Disruption of iron homeostasis sensitizes pancreatic cancer to irreversible electroporation

Domain: chemistry
Published: 2026-01-19
Editorial outcome: major_revision

Abstract
Irreversible electroporation (IRE) is an ablative treatment for pancreatic cancer. It utilizes high-intensity pulsed electric field (PEF) to eliminate cancer cells by irreversibly disrupting cell membranes. However, PEF intensity is distributed unevenly; and cancer cells may survive in regions where it falls below the threshold of complete ablation. We find that iron-base metal organic framework nanoparticles (MOF-Fe) sensitize pancreatic cancer cells to PEF by inducing iron overload and ferroptosis. But their efficacy is diminished by the upregulation of ferritin heavy chain 1 (FTH1), a cellular response to restore iron homeostasis. C20U4V, a proteolysis targeting chimera (PROTAC) derived from arachidonic acid, degrades FTH1 and potentiates MOF-Fe-induced ferroptosis. It is then encapsulated in reactive oxygen species (ROS)-responsive micelles. The resulting M-C20U4V, when combined with MOF-Fe, efficiently induces ferroptosis and boosts PEF ablation efficacy. Therefore, disruption of iron homeostasis represents a potential strategy to lower the risk of tumor recurrence after IRE.

Introduction
Irreversible electroporation (IRE) is a local ablative technique for treating patients with solid tumors, including pancreatic cancer1. During ablation, electrodes are placed in or around the tumor to deliver high-intensity electric pulses. The resulting pulsed electric field (PEF) then eliminates cancer cells by inducing irreversible disruption of their cell membranes2, and promotes systemic anti-tumor immunity through the release of damage-associated molecular patterns3. However, the PEF intensity is unevenly distributed in the tumor4,5. In regions where PEF intensity falls below the threshold for completion of cell eradication, residual cancer cells may survive and ultimately drive tumor recurrence. Current approaches to eliminating these residual cancer cells often rely on inducing apoptosis6–10. Therefore, their efficacy may be compromised when cancer cells harbor mutations in the apoptotic cascades. Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death that can overcome resistance to conventional chemotherapies11,12. During ferroptosis, intracellular iron catalyzes the Fenton reaction to generate reactive oxygen species (ROS); these ROS then oxidize phospholipids in cellular or organelle membranes. The excessive accumulation of oxidized phospholipids ultimately triggers cell membrane rupture and subsequent cell death. Cells have evolved sophisticated mechanisms to counteract ferroptosis via glutathione peroxidase 4 (GPX4)-dependent and -independent pathways13,14. Correspondingly, inhibitors that target these defensive mechanisms, such as erastin and 1S,3R-RLS-3 (RSL3), can induce ferroptosis in cancer cells. The clinical translation of these inhibitors, however, has yet to be achieved15.

Given that iron overload is the primary driver of ferroptosis16, numerous iron-containing nanoparticles have been developed to induce this form of cell death17. These nanoparticles are endocytosed by cancer cells and trafficked to acidic lysosomes, where they degrade and release ferrous or ferric ions. Notably, the activation of lysosomal iron has recently been shown to catalyze ROS production and initiate ferroptosis18. MIL-101 (Fe) metal-organic framework (MOF-Fe) nanoparticles could release iron in a flash fashion19, and therefore are promising candidates to induce iron overload. On the other hand, intracellular iron levels are tightly regulated by iron homeostasis20, and excess iron can be exported out of cells by ferroportin or sequestered in ferritin. Ferritin is a 24-mer protein composed of two subunits: a heavy chain (FTH1) and a light chain (FTL). FTH1 catalyzes the oxidation of ferrous into ferric ions, while FTL facilitates ferric ion biomineralization and long-term storage within ferritin21. Conversely, autophagic degradation of ferritin disrupted iron homeostasis and induced ferroptosis22, while FTH1-targeted siRNA enhanced iron nanoparticle-induced lipid oxidation23.

RNA interference reduces protein expression by disrupting mRNA translation. Changes in protein expression typically occur 24 h after siRNA transfection24. In contrast, pre-existing proteins can be degraded using proteolysis-targeting chimeras (PROTACs)25. PROTACs are bifunctional molecules composed of a target-binding moiety and an E3 ligase-binding moiety. They can recruit E3 ligases to target proteins, leading to their ubiquitylation and subsequent degradation in proteasomes. Mono- or poly-unsaturated fatty acids (MUFA and PUFA, respectively) have been reported to bind to a U-shaped pocket in ferritin and promote iron mineralization26,27. A ferritin degrader was generated as a pyroptosis inducer from oleic acid, an MUFA28. However, MUFAs confer resistance to ferroptosis29. In contrast, PUFAs can promote ferroptosis once incorporated into phospholipids and peroxidized by lipoxygenases30.

We hypothesized that disrupting iron homeostasis using MOF-Fe and a PUFA-derived ferritin-degrading PROTAC could sensitize pancreatic cancer cells to PEF. To test this hypothesis, we assessed the capacity of MOF-Fe to induce ferroptosis and enhance the efficacy of PEF ablation. FTH1 expression was genetically modulated to elucidate its role in MOF-Fe-induced ferroptosis. A PROTAC library was synthesized by conjugating PUFAs, directly or indirectly through different linkers, to a ligand for von-Hippo-Lindau (VHL) E3 ligase, (2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, abbreviated as (S,R,S)-AHPC. The chimeras were screened for their ability to degrade FTH1 in the presence of MOF-Fe. C20U4V, the lead compound derived from arachidonic acid, was assessed for its ability to potentiate MOF-Fe-induced ferroptosis. Lastly, a ROS-responsive polymeric micelles formulation, M-C20U4V, was prepared. The anti-tumor efficacy of PEF, MOF-Fe, and M-C20U4V was evaluated in murine pancreatic tumor models. Our findings suggest that disruption of iron homeostasis is a potential strategy to lower the risk of tumor recurrence after IRE.

Results
PEF upregulated ferroptosis suppressor genes
Although PEF has been reported to generate ROS and cause lipid oxidation31, it is unclear whether such oxidative stress causes ferroptotic cell death. Panc-1 cells were treated with PEF (600 V/cm, 99 pulses), a setting that induced biological alterations without complete elimination of cancer cells32. C11-BODIPY is an established probe for lipid oxidation; malondialdehyde (MDA) is a product of lipid peroxidation33. PEF did not enhance C11-BODIPY oxidation34 or increase MDA concentration (Fig. 1a, b). PEF-treated cells were incubated with ferroptosis inhibitors (liproxstatin-1, abbreviated as Lip-1; ferrostatin-1, abbreviated as Ferr-1) or a reducing agent (tocopherol) at concentrations below their respective half-inhibitory concentrations (IC50, Fig. 1c and Supplementary Fig. 1a). None of these agents rescued the viability of Panc-1 or MIAPaCa-2 cells following PEF (Fig. 1d and Supplementary Fig. 1b). In terms of other cell death modes (Supplementary Fig. 1c–e), PEF-treated cells showed a higher frequency of Annexin V+ populations, an indicator of apoptosis. They also released lactate dehydrogenase (LDH) and expressed a slightly higher level of cleaved-gasdermin D (N-terminal), suggesting the occurrence of pyroptosis. There was no significant change in the expression of phosphor-MLKL and phosphor-RIPK1, two necroptosis markers.

Residual cancer cells following IRE therapy have been reported to be affected by both PEF and the debris of IRE-killed cells35. Panc-1 cells were accordingly treated and their transcriptome analyzed. Upregulated genes in the treated cells were significantly enriched in gene ontology (GO) terms including “regulation of ferroptosis” and “ferroptosis (Fig. 1e). Seven genes were upregulated, and three were downregulated, in the ferroptosis pathway (WikiPathways, WP4313, Fig. 1f). Six of the upregulated genes (HMOX1, SLC7A11, GCLM, GCLC, FTL, and FTH1) are ferroptosis suppressors, and two of the downregulated ones (LPCAT3 and ACSL5) are ferroptosis drivers36. Gene set enrichment analysis (GSEA) unveiled the nuclear factor erythroid 2-related factor 2 (NRF2)-related pathways among the top-upregulated ones (Fig. 1g). NRF2 pathway (WikiPathways, WP2884) regulates cellular response to oxidative stress, and 16 genes were upregulated (Fig. 1h). The protein expression levels of NRF2, heme oxygenase 1 (HMOX1), and FTH1 gradually increased following PEF treatment in Panc-1 cells (Fig. 1i and Supplementary Fig. 1f).

These findings were further verified in KPC-A719 pancreatic tumor models. Tumors treated with PEF ablation presented a necrotic core surrounded by viable tumor regions (Fig. 1j). 4-Hydroxynonenal (4-HNE) is a marker of lipid peroxidation. The frequency of 4-HNE+ pixels was similar between the viable regions of treated tumors and those of sham controls (Fig. 1k). In a separate experiment, KPC-A719 tumors were treated with PEF at a lower voltage (500 V). On day 2 post-PEF, the expression levels of HMOX1 and FTH1 increased by 13.6 and 2.6-fold, respectively (Fig. 1l and Supplementary Fig. 1g). Collectively, these results indicate that low-intensity PEF upregulated ferroptosis suppressor genes in pancreatic cancer cells instead of inducing ferroptotic cell death.

MOF-Fe transiently suppressed tumor recurrence after PEF ablation
We next asked whether MOF-Fe could induce ferroptosis and potentiate PEF. MOF-Fe nanoparticles were octahedral-shaped with a diameter of 98.4 ± 2.0 nm. Their surface ζ-potential was 31.0 ± 2.5 mV after coating with polyethylene imine (PEI) (Fig. 2a–c). Superparamagnetic iron oxide (SPIO) nanoparticles represent another class of iron-containing nanoparticles for theranostic applications and are well-documented to induce ferroptosis37,38. MOF-Fe released iron more rapidly than SPIO at pH 5.2 (Fig. 2d). MOF-Fe degradation was accompanied by size reduction, surface ruffling, and particle agglomeration (Supplementary Fig. 2a). Accordingly, Panc-1 cells formed fewer colonies with MOF-Fe than with SPIO (Fig. 2e and Supplementary Fig. 2b). FerroOrange is a ferrous ion-specific fluorescence probe39. Panc-1 incubated with MOF-Fe showed stronger FerroOrange and oxidized C11-BODIPY staining, along with higher intracellular iron concentrations (Fig. 2f–h). Lipidomics revealed that MOF-Fe elevated the levels of two oxidized and three peroxidized phosphatidylethanolamine (Fig. 2i and Supplementary Data). Conversely, Lip-1 diminished MOF-Fe-induced ROS elevation (DCFA-DA staining) and C11-BODIPY oxidation (Supplementary Fig. 2c, d). These results indicate the MOF-Fe-induced changes were indeed ferroptotic events.

Panc-1 cells treated with PEF plus MOF-Fe formed fewer colonies than those with PEF or MOF-Fe alone (Fig. 2j and Supplementary Fig. 2e). The anti-tumor efficacy of PEF ablation and MOF-Fe was then investigated in vivo. PEF ablation significantly enhanced MOF-Fe retention in KPC-A719 tumors: at 24 h post-injection, tumors in the PEF ablation + MOF-Fe group contained 3.9-fold more iron than those in the MOF-Fe alone group (Fig. 2k). Tumor-bearing mice were treated as outlined in Supplementary Fig. 2f. No significant change of body weight was observed (Supplementary Fig. 2g). The combination of PEF ablation + MOF-Fe was more efficacious in tumor suppression than either monotherapy for up to 20 days from enrollment (Fig. 2l and Supplementary Fig. 2h). Tumor sections were collected on day 4 after enrollment and analyzed (Supplementary Fig. 3). H&E staining displayed extensive necrosis in the tumor of the combination group. Tumors injected with MOF-Fe showed positive staining of iron (Prussian blue). The combination group also exhibited stronger staining of 4-HNE, FTH1, and HMOX1, along with fewer Ki67+ proliferating cells than those from the other groups. These data suggest that MOF-Fe induced ferroptosis and potentiated PEF ablation in pancreatic cancer.

KPC-A719 tumors in the PEF ablation + MOF-Fe group rebounded quickly after day 20 after enrollment (Fig. 2l), indicating the presence of acquired resistance. The underlying mechanism was then investigated. NRF2-related pathways were significantly upregulated in MOF-Fe-treated Panc-1 cells (Fig. 2m), which was corroborated by the enrichment of GO term “response to oxidative stress” (Supplementary Fig. 4a) and NRF2 translocation from the cytosol into the nucleus (Supplementary Fig. 4b). Quantitative proteomics showed that MOF-Fe elevated the expression levels of six proteins in the ferroptosis pathway, all of which belong to ferroptosis suppressors (Fig. 2n). Four proteins in the ferroptosis pathway were downregulated, three of which are ferroptosis drivers. Increase in the mRNA expression levels of HMOX1 and FTH1 were verified with RT-PCR (Supplementary Fig. 4c). Immunoblots confirmed a time-dependent increase in NRF2, FTH1, and HMOX1 protein expression in MOF-Fe-treated Panc-1 and KPC-A719 cells, as well as a dose-dependent increase in FTH1 expression in Panc-1 cells (Fig. 2o and Supplementary Fig. 4d–f). Given that many NRF2-mediated genes are ferroptosis suppressors, we surmised that NRF2 signaling might underlie acquired resistance to the PEF + MOF-Fe combination treatment.

FTH1 promoted resistance to MOF-Fe-induced ferroptosis
FTH1, which sequesters excess iron in cells, belong to both the ferroptosis and NRF2 pathways. Analysis of the GEPIA2 database40 revealed higher FTH1 expression in human pancreatic tumors than in the adjacent normal tissues. Higher FTH1 expression also correlated with worse prognosis in patients with pancreatic cancer (Supplementary Fig. 4g, h). We went on to explore the role of FTH1 in ferroptosis resistance. Deferoxamine (DFO, an iron chelator), Lip-1, and brusatol (an NRF2 inhibitor) suppressed MOF-Fe-induced FTH1 upregulation, suggesting that FTH1 was involved in NRF2-mediated anti-ferroptosis response (Supplementary Fig. 4i–l).

FTH1-overexpressing (OE) and -knockdown (KD) Panc-1 cells were established, respectively (Supplementary Fig. 5a and Fig. 3a). FTH1-overexpression diminished MOF-Fe-induced elevation of ROS (DCFH-DA+ cells %) and C11-BODIPY oxidation, and facilitated colony formation in the presence of MOF-Fe (Supplementary Fig. 5b–d). A reverse trend was observed in FTH1-KD cells (Supplementary Fig. 5e–g). Interestingly, FTH1-OE Panc-1 cells were less sensitive to erastin and RSL3 than the vector control (Supplementary Fig. 6a). FTH1 expression was then restored in FTH1-KD cells (Fig. 3b) using a modified FTH1-overexpressing plasmid, abbreviated as FTH1-OE (m). The coding sequence of this plasmid had synonymous mutations, preventing recognition by the FTH1-targeting shRNAs (Supplementary Tables 6, 8). FTH1 restoration alleviated MOF-Fe-induced ROS and C11-BODIPY oxidation, and increased the number of colonies in the presence of MOF-Fe (Fig. 3c–e and Supplementary Fig. 6b–d). Taken together, these data suggest that FTH1 promoted resistance to MOF-Fe-induced ferroptosis.

Arachidonic acid-derived PROTAC degraded MOF-Fe-induced FTH1
An exploratory PROTAC library was synthesized by conjugating (S,R,S)-AHPC to six fatty acids (oleic acid C18:1, linoleic acid C18:2, linolenic acid C18:3, arachidonic acid C20:4, eicosapentaenoic acid C20:5, and erucic acid C22:1) directly or through five linkers (Fig. 4a). Figure 4b illustrates the structure of C20U4V, a representative chimera prepared by directly conjugating arachidonic acid (AA) to (S,R,S)-AHPC. Chemical characterization of the chimeras is included in Supplementary Figs. 16–73, 81–109. Chimeras with IC50 values lower or close to 10 μM in both Panc-1 and MIAPaCa-2 cells were first identified using CCK-8 assay (Fig. 4c and Supplementary Table 1. C20U4V emerged as the candidate compound after another round of screening for FTH1 degradation in the presence of MOF-Fe (Fig. 4d and Supplementary Fig. 7a). C20U4V was efficiently taken up by Panc-1 cells, elevating HMOX1 and FTH1 mRNA transcription and increasing NRF2 and HMOX1 protein expression (Supplementary Fig. 7b–d). This observation was in consistence with the AA moiety in C20U4V, which has been reported to induce oxidative stress30. In the presence of MOF-Fe, C20U4V degraded FTH1 in a time- and dose-dependent manner in Panc-1, MIAPaCa-2, and KPC-A719 cells (Fig. 4e and Supplementary Fig. 7e–h).

