Dear Editor,
We appreciate the insightful editorial interest and in-depth interpretations regarding our recent study entitled “Novel near-infrared probe for monitoring lipid peroxidation-mediated viscosity change in ferroptotic hepatocytes” [
1-
3]. In this correspondence, we discuss the comments of the authors.
Lipid peroxidation, a defining hallmark of ferroptosis, has recently been linked to pronounced alterations in cellular viscosity. During ferroptosis, oxygen radicals attack polyunsaturated fatty acids, generating primary lipid peroxides and secondary products, particularly electrophilic aldehydes such as malondialdehyde and 4-hydroxynonenal. These highly reactive species form covalent adducts with nearby lipids and proteins, thereby compromising membrane integrity and altering physical properties such as molecular packing, fluidity, and viscosity [
4]. The spatiotemporal progression of lipid peroxidation during ferroptosis appears to involve multiple organelles. Our study indicates that lipid peroxidation is initially detected in cytosolic lipid droplets (cLDs), and subsequently propagates toward the nucleus via the endoplasmic reticulum (ER), as evidenced by spatiotemporal fluorescent responses. While our findings suggest a prominent role for LDs under our experimental conditions, other studies have proposed distinct subcellular origins for lipid peroxidation during ferroptosis. Von Krusenstiern et al. (2023) identified the ER membrane as the primary site of lipid peroxide accumulation prior to mitochondrial or plasma membrane damage using stimulated Raman scattering imaging [
5]. Furthermore, Cañeque et al. (2025) recently highlighted lysosomes as critical initiation sites, where redox-active iron and ROS in acidic conditions promote a peroxidation cascade during ferroptosis induced by the lysosomal iron activator fentomycin-1 [
6]. Collectively, these findings suggest that ferroptosis initiation may be organelle-specific, influenced by lipid composition, iron distribution, and the nature of the triggering stimulus.
Beyond these mechanistic insights, numerous studies have demonstrated the role of ferroptosis in metabolic dysfunction-associated steatotic liver disease (MASLD), where excessive hepatic iron accumulation drives oxidative injury. Notably, alterations in liver viscosity have been reported in patients with chronic liver disease. Previous studies have explored liver stiffness and viscosity for staging steatosis and fibrosis [
7], and liver viscosity has recently been integrated into diagnostic frameworks for assessing MASLD severity in clinical practice [
8]. In this context, our work provides a complementary cellular-level perspective by enabling direct visualization of lipid peroxidation–mediated viscosity changes during ferroptosis. These insights may inform the interpretation of macroscopic viscosity measurements and support the development of ferroptosis-targeted diagnostic and therapeutic approaches for MASLD.
We also appreciate the insightful comments regarding the potential link between Cell Death Inducing DFFA Like Effector C (CIDEC) and ferroptosis. While our study primarily utilized CIDEC as a screening purpose rather than exploring its precise mechanistic pathways, we acknowledge its potential relevance. As a key regulator of LD size, we hypothesize that CIDEC may influence LD-dependent ferroptosis by regulating LD dynamics. Although CIDEC is predominantly expressed in adipocytes, it distinguishes itself from other CIDE family members by showing a stepwise and significant increase in hepatocytes during MASLD progression. A very recent study published in the Journal of Hepatology (Zeng et al. 2026) demonstrated that suppression of CIDEB expression reduces LD size and enhances fatty acid oxidation (FAO), thereby ameliorating MASLD [
9]. In this context, the application of TTM-4 could serve as a valuable tool to assess whether ferroptosis is mitigated under such conditions. However, a crucial nuance must be addressed. As shown in Supplementary Fig. 16, downregulation of CIDEC actually exacerbated ferroptosis-induced toxicity under palmitate treatment. This finding suggests that our interpretation should not focus solely on the suppression of LD formation but must also account for the metabolic fate and pathway of released free fatty acids. For instance, liver-specific knockout of CIDEB increases FAO of unsaturated fatty acids, while paradoxically reducing FAO of saturated fatty acids [
9]. This discrepancy implies that the intensity of ferroptosis detected by TTM-4 may vary depending on the specific type of fatty acids treated. Therefore, we believe that further studies on the CIDE family are necessary to reveal these complex interactions and their implications for ferroptosis in MASLD.
A similar paradigm applies to the formation of nuclear LDs (nLDs). While CIDEC is known to regulate the expansion of LDs in the cytosol, we focused on Microsomal Triglyceride Transfer Protein (MTTP) and Apolipoprotein B (ApoB) as key factors of lipid translocation into the nucleus. Sołtysik et al. (2019) suggested that nLD biogenesis occurs not via direct transport from the cytosol, but through the interaction between MTTP and ApoB within the ER [
10]. Specifically, under conditions of sufficient MTTP, ApoB deficiency impairs VLDL secretion from the ER, which subsequently induces the flux of lipids from the ER into the nucleus. Conversely, depletion of MTTP inhibits lipid transport into the ER, thereby preventing nLD formation. This mechanism was further supported by our ability to track the trafficking of oxidized nLDs using TTM-4. Specifically, our data suggest that lipid peroxidation is initiated in the cytosolic compartment and subsequently propagates to the nucleus through the ER, as evidenced by the spatiotemporal fluorescence pattern of TTM-4.
Undoubtedly, ferroptosis is a pivotal cell death mechanism in the pathogenesis of MASLD; however, current diagnostic modalities remain insufficient to detect and monitor this process effectively. In this context, TTM-4, a viscosity-responsive fluorescent probe, holds immense potential for both basic research and clinical applications. From a research perspective, TTM-4 can be employed to identify ferroptosis-related markers associated with lipid droplet accumulation and the generation of oxidized LDs even in the nucleus. Clinically, its application to patient-derived tissues could enable precise characterization of disease stage, thereby informing future therapeutic strategies. We anticipate that TTM-4 will serve as a valuable translational tool, helping to bridge the gap between basic investigations of ferroptosis and the clinical management of MASLD.
FOOTNOTES
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Authors’ contribution
Manuscript drafting: Taeeung Kim, Le Bich Hang Pham. Revision and supervision: Keon Wook Kang, Jeeyeon Lee. Final approval: All authors.
-
Acknowledgements
This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (RS-2021-NR059390 and RS-2026-25476750 to J. Lee), and the Bio&Medical Technology Development Program of the NRF funded by the Korea government (MSIP) (RS-2026-25517589 to KW. Kang).
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Conflicts of Interest
The authors have no conflicts to disclose.
Abbreviations
Cell Death Inducing DFFA Like Effector C
metabolic dysfunction-associated steatotic liver disease
Microsomal Triglyceride Transfer Protein
very low-density lipoprotein
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Yunseo Bong, Wonhyo Seo
Clinical and Molecular Hepatology.2026; 32(3): e437. CrossRef
