor-promoting process and contrasts it with cuproptosis as a copper-dependent cell death mechanism. While conceptually appealing, the biochemical boundary between these two states remains insufficiently defined. Is cuproplasia simply a physiological extension of copper-dependent enzymatic activity, or does it represent a distinct metabolic state with identifiable molecular markers? At what quantitative or qualitative threshold does copper signaling shift from supporting mitochondrial metabolism to inducing proteotoxic stress? The authors are encouraged to propose biochemical criteria (e.g., mitochondrial copper pools, lipoylation status, Fe–S cluster integrity, redox buffering capacity) that may distinguish cuproplasia from cuproptosis.

 

Response: We thank the reviewer for this insightful critique regarding the biochemical boundary between cuproplasia and cuproptosis. We agree that defining this transition is essential for mechanistic rigor. We have revised Section 1 to propose specific biochemical criteria for this threshold. We now define the shift from cuproplasia (physiological signaling) to cuproptosis (proteotoxic stress) as a metabolic “inflection point” characterized by the transition of DLAT from a functional monomer to a toxic high-molecular-weight oligomer, and the qualitative loss of Fe-S cluster integrity. These revisions clarify that the cuproptotic threshold is reached when the rate of FDX1-mediated copper reduction surpasses the mitochondrial thiol-buffering capacity, providing a clear roadmap for distinguishing these two states.

 

 

10. While the focus on mitochondria is justified given the central role of lipoylated TCA cycle proteins in cuproptosis, the authors underrepresent cytosolic copper buffering systems, including metallothioneins, glutathione, and copper chaperones. It is unclear whether cuproptosis reflects: a failure of cytosolic copper sequestration, excessive mitochondrial import, or altered copper redox cycling. A more balanced discussion of compartment-specific copper chemistry would strengthen the mechanistic framework.

 

Response: We appreciate the reviewer’s emphasis on compartment-specific copper chemistry. We agree that the failure of cytosolic sequestration is the necessary precursor to mitochondrial proteotoxic stress. We have added a discussion to Section 1 acknowledging that cuproptosis is not merely a mitochondrial event but a systemic failure of cellular copper buffering, involving chaperones, metallothioneins, and GSH. This balanced framing clarifies that the pathway represents a breakdown in the transition between cytosolic storage and mitochondrial import.

 

11. FDX1 is repeatedly described as a central regulator of cuproptosis. However, several biochemical questions remain unresolved and should be acknowledged more explicitly: Is FDX1’s role primarily catalytic (Cu²⁺ → Cu⁺ reduction), structural (protein–protein interactions), or indirect via lipoylation control? How does FDX1-dependent copper reduction differ from other mitochondrial reductive pathways? Are there tumor types in which cuproptosis occurs independently of FDX1? A clearer distinction between established biochemical evidence and inferred regulatory roles is required.

 

Response: The reviewer correctly identifies several critical, unresolved questions regarding FDX1. In accordance with this suggestion, we have revised Section 1 to more explicitly distinguish between the established regulatory role of FDX1 and the ongoing biochemical debates regarding its catalytic versus structural functions. We have also added a note regarding the potential for FDX1-independent pathways, characterizing these as current gaps in the literature. By framing these points as active areas of research, we believe the manuscript now offers a more rigorous and scientifically cautious perspective on FDX1-mediated regulation.

 

12. The authors frequently reference Cu⁺ and Cu²⁺ but does not sufficiently discuss the redox chemistry of copper in biological systems. From a biochemical perspective, copper toxicity is inseparable from its redox activity. How does copper redox cycling contribute to Fe–S cluster destabilization independently of lipoylated protein aggregation? To what extent is cuproptosis driven by redox imbalance versus direct metal–protein coordination chemistry? A short subsection or few paragraphs explicitly addressing copper redox biochemistry would significantly enhance mechanistic clarity.