Quantitative proteomics revealed significant expression changes in eight ferroptosis-related proteins in MOF-Fe + C20U4V-treated Panc-1 cells versus MOF-Fe-treated cells (Fig. 4f), including FTH1 and FTL (Supplementary Fig. 7i). Given the presence of AA moiety in C20U4V, we analyzed the AA interactome in MOF-Fe-treated Panc-1 cells using BPAA, a biotin-conjugated AA derivative (Fig. 4g and Supplementary Figs. 8a, 74, 75, 110). Pull-down assay confirmed the interaction between FTH1 and BPAA. Proteomics revealed 648 proteins, including FTH1, that might have interacted with BPAA (Supplementary Fig. 8b). GO analyses of the interactome across biological process (BP), molecular function (MF), and cellular components (CC) yielded enrichment of a broad span of GO terms such as “DNA replication initiation”, “DNA helicase activity”, and “podosome” (Supplementary Fig. 8c–e). This observation was consistent with the ubiquitous roles of AA in cellular physiology. Seven ferroptosis-related and eight NRF2-related proteins were identified in the interactome, among which NQO1 and GCLM was also degraded by C20U4V (Supplementary Fig. 8f–h).

We then analyzed whether C20U4V degraded FTH1 as a PROTAC. C20U4V enhanced FTH1 ubiquitylation in HEK-293T cells transfected with HA-tagged ubiquitin (HA-Ub) and Flag-labeled FTH1 (Flag-FTH1, Fig. 4h and Supplementary Fig. 9a). No FTH1 degradation was achieved by AA or (S,S,S)-C20U4V prepared from (S,S,S)-AHPC, the inactive stereoisomer of (S,R,S)-AHPC (Fig. 4i, j and Supplementary Fig. 9b, c). Neither did C20U4V degrade MOF-Fe-induced FTH1 in VHL-KD cells (Fig. 4k, l and Supplementary Fig. 9d). In FTH1-OE Panc-1 cells (Supplementary Fig. 9e, f), C20U4V degraded FTH1 when cycloheximide (CHX) was added to inhibit de novo protein synthesis. Conversely, (S,S,S)-C20U4V abolished this FTH1 degradation. Collectively, we have demonstrated that C20U4V functioned as a PROTAC and degraded MOF-Fe-induced FTH1.

C20U4V potentiated MOF-Fe and PEF in vitro
The MOF-Fe + C20U4V combination inhibited colony formation in Panc-1, MIAPaCa-2, and KPC-A719 cells with HSA synergy scores above 10 (Fig. 5a and Supplementary Fig. 10a–f). The dual-treatment induced stronger C11-BODIPY oxidation and a higher frequency of DCFH-DA+ cells than MOF-Fe alone (Fig. 5b, c). MDA concentration was similar between the MOF-Fe and dual-treatment groups, both of which were higher than that in the vehicle control (Fig. 5d). Compared to the vehicle control, the dual-treatment upregulated 14 oxidized and 20 peroxidized phospholipids, including PC18:0-22:6-OOH, PE18:0-22:6-OOH, and PE16:0-22:6-OOH (Fig. 5e). Compared to MOF-Fe, the dual treatment also upregulated nine oxidized and 14 peroxidized phospholipids (Supplementary Fig. 10g). Ferroptosis is often accompanied by mitochondria changes41. As shown in Fig. 5f–h, the aggregate (red)-to-monomer (green) ratio of JC-1 staining characterizes mitochondrial membrane potential, which was significantly lowered by all the treatments. Adenosine triphosphate (ATP) concentration was reduced by MOF-Fe or the dual-treatment. The dual-treatment also caused smaller mitochondria with disorganized cristae (yellow arrows).

The dual-treatment sensitized pancreatic cancer cells to PEF. Panc-1 and KPC-A719 cells treated with the “PEF + MOF-Fe + C20U4V” triple-treatment formed fewer colonies than all the other groups (Fig. 5i and Supplementary Fig. 10h, i). Panc-1 cells in the triple-treatment group exhibited significantly higher NRF2 and HMOX1 protein expression than those in the dual-treatment group (Fig. 5j and Supplementary Fig. 10j). Differentially expressed genes (DEGs) between the triple-treatment group and the vehicle control were analyzed via GO analysis. Upregulated genes in the triple-treatment group were enriched in stress response-related GO terms; whereas downregulated genes were enriched in those linked to cell division and DNA replication (Fig. 5k, l). GSEA revealed ten significantly changed Hallmark gene sets (gsea-msigdb.org) when comparing the triple-treatment groups to the vehicle control (Fig. 5m and Supplementary Table 2). The “Hallmark E2F targets” gene set, which is related to the progression of the cell cycle, was downregulated with the lowest NES score (NES = −2.50, p.adjust = 1.67 × 10−9). By contrast, the “Hallmark unfolded protein response” was upregulated with the highest NES score (NES = 2.25, p.adjust = 1.21 × 10−8), indicating a high level of cellular stress.

In pancreatic cancer patient-derived organoids, the dual-treatment significantly reduced organoid number compared to the vehicle control and yielded smaller organoids than all other groups (Fig. 6a). Organoids in the vehicle control exhibited glandular structures consisting of simple cuboidal epithelium with enlarged nuclei. Those treated with MOF-Fe or C20U4V formed fewer and smaller glandular structures, which consisted of simple or stratified epithelial cells of variant sizes. In contrast, the dual-treatment abolished glandular formation, resulting in cell clusters with newly-formed lumens (Fig. 6b). Prussian Blue staining was observed in MOF-Fe and the dual-treatment groups (Fig. 6c), and organoids in the dual-treatment group exhibited significantly fewer Ki67+ cells than all other groups (Fig. 6d). The efficacy of “PEF + MOF-Fe + C20U4V” triple-treatment was then examined in organoids derived from another patient. The triple-treatment produced fewer and smaller organoids than all other groups (Fig. 6e). We also evaluated C20U5V to determine whether the sensitization effect was specific to C20U4V. C20U5V was prepared by directly conjugating eicosapentaenoic acid to (S,R,S)-AHPC. It moderately degraded FTH1 in the presence of MOF-Fe (Supplementary Fig. 7a). As shown in Supplementary Fig. 11, Panc-1 cells treated with the MOF-Fe + C20U5V dual-treatment or MOF-Fe alone showed similar levels of C11-BODIPY oxidation. MOF-Fe and C20U5V synergistically inhibited colony formation in Panc-1 cells. The PEF + MOF-Fe + C20U5V triple-treatment also yielded fewer colonies than PEF or the dual-treatment.

C20U4V encapsulation in ROS-responsive polymeric micelles
C20U4V was insoluble in water and thus cannot be directly used in vivo. We previously reported that ROS levels were elevated in PEF-treated tumors42, and therefore prepared ROS-responsive polymeric micelles to encapsulate C20U4V. Methoxypolyethylene glycol was extended with ε-caprolactone via controlled ring polymerization and capped with 2-bromoisobutyryl bromide. The macroinitiator was then reacted with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acrylate via the atom radical polymerization to yield the resultant triblock copolymer, abbreviated as PEG-PCL-PB (Fig. 7a). Chemical characterization of the products is included in Supplementary Figs. 76–80. The average degree of polymerization of the PCL and PB blocks was about 11 and 15, respectively, which was calculated from comparing the integrals of methylene protons of the PEG block (δ = 3.38, 3.64 ppm), the methylene protons of the PCL block (δ = 1.65, 2.31, and 4.06 ppm), and the aromatic protons of the PB block (δ = 7.21, 7.73 ppm, Supplementary Fig. 80).

C20U4V was encapsulated using the nanoprecipitation method with a loading efficiency of 90% and a loading percentage of 9% by weight. M-C20U4V, the resulting formulation, exhibited an average hydrodynamic size of 78.6 ± 14.5 nm and a surface ζ-potential of −0.2 ± 4.5 mV (Fig. 7b, c). Incubation of M-C20U4V with 100 μM H2O2 accelerated the release of payload (Fig. 7d). C20U4V and M-C20U4V showed similar IC50 values in Panc-1, MIAPaCa-2, and KPC-A719 cells (<6 μM, Supplementary Fig. 12a). Blank micelles were non-toxic to Panc-1 or MIAPaCa-2 cells at concentrations up to 268.5 μg/mL, equivalent to 30 μM C20U4V (Supplementary Fig. 12b). M-C20U4V degraded FTH1 in MOF-Fe-treated Panc-1, MIAPaCa-2, and KPC-A719 cells over a 12–48 h time course (Fig. 7e and Supplementary Fig. 12c, d). M-C20U4V and MOF-Fe inhibited Panc-1 colony formation with an HSA synergy score above 20 (Fig. 7f, g). After labeling with DiR, a near-infrared fluorescence dye, M-C20U4V was intravenously injected into mice bearing KPC-A719 tumors (Fig. 7h, i). Its uptake value in untreated tumors was 11.2 ± 2.4% ID/g (percent of injected dose per gram of tissue). PEF ablation at 4 h after M-C20U4V injection did not significantly change its tumor uptake (14.1 ± 2.0% ID/g). Taken together, we have prepared a ROS-responsive formulation in M-C20U4V for in vivo evaluation.

MOF-Fe and M-C20U4V enhanced PEF ablation in vivo
The anti-tumor efficacy of PEF ablation, MOF-Fe, and M-C20U4V was first evaluated in subcutaneous KPC-A719 tumor models, using a treatment schedule outlined in Supplementary Fig. 12e. Growth curves of individual tumors are shown in Supplementary Fig. 12g. As shown in Fig. 7j, tumors of the MOF-Fe + M-C20U4V dual-treatment grew at a similar rate to that of the sham control. Tumors in the PEF ablation group experienced a brief growth delay for about 8 days, but then bounced back quickly. In contrast, tumors receiving the PEF ablation + MOF-Fe + M-C20U4V triple-treatment shrank to a minimal volume for about 22 days, and then grew at a slower rate than those of all other groups. Three out of 14 mice in the triple-treatment group had no visible tumors at the study’s conclusion (day 60). The average tumor size in the triple-treatment group on day 60 was significantly smaller than those in all the other groups (p < 0.0001). A similar trend of therapeutic efficacy was observed in KPC-A719 orthotopic models (Fig. 7k), which are more relevant to human malignancy43. Mice in the triple-treatment group exhibited a median survival of 27 days, significantly longer than those in the control (11 days, p < 0.0001), PEF ablation (12 days, p = 0.001), or dual-treatment (14.5 days, p = 0.005) groups. No significant change in body weight was observed in either model (Supplementary Fig. 12f, h).

Intratumoral immune cells in orthotopic KPC-A719 tumors were analyzed with flow cytometry on day 4 after enrollment (Fig. 8a–j). The gating strategy is shown in Supplementary Fig. 13a–c. Tumors in the triple-treatment group contained significantly more CD45+, CD3+ T, and CD4+ T cells than those in the other groups. The abundance of CD8+ and Interferon-γ+ (IFNγ+) CD8+ T cells were similar in PEF- and triple-treatment groups, both of which were higher than those in the other two groups. There was no significant difference in the CD8+ T cell-to-Treg ratio among all groups. CD8+ T cell depletion alongside the triple-treatment resulted in significantly larger tumors on day 11 post-enrollment (Supplementary Fig. 13d–h). Thus, CD8+ T cells at least partially contributed to the therapeutic efficacy of the triple-treatment.

Short-term response of KPC-A719 orthotopic tumors were also examined on day 9 after enrollment. Tumors of the sham control presented a mixture of small or medium-sized irregular glands and poorly differentiated sarcoma-like tumor cells. Some tumor glands formed a cancer nest surrounded by desmoplastic stroma. Tumor cells exhibited enlarged and hyperchromatic nucleus, multiple nucleoli, and pathological mitosis (Supplementary Fig. 14). Necrotic regions were visible in tumors treated with MOF-Fe + M-C20U4V, especially around the sites of MOF-Fe accumulation. Infiltration by macrophages and neutrophils were present at the borders between the necrotic and viable regions. Both PEF and the triple treatment induced extensive areas of necrosis. Residual cancer cells were present outside the ablation zone of PEF-ablated tumors, whereas extensive infiltration by neutrophils and macrophages were observed in the triple treatment group. Tumors in the triple-treatment groups had the lowest proportion of Ki67+ proliferating cells, and the highest percentage of FTH1+, 4-HNE+ or HMOX1+ pixels among all four groups (Fig. 8k–n). The triple treatment induced IFNγ levels comparable to those from PEF ablation alone (Fig. 8o). It also resulted in a massive infiltration of CD68+ macrophages, accompanied by a reduction in CD206+ M2 macrophages (Supplementary Fig. 15a, b). Together, we have shown that the triple-treatment exhibited satisfactory anti-tumor efficacy and activated anti-tumor immunity.

Lastly, we investigated the acute toxicity of the triple treatment in mice bearing KPC-A719 tumors (Supplementary Fig. S15c, d). No pathological change was found in the heart, liver, spleen, lung, or kidneys. The indexes of liver function (alanine transaminase, aspartate transaminase), kidney function (creatine and blood urea nitrogen), and heart (creatine kinase) function were similar between treated and control mice.

Methods
Research compliance
All animal studies were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology, complied with the ARRIVE guidelines, and were conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals at specific pathogen-free facilities. Informed consent was obtained from all patients involved in the study. Information on sex and gender was obtained by self-reporting from human participants with consent to share at the individual level. The study on patient-derived organoids was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Approval number: [2021] IEC (284), approval date: March 3, 2021. All experiments were conducted in compliance with the Helsinki Declaration.

Chemicals and reagents
All chemicals were purchased from Aladdin Biochemical Technology (Shanghai, China) or Sigma-Aldrich (MO, USA) and used as received. Ferroptosis inhibitors were obtained from TOPSCIENCE (Shanghai, China). Superparamagnetic iron oxide (SPIO) nanoparticles (average size = 7 nm, carboxylate modified) were purchased from XFNANO (Nanjing, China). All small-molecule probes used in the manuscript meet the community requirements in https://www.chemicalprobes.org/information-centre.

Synthesis and characterization of MOF-Fe
MOF-Fe was synthesized using the hydrothermal method via previous reports. Briefly, ferric chloride hexahydrate (FeCl3·6H2O, 0.675 g, 2.5 mmol) and aminoterephthalic acid (0.226 g, 1.25 mmol) were dissolved in dimethyl formamide (DMF, 25 mL) along with acetic acid (1 mL). The mixture was heated in a Teflon-coated autoclave at 120 °C for 20 h. The product was collected by centrifuge, washed with DMF and ethanol, and then dried under vacuum. The particles were then dispersed in an aqueous solution of PEI (MW + 25,000, 10 mg/mL), and uncoated PEI was removed by centrifuging. The resulting nanoparticles, termed MOF-Fe, were stored at 4 °C and used within one week. Iron content was determined with an iron analysis kit (Solarbio, Beijing, China). MOF-Fe morphology and size were examined using scanning electron microscopy (SEM, Clara, TESCAN, Shanghai, China). Surface ζ-potential was measured on Malvern Zetasizer Nano-ZS (Malvern Instruments, MA, USA).

Synthesis of ferritin-degrading PROTACs
C20U4V synthesis is described as an example. (S,R,S)-AHPC mono-hydrochloride (70 mg, 0.16 mmol) and arachidonic acid (48 mg, 0.16 mmol) were added to a round-bottom flask and dissolved with N,N-dimethylformamide (DMF, 2 mL). After adding N, N-diisopropylethylamine (115 μL, 0.64 mmol) and HATU (124 mg, 0.32 mmol), the mixture was stirred for 2 h at room temperature and diluted with distilled water (20 mL). After extraction with ethyl acetate, the organic phase was collected, washed with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated at 40 °C under reduced pressure. The crude product was purified using flash column chromatography on silica gel (dichloromethane/methanol = 20/1, vol/vol) to yield a colorless oil. The synthesis procedures and structure characterization (1H-NMR, 13C-NMR, and ESI mass spectra) of all the chimeras are included in the Supplementary Information.

Synthesis of ROS-sensitive MPEG-PCL-PB
Step 1: Methoxypolyethylene glycol (1.00 g, 0.2 mmol) was dissolved in anhydrous toluene (1 mL) in a round-bottom flask. ε-Caprolactone (0.23 mL, 2.2 mmol) and Sn(Oct)2 (15.2 μL, 4.7 mmol) were added, and the solution was stirred at 105 °C for 12 h under a nitrogen atmosphere. The mixture was precipitated in petroleum ether and dried in a vacuum. The product, PEG-PCL, was obtained as a white solid (1.2 g, yield = 96%).
Step 2: PEG-PCL (1.2 g, 0.2 mmol) was dissolved in DCM (5 mL) along with triethylamine (0.38 mL, 3 mmol) in an ice-water bath under a nitrogen atmosphere. 2-Bromoisobutyrl bromide (0.11 mL, 2 mmol) was dropped in. The reaction was gradually warmed to room temperature and stirred overnight. The mixture was concentrated at 40 °C under reduced pressure. The crude product was redissolved in tetrahydrofuran (THF) and reprecipitated in hexane to afford PEG-PCL-BIBB as a pale-yellow solid (1.1 g, yield = 90%).
Step 3: (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) methanol (8.6 g, 37 mmol) and triethylamine (6.2 mL, 44 mmol) was dissolved in dichloromethane (DCM, 30 mL) in an ice-water bath under a nitrogen atmosphere. Acryloyl chloride (3.6 mL, 44 mmol) was added dropwise. The reaction was stirred at room temperature for 2 h and quenched with water. The mixture was extracted with ethyl acetate (3 × 50 mL), and then the organic phase was washed with saturated aqueous sodium chloride solution (50 mL). The mixture was concentrated at 40 °C under reduced pressure. The crude product was purified by silica gel column chromatography (hexane/EA = 10:1, v/v) to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acrylate as a white solid (11 g, yield = 92%).
Step 4: PEG-PCL-BIBB (0.5 g, 0.077 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acrylate (1.2 g, 3.85 mmol) and tris(2-dimethylaminoethyl)amine (Me6TREN, 40 μL, 0.15 mmol) was dissolved in anisole (2 mL). The mixture was evacuated and refilled with nitrogen for three cycles. Cuprous bromide (CuBr, 22 mg, 0.15 mmol) was added under nitrogen protection, and the mixture was stirred at 60 °C for 24 h. Anisole was removed under reduced pressure. The residue was redissolved in DCM, passed through a neutral alumina column to remove the catalyst, condensed under reduced pressure to afford PEG-PCL-PB as a pale gray solid (0.45 g). The chemical structures were confirmed by 1H-NMR (Supplementary Information).

M-C20U4V polymeric micelles
Drug-loaded micelles were prepared using the nanoprecipitation method. Briefly, MPEG-PCL-PB (100 mg) and C20U4V (10 mg) was dissolved in acetone (1 mL) and rotated in the dark for 1 h. Ultrapure water (2 mL) was added quickly to the acetone solution under vigorous vortexing, and the mixture was rotated at 100 mBar, 45 °C for 10 min to remove acetone. After dialyzing against ultrapure water at 4 °C, the final dispersion was filtered through a 0.22-μm syringe filter to yield the drug-loaded micelles M-C20U4V. Drug loading capacity was measured using high-performance liquid chromatography (E Classical 3100, Elite HPLC, Dalian, China) equipped with an ultraviolet-visible detector (wavelength = 254 nm). The micelles were dissolved in dimethyl sulfoxide (DMSO) to release the payload, filtered with a 0.22-μm filter, and eluted through a C18 reverse-phase column (Ø4.6 × 200 mm, 5-μm particles). The mobile phase was a gradient mixture of (A) ultrapure water and (B) acetonitrile, from 10% B at 0 min to 90% B at 20 min. Size and surface ζ-potential of M-C20U4V were examined on Malvern Zetasizer Nano-ZS (Malvern Instruments, MA, USA).

Cell lines and animal models
Panc-1 and MIAPaCa-2 human pancreatic cancer cell lines were purchased from the Cell Resource Center, Peking Union Medical College, the Headquarters of the National Infrastructure of Cell Line Resources. All cell lines were authenticated with short-tandem repeat (STR) profiling. KPC-A719 cells were isolated from the established tumor of a female KrasG12D/+; p53R172H/+; Pdx-1-Cre (KPC) genetically engineered mouse. Gene mutations were verified by Sanger sequencing. All cells were routinely tested for mycoplasma contamination.
Subcutaneous tumor models were established by inoculating 2 × 106 KPC-A719 cells at the lower left back of 8-week-old C57BL/6 female mice (Hubei Biont Biological Technology, Wuhan, China). Orthotopic models were established by directly injecting 5 × 105 KPC-A719 cells in 15 μL Hank’s balanced salt solution (HBSS) into the head of the pancreas10. Tumor growth was monitored using a caliper (subcutaneous model) or by palpation under anesthesia (orthotopic model). Mice were enrolled for treatment once the tumors reached 7 mm in one dimension, and those with orthotopic tumors exceeding 10 mm in one dimension were not enrolled. The maximal tumor burden for subcutaneous models permitted by the institutional review board is: maximal diameter exceeding 20 mm, the calculated tumor size above 3000 mm3 as determined by the formula: size = 0.5 × length × width2, or the tumor weight more than 15% of mouse body weight, whichever is reached first. The maximal tumor size/burden was not exceeded in the study. Mice bearing orthotopic tumors were euthanized once they reached humane endpoints defined by the National Institute of Health, including rapid weight loss, anorexia, lethargy, abnormal posture, etc.

Pancreatic cancer patient-derived organoids
Patient information, including sex and gender, is summarized in Supplementary Table 3. Sex and gender of participants were determined based on self-report. The information was collected with consent for sharing individual-level data. Disaggregated number for the individual experiments was provided in the source data. Sex or gender were not considered in the study design because pancreatic cancer occurs across the whole population. Patient-derived organoids were cultured according to a previous report69. Briefly, patient tumor tissues were minced into fragments and digested with organoid tissue digestion solution (D23013, D1 Medical Technology, Shanghai, China). Phosphate-buffered saline (PBS) was added to stop the digestion, and the cell suspension was passed through a 100-μm-mesh and centrifuged. The cell pellet was re-dispersed in PBS and mixed with diluted Matrigel (D23016, D1 Medical Technology) to a density of 100–1000 cells/μL. Fifty μL of the mixture was added to the center of one well of a 24-well plate, and incubated at 37 °C for 15 min. Each well was then added with 500 μL of organoid culture medium (K211M05, D1 Medical Technology), and the plate was returned to incubation at 37 °C.

Treatment with pulsed electric field (PEF)
Electric pulses were generated from an HW-E01 electroporation system (Huiwei Medical, Ningbo, China). For in vitro studies, electric pulses were delivered to adherent cells using a Petri Pulse electrode composed of 13 gold-plated electrodes 2 mm apart (Catalog #45-0130, BTX Harvard Apparatus, MA, USA), or to cell suspension in an electroporation cuvette fitted with two aluminum plate electrodes 4 mm apart (Catalog #1652081, Bio-Rad, Hercules, CA, USA). PEF parameters were: pulse intensity = 0 to 600 V/cm, pulse duration = 100 μs, pulse repetition frequency = 1 Hz, number of pulses = 99.
For in vivo studies, electric pulses were delivered using a two-needle array electrode with a 5-mm gap (Catalog #45-0168, BTX Harvard Apparatus). PEF parameters were: pulse voltage = 500 or 1150 V, pulse duration = 100 μs, pulse repetition frequency = 1 Hz, number of pulses = 99. The electrode was inserted to completely penetrate the tumor nodule. After one round of PEF treatment, electrodes were retrieved and inserted along the orthogonal direction to deliver 99 more pulses with the same parameters. PEF in vivo at 1150 V was denoted as PEF ablation throughout the manuscript, while low-intensity PEF in other experiments was specified with the exact pulse intensity or voltage.

RNA sequencing and data analysis
RNA sequencing was performed at Novogene Co. Ltd. Total RNA was extracted from treated cells and assessed for quantity and quality using RNA Nano 6000 assay kit for the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). mRNA was isolated using poly-T oligo-attached magnetic beads and fragmented with divalent cations at elevated temperature in a buffer for first-strand synthesis. First-strand cDNA was prepared using random hexamer primers and M-MuLV reverse transcriptase. Second-strand cDNA was synthesized using DNA polymerase I and dNTPs. The cDNAs were then end-processed, purified on an AMPure XP system (Beckman Coulter, Beverly, CA, USA), amplified through PCR reactions, and further purified with AMPure XP beads to generate the libraries. Qualified libraries were pooled and sequenced on an Illumina NovaSeq 6000 platform. After cleaning and quality validation, paired-end clean reads of the sequencing data were aligned to the reference genome using Hisat2 (v2.05). The number of reads mapped to each gene was counted using featureCounts (v1.5.0-p3).
Differential expression analysis was performed using the edegR R package (v4.4.2). P values were adjusted via Benjamini and Hochberg’s algorithm to control the false discovery rate (FDR). Differentially expressed genes (DEGs) with a p.adjusted < 0.05 and a |fold-change| > 1.5 was considered statistically significant. Gene ontology (GO) enrichment analysis of DEGs were conducted using clusterProfiler R package (v4.14.4). Gene set enrichment analysis (GSEA) was performed using GSEABase R package (v1.68.0).

Quantitative proteomics analysis
Quantitative proteomics was performed at Novogene Co. Ltd. Panc-1 cells (n = 2 or 3 independent experiments specified in corresponding figure legends) were lysed under ultrasonication with DB lysis buffer composed of 8 M urea and 100 mM triethylammonium bicarbonate (TEAB) at pH 8.5, and centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was incubated with dithiothreitol (DTT) at 56 °C for 1 h, and alkylated with iodoacetamide for 1 h. Protein quality was verified using electrophoresis. The samples were digested with trypsin in the DB lysis buffer overnight, acidified with formic acid to pH 3, and centrifuged at 12,000 × g for 5 min. The supernatant was eluted through a C18 desalting column and lyophilized.
The lyophilized powder was dissolved in 0.1% formic acid, and injected into a Vanquish Neo upgraded UHPLC system equipped with a C18 pre-column (5 mm × 300 μm, 5 μm, Thermo 174500) heated at 50 °C and a C18 analytic column (PepMapTM Neo UHPLC 150 μm × 15 cm, 2 μm, Thermo ES906). The mobile phases were: A (0.1% formic acid in 100% water) and B (0.1% formic acid in 80% acetonitrile). The elution conditions were: flow rate = 2.5 mL/min, gradient = 4% B (0 min), 5% B (0.2 min), 75% B (4 min), 65% B (5.8 min), 99% B (6.2 min). The eluent was analyzed on a Thermo Orbitrap Astral mass spectrometer operated in a data-independent acquisition mode. The full scan was performed with the following parameters: scanning range = 380 to 980 m/z, primary resolution = 240,000 (200 m/z), AGC = 500%, parent ion window size = 2 m/z, number of DIA window = 300, NCE = 25%. The second scan was then acquired with the following parameters: scanning range = 150 to 2000 m/z, sub-ion resolution Astral = 80,000, maximal injection time = 3 ms.
All the raw files were analyzed using the DIA-NN library search software (version 1.9.2). The library search parameters were: a mass tolerance of 10 ppm for precursor ions and 0.02 Da for fragment ions. Cysteine was modified by alkylation, methionine by oxidation, and N-terminal modifications include acetylation, loss of methionine, and loss of both methionine and acetylation. Two missed cleavage sites were allowed at most. The search results were further filtered by retaining only credible peptide spectrum matches with a confidence level above 99%. Differential expression of proteins was analyzed using EdgeR R package (v 4.2.2), and adjusted with Benjamini–Hochberg (BH) algorithm. Differentially expressed proteins with a p value < 0.1 and |fold-change| > 1.3 were considered statistically significant.

Lipidomics
The lipidomics study was conducted at LipidALL Technology. Panc-1 cells were treated with MOF-Fe and/or C20U4V for 48 h (n = 5 independent experiments), homogenized in a mixture of chloroform/methanol/H2O (v/v/v = 3/6/1), and incubated at 1500 RPM for 1 h at 4 °C. Lipids were twice extracted from the mixture using chloroform, and the combined organic phase was pooled and dried in a SpeedVac under OH mode. The aqueous phase and cell pellet were also dried in a SpeedVac vacuum concentrator under H2O mode for protein quantification. Lipidomics analyses were conducted using an ExionLC-AD-Sciex QTRAP 6500 PLUS as previously reported70. Oxidized phospholipids were separated by normal phase-HPLC using a TUP-HB silica column (150 mm × 2.1 mm, 3 μm) with the following conditions: mobile phase A = chloroform/methanol/ammonium hydroxide = 89.5/10/0.5, mobile phase B = chloroform/methanol/ammonium hydroxide/water = 55/39/0.5/5.5. Individual lipid species were quantified by referencing to spiked internal standards. For each phospholipid, the statistical significance of the change between two comparison groups was determined using multiple two-tailed unpaired t-tests without correction. Differentially expressed phospholipids with a p value < 0.05 and |fold-change| > 1.3 were considered statistically significant.

Quantitative RT-PCR and immunoblotting
Total RNA was extracted using a FastPure cell/tissue total RNA isolation kit (Catalog #RC101-01, Vazyme, Nanjing, China), and transcribed to cDNA using HiScript II RT supermix (Vazyme). Quantitative RT-PCR was conducted using ChamQ SYBR qPCR mastermix (Vazyme) with primers listed in Supplementary Table 4 on a Bio-Rad CFX96 real-time PCR system (Bio-Rad). The relative mRNA expression was calculated using the 2−ΔΔCt algorithm and normalized to that of 18S endogenous control. The original Ct values are included in the Source Data file.
Cells were lysed in radio-immunoprecipitation analysis (RIPA) buffer supplemented with protease/phosphatase inhibitors. Protein lysate was denatured, fractionated on tris-glycine polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk, and sequentially incubated with primary antibodies (Supplementary Table S5), horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence substrate. The blots were scanned using a Tanon 5200 Chem-Image system (Tanon Science & Technology, Shanghai, China) and analyzed using ImageJ (v1.54g).

Generation of gene knockdown and overexpression cell lines
Gene knockdown plasmids were constructed by ligating pLKO.1 vector with non-targeting shRNA (NC) or shRNAs targeting human FTH1 (shFTH1) or VHL (shVHL, Supplementary Table 6). The coding sequences of human FTH1 were amplified by RT-PCR using the corresponding primers (Supplementary Table 7) and cloned into pHAGE_puro vector. The coding sequence for FTH1 restoration (Supplementary Table 8) was cloned into the pTSBX-CMV-EF1-BSD vector. The obtained recombinant lentiviral plasmids were verified with sequencing and co-transfected with pMD2.G envelope plasmid and psPAX2 packaging plasmid into 293T cells using polyethylene imide (PEI; Polysciences, PA, USA). After 24 h, the transfection medium was changed to complete growth medium, which was collected after another 48 h, sterile-filtered, and used as lentivirus supernatant. Panc-1 cells were transduced with the lentivirus supernatant in the presence of polybrene (4 μg/mL, Yeasen Biotechnology, Shanghai, China) and selected with puromycin (2.5 μg/mL) or blasticidin (10 μg/mL). Efficiency or gene knockdown or overexpression was confirmed by immunoblotting.

FTH1 Ubiquitylation determined with immunoprecipitation
The coding sequences of human FTH1 and ubiquitin (Ub) were amplified by RT-PCR using the corresponding primers (Supplementary Table 7) and cloned into pRK-Flag and pCMV-HA vectors, respectively. The obtained plasmids were verified by sequencing and co-transfected into HEK-293T cells for 48 h. The cells were treated with C20U4V (10 μM) for 6 h in the presence of the proteosome inhibitor MG132 (10 μM). Protein lysates were extracted using the immunoprecipitation lysis buffer. Protein A+G magnetic beads were incubated with anti-Flag antibody at 4 °C for 4 h, washed, and mixed with the protein lysate at 4 °C overnight (P2179S, Beyotime Biotech, Shanghai, China). The beads were collected on a magnetic separation rack and washed. Target proteins were dissociated from the beads and denatured by heating in 2× SDS buffer at 95 °C for 5 min, and analyzed by immunoblotting.