 

Response: We thank the reviewer for the suggestion to expand on copper redox biochemistry; however, we respectfully maintain that a dedicated subsection on this topic would detract from the core focus of the manuscript, which is the comparative therapeutic application of metal-driven RCD. We have already addressed the necessity for mechanistic rigor by integrating specific biochemical markers into the revised Section 1. Specifically, we have defined the transition from cuproplasia to cuproptosis as a metabolic inflection point characterized by the qualitative loss of Fe-S cluster integrity and the FDX1-dependent transition of DLAT to toxic oligomers. Furthermore, we have already clarified in the text that FDX1-mediated copper reduction (Cu2+ to Cu+) is the critical redox event that triggers this proteotoxic collapse. By weaving these technical details, distinguishing coordination-driven aggregation from redox-mediated cluster destabilization, into the existing mechanistic framework, we believe the manuscript provides sufficient clarity without disrupting the narrative flow or excessively increasing the manuscript length.

 

 

13. Figures are informative but would benefit from clearer labeling distinguishing physiological copper signaling from pathological copper overload.

 

Response: We thank the reviewer for this suggestion. To improve the clarity of the mechanistic transitions, we have overhauled the legends and schematic logic of Figures 4 and 7 to explicitly distinguish physiological metal signaling from pathological RCD cascades. These modifications are supported by the expanded discussion in Section 2 on cell-type specificity, where we delineate the causal hierarchy leading to death, specifically how ATP depletion and the inhibition of efflux transporters (like ATP7A) transform regulatory metal flux into lethal trapping. We believe this integrated approach provides a clearer visual and narrative roadmap of the transition from homeostasis to cell death.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive and timely review of ferroptosis and cuproptosis, with a particular emphasis on the interplay between iron and copper metabolism in cancer and neurodegenerative diseases. The authors integrate a large body of recent literature, including advanced nanoplatforms, metal chelators, and molecular regulators (e.g., GPX4, FDX1, ATP7A), and propose a clear dual-paradigm framework in which metal-driven regulated cell death is exploited either as a therapeutic weapon in oncology or as a therapeutic target in neuroprotection.Overall, the manuscript is well-organized, mechanistically detailed, and conceptually ambitious, and it has the potential to be a valuable resource for researchers in redox biology, cell death, cancer therapy, and neurodegeneration. However, several major issues related to balance, clarity, critical evaluation, and translational relevance should be addressed to strengthen the manuscript. My comments are described as follows:

 

Comments:

1. Although the review is exceptionally detailed, much of the content—particularly in Sections 3 and 4—remains largely descriptive, with numerous nanoplatforms and small molecules presented sequentially without sufficient critical comparison; incorporating a more analytical perspective that explicitly addresses limitations such as off-target toxicity, risks associated with metal overload, batch-to-batch variability of nanomaterials, and challenges in reproducibility and scalability would substantially strengthen the manuscript, and the inclusion of a concise comparison highlighting the respective advantages and drawbacks of small-molecule approaches versus nanoplatform-based strategies would further improve clarity, readability, and overall impact.
2. The manuscript repeatedly emphasizes “clinical potential,” yet most strategies discussed remain preclinical; could the authors more clearly distinguish between proof-of-concept studies, early translational research, and clinically evaluated interventions (such as CuII(atsm) and deferiprone), and consider adding a concise discussion of the major barriers to clinical implementation, including pharmacokinetics, metal specificity, long-term safety, and regulatory challenges.
3. The review convincingly highlights crosstalk between ferroptosis and cuproptosis, but in several sections the conceptual boundaries between these pathways appear blurred; could the authors clarify the experimental criteria used to define cuproptosis versus ferroptosis, particularly in nanomaterial-based systems where ROS generation, ATP depletion, and mitochondrial damage overlap, and consider adding a concise table or schematic summarizing key diagnostic markers and experimental readouts to strengthen conceptual rigor?
4. Although the review provides an in-depth and technically sophisticated overview of nanoplatform-based therapeutic strategies, the prominence of this focus occasionally comes at the expense of a more thorough consideration of intrinsic biological regulatory mechanisms, including ferritinophagy, mitophagy, transcriptional regulation of metal transporters, and immune-dependent control of ferroptosis; a more integrated discussion of cell-type specific differences particularly among neurons, oligodendrocytes, and malignant cells in terms of metal homeostasis, redox buffering capacity, and susceptibility to regulated cell death would further strengthen the mechanistic rigor and academic completeness of the manuscript.
5. The figures are generally informative and conceptually strong, but several appear overly complex and may be challenging for non-specialist readers, particularly Figures 4 and 7; simplifying visual elements, providing clearer stepwise explanations in the legends, and ensuring that all abbreviations are explicitly defined in the figure captions would substantially improve clarity and accessibility.
6. Several mechanistic pathways, such as the cascade from GSH depletion to GPX4 inactivation and subsequent lipid peroxidation, are reiterated across multiple sections, and a more strategic consolidation of these descriptions would improve narrative flow while preserving conceptual clarity.
7. The manuscript is generally well written, but some sentences are excessively long and dense.