FTH1 pull-down assay
Biotin-PEG3-arachidonic acid (BPAA) was first synthesized. Briefly, arachidonic acid (87.3 mg, 0.29 mmol) was first activated with HATU (181.7 mg, 0.48 mmol) and DIPEA (124 mg, 0.96 mmol) in dichloromethane (DCM, 3 mL) for 10 min at room temperature. Biotin-PEG3-NH2 (100 mg, 0.24 mmol) was then added, and the mixture was stirred overnight in the dark. The crude product was condensed in vacuo and purified via flash column chromatography on silica gel (petroleum ether then DCM) to yield a brown solid (yield = 90%). The chemical structure was confirmed by 1H-NMR, 13C NMR and ESI mass spectrum (Supplementary Figs. 74, 75, 110).
For the pull-down assay, Panc-1 cells were seeded and incubated with MOF-Fe and different concentrations of BPAA for 24 h. The cells were lysed, and incubated with streptavidin-coated magnetic beads for 4 h at 4 °C. After washing, the absorbed proteins were dissociated from the beads and denatured by heating in 2× SDS buffer at 95 °C for 5 min, and analyzed by immunoblotting.

Qualitative proteomics analysis
Qualitative proteomics was conducted at Novogene Co. Ltd. Proteins were eluted off the magnetic beads from the pull-down assay, and processed in a similar procedure as in the section “Quantitative proteomics analysis”. The lyophilized samples were dissolved in 0.1% formic acid, and injected into an EASY-nLCTM 1200 nano-upgraded UHPLE system with a homemade C18 Nano-Trap column (4.5 cm × 75 μm, 3 μm). The mobile phases were: A (0.1% formic acid in 100% water) and B (0.1% formic acid in 80% acetonitrile). The elution conditions were: flow rate = 800 nL/min, gradient = 2% B (0 min), 6% B (2.5 min), 6% B (5 min), 25% B (25 min), 40% B (27 min), 80% B (29 min), 80% B (30 min). The eluent was analyzed on a timsTOF Pro 2 mass spectrometer with a Captive Spray ion source (ion spray voltage = 1.4 kV, drying gas temperature = 180 °C) in a data-independent acquisition mode. The full scan was performed using the following parameters: m/z = 100–1700, ramp time = 100 ms, lock duty cycle = 100%, scan window size = 25 Da, and number of mobility window = 2. The resulting spectra were searched using Spectronaut-Pulsar with the following parameters: mass tolerance for precursor ion = 10 ppm, mass tolerance = 0.02 Da, maximum missed cleavage sites = 2. Identified proteins were analyzed using Venn Diagram and GOR packages (v. 4.2.2).

Immunofluorescence (IF) and Immunohistochemical (IHC), and multiplex IF staining
For IF staining, cells seeded on glass coverslips were fixed with 4% paraformaldehyde, and incubated with primary antibodies (Supplementary Table S5) and fluorescence-labeled secondary antibodies. Cell nuclei were counterstained with Hoechst 33342 or DAPI. Fluorescence images were acquired under a confocal fluorescence microscope, and analyzed with ImageJ. For IHC staining, formalin-fixed paraffin-embedded (FFPE) tumor sections were deparaffinated, hydrated, and heated in antigen-retrieval buffer (10 mM sodium citrate with 0.1% Tween-20, pH 6.0) at 95 °C for 20 min. The tumor sections were blocked with 5% bovine serum albumin (BSA), incubated with primary antibodies (Supplementary Table S5), anti-rabbit poly(horseradish peroxidase) (Catalog # SV0002, Boster Bio, Wuhan, China), and visualized via 3,3’-diaminobenzidine (DAB) reaction. Cell nuclei were counterstained with hematoxylin. Images were captured under a bright-field microscope, and analyzed using ImageJ. For multiplex IF, rehydrated FFPE sections were heated in an antigen-retrieval buffer (EDTA, pH 9.0) in a pressured heater, and blocked with 3% H2O2 and 10% goat serum. Each target was stained by sequential incubation with the respective primary antibody, goat anti-rabbit IgG H&L (HRP) (Catalog # ab205718, Abcam), and iFluor® tyramide (Supplementary Table S5). Cell nuclei were counterstained with DAPI. Images were scanned using a fluorescence scanner (Pannoramics SCAN II, 3D HISTECH, Budapest, Hungary) and analyzed.

Flow cytometry
Tumors were collected, weighed, and digested in RPMI-1640 medium supplemented with collagenase IV (1 mg/mL), hyaluronidase (0.4 mg/mL), DNase I (0.1 mg/mL), and BSA (10 mg/mL) in a 37 °C water bath for 30 min with constant shaking. The cell suspension was filtered through a 40-μm-mesh, treated with red blood cell lysis buffer, and resuspended in phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS) and 1 mM EDTA. After staining with Zombie NIR fixable viability kit (Catalog #423106, BioLegend, San Diego, CA, USA), the cells were fixed and permeabilized by adding methanol pre-chilled to −20 °C during vortexing and incubating on ice for 10 min. After washing, the cells were stained with corresponding antibodies (Supplementary Table S9), and analyzed on Beckman DxFLEX cytometer (Beckman Coulter, Brea, CA, USA). Data were processed using FlowJo Software V10 (BD Biosciences, San Jose, CA, USA).

Biodistribution of MOF-Fe and M-C20U4V
Tumors and major organs were collected 24 h after the intratumoral injection of MOF-Fe. After weighing, the samples were digested with nitric acid and dried. Iron content was measured using atomic absorption spectroscopy (Varian AA-240FS, Agilent, Wuhan, China). DiR-labeled M-C20U4V micelles were prepared in the same manner as M-C20U4V except that DiR (25 mg/mL in DMSO, 10 μL) was also added to the acetone solution. Micelles were intravenously injected into tumor-bearing mice 4 h before PEF ablation. At 48 h post-injection, major organs and tumors were collected, weighed, and measured for their fluorescence intensity on a fluorescence scanner (Pearl Trilogy, LI-COR, Shanghai, China).