Comments on the Quality of English Language

The manuscript is generally well written and demonstrates a high standard of scientific English; however, certain sentences are overly long and syntactically dense, which may affect readability, and minor language editing to improve conciseness, sentence structure, and flow would further enhance clarity and overall presentation.

Author Response

Reviewer 3

This manuscript provides a comprehensive and timely review of ferroptosis and cuproptosis, with a particular emphasis on the interplay between iron and copper metabolism in cancer and neurodegenerative diseases. The authors integrate a large body of recent literature, including advanced nanoplatforms, metal chelators, and molecular regulators (e.g., GPX4, FDX1, ATP7A), and propose a clear dual-paradigm framework in which metal-driven regulated cell death is exploited either as a therapeutic weapon in oncology or as a therapeutic target in neuroprotection.Overall, the manuscript is well-organized, mechanistically detailed, and conceptually ambitious, and it has the potential to be a valuable resource for researchers in redox biology, cell death, cancer therapy, and neurodegeneration. However, several major issues related to balance, clarity, critical evaluation, and translational relevance should be addressed to strengthen the manuscript.

Response: We thank the reviewer for the highly constructive and thorough evaluation of our manuscript. We are pleased that the reviewer found the dual-paradigm framework and mechanistic details to be conceptually ambitious and well-organized. Following the reviewer's suggestions, we have made targeted improvements to distinguish clinical from preclinical data and to clarify the boundaries between ferroptosis and cuproptosis.

My comments are described as follows:

Comments:

1. Although the review is exceptionally detailed, much of the content—particularly in Sections 3 and 4—remains largely descriptive, with numerous nanoplatforms and small molecules presented sequentially without sufficient critical comparison; incorporating a more analytical perspective that explicitly addresses limitations such as off-target toxicity, risks associated with metal overload, batch-to-batch variability of nanomaterials, and challenges in reproducibility and scalability would substantially strengthen the manuscript, and the inclusion of a concise comparison highlighting the respective advantages and drawbacks of small-molecule approaches versus nanoplatform-based strategies would further improve clarity, readability, and overall impact.
2. The manuscript repeatedly emphasizes “clinical potential,” yet most strategies discussed remain preclinical; could the authors more clearly distinguish between proof-of-concept studies, early translational research, and clinically evaluated interventions (such as CuII(atsm) and deferiprone), and consider adding a concise discussion of the major barriers to clinical implementation, including pharmacokinetics, metal specificity, long-term safety, and regulatory challenges.

 

Response to 1 and 2: We agree with the reviewer that a clear distinction between preclinical proof-of-concept and actual clinical status is essential. To address this, we have introduced a new section, “5. Translational perspectives and clinical landscape”, which provides the requested analytical perspective by critically evaluating clinical failures and the current bifurcation of the field. In this section, we contrast the success of Cu^II(atsm) with the clinical worsening observed in the FAIRPARK-II trial for DFP and the metabolic constraints identified in Elesclomol phase 3 trials. Furthermore, we explicitly discuss major translational barriers including off-target toxicity, such as the inhibition of iron-dependent dopamine synthesis, biological delivery hurdles related to the BBB and TME, and the industrial challenges of GMP manufacturing and batch-to-batch reproducibility for complex nanomedicines. To further clarify the clinical status of these interventions, we have updated Table 4 with a “Developmental stage” column to more explicitly categorize therapeutic agents, ensuring a clear separation between clinically evaluated interventions and preclinical xenograft models.

 

3. The review convincingly highlights crosstalk between ferroptosis and cuproptosis, but in several sections the conceptual boundaries between these pathways appear blurred; could the authors clarify the experimental criteria used to define cuproptosis versus ferroptosis, particularly in nanomaterial-based systems where ROS generation, ATP depletion, and mitochondrial damage overlap, and consider adding a concise table or schematic summarizing key diagnostic markers and experimental readouts to strengthen conceptual rigor?

 

Response: We thank the reviewer for this insightful suggestion to strengthen the conceptual rigor of the manuscript. We agree that in nanomaterials-based systems, where ROS generation, ATP depletion, and mitochondrial damage often overlap, the conceptual boundaries between death pathways can appear blurred. To address this, we have expanded Table 1 to include specific Diagnostic markers and Metabolic impacts that serve as experimental criteria. We clarify that while mitochondrial stress is a shared feature, cuproptosis is uniquely defined by FDX1-dependent DLAT oligomerization and direct/severe ATP depletion, whereas ferroptosis is primarily characterized by GPX4 downregulation and ROS-mediated lipid peroxidation. Furthermore, the caption for Figure 1 has been updated to explicitly state these experimental criteria, ensuring that the visual representation and the diagnostic roadmap are fully synchronized. This establishes a rigorous framework for identifying the dominant cell death mode within the complex microenvironments created by nanomaterials-based systems.

 

4. Although the review provides an in-depth and technically sophisticated overview of nanoplatform-based therapeutic strategies, the prominence of this focus occasionally comes at the expense of a more thorough consideration of intrinsic biological regulatory mechanisms, including ferritinophagy, mitophagy, transcriptional regulation of metal transporters, and immune-dependent control of ferroptosis; a more integrated discussion of cell-type specific differences particularly among neurons, oligodendrocytes, and malignant cells in terms of metal homeostasis, redox buffering capacity, and susceptibility to regulated cell death would further strengthen the mechanistic rigor and academic completeness of the manuscript.

 

Response: We agree that a more integrated discussion of intrinsic biological mechanisms enhances the manuscript's rigor. Accordingly, we have added a dedicated paragraph to Section 2 that synthesizes the role of ferritinophagy, mitophagy, and the transcriptional regulation of transporters. This addition specifically contrasts the redox buffering capacity and metal homeostasis of neurons, oligodendrocytes, and malignant cells, providing the requested cell-type specific context for the subsequent discussion of nanomedicine strategies.

 

5. The figures are generally informative and conceptually strong, but several appear overly complex and may be challenging for non-specialist readers, particularly Figures 4 and 7; simplifying visual elements, providing clearer stepwise explanations in the legends, and ensuring that all abbreviations are explicitly defined in the figure captions would substantially improve clarity and accessibility.

 

Response: We appreciate the reviewer’s perspective on the visual complexity of the manuscript. To enhance accessibility for a broad readership, we have comprehensively revised the captions for Figures 4 and 7 to follow a clear, stepwise narrative format. By breaking down the intricate biochemical cascades into sequential stages, from initial nanoparticle internalization and metal release to the final execution of cell death, we have transformed these complex models into intuitive, logical roadmaps. Furthermore, to ensure each figure remains self-contained and accessible, we have added explicit abbreviation keys directly within the legends. This allows non-specialist readers to decipher the metabolic and transport proteins involved without constant cross-referencing to the main text, thereby significantly improving the clarity and overall narrative utility of our visual data.

 

6. Several mechanistic pathways, such as the cascade from GSH depletion to GPX4 inactivation and subsequent lipid peroxidation, are reiterated across multiple sections, and a more strategic consolidation of these descriptions would improve narrative flow while preserving conceptual clarity.