Statistics and reproducibility
The details of data presentation, sample size, statistical analysis method, and significance of differences are specified in figure legends. Sample size was determined with reference to the experiment designs reported in similar previous studies. Randomization was adopted for all experiments. Researchers were blinded during data analyses since the samples were code-named. Experiments were performed in a non-blinded manner because the experiments involved multiple treatments that required clear labeling for operation and data collection. No data were excluded from the analysis. All experiments were independently repeated at least three times with consistent results. Significant difference was assessed using one-tailed or two-tailed unpaired Student’s t-test, or one-tailed ratio-paired t-test for the comparison of two data sets, and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison for three or more datasets. Log-rank test was used for Kaplan–Meier survival analysis. Synergy score was calculated using Synergistic effects were determined using SynergyFinder Plus71. A p value less than 0.05 was considered statistically significant. Data were analyzed using Prism GraphPad 9 (GraphPad Software, San Diego, CA, USA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled \"Disruption of Iron Homeostasis Sensitizes Pancreatic Cancer to Irreversible Electroporation\" offers valuable insights into pancreatic cancer therapy using a triple-combination strategy (IRE, MOF-Fe, and M-C20U4V). In particular, the authors synthesized C20U4V, a novel PROTAC, to target FTH1. Overall, the work is well-executed, but a few additions and clarifications are necessary prior to publication. The authors are requested to carefully address the following comments to improve the manuscript.\nMAJOR CONCERNS\n1. In Figure 2, the experiments do not clearly demonstrate that MOF-Fe induces ferroptosis.\n1-1) In Figure 2D, the correlation (R-value) between FTH1 expression and the AUCs of three chemicals (Erastin, 1S,3RSL-3, and ML162) ranges between 0.114 and 0.152. Are these values truly meaningful? Please discuss their significance.\n1-2) In Figure 2E, the authors show data from two cell lines transfected with an FTH1 vector. What is the difference between #1 and #2? The distinction is unclear and appears illogical.\n1-3) In Figures 2F and 2H, FTH1 was more effectively suppressed in FTH1KD#2 than in FTH1KD#3. Please use the KD#2 cell line for consistency. Additionally, even without treatment, the FTH1KD#3 group exhibited elevated ROS levels. This finding is unclear—please explain why this cell line was used.\n1-4) To strengthen the biological significance of FTH1 in Figure 2, the reviewer suggests performing rescue experiments. The authors should restore FTH1 expression in the KD cell lines using the FTH1 vector, then treat these cells with MOF-Fe.\n2. In Figure 4, the novel PROTAC C20U4V requires further characterization.\n2-1) Please confirm the localization or cellular uptake of C20U4V in pancreatic cancer cell lines.\n2-2) To clarify the role of C20U4V in targeting FTH1, the authors should assess FTH1 protein levels following C20U4V treatment in FTH1-overexpressing (FTH1OE) cell lines.\n3. In contrast to the enhanced tumor retention of MOF-Fe observed in Figure 2M, M-C20U4V did not show improved retention following IRE treatment in Figure 7H. Please discuss this discrepancy.\n4. In Figure 8, the relationship between the triple-combination treatment and anti-tumor immune responses is unclear. Please specify the main immunological factors or immune cell types involved. Additionally, IFN-γ expression should be analyzed in the other treatment groups in Figure 8J.\nMINOR CONCERNS\n1. The authors should verify that all figure labels correspond accurately with the manuscript text, including those in the supplementary figures.\n2. In Supplementary Figures 2c–e, please explain the rationale and biological relevance of using DFO.", "reviewer_2": "In this study the authors develop a strategy to degrade FTH1 in pancreatic cancer cells via an arachidonic-acid-derived PROTAC. They argue that this sensitizes the cells to ferroptosis, allowing the eradication of persister cells after irreversible electroporation treatment. This study is timely, interesting and well-performed. This reviewer is favourable that this manuscript should be considered for publication, but recommends that the authors consider the following points to strengthen their findings.\nMajor points :\n(1) The authors talk about ferroptosis without citing the key original references, and this oversight should be corrected (Dixon et al. Cell. 2012 May 25;149(5):1060-72, Cell Metab. 2008 Sep;8(3):237-48).\n(2) Some Western blots look odd and the authors should check the raw data if everything is correctly labelled, and if loading controls are on the same gels or not, i.e. sample loading controls or sample processing controls. For instance Fig. 3i: why is the last band of FTH1 pointing down, whereas the actin points up? Was the band inversed? Another example Fig 5i: The band widths of NRF2 and actin are not the same and look like from different gels. Figure 7e: why are FTH1 bands pointing down, whereas actin is pointing up (very visible in the last band on the right). This reviewer did not check all the blots, but recommends to check throughout to avoid any mistakes that can happen during Figure preparation.\n(3) A recent paper showed that ferroptosis can be induced by targeting and activating lysosomal iron in PDAC and other cancers and specifically kill iron-rich cells (Nature. 2025 May 7. doi: 10.1038/s41586-025-08974-4). This work is directly relevant to this manuscript and the authors should consider putting their work and this work in perspective in the introduction or discussion. This work also describes ferroptosis initiation in lysosomes, where the chemical environment and iron abundance favour the Fenton reaction. This should be discussed in the introductory paragraph between references 14 and 16.\n(4) Two key papers showed that iron overload on persister cancer cells characterizes these cells, and can be exploited therapeutically. Even though these studies were on breast cancer and ovarian cancer, they are directly relevant for this work here (Oncogene. 2017 Jul 20;36(29):4089-4099, Nat Chem. 2017 Oct;9(10):1025-1033)\n(5) It is true that ferritin can act as an iron storage protein, but only in its oligomeric 24-mer form. Ferritin also has other functions and properties, and this might be interesting to discuss for the authors in light of their work (for a comprehensive review see: Metallomics. 2021 May 12;13(5):mfab021).\n(6) Lines 103-108: The authors describe experiments where they used sublethal doses of PFE and then talk about ferroptosis inhibitors not rescuing cell viability. The design of these experiments makes no sense by definition, as lethality describes death. So it is not a sublethal dose of PFE? Also, unless the authors identify the specific cell death mechanism, they cannot claim that it is not ferroptosis by using one condition and concentration of Ferrostatin or Liproxstatin. They should establish concentration curves, with several time points and concentrations, and also use tocopherol, a strong anti-oxidant, that is the best ferroptosis inhibitor known to-date. This part of the study needs some serious revisiting (I suggest to look at the experimental details of the Conrad, Friedman-Angeli and Rodriguez labs, to get inspired how best design these experiments).\n(7) BODIPY-C11 does not measure lipid oxidation, but it is a surrogate or proxy to infer lipid oxidation. To show lipid oxidation, the authors should perform lipidomics.\n(8) The authors present small heat maps of cherry-picked genes in their RNA-seq data. Whereas this reviewer understands that presenting omics data is challenging, this is a highly biased approach. Bubble plots of GO pathways could be a good way to start with a non-biased analysis. For instance, do the authors identify GO for ferroptosis in their PEF or MOF-Fe experiments? (Please look at these papers for an idea of how to present RNA-seq data: Nature. 2023 May;617(7960):386-394, Nature. 2025 May 7. doi: 10.1038/s41586-025-08974-4).\n(9) Line 217-218. Mitochondrial alteration is also indicative of other cell death modalities like apoptosis (review: Nat Rev Mol Cell Biol. 2020 Feb;21(2):85-100). Even ferrotposis inducers can induce non-ferroptotic non-apoptotic cell death in some cell types (Cancer Discov. 2022 Mar 1;12(3):774-791). Maybe the authors could be more careful with these semantics?\n(10) Since the authors claim that FTH1 downregulation sensitizes the cells to ferrptosis, they should test if these cells are mores sensible to ferroptosis inducers like erastin/ RSL3 etc.\nMinor points:\n(1) Flow cytometry data histograms, the y-axes are not labelled throughout. This is normalized to the mode? Please indicate scale and annotate the axes.\n(2) The authors write peroxidation throughout, whereas iron and hydrogen peroxide can generate hydroxyl and hydroperoxyl radicals, that can lead to peroxidation and oxidation. It is better to talk about lipid oxidation rather than lipid peroxidation, even if this semantic mistake is used aplenty in the literature.\n(3) Line 114. Significantly instead of significant.\n(4) Line 173/174: very interesting result. This goes hand in hand with the finding that tumors are higher in iron content compared to healthy tissue from human biopsies (Nature. 2025 May 7. doi: 10.1038/s41586-025-08974-4).", "reviewer_3": "Summary: A combination of IRE, MOF-Fe, and C20U4V is used as a collective therapeutic approach to make pancreatic cancer cell lines decrease tumor and cell colony recurrence commonly seen with the use of only one or two of these interventions. The authors found that FTH1, a gene associated with ferroptosis, presented a repeated blockage to treatment efficacy and contributed to tumor regression. Through the experimentation of varying combinations of these three individual treatments, they found that they could regulate this gene with combination therapy to make pancreatic cancer more susceptible to IRE treatment. The authors provide strong justification of the methods used and discuss each aspect of experimentation in detail. My comments address only a few points concerning the discussion of cell death mechanisms. Overall, this paper is strong and only requires minor revisions.\nMajor Comments:\n- You discuss the fact that IRE intends to induce apoptotic cell death, but I think it is important to acknowledge that a significant portion of the ablation area can be allotted to necrotic cell death dependent on the pulse parameters. You perform H&E staining for necrosis but don’t discuss it further.\n- Studies have also identified the presence of pyroptosis and necroptosis contributing as modes of cell death. While there has been a significant amount of apoptotic and necrotic research, it is important to address the contribution of these other forms of cell death and how they may impact the overall ablation.\n- As irreversible electroporation for tissue ablation was founded by Dr. Rafael Davalos, there should be at least a few of his papers cited when addressing the background of the technology.\n- Mitochondrial alteration can also be due to apoptotic cell death. How are you able to conclude that it is only characteristic of ferroptosis?\nMinor Comments:\n- The DEG acronyms are not defined in text or figure captions.\n- Generally, review the paper for minor grammatical errors.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "This is a highly innovative study aiming to improve the efficacy of pulse electrical fields (PEF) by inducing ferroptosis. The first layer of innovation is a fast-release iron nanoparticle (MOF) only modestly delayed growth of subcutaneous KPC-derived tumors. The data that resistance to this iron therapy was driven by increased expression of ferritin are compelling, including genetic knockout studies FTH1. The next and even greater innovation was the development of a PROTAC targeting fatty acids, based on rationale that FTH1 is known to bind multiple fatty acids. The winner (C20U4V) was based on arachidonic acid (AA) and chosen on the basis of growth inhibition in vitro. It is confusing that C20U4V increases FTH on its own in proteomic studies in vitro. It is only when given in combination with MOF that C20U4V decreases FTH relative to FTH. The combo treatment increases FTH in multiple experiments compared to control or IRE alone. A potential explanation is offered that this may be due to effects of AA, but it seems inappropriate to refer to this as an “FTH1-targeting” PROTAC, when it clearly affects multiple proteins. The efficacy of the PROTAC for degrading FTH is not extremely high (Fig 5i)—1.3-fold by proteomics in vitro and FTH1 was not significantly decreased (p=0.07) in treated tumors in vivo (relative to combo of PEF + MOF without PROTAC). Despite this, there is impressive growth delay with the triple combination in subcutaneous tumor model and survival improvement in an orthotopic model.\nMy other significant criticism is the use of sublethal PEF. It is understandable why this is necessary for in vitro studies, because it is too easy to kill cell in vitro, and you won’t have any cells to study if you use lethal PEF. However, the conditions for intentionally sublethal PEF are very different than what are used clinically for IRE (or what have been used in mouse models of IRE). How do the effects of IRE on ferroptosis-associated proteins compare to sublethal PEF? Would there still be synergy? If perhaps this is not feasible to do for manuscript, this limitation needs to be acknowledged. A sign that the conditions used are perhaps insufficient is that “IRE” in this study (which should perhaps be referred to as either PEF or incomplete IRE or “reversible electroporation”) did not increase uptake of M-C20U4V in tumors.\nFinally, the reasons for “resistance” to IRE are many and complex, many are likely technical or anatomical related to incomplete physical ablation. While mutations in apoptosis genes might contribute, this is an unnecessary over-simplification of the issue. It is certainly true that local recurrence rates are higher than we would like, but the statement that “the rate of local recurrence is higher than other ablative therapies” is not supported by data. Two studies were referenced that had amongst the highest recurrence rates in the literature but are not representative of the field, particularly open surgical IRE, and did not compare IRE to other forms of ablation, which have equally high, if not higher rates of local recurrence for pancreatic cancer. This statement needs to be amended/softened.\nMinor things is that there are too many non-standard, undefined abbreviations in graphic abstract.", "reviewer_6": "Review\nThis manuscript by Linrong Li, Shihao Su et al. presents an novel study investigating a strategy to enhance the efficacy of irreversible electroporation (IRE) in primarily pancreatic cancer by sensitizing residual tumour cells to ferroptosis. During IRE, residual viable cancer cells can remain a significant cause of tumour recurrence. The authors demonstrate that iron-based metal-organic framework nanoparticles (MOF-Fe) can suppress recurrence by inducing iron overload and subsequently ferroptosis; however, this effect is attenuated by IRE-induced upregulation of ferritin heavy chain 1 (FTH1). To overcome this limitation, the authors employ an arachidonic acid-derived PROTAC, C20U4V, to selectively degrade FTH1, thereby amplifying ferroptotic activity in both pancreatic cancer cell lines and organoids. Encapsulation of C20U4V within a ROS-responsive micelles (called M-C20U4V) enables in vivo delivery, and in combination with MOF-Fe, they show enhanced ferroptosis and therapeutic efficacy in murine models. This study is conceptually novel, supported by experimental data, and of clear translational relevance. While a few minor points requiring clarification, the manuscript is of sound quality and in my opinion would be suited publication in Nature Communications.\nComments\nSublethal PEF Induces Anti-Ferroptotic Gene Expression\nAs shown pulsed electric fields (PEF) upregulate anti-ferroptotic genes in pancreatic cancer cells. However, in Figure 1B, immunohistochemical staining of 4-Hydroxynonenal (4-HNE) in KPC tumours appears non-significant. It may be more appropriate to include this panel as a supplementary figure, especially since the subsequent panels (PANC-1 data) clearly demonstrate significant effects.\nFigure Labeling and Misreferencing in the Text:\nThere are multiple inconsistencies between figure panels and their citations in the text:\nFigure 1C is incorrectly labelled as Figure 1B.\nFigures 1I–K are referred to in the text as Figures 1E–G.\nFigure 1D in the text corresponds to Figure 1H.\nFigure 1E in the text is Figure 1I.\nFigure 1H in the text refers to Figure 1L.\nFigures 1J and 1K are not referenced in the text at all. They appear to be extensions of earlier panels and may benefit from reordering to align with their corresponding experiments and improve overall clarity.\nAnti-Ferroptotic Gene Upregulation and Ferroptosis Resistance\nThe demonstration that PANC-1 cells upregulate anti-ferroptosis/antioxidative stress genes post-PEF is convincing. The data showing that Ferrostatin-1 (Ferr-1) and Liproxstatin-1 (Lip-1) do not restore viability suggests that PEF does not sensitize cells to ferroptosis. However, a useful addition could be to investigate whether PEF-treated cells gain resistance to ferroptosis. For example, treating both PEF and non-PEF cells with erastin and comparing their sensitivities could yield informative insights about sensitisation/desensitisation of model systems to ferroptosis post electroporation.\nArachidonic Acid-Derived PROTACs Targeting FTH1\nC20U4V is the chosen molecule for FTH1 degradation. However, C20U5V also appears to have very similar activity. What was the rationale for selecting C20U4V over C20U5V? Furthermore, why was C20U5V excluded from the CCK-8 assay? I imagine that the EPA will have a different interactome to AA and therefore it would be interesting to perform some of the later experiments with MOF-FE and PEF to determine what difference are present between compounds.\nAlthough the degradation is shown to be VHL-dependent, arachidonic acid (AA) is not likely to be inherently selective for FTH1. It would therefore strengthen the conclusion to demonstrate that FTH1 degradation is a result of specific binding of VHL and FTH1 rather than a by-product of a loss of stability of FTH1 through non specific targeting of C20U4V. A suggested approach is a competition assay, where FTH1 is pre-saturated with (S,S,S)-C20U4V prior to treatment with C20U4V to confirm target specificity.\nA pull-down assay combined with Mass spectrometry could establish to us that C20U4V is binding to FTH1 and also could help define the interactome of the arachidonic acid and further support target engagement of C20U4V.\nA DC₅₀ titration assay for C20U4V would also be beneficial to quantify compound potency and the optimal therapeutic window of C20U4V.