 

Response: We appreciate the reviewer’s suggestion to streamline the narrative flow. To ensure conceptual clarity without losing the technical detail required for each specific section, we have implemented structural cross-referencing throughout the manuscript. Specifically, at the beginning of Sections 3.1 and 4.1, we added signpost statements that link the discussion back to the foundational GSH/GPX4/LPO biochemical framework established in Section 1 and Figure 1. This framing allows the reader to maintain a cohesive understanding of the core mechanism while the text focuses on the unique therapeutic applications in oncology and neurodegeneration. By explicitly identifying these pathways as established prerequisites, we have refined the narrative structure to avoid the appearance of redundant descriptions while preserving the mechanistic rigor of each chapter.

 

7. The manuscript is generally well written, but some sentences are excessively long and dense.

 

Response: We thank the reviewer for this observation. To improve the accessibility and readability of the manuscript, we have performed a comprehensive linguistic revision focusing on decomposing excessively long and conceptually dense sentences. Specifically, in Sections 1 and 2, we have restructured compound technical statements, such as those defining the Fe²⁺-driven Fenton reaction, the Cu⁺-mediated proteotoxic stress, and the LAT1-mediated delivery mechanisms, into shorter, standalone sentences. By isolating distinct biochemical steps and ensuring each statement contains a clear, active subject, we have streamlined the narrative flow while preserving the technical and mechanistic rigor required for the discussion. These edits ensure that the complex interplay of metal homeostasis remains clear to a multi-disciplinary audience.

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive and mechanistically detailed review of ferroptosis and cuproptosis, emphasizing the therapeutic modulation of iron and copper homeostasis in oncology and neurodegenerative diseases. The authors provide an ambitious synthesis that integrates classical metal chelation strategies with emerging nanotechnological and genetic approaches. The conceptual framing of regulated cell death (RCD) as either a therapeutic weapon or a therapeutic target depending on disease context is original and compelling. The review is well-written, well referenced, and rich in mechanistic insight.

The review strongly emphasizes therapeutic promise but provides relatively limited discussion of unresolved challenges and limitations. The authors should include a dedicated subsection addressing: toxicity and off-target effects of Fe/Cu nanoplatforms, challenges in clinical translation (delivery, clearance, immunogenicity), context-dependence of cuproptosis and its relevance beyond highly metabolic cancer cells, limitations of current biomarkers for cuproptosis in vivo.... This would significantly strengthen the manuscript’s critical depth.

Cuproptosis is presented as a robust therapeutic opportunity, but the field is still emerging, and its physiological relevance across tissues remains incompletely defined. The authors should more clearly distinguish between well-established ferroptosis mechanisms and emerging cuproptosis pathways, and alo explicitly discuss uncertainties, such as cell-type specificity, copper thresholds, and overlap with other forms of proteotoxic stress.

Figures are conceptually strong but some legends are overly long and repeat text verbatim from the manuscript. Consider simplifying figure legends and focusing on what the figure uniquely adds beyond the text.

Author Response

Reviewer 4

This manuscript presents a comprehensive and mechanistically detailed review of ferroptosis and cuproptosis, emphasizing the therapeutic modulation of iron and copper homeostasis in oncology and neurodegenerative diseases. The authors provide an ambitious synthesis that integrates classical metal chelation strategies with emerging nanotechnological and genetic approaches. The conceptual framing of regulated cell death (RCD) as either a therapeutic weapon or a therapeutic target depending on disease context is original and compelling. The review is well-written, well referenced, and rich in mechanistic insight.

Response: We are grateful for the reviewer’s positive assessment of our conceptual framing and mechanistic depth. To address the request for greater critical depth, we have integrated a nuanced discussion of the challenges, uncertainties, and emerging nature of the cuproptosis field.

The review strongly emphasizes therapeutic promise but provides relatively limited discussion of unresolved challenges and limitations. The authors should include a dedicated subsection addressing: toxicity and off-target effects of Fe/Cu nanoplatforms, challenges in clinical translation (delivery, clearance, immunogenicity), context-dependence of cuproptosis and its relevance beyond highly metabolic cancer cells, limitations of current biomarkers for cuproptosis in vivo.... This would significantly strengthen the manuscript’s critical depth.

Response: We agree that a balanced perspective is essential for a high-impact review. We have added Section 5 (Translational perspectives and clinical landscape) to explicitly address these hurdles. This section provides a critical analysis of the narrow therapeutic window, citing the FAIRPARK-II trial to illustrate how off-target effects (such as the inhibition of iron-dependent enzymes like tyrosine hydroxylase) can lead to clinical worsening despite successful metal sequestration. We also discuss the biological barriers of the BBB and TME, the metabolic constraints of agents like Elesclomol, and the industrial challenges of GMP scale-up for complex nanopharmaceuticals. Regarding biomarkers, we have emphasized the shift toward identifying reliable circulatory indicators, such as serum FDX1 or lipidomic profiles, to monitor RCD activation in vivo.

Cuproptosis is presented as a robust therapeutic opportunity, but the field is still emerging, and its physiological relevance across tissues remains incompletely defined. The authors should more clearly distinguish between well-established ferroptosis mechanisms and emerging cuproptosis pathways, and alo explicitly discuss uncertainties, such as cell-type specificity, copper thresholds, and overlap with other forms of proteotoxic stress.

Response: Regarding the request to more clearly distinguish between established ferroptosis and the nascent field of cuproptosis, we agree that providing clear diagnostic boundaries is essential for the field's advancement. We have implemented several targeted revisions to address this. In Table 1, we expanded the comparison by adding two specific rows for Diagnostic marker and Metabolic impact. This clarifies the contrast between GPX4-regulated lipid peroxidation and ROS levels in ferroptosis versus the FDX1-dependent DLAT oligomerization and direct, severe ATP depletion characteristic of cuproptosis. Furthermore, we updated the legend for Figure 1 to explicitly state that these pathways are distinguished in nanomaterials-based systems by the specific detection of FDX1-dependent DLAT oligomerization versus GPX4-regulated lipid peroxidation. This addition provides a definitive experimental roadmap for differentiation. Finally, in Section 2, we integrated a comprehensive discussion on the biological and metabolic constraints of these pathways. We now explicitly describe how susceptibility is governed by cell-type specific mechanisms, such as NCOA4-mediated ferritinophagy in “iron-addicted” malignant cells versus the unique vulnerability of CNS oligodendrocytes due to high myelin-related iron requirements and low redox buffering capacity. By also utilizing the LDH-dependent clinical outcomes of Elesclomol to demonstrate how mitochondrial respiration versus glycolysis governs copper-induced death, we ensure the manuscript clearly distinguishes between well-established lipid-driven membrane failure and the emerging protein-driven metabolic catastrophe.

Figures are conceptually strong but some legends are overly long and repeat text verbatim from the manuscript. Consider simplifying figure legends and focusing on what the figure uniquely adds beyond the text.

Response: Regarding the concern that figure legends were overly long or repeated the main text, we have conducted a targeted restructuring of our visual documentation. For the more complex diagrams in Figures 4 and 7, we have completely overhauled the captions, replacing long descriptive blocks with a concise, stepwise format. This new structure focuses on the visual logic of the illustrated mechanisms, such as the temporal sequence of nanoparticle degradation and the “vicious cycle” of metal accumulation, rather than re-detailing the biochemical theory. We also appended explicit abbreviation keys to these captions to reduce narrative density. For Figure 1, we have streamlined the existing text and added a specific concluding statement that distinguishes these pathways in nanomaterials-based systems via the detection of FDX1-dependent DLAT oligomerization versus GPX4-regulated lipid peroxidation. These revisions ensure that the legends now focus on what the figures uniquely add to the manuscript as standalone pedagogical tools while significantly improving the overall narrative flow.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All of my concerns were cleared.

Author Response

We thank the reviewer for their positive assessment and are pleased that our revisions have addressed all concerns.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for your revision.

Author Response

We thank the reviewer for their positive assessment of our revised manuscript.