\nMicelle-Loaded C20U4V (M-C20U4V)\nIn Figure 5, degradation kinetics for C20U4V are measured at 12 hours, whereas M-C20U4V is measured at 48 hours. For proper comparison, these should be performed at the same timepoint. In the supplementary file they do perform a 48 Hour titration of C20U4V and show that it is no longer capable of degradation, is this due to stimulation of FTH1 expression of FTH1 or that they’re compound isn’t potent? Could the authors clarify this\nAre they authors able to treat at even higher concentrations given the low level of activity or are they constrained by solubility of the molecule?\nDual Treatment with MOF-Fe and M-C20U4V in vivo\nThere are some referencing issues in this section:\nFigure 8 is mistakenly cited as Figure 6 in the text.\nFigure 8K should say M-C20U4V, not C20U4V.\nConclusion\nThis study presents a promising strategy for enhancing ferroptotic cancer therapy following IRE via targeted degradation of FTH1 using a PUFA-based PROTAC. The results are compelling, but improving figure organization, correcting some references, and clarifying compound selection and specificity would be advisable."}, "author_response": "Point-by-point Responses to Reviewers’ Comments\nTable of Contents\nResponses to Reviewer #1 ························································································ Page 2\nResponses to Reviewer #2 ······················································································ Page 12\nResponses to Reviewer #3 ······················································································ Page 23\nResponses to Reviewer #4 ······················································································ Page 26\nResponses to Reviewer #5 ······················································································ Page 27\nResponses to Reviewer #6 ······················································································ Page 34\nReferences ·············································································································· Page 46\n1 / 47\nReviewer #1 (Remarks to the Author); expert in iron NP, therapy:\nThe manuscript titled \"Disruption of Iron Homeostasis Sensitizes Pancreatic Cancer to Irreversible\nElectroporation\" offers valuable insights into pancreatic cancer therapy using a triple-combination strategy\n(IRE, MOF-Fe, and M-C20U4V). In particular, the authors synthesized C20U4V, a novel PROTAC, to\ntarget FTH1. Overall, the work is well-executed, but a few additions and clarifications are necessary prior\nto publication. The authors are requested to carefully address the following comments to improve the\nmanuscript.\nAnswer: We appreciate the reviewer’s positive feedback. Detailed responses are provided as follows\nMAJOR CONCERNS\n1. In Figure 2, the experiments do not clearly demonstrate that MOF-Fe induces ferroptosis.\n1-1) In Figure 2D, the correlation (R-value) between FTH1 expression and the AUCs of three chemicals\n(Erastin, 1S,3RSL-3, and ML162) ranges between 0.114 and 0.152. Are these values truly meaningful?\nPlease discuss their significance.\nAnswer:\nWe agree with the reviewer that a R value < 0.3 implies little correlation. Therefore, these three panels\nare removed from the revised manuscript. The cytotoxicity of erastin and (1S,3R)-RSL-3 (RSL3), two\nferroptosis inducers, were instead evaluated in FTH1-overexpressing (OE) and -knockdown (KD) Panc-1\ncells (Supplementary Fig. 6a). The IC values of both molecules were increased by FTH1\n50\noverexpression, but were not significantly changed by FTH1 knockdown. In addition, C20U4V at 5 μM\nconcentration significantly decreased the IC values of both erastin (12.6 μM vs. 1.6 μM) and RSL3 (5.9\n50\nμM vs. 0.15 μM).\n2 / 47\nSupplementary Fig. 6a, Cell viability of FTH1-OE and -KD cells after 72h treatment with erastin or RSL3. N = 6.\nAdditional data for review, Panc-1 viability after 72 h treatment with erastin or RSL 3 in the presence of 5 μM\nC20U4V. N = 6.\n1-2) In Figure 2E, the authors show data from two cell lines transfected with an FTH1 vector. What is the\ndifference between #1 and #2? The distinction is unclear and appears illogical.\nAnswer:\nThe two FTH1-OE cell lines were generated using plasmids extracted from two monoclonal colonies\nof competent E. coli. These E. coli colonies were derived by transforming competent cells with the\npurchased FTH1-OE plasmids. We agree that it is unnecessary to include both cell lines in the manuscript.\nOnly one cell line is included in the revised manuscript.\n1-3) In Figures 2F and 2H, FTH1 was more effectively suppressed in FTH1KD#2 than in FTH1KD#3.\nPlease use the KD#2 cell line for consistency. Additionally, even without treatment, the FTH1KD#3 group\nexhibited elevated ROS levels. This finding is unclear—please explain why this cell line was used.\nAnswer:\nWe agree. All three FTH1-KD cells were evaluated as suggested. FTH1-knockdown enhanced MOF-\nFe-induced ROS elevation (DCFH-DA+ cells%) most prominently in shFTH1#3 (Supplementary Fig.5e).\nAll three KD cell lines exhibited higher level of C11-BODIPY and formed fewer colonies than NC after\nMOF-Fe treatment (Supplementary Fig.5f, g).\nIt is common in the field of molecular biology to use multiple shRNA sequences targeting the same\ngene, so that the biological consequences are not due to the non-specific silencing of off-target genes [1].\nThree KD cell lines were used in this study. It is unclear why KD#3 exhibited high ROS levels at baseline.\nIt is possible that shFTH1#3 may have other off-target effects, or the cells evolved compensatory\nmechanisms during puromycin selection. Still, the results from all three KD cell liens were mostly consistent,\nand supported that FTH1-knockdown sensitized Panc-1 cells to MOF-Fe.\n3 / 47\nSupplementary Fig. 5 | Effects of FTH1 expression of ferroptosis (additional data 1)\ne, Immunofluorescence images and quantification of the percentage of DCFH-DA+ cells per FOV. N = 6. Scale bar\n= 100 μm. f, Flow cytometry histograms and quantification of C11-BODIPY oxidation. g, Representative images of\nPanc-1 colonies and quantification. N = 3. Data are presented as mean ± SEM. Statistical significance was\ndetermined using one-away ANOVA followed by Dunnett’s multiple comparison or ratio-paired t-test. n.s. = not\nsignificant. *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.\n4 / 47\n1-4) To strengthen the biological significance of FTH1 in Figure 2, the reviewer suggests performing rescue\nexperiments. The authors should restore FTH1 expression in the KD cell lines using the FTH1 vector, then\ntreat these cells with MOF-Fe.\nAnswer:\nWe thank the reviewer for this excellent suggestion. FTH1 expression was restored in all three FTH1-\nKD cell lines (Fig. 3b) using a modified FTH1-overexpressing plasmid, abbreviated as FTH1-OE (m). The\ncoding sequence of this plasmid had synonymous mutations so it was not recognized by the FTH1-\ntargeting shRNAs (Supplementary Table 8). In all three KD cells lines, the restoration of FTH1 mitigated\nMOF-Fe-induced total ROS and C11-BODIPY oxidation, and increased the number of colonies in the\npresence of MOF-Fe (shFTH1#1, #3 in Fig. 3c-e, shFTH1#2 in Supplementary Fig6. b-d). Taken\ntogether, these data suggest that FTH1 promoted the resistance to MOF-Fe-induced ferroptosis.\n5 / 47\nFig. 3 | FTH1 promoted resistance to MOF-Fe (see previous page)\na, b, Immunoblots of FTH1-KD (a) and -rescue (b) Panc-1 cells. c, Immunofluorescence images and quantification\nof the percentage of DCFH-DA+ cells per field-of-view (FOV). N = 6. Scale bar = 100 μm. d, Flow cytometry\nhistograms and quantification of C11-BODIPY oxidation. N = 3. Panc-1 cells in c, d were treated with MOF-Fe (10\nμg Fe/mL) for 48 h. e, Representative images of Panc-1 colonies and quantification. N = 3. Data are presented as\nmean ± SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple\ncomparison or ratio-paired t-test. *p < 0.05. p < 0.01. ***p < 0.001. ****p < 0.0001.\nSupplementary Fig. 6 | Effects of FTH1 expression of ferroptosis (additional data 2).\nb, Immunofluorescence images and quantification of the percentage of DCFH-DA+ cells per FOV. N = 6. Scale bar\n= 100 μm. c, Flow cytometry histograms and quantification of C11-BODIPY oxidation. N = 3. d, Representative\nimages of Panc-1 colonies and quantification. N = 3. Data are presented as mean ± SEM. Statistical significance\nwas determined using one-away ANOVA followed by Dunnett’s multiple comparison or ratio-paired t-test. *p <\n0.05, ****p < 0.0001.\n6 / 47\n2. In Figure 4, the novel PROTAC C20U4V requires further characterization.\n2-1) Please confirm the localization or cellular uptake of C20U4V in pancreatic cancer cell lines.\nAnswer:\nThe cellular uptake of C20U4V was measured using liquid chromatography-mass spectrometry\nLC/MS. Panc-1 cells were incubated with 20 μM C20U4V for 6 and 12 h, and then subjected to test. The\ncellular uptake (μg/mg protein) was 80.4 ± 2.1 at 6 h and 103.3 ± 6.3 at 12 h (Supplementary 7b).\nUnfortunately, we were unable to pinpoint the exact subcellular location of C20U4V because it is not\nfluorescent. Conjugation with a fluorescent moiety would probably change its structure and alter its\nsubcellular distribution. The equipment for single-cell spatial mass spectrometry imaging is not available\nto us. Still, we have shown that C20U4V was taken up by Panc-1 cells, and the successful degradation of\nFTH1 supports that it functioned as expected.\nSupplementary Fig. 7 | Evaluation of PUFA-derived PROTACs (additional data 1)\nb, Quantification of intracellular uptake of C20U4V. Cells were incubated with 20 μM C20U4V. N = 4.\n2-2) To clarify the role of C20U4V in targeting FTH1, the authors should assess FTH1 protein levels\nfollowing C20U4V treatment in FTH1-overexpressing (FTH1OE) cell lines.\nAnswer:\nWe agree. Cycloheximide (CHX) was added to FTH1-OE cells to inhibit de novo protein synthesis,\nand then the cells were incubated with C20U4V for 12 h. Significant FTH1 degradation was observed with\n20 μM C20U4V (Supplementary Fig. 9e). CHX is commonly used to study protein stability. A similar\nassay to study protein degradation by PROTACs was recently reported [2].\nSupplementary Fig. 9 | Evaluation of PUFA-derived PROTACs (additional data 1).\ne, Immunoblots and quantification of FTH1 in FTH1-OE Panc-1 cells after treatment with C20U4V in the presence\nof CHX. N = 3.\n7 / 47\n3. In contrast to the enhanced tumor retention of MOF-Fe observed in Figure 2M, M-C20U4V did not show\nimproved retention following IRE treatment in Figure 7H. Please discuss this discrepancy.\nAnswer:\nWe apologize for the confusion. MOF-Fe was injected intratumorally while M-C20U4V was injected\nintravenously through tail vein. IRE ablated a substantial portion of tumor cells and reduced tumor\ninterstitial pressure, which may have facilitated MOF-Fe retention within the tumor. Tumor uptake of M-\nC20U4V, on the other hand, is affected by the micelles’ pharmacodynamic properties as well as the\nfunction of intratumoral vasculature.\nIRE and low-intensity reversible electroporation exert distinct effects on vasculatures [3]. Significant\napoptosis of endothelial cells and microvessel thromboses were observed in tumors at 30 min after IRE\n[4]. IRE also disrupted blood-brain barrier [5]. Reversible electroporation, on the other, induced a\ntemporary “vasculature lock” that is often resolved within 48 h [6]. The vasculature lock could be divided\ninto two phases. During the first phase, electroporation induced a reflexory vasoconstriction of afferent\narterioles mediated by the sympathetic nervous system. During the second phase, the increase in vascular\npermeability triggered fluid leakage from the vascular lumen into the adjacent interstitial space of the tumor.\nThe subsequent elevation of tumor interstitial pressure, along with the reduction in intravascular pressure,\nultimately converged to cause vasculature collapse.\nThese early studies suggested that electroporation may have dichotomous effects on tumor uptake of\nnanoparticles. On the one hand, increase of vasculature permeability facilitated nanoparticle leakage into\nthe interstitial space, and thus favored their tumor uptake. On the other hand, IRE-induced thromboses\nand reversible electroporation-induced vasculature lock may hinder tumor blood perfusion, and\nconsequently was unfavorable for the tumor uptake of nanoparticles.\nSeveral studies have investigated the effects of IRE on the tumor uptake of chemotherapy drugs or\nnanoparticles (summarized in the table below). Bhutiani et al discovered that IRE enhanced gemcitabine\ndelivery to the zone of reversible electroporation in pancreatic tumor models [7]. Peng et al. reported that\nthe combination of microsecond and nanosecond PEF increased nano-gel uptake in murine colon tumor\nmodels [8]. We previously showed that tumor uptake of fluorescence-labeled dextran was elevated on day\n4 post-IRE [9].\nWe noticed that the sequence of electroporation and nanoparticle injection was different in the current\nstudy: M-20U4V was injected 4 h before electroporation, while nanoparticles/gemcitabine was injected\nafter electroporation in the other three studies. Such discrepancy in schedule might have shifted the peak\nof plasma concentration-time curve of M-C20U4V relative to the behavior change of tumor vasculature.\nNo improvement of M-C20U4V retention was observed as a result. Further experiments will be performed\nto validate this hypothesis.\n8 / 47\nTable. Summary on tumor uptake of chemotherapy drugs or nanoparticles after PEF (for\nreview only)\nRef. Pulse Parameters Agent and Schedule Findings\n[7] Pulse intensity = 1600~2000 Gemcitabine injected twice with 2 h in between; IRE increased\nV/cm IRE between two injections of Gemcitabine; gemcitabine delivery in\nPulse duration = 90 μs Samples collected from the reversible the zone of reversible\nPulse number = 10 per group electroporation zone. electroporation\nPulse interval = 250 ms\n[8] Combination of μs and ns pulses Cell membrane-coated nanogel about 100 nm in PEF improved the whole\nμ pulse intensity = 1500 V/cm size; tumor uptake of nanogel\nμ pulse duration = 100 μs Tumors first treated with PEF, then intravenously for up to 5 times.\nnano pulse intensity = 10,000 injected with the nanogel;\nV/cm Tumor uptake measured by fluorescence from 1\nnano pulse duration = 500 ns to 48 h after PEF.\n[9] Pulse intensity = 2400 V/cm FITC-conjugated dextran (70 kD) was injected IRE increased the whole\nPulse duration = 100 μs immediately after IRE tumor uptake of dextran\nPulse number = 99 Tumor uptake measured by fluorescence on day on day 4 after IRE\nPulse interval = 1 s 4 and 6 after IRE\nCurrent Pulse intensity = 2300 V/cm Fluorescence-labeled polymeric micelles about 80 No difference in\nstudy Pulse duration = 100 μs nm in size; polymeric micelles\nPulse number = 99 Polymeric micelles were injected 4 h before IRE. uptake between IRE-\nPulse interval = 1 s Tumor uptake measured by fluorescence at 48 h treated and sham treated\nafter injection (44 h after IRE) tumors\n9 / 47\n4. In Figure 8, the relationship between the triple-combination treatment and anti-tumor immune responses\nis unclear. Please specify the main immunological factors or immune cell types involved. Additionally, IFN-\nγ expression should be analyzed in the other treatment groups in Figure 8J.\nAnswer:\nWe agree. CD8+ T cell depletion was performed using anti-CD8 antibodies in the triple-treatment\ngroup (Supplementary Fig. 13d-h). On day 11 after enrollment, the average weight of tumors in the “triple-\ntreatment + anti-CD8 antibody” group was significantly higher than those in the triple-treatment group.\nThe successful depletion of CD8+ T cells was validated using IHC. These results suggested that therapeutic\nefficacy of the triple-treatment could partially be attributed to CD8+ T cells.\nSupplementary Fig. 13 | Anti-tumor efficacy of IRE, MOF-Fe, and M-C20U4V (additional data).\nd, Treatment schedule of CD8+ T cells depletion. e, f, Photographs of KPC-A719 tumors (e) and quantification of\ntumor weight (f) on day 11 after enrollment. g, Change of body weight. h, Representative IHC images of CD8+ T\ncells to verify successful depletion. Data are presented as mean ± SEM. Statistical significance was determined\nusing one-away ANOVA followed by Dunnett’s multiple comparison.\nWe also performed additional flow cytometry experiments to analyze the frequency of intratumoral\nIFNγ+CD8T+ cells. The digested tumors were incubated with leukocyte activation cocktail (BD Pharmingen\nCatalog# 550583) containing PMA, Ionomycin and protein transport inhibitor Brefeldin A for 6 h. The cell\nsuspensions were then fixed, permeabilized, and stained with FITC-anti-IFNγ antibody and lineage\nmarkers. As shown in Fig. 8e, the frequency of IFNγ+CD8T+ cells was similar in IRE- and the triple-\ntreatment groups, both of which were higher than those in the other groups. Multiplex immunofluorescence\nstaining was also performed in tumors from all four groups (Fig. 8o). The mean fluorescence intensity of\nIFNγ was slightly higher in the triple-treatment group than in the IRE group.\n10 / 47\nFig. 8e, Frequency of intratumoral IFNγ+CD8+ T cells: CD45+CD3+CD8+IFNγ+,\nFig. 8o, Multiplex immunofluorescence staining of CD8 and IFN-γ and quantification. N = 20. Scale bar = 100 μm.\nTumors were collected on day 9 after enrollment.\nMINOR CONCERNS\n1. The authors should verify that all figure labels correspond accurately with the manuscript text, including\nthose in the supplementary figures.\nAnswer:\nWe apologize for the mistakes. The manuscript has been carefully examined, and typos are corrected.\n2. In Supplementary Figures 2c–e, please explain the rationale and biological relevance of using DFO.\nAnswer:\nDeferoxamine (DFO) is an iron chelator that form a stable complex with ferric ions. It is clinically useful\nfor treating iron overload. DFO has been used to inhibit ferroptosis induced by iron overload [10]. We\nincluded DFO to elucidate whether free iron released from MOF-Fe was responsible for FTH1 upregulation.\nAs shown in Supplementary Fig. 4j in the revised manuscript, FTH1 expression was indeed reduced by\nDFO.\nSupplementary Fig. 4 j, FTH1 immunoblots and quantification after treatment with MOF-Fe (3 μg Fe/mL) and/or\nDFO. N = 3.\n11 / 47\nReviewer #2 (Remarks to the Author); expert in ferroptosis:\nIn this study the authors develop a strategy to degrade FTH1 in pancreatic cancer cells via an arachidonic-\nacid-derived PROTAC. They argue that this sensitizes the cells to ferroptosis, allowing the eradication of\npersister cells after irreversible electroporation treatment. This study is timely, interesting and well-\nperformed. This reviewer is favourable that this manuscript should be considered for publication, but\nrecommends that the authors consider the following points to strengthen their findings.\nAnswer:\nWe appreciate the reviewer’s positive feedback. Detailed responses are provided as follows\nMajor points :\n(1) The authors talk about ferroptosis without citing the key original references, and this oversight should\nbe corrected (Dixon et al. Cell. 2012 May 25;149(5):1060-72, Cell Metab. 2008 Sep;8(3):237-48).\nAnswer:\nWe apologize for the mistake. These two ground-breaking papers have been added to the reference.\n(2) Some Western blots look odd and the authors should check the raw data if everything is correctly\nlabelled, and if loading controls are on the same gels or not, i.e. sample loading controls or sample\nprocessing controls. For instance, Fig. 3i: why is the last band of FTH1 pointing down, whereas the actin\npoints up? Was the band inversed? Another example Fig 5i: The band widths of NRF2 and actin are not\nthe same and look like from different gels. Figure 7e: why are FTH1 bands pointing down, whereas actin\nis pointing up (very visible in the last band on the right). This reviewer did not check all the blots, but\nrecommends to check throughout to avoid any mistakes that can happen during Figure preparation.\nAnswer:\nWe greatly appreciate the reviewer’s efforts in meticulously examining the immunoblots. All\nimmunoblots in the revised manuscript have been carefully examined. The original, uncropped images of\nmembranes are attached as a PDF file titled “WB Raw Images” in the “Source Data” zipped folder.\nChemiluminescence images used for protein quantification are also attached if the merged image\ncorresponds to a different exposure time.\nUncropped raw images of the blots specifically mentioned in the reviewer’s comments are summarized\nin the figure next page. (1) Per suggestion from Reviewer #1, only one clone of FTH1-OE Panc-1 cells is\nincluded in the revised manuscript. The immunoblots is shown in Supplementary Fig. 5. (2) The NRF2\nband in Fig. 5j and its corresponding vinculin loading control were run on one gel. The bands of HMOX1,\nFTH1, and β-actin were run on another gel. The β-actin band was included in Fig. 5j to represent the\nloading levels. Quantification was performed using loading controls specific to respective target. (3) Data\nin Fig. 7e was updated to show the time course of FTH1 expression after treatment with MOF-Fe and M-\nC20U4V.\n12 / 47\n13 / 47\n(3) A recent paper showed that ferroptosis can be induced by targeting and activating lysosomal iron in\nPDAC and other cancers and specifically kill iron-rich cells (Nature. 2025 May 7. doi: 10.1038/s41586-\n025-08974-4). This work is directly relevant to this manuscript and the authors should consider putting\ntheir work and this work in perspective in the introduction or discussion. This work also describes\nferroptosis initiation in lysosomes, where the chemical environment and iron abundance favour the Fenton\nreaction. This should be discussed in the introductory paragraph between references 14 and 16.\nAnswer:\nWe thank the reviewer for bringing this excellent work to our attention. Most iron-based nanoparticles\nare endocytosed through the endosome-lysosome route, and the acidic environment of lysosomes is\ncommonly utilized for the stimulus-responsive degradation and payload release of nano-formulations.\nTherefore, the paper by Cañeque et al. provides a strong rationale why iron-based nanoparticles could be\nattractive candidates to induce ferroptosis.\nThe following paragraph is included to the introduction between references 14 and 16 of the original\nmanuscript.\n…These nanoparticles are endocytosed by cancer cells and trafficked to acidic lysosomes, where they degrade\nand release ferrous or ferric ions. Notably, the activation of lysosomal iron has recently been shown to\ncatalyze ROS production and initiate ferroptosis18. MIL-101 (Fe) metal-organic framework (MOF-Fe)\nnanoparticles could release iron in a flash fashion19, and therefore are promising candidates to induce iron overload.\nThe topic is further expanded in the discussion section as follows:\n…Recently, activation of lysosomal iron has emerged as an effective strategy for triggering ferroptosis and thereby\neliminating drug-resistant persister cancer (DTP) cells18. Salinomycin sequestered iron in lysosomes to the point of\niron overload, ultimately causing ferroptotic death in breast cancer stem cells56. These results underscore the\npotential of using iron-based nanoparticles to eliminate drug resistant cancer cells, as these nanoparticles are\nendocytosed via the endosome-lysosome route and are often design to release their payloads within acidic\nlysosomes.\n14 / 47\n(4) Two key papers showed that iron overload on persister cancer cells characterizes these cells, and can\nbe exploited therapeutically. Even though these studies were on breast cancer and ovarian cancer, they\nare directly relevant for this work here (Oncogene. 2017 Jul 20;36(29):4089-4099, Nat Chem. 2017\nOct;9(10):1025-1033)\nAnswer:\nWe agree. Similar to pancreatic cancer, both breast and ovarian cancers are solid tumors that exhibit\nan immunosuppressive microenvironment and become refractory to conventional treatments at advanced\nstages. Therefore, it would be very helpful to extrapolate the therapeutic interventions on persister cells in\nbreast or ovarian cancers to pancreatic cancer. These two papers are added to the reference in the revised\nmanuscript. The following paragraph is added to the discussion section:\n…Recently, activation of lysosomal iron has emerged as an effective strategy for triggering ferroptosis and thereby\neliminating drug-resistant persister cancer (DTP) cells18. Salinomycin sequestered iron in lysosomes to the\npoint of iron overload, ultimately causing ferroptotic death in breast cancer stem cells56. These results\nunderscore the potential of using iron-based nanoparticles to eliminate drug resistant cancer cells, as these\nnanoparticles are endocytosed via the endosome-lysosome route and are often design to release their payloads\nwithin acidic lysosomes. In addition, certain types of cancer cells exhibit iron addiction, a trait that renders\nthem particularly vulnerable to ferroptosis. Indeed, ovarian tumor initiating cells have been reported to\naccumulate excess iron and reply on iron for proliferation. Treatment with erastin effectively induced\nferroptotic cell death in these cells and reduced tumor burden in vivo57…\n(5) It is true that ferritin can act as an iron storage protein, but only in its oligomeric 24-mer form. Ferritin\nalso has other functions and properties, and this might be interesting to discuss for the authors in light of\ntheir work (for a comprehensive review see: Metallomics. 2021 May 12;13(5):mfab021).\nAnswer:\nWe thank the reviewer for bringing this elegant review to our attention. We have included this paper\nin the reference of revised manuscript, and re-wrote the corresponding paragraph in the discussion.\n…extracellular ferric ions bound to transferrin or hyaluronate are recognized by their respective receptors, i.e.,\ntransferrin receptor and CD44, before being endocytosed and transported into lysosomes53…\n...In addition to iron storage, ferritin and its two subunits also regulated cancer cell plasticity via epigenetic\nmodulation of epithelial-to-mesenchymal (EMT)-related genes21.…\n15 / 47\n(6) Lines 103-108: The authors describe experiments where they used sublethal doses of PFE and then\ntalk about ferroptosis inhibitors not rescuing cell viability. The design of these experiments makes no sense\nby definition, as lethality describes death. So, it is not a sublethal dose of PFE? Also, unless the authors\nidentify the specific cell death mechanism, they cannot claim that it is not ferroptosis by using one condition\nand concentration of Ferrostatin or Liproxstatin-1. They should establish concentration curves, with several\ntime points and concentrations, and also use tocopherol, a strong anti-oxidant, that is the best ferroptosis\ninhibitor known to-date. This part of the study needs some serious revisiting (I suggest to look at the\nexperimental details of the Conrad, Friedman-Angeli and Rodriguez labs, to get inspired how best design\nthese experiments).\nAnswer:\nWe apologize for the confusion. The term “sublethal PEF” is removed from the revised manuscript.\nPEF with specified pulse intensity (e.g., 600 V/cm) is now used to describe a given experiment. We chose\neliminating all cancer cells. A recent study also reported that PEF of 500 to 700 V/cm intensity induced\nregulated cell death [11].\nAccording to the reviewer’s suggestion, the half-inhibitory concentrations (IC ) of liproxtatin-1 (Lip-1),\n50\nferrostatin-1 (Ferr-1), and tocopherol were first measured in Panc-1 and MIAPaCa-2 cells\n(Supplementary Fig. 1a). These agents were then added to PEF-treated cells to investigate their impact\non cell viability (experiment scheme shown in Fig. 1c). Different concentrations of the agents and durations\nof incubation were both examined (Fig. 1d, Supplementary 1b). None of the three agents, within the\ntested conditions, improved the viability of Panc-1 or MIAPaCa-2 cells after PEF. These results suggested\nthat it was unlikely for PEF at this setting to induce ferroptosis in the two cell lines.\n16 / 47\nSupplementary Fig. 1a, Cell viability after treatment with Lip1, Ferr-1, and tocopherol. N = 6.\nFig. 1c, Experiment scheme for panel d.\nFig. 1d, Panc-1 viability after PEF and incubation with Lip-1, Ferr-1, or tocopherol. N = 6.\nSupplementary Fig. b, MIAPaCa-2 viability after PEF and incubation with Lip-1, Ferr-1, or tocopherol. N = 6.\nCell viabilty was normalized to those of untreated cells. Data are presented as mean ± standard error of mean\n(SEM). Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison,\nStudent’s two-sided t-test, or ratio paired t-test. n.s. = not significant. *p < 0.05. **p < 0.01.\nPEF-induced cell death involves multiple regulated pathways. In a review by Napotnik et al, the\nauthors summarized the types of cell death after electroporation, including apoptosis, necrosis, pyroptosis,\nand necroptosis [12]. This paper has been added to the reference of the revised manuscript.\n17 / 47\n(7) BODIPY-C11 does not measure lipid oxidation, but it is a surrogate or proxy to infer lipid oxidation. To\nshow lipid oxidation, the authors should perform lipidomics.\nAnswer:\nWe agree. Lipidomics of representative phosphatidylcholines (PC) and phosphatidylethanolamines\n(PE) were performed on Panc-1 cells treated with vehicle control, MOF-Fe or MOF-Fe + C20U4V for 48\nh. The raw data are included in Supplementary Data.\nMOF-Fe elevated the levels of two oxidized and three peroxidized PE relative to the vehicle control\n(Fig. 2i). Compared to the vehicle control, the “MOF-Fe + C20U4V” dual-treatment upregulated 14\noxidized and 20 peroxidized phospholipids, including PC 18:0-22:6-OOH, PE 18:0-22:6-OOH, and PE\n16:0-22:6-OOH (Fig. 5e). Compared to MOF-Fe, the dual treatment upregulated nine oxidized and 14\nperoxidized phospholipids (Supplementary Fig. 10g). These results supported that MOF-Fe and C20U4V\ninduced both the oxidation and peroxidation of phospholipids.\nSummary of Lipidomics data\nFig. 2i, Volcano plots of lipidomics after treatment with MOF-Fe relative to vehicle control. N = 5. Fig. 4e, Volcano\nplots of lipidomics after treatment with MOF-Fe (3 μg Fe/mL) and C20U4V (20 μM) relative to vehicle control. N =\n5. Supplementary Fig. 10g, Volcano plots of lipidomics after treatment with MOF-Fe + C20U4V relative to MOF-\nFe. N = 5. For each phospholipid, the statistical significance of the change between two comparison groups was\ndetermined using Student’s t-test. Differentially expressed phospholipids with a p-value < 0.05 and |fold-change| >\n1.3 were considered statistically significant.\n18 / 47\n(8) The authors present small heat maps of cherry-picked genes in their RNA-seq data. Whereas this\nreviewer understands that presenting omics data is challenging, this is a highly biased approach. Bubble\nplots of GO pathways could be a good way to start with a non-biased analysis. For instance, do the authors\nidentify GO for ferroptosis in their PEF or MOF-Fe experiments? (Please look at these papers for an idea\nof how to present RNA-seq data: Nature. 2023 May;617(7960):386-394, Nature. 2025 May 7. doi:\n10.1038/s41586-025-08974-4).\nAnswer:\nWe thank the reviewer for the helpful instruction. We agree that RNAseq data should be presented in\nan un-biased manner. Bubble plots of GO analysis, volcano plots of GSEA enrichment, or volcano plots\nof gene expression in a specific pathway are used throughout the revised manuscript.\nPanc-1 cells were treated with PEF and cell debris. Upregulated genes in the treated cells were\nsignificantly enriched in gene ontology (GO) terms of “regulation of ferroptosis” and “ferroptosis (Fig. 1e).\nFurther look into the ferroptosis pathway (WikiPathways, WP4313) revealed seven upregulated genes and\nthree downregulated ones (Fig. 1f). Six upregulated genes (HMOX1, SLC7A11, GCLM, GCLC, FTL, FTH1)\nare ferroptosis suppressors, and two downregulated ones (LPCAT3, ACSL5) are ferroptosis drivers [13].\nGene set enrichment analysis (GSEA) unveiled the nuclear factor erythroid 2-related factor 2 (NRF2)-\nrelated pathways as the top-upregulated ones (Fig. 1g). NRF2 pathway (WikiPathways, WP2884)\nregulates cellular response to oxidative stress, and 16 genes were upregulated (Fig. 1h).\n19 / 47\nFig. 1 | PEF upregulated ferroptosis suppressor genes in pancreatic cancer cells.\ne, GO analysis of biological process (GO_BP) for genes upregulated in the PEF + cell debris group relative to the\n to the\ncontrol group. f, Volcano plots of transcriptome. Differentially expressed genes (DEGs) in the ferroptosis pathway\nare labeled with red (upregulated) or blue (downregulated). g, GSEA on the WikiPathway gene set. h, Volcano plots\nof transcriptome. DEGs in the NRF2 pathway are labeled with red (upregulated) or blue (downregulated). Cells in\npanels e-f were treated with PEF and incubated with cell debris for 24 h. N = 3.\nUpregulated genes in the MOF-Fe treated Panc-1 cells were enriched in gene ontology (GO) terms of\n“regulation of ferroptosis” and “ferroptosis (Supplementary Fig. 4a). NRF2-related pathways were also\nsignificantly upregulated (Fig. 2m), which was corroborated by the enrichment of GO term “response to\noxidative stress” (Supplementary Fig. 4a).\nSupplementary Fig. 4 | Effects of MOF-Fe on FTH1 (additional data).\na, GO_BP analysis for genes upregulated in the MOF-Fe group relative to the vehicle control. N = 3.\nFig. 2 | MOF-Fe induced ferroptosis and temporarily suppressed tumor recurrence after IRE.\ne, GSEA on the WikiPathway gene set. N = 3.\n20 / 47\n(9) Line 217-218. Mitochondrial alteration is also indicative of other cell death modalities like apoptosis\n(review: Nat Rev Mol Cell Biol. 2020 Feb;21(2):85-100). Even ferroptosis inducers can induce non-\nferroptotic non-apoptotic cell death in some cell types (Cancer Discov. 2022 Mar 1;12(3):774-791). Maybe\nthe authors could be more careful with these semantics?\nAnswer:\nWe apologize for the inaccurate statement. Mitochondria is not only a central hub for apoptosis, but is\nalso involved in other forms of cell death including necroptosis, ferroptosis, and pyroptosis. We thank the\nreviewers for bringing to our attention the elegant review “Mitochondria as multifaceted regulators of cell\ndeath”, Nature Reviews Molecular Cell Biology 2020, 21:85-100. This paper is added to the reference of\nthe revised manuscript.\nThe original sentence “Mitochondria alteration is characteristic of ferroptosis” is corrected to the\nfollowing: “Ferroptosis is often accompanied by mitochondria changes” in the revised manuscript.\n(10) Since the authors claim that FTH1 downregulation sensitizes the cells to ferroptosis, they should test\nif these cells are more sensible to ferroptosis inducers like erastin/ RSL3 etc.\nAnswer:\nWe agree. FTH1-OE and FTH1-KD Panc-1 cells were treated with erastin and RSL3 for 72 h, and\ntheir viability was measured using CCK-8 assay (Supplementary Fig. 6a). The IC values of both\n50\nferroptosis inducers were increased by FTH1 overexpression, but were not significantly changed by FTH1\nknockdown. These data suggest that FTH1 upregulation contributed to the resistance to erastin or RSL3.\nIn addition, C20U4V at 5 μM concentration significantly decreased the IC values of both erastin (12.6\n50\nμM vs. 1.6 μM) and RSL3 (5.9\nμM vs. 0.15 μM).\nSupplementary Fig. 6a, Cell viability of FTH1-OE and -KD cells after 72h treatment with erastin or RSL3. N = 6.\nAdditional data for review, Panc-1 viability after 72 h treatment with erastin or RSL 3 in the presence of 5 μM\nC20U4V. N = 6.\n21 / 47\nMinor points:\n(1) Flow cytometry data histograms, the y-axes are not labelled throughout. This is normalized to the mode?\nPlease indicate scale and annotate the axes.\nAnswer:\nWe apologize for the mistake. All flow cytometry plots of the C11-BODIPY staining are labeled with\n“#Cell (Normalized to Mode)” along the y-axes. Oxidation index was used for quantification per the\nreviewer’s suggestion. An example is shown in Fig. 1a.\n𝑂𝑥𝑖𝑑𝑎𝑡𝑖𝑜𝑛 𝐼𝑛𝑑𝑒𝑥 = 𝑀𝐹𝐼𝑠𝑎𝑚𝑝𝑙𝑒 (𝑜𝑥𝑖𝑑𝑖𝑧𝑒𝑑)−𝑀𝐹𝐼𝑏𝑙𝑎𝑛𝑘 (𝑜𝑥𝑖𝑑𝑖𝑧𝑒𝑑),𝑀𝐹𝐼 = 𝑚𝑒𝑎𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑖𝑛𝑑𝑒𝑥 [14]:\n𝑀𝐹𝐼𝑠𝑎𝑚𝑝𝑙𝑒 (𝑟𝑒𝑑𝑢𝑐𝑒𝑑)−𝑀𝐹𝐼𝑏𝑙𝑎𝑛𝑘 (𝑟𝑒𝑑𝑢𝑐𝑒𝑑)\nFig. 1a, Flow cytometry histograms and quantification of C11-BODIPY oxidation after treatment with PEF.\n(2) The authors write peroxidation throughout, whereas iron and hydrogen peroxide can generate hydroxyl\nand hydroperoxyl radicals, that can lead to peroxidation and oxidation. It is better to talk about lipid\noxidation rather than lipid peroxidation, even if this semantic mistake is used aplenty in the literature.\nAnswer:\nWe thank the reviewer for the clarification. Most “peroxidation” is changed to “oxidation” in the revised\nmanuscript, except those that are directly quoted from reference.\n(3) Line 114. Significantly instead of significant.\nAnswer:\nWe apologize for the typo. Grammatic errors are carefully checked throughout the revised manuscript.\n(4) Line 173/174: very interesting result. This goes hand in hand with the finding that tumors are higher in\niron content compared to healthy tissue from human biopsies (Nature. 2025 May 7. doi: 10.1038/s41586-\n025-08974-4).\nAnswer:\nIt is great to know that our findings corroborate the data from a top-tier study published in Nature.\n22 / 47\nReviewer #3 (Remarks to the Author); expert in IRE:\nSummary: A combination of IRE, MOF-Fe, and C20U4V is used as a collective therapeutic approach to\nmake pancreatic cancer cell lines decrease tumor and cell colony recurrence commonly seen with the use\nof only one or two of these interventions. The authors found that FTH1, a gene associated with ferroptosis,\npresented a repeated blockage to treatment efficacy and contributed to tumor regression. Through the\nexperimentation of varying combinations of these three individual treatments, they found that they could\nregulate this gene with combination therapy to make pancreatic cancer more susceptible to IRE treatment.\nThe authors provide strong justification of the methods used and discuss each aspect of experimentation\nin detail. My comments address only a few points concerning the discussion of cell death mechanisms.\nOverall, this paper is strong and only requires minor revisions.\nAnswer:\nWe appreciate the reviewer’s favorable evaluation. Detailed responses are included as follows.\nMajor Comments:\n- You discuss the fact that IRE intends to induce apoptotic cell death, but I think it is important to\nacknowledge that a significant portion of the ablation area can be allotted to necrotic cell death dependent\non the pulse parameters. You perform H&E staining for necrosis but don’t discuss it further.\nAnswer:\nWe apologize for the incorrect statement. A recent study by Mercadal et al. demonstrated that IRE\ninduced both accidental cell death (likely necrosis) and delayed cell death [11]. In this study, it was shown\nthat IRE with pulse intensity of 500 to 700 V/cm induced delayed cell death, which is probably executed\nthrough various regulated mechanisms. Their findings are in line with our experiments, in which PEF at\nhas been added to the reference in the revised manuscript.\nDetailed discussion on the H&E staining of Supplementary Fig. 14 is included as follows:\nTumors of the sham control presented a mixture of small or medium-sized irregular glands and poorly\ndifferentiated sarcoma-like tumor cells. Some tumor glands formed a cancer nest surrounded by desmoplastic\nstroma. Tumor cells exhibited enlarged and hyperchromatic nucleus, multiple nucleoli, and pathological mitosis\n(Supplementary Fig. 14). Necrotic regions were visible in tumors treated with MOF-Fe + M-C20U4V, especially\naround the sites of MOF-Fe accumulation. Infiltration by macrophages and neutrophils were present at the borders\nbetween the necrotic and viable regions. Both PEF and the triple treatment induced extensive areas of necrosis.\nResidual cancer cells were present outside the ablation zone of PEF-ablated tumors, whereas extensive infiltration\nby neutrophils and macrophages were observed in the triple treatment group.\n23 / 47\n- Studies have also identified the presence of pyroptosis and necroptosis contributing as modes of cell\ndeath. While there has been a significant amount of apoptotic and necrotic research, it is important to\naddress the contribution of these other forms of cell death and how they may impact the overall ablation.\nAnswer:\nWe agree. Cell death by pulsed electric fields involves multiple regulated pathways. In a review by\nNapotnik et al, the authors summarized the types of cell death after electroporation, including apoptosis,\nnecrosis, pyroptosis, and necroptosis [12]. This paper has been added to the reference of the revised\nmanuscript.\nAdditional experiments were performed to explore the possible routes of cell death in Panc-1 and\nMIAPaCa-2 cells at 24 h after treatment with PEF (pulse intensity = 600 V/cm, pulse duration = 100 μs,\nnumber of pulses = 99, pulse repetition frequency = 1 Hz). As shown in Supplementary Fig. 1c-e, PEF-\ntreated cells showed higher frequency of Annexin V+ populations, an indicator of apoptosis. They also\nexhibited a dramatic release of lactate dehydrogenase (LDH) and slightly higher expression of cleaved-\ngasdermin D (N-terminal), suggesting the occurrence of pyroptosis. The expression of phosphor-MLKL\nand phosphor-RIPK1, two necroptosis markers, was not affected.\nIt should be cautioned that these results may be restricted to the current PEF setting. A systematic\nevaluation through different PEF settings would provide useful information, but is unfortunately beyond the\nscope of current study.\nSupplementary Fig. 1 | Effects of PEF on pancreatic cancer cells (addition data).\nc, Flow cytometry pseudo-color plots and quantification. N = 3. d, Quantification of LDH release. N = 6.\ne, Immunoblots and quantification of p-MLKL, p-RIPK1, and cleaved-GSDMD (N terminal). N =6.\n24 / 47\n- As irreversible electroporation for tissue ablation was founded by Dr. Rafael Davalos, there should be at\nleast a few of his papers cited when addressing the background of the technology.\nAnswer:\nWe apologize for this mistake. We are grateful for the many ground-breaking works Dr. Davalos has\nbrought to this field. The following papers from Dr. Davalos’ group are added to the reference of the revised\nmanuscript.\n⚫ Ringel-Scaia VM, et al. High-frequency irreversible electroporation is an effective tumor ablation\nstrategy that induces immunologic cell death and promotes systemic anti-tumor immunity.\nEBioMedicine 44, 112-125 (2019).\n⚫ Edd JF, Davalos RV. Mathematical modeling of irreversible electroporation for treatment planning.\nTechnol Cancer Res Treat 6, 275-286 (2007)\n⚫ Mercadal B, Beitel-White N, Aycock KN, Castellvi Q, Davalos RV, Ivorra A. Dynamics of Cell Death\nAfter Conventional IRE and H-FIRE Treatments. Ann Biomed Eng 48, 1451-1462 (2020).\n- Mitochondrial alteration can also be due to apoptotic cell death. How are you able to conclude that it is\nonly characteristic of ferroptosis?\nAnswer:\nWe apologize for the inaccurate statement. Mitochondria is not only a central hub for apoptosis, but is\nalso involved in other forms of cell death including necroptosis, ferroptosis, and pyroptosis [15]. This paper\nis added to the reference of the revised manuscript.\nThe original sentence “Mitochondria alteration is characteristic of ferroptosis” is corrected to the\nfollowing: “Ferroptosis is often accompanied by mitochondria changes” in the revised manuscript.\nMinor Comments:\n- The DEG acronyms are not defined in text or figure captions.\nAnswer:\nWe apologize for the mistake. DEG is defined as differentially expressed genes, and the full name is\ndisplayed at first use of DEG in the results section “C20U4V augmented MOF-Fe-induced ferroptosis and\nenhanced the efficacy of PEF in vitro”.\n- Generally, review the paper for minor grammatical errors.\nAnswer:\nWe thank the reviewer for the reminder. The revised manuscript has been carefully examined for typos\nand grammatical errors.\n25 / 47\nReviewer #4 (Remarks to the Author);\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nAnswer:\nWe appreciate the reviewer’s efforts in assessing the manuscript.\n26 / 47\nReviewer #5 (Remarks to the Author); expert in PDAC:\nThis is a highly innovative study aiming to improve the efficacy of pulse electrical fields (PEF) by inducing\nferroptosis. The first layer of innovation is a fast-release iron nanoparticle (MOF) only modestly delayed\ngrowth of subcutaneous KPC-derived tumors. The data that resistance to this iron therapy was driven by\nincreased expression of ferritin are compelling, including genetic knockout studies FTH1. The next and\neven greater innovation was the development of a PROTAC targeting fatty acids, based on rationale that\nFTH1 is known to bind multiple fatty acids. The winner (C20U4V) was based on arachidonic acid (AA) and\nchosen on the basis of growth inhibition in vitro.\nAnswer:\nWe thank the reviewer for the positive evaluation. Detailed responses are as follows.\nIt is confusing that C20U4V increases FTH1 on its own in proteomic studies in vitro. It is only when given\nin combination with MOF that C20U4V decreases FTH relative to FTH. The combo treatment increases\nFTH in multiple experiments compared to control or IRE alone. A potential explanation is offered that this\nmay be due to effects of AA, but it seems inappropriate to refer to this as an “FTH1-targeting” PROTAC,\nwhen it clearly affects multiple proteins.\nAnswer:\nThe expression level of a specific protein is affected by the rate of protein synthesis, i.e., translation\nusing mRNA as the template, as well the rate of protein degradation through the ubiquitin-proteosome\nsystem or the autophagosome-lysosome routes, etc. In our opinion, C20U4V alone did not reduce FTH1\nexpression in Panc-1 cells (Supplementary Fig. 7d) because it also elevated the level of FTH1 mRNA\n(Supplementary Fig. 7c). This observation is in line with the upregulation of NRF2, which is a central\nmediator of anti-oxidative responses and is known to stimulate the transcription of FTH1 mRNA. The\noxidative stress induced by C20U4V is consistent with a previous report that exogenous arachidonic acid\npromoted RSL3-induced ferroptosis [16].\nIn the revised manuscript, we used FTH1-OE Panc-1 cells to evaluate the standalone capacity of\nC20U4V for FTH1 degradation. Cycloheximide (CHX), an inhibitor of protein synthesis, was added to stop\nthe de novo synthesis of FTH1. C20U4V was added at 10 and 20 μM and incubated with the cells for 12\nh. As shown in Supplementary 9e, around 70% of FTH1 was degraded by 20 μM C20U4V. CHX is\ncommonly used to study protein stability. A similar assay was reported to study protein degradation by\nPROTACs [2]. We did not apply this experiment setting to the parental Panc-1 cells, because their FTH1\nexpression would be furthered lowered by CHX, making the results unreliable.\nWe agree that C20U4V also targeted other proteins. When examining several other targets in the\nferroptosis and NRF2 pathway (Supplementary Fig. 7j, 8h), the expression of ferritin light chain (FTL),\nNAD(P)H quinone dehydrogenase 1 (NQO1), and Glutamate-cysteine ligase modifier subunit (GCLM) was\nalso reduced by C20U4V in the presence of MOF-Fe. It should be noted that concurrent degradation of\nmultiple proteins by a PROTAC is commonly encountered in literature [17]. Per the reviewer’s suggestion,\n27 / 47\nwe have changed the term “FTH1-targeting PROTAC” to “FTH1-degrading PROTAC” throughout the\nrevised manuscript.\nSupplementary Fig. 7c, mRNA expression of NRF2, HMOX1, and FTH1 after C20U4V treatment. N = 3.\nSupplementary Fig. 7d, Immunoblots and quantification of NRF2, HMOX1, and FTH1 after C20U4V treatment.\nN= 3 to 5.\nSupplementary Fig. 9e, Immunoblots and quantification of FTH1 in FTH1-OE Panc-1 cells after treatment with\nC20U4V in the presence of CHX. N = 3.\nSupplementary Fig. 7j, Immunoblots and quantification of FTL after treatment with MOF-Fe and C20U4V. N= 3.\nSupplementary Fig. 8h, Immunoblots and quantification of NQO1, SLC7A11, GCLM, and TP53 after treatment\nwith MOF-Fe and C20U4V. N = 4 to 8.\n28 / 47\nThe efficacy of the PROTAC for degrading FTH is not extremely high (Fig 5i)—1.3-fold by proteomics in\nvitro and FTH1 was not significantly decreased (p=0.07) in treated tumors in vivo (relative to combo of\nPEF + MOF without PROTAC). Despite this, there is impressive growth delay with the triple combination\nin subcutaneous tumor model and survival improvement in an orthotopic model.\nAnswer:\nWe agree that the ability of C20U4V to degrade FTH1 was not as potent as we wished. One\nexplanation could be that the binding affinity of arachidonic acid to ferritin is around 60 μM [18], which is\nmuch higher than the typical ligands for protein-of-interest in PROTACs. Therefore, C20U4V may exhibit\nlimited efficiency in bridging FTH1 with VHL, and inducing FTH1 degradation. Unfortunately, to our best\nknowledge, there currently lacks a highly specific ligand for FTH1. We believe that the data obtained with\nC20U4V still supports our hypothesis that disruption of iron homeostasis could sensitize pancreatic cancer\ncells to IRE.\nThe suboptimal fold-change in the proteomics of original Fig. 4e was probably due to the factor that\nthe samples were collected 48 h after treatment with MOF-Fe and C20U4V. Additional experiments have\nshown that maximal degradation efficiency was achieved at 12 h (Supplementary Fig. 7e). The\nproteomics experiment was thus repeated after 12 h treatment with MOF-Fe and C20U4V (Fig. 4f in the\nrevised manuscript). FTH1 expression was decreased by 35% (Log FC = -0.6049, p = 0.001644). The\n2\ndiscrepancy in degradation efficiency between the immunoblot and proteomics may be attributed to the\ndifference in detection sensitivity. We also tested different C20U4V concentrations, and observed that\nFTH1 was substantially degraded by 90% with 20 and 50 μM C20U4V (Supplementary Fig. 7f).\n29 / 47\nSupplementary Fig. 7e, Quantification of FTH1 expression after treatment with MOF-Fe (3 μg Fe/mL) and C20U4V\n(10 μM) at different time points. N = 3.\nFig. 4f, , Volcano plots of protein expression in Panc-1 cells treated for 12 h with MOF-Fe + C20U4V vs. MOF-Fe.\nDifferentially expressed proteins in the ferroptosis pathway are labeled. N = 3.\nSupplementary Fig. 7f, Immunoblots and quantification of FTH1 after treatment with MOF-Fe and different\nconcentrations of C20U4V. N = 4.\nWe apologize for the confusion on the comparison in the original Fig. 5i. The comparison was\nperformed in vitro between the “MOF-Fe + C20U4V” dual-treatment group and the “PEF + MOF-Fe +\nC20U4V” triple-treatment group, and was intended to show that adding PEF further lowered FTH1\nexpression although the p-value (0.074) was marginal. Since this comparison is not essential for the\nmanuscript, it has been removed in the revised version.\nMy other significant criticism is the use of sublethal PEF. It is understandable why this is necessary for in\nvitro studies, because it is too easy to kill cell in vitro, and you won’t have any cells to study if you use\nlethal PEF. However, the conditions for intentionally sublethal PEF are very different than what are used\nclinically for IRE (or what have been used in mouse models of IRE). How do the effects of IRE on\nferroptosis-associated proteins compare to sublethal PEF? Would there still be synergy? If perhaps this is\nnot feasible to do for manuscript, this limitation needs to be acknowledged. A sign that the conditions used\nare perhaps insufficient is that “IRE” in this study (which should perhaps be referred to as either PEF or\nincomplete IRE or “reversible electroporation”) did not increase uptake of M-C20U4V in tumors.\nAnswer:\nWe agree that the term “IRE” in the original manuscript was confusing. In clinics, “IRE” refers to the\nprocedure of using high-intensity pulsed-electric field (PEF) to ablate solid tumors, and the main goal is to\nablate as much tumor as possible. However, residual cancer cells may survive due to many different\nreasons. Our study was designed to mimic the clinical setting, and the pulse intensity for in vivo ablation\n(1150 V, 5 mm between electrodes) was in the “irreversible” range.\nTherefore, we propose using PEF in the revised manuscript. The description of in vivo experimental\nmethods is revised as follows:\n…For in vivo studies, the electric pulses were delivered using a two-needle array electrode with a 5-mm gap\n(Catalog# 45-0168, BTX Harvard Apparatus). The PEF parameters were: pulse voltage = 500 or 1150 V, pulse\nduration = 100 μs, pulse repetition frequency = 1 Hz, number of pulses = 99. The electrode array was inserted to\ncompletely penetrate the tumor nodule. After one round of PEF treatment, the electrodes were retrieved and\ninserted along the orthogonal direction to deliver 99 more pulses with the same parameters. PEF in vivo at 1150\nV was denoted as ablative PEF or PEF ablation throughout the manuscript, while low-intensity PEF used in\nother experiments was specified with the exact pulse intensity or voltage in the corresponding figure\nlegends.\nThe impact of true “IRE” on ferroptosis-related markers was investigated by comparing the viable\nregion and ablation zone (where true “IRE” was applied to) in KPC-A719 tumors (see figure next page).\n30 / 47\n4-HNE staining was examined on day 1 after ablative PEF since it is a ROS-induced chemical modification.\nThe viable regions exhibited moderate level of cytoplasmic staining, which was absent in the ablation zone.\nFTH1 and HMOX1 staining were evaluated on day 9. Both targets were positively stained in the viable\nregions, which was not observed in the ablation zone. The faint colorization of the ablation zone probably\narose from non-specific staining. In summary, it would be safe to claim that the ferroptosis-related proteins\nwere not upregulated in the ablation zone after true “IRE”.\nRepresentative IHC staining of 4-HNE, FTH1, and HMOX1 (for review only). Microscopic images were taken\nfrom the viable region or ablation zone of the same tumor. Scale bar = 100 μm.\nWe acknowledge that the tumor uptake of M-20U4V was not increased after the PEF ablation. M-\nC20U4V was intravenously injected via tail vein. Therefore, its tumor uptake is affected by its\npharmacodynamic properties as well as the function of intratumoral vasculature.\nIRE and low-intensity reversible electroporation exert distinct effects on vasculatures [3]. Significant\napoptosis of endothelial cells and microvessel thromboses was observed in tumor models at 30 min after\nIRE [4]. IRE could also disrupt the blood-brain barrier [5]. Reversible electroporation, on the other, induced\na temporary “vasculature lock” that is often resolved within 48 h [6]. The vasculature lock could be divided\ninto two phases. During the first phase, electroporation induced a reflexory vasoconstriction of afferent\narterioles mediated by the sympathetic nervous system. During the second phase, the elevation of\nvasculature permeability led to the leakage of fluids from the vasculature lumen into the surrounding\ninterstitial space. The subsequent increase in the tumor interstitial pressure and the decrease in\nintravascular pressure converged to cause vasculature collapse.\nThese studies suggested that electroporation might have dichotomous effects on the tumor uptake of\nnanoparticles. On the one hand, the increase of vasculature permeability facilitated the leakage of\nnanoparticles into the interstitial space, and thus favored their tumor uptake. On the other hand, the IRE-\ninduced thromboses and reversible electroporation-induced vasculature lock could hinder tumor blood\nperfusion, and consequently was unfavorable for the tumor uptake of nanoparticles.\n31 / 47\nSeveral studies have investigated the effects on IRE on the tumor uptake of chemotherapy drugs or\nnanoparticles (summarized in the table below). Bhutiani et al discovered that IRE enhanced gemcitabine\ndelivery to the zone of reversible electroporation in pancreatic tumor models [7]. Peng et al. reported that\nthe combination of microsecond and nanosecond PEF increased the uptake of nano-gel in murine colon\ntumor models [8]. We previously showed that the tumor uptake of fluorescence-labeled dextran was\nelevated on day 4 post-IRE [9].\nTable. Study summary on tumor uptake of chemotherapy drugs or nanoparticles after PEF (for\nreview only)\nRef. PEF Parameters Agent and Schedule Findings\n[7] Pulse intensity = 1600~2000 Gemcitabine injected twice with 2 h in between; IRE increased\nV/cm IRE between two injections of Gemcitabine; gemcitabine delivery in\nPulse duration = 90 μs Samples collected from the reversible the zone of reversible\nPulse number = 10 per group electroporation zone. electroporation\nPulse interval = 250 ms\n[8] Combination of μs and ns pulses Cell membrane-coated nanogel about 100 nm in PEF improved the whole\nμ pulse intensity = 1500 V/cm size; tumor uptake of nanogel\nμ pulse duration = 100 μs Tumors first treated with PEF, then intravenously for up to 5 times.\nnano pulse intensity = 10,000 injected with the nanogel;\nV/cm Tumor uptake measured by fluorescence from 1\nnano pulse duration = 500 ns to 48 h after PEF.\n[9] Pulse intensity = 2400 V/cm FITC-conjugated dextran (70 kD) was injected IRE increased the whole\nPulse duration = 100 μs immediately after IRE tumor uptake of dextran\nPulse number = 99 Tumor uptake measured by fluorescence on day on day 4 after IRE\nPulse interval = 1 s 4 and 6 after IRE\nCurrent Pulse intensity = 2300 V/cm Fluorescence-labeled polymeric micelles about 80 No difference in\nstudy Pulse duration = 100 μs nm in size; polymeric micelles\nPulse number = 99 Polymeric micelles were injected 4 h before IRE. uptake between IRE-\nPulse interval = 1 s Tumor uptake measured by fluorescence at 48 h treated and sham treated\nafter injection (44 h after IRE) tumors\nWe noticed that the sequence of PEF ablation and nanoparticle injection was different in the current\nstudy: M-20U4V was injected 4 h before electroporation, while nanoparticles/gemcitabine was injected\nafter electroporation in the other three studies. Such discrepancy in schedule might have shifted the peak\nof plasma concentration-time curve of M-C20U4V relative to the behavior change of tumor vasculature.\nNo improvement of M-C20U4V retention was observed as a result. Further experiments will be performed\nto validate this hypothesis.\n32 / 47\nFinally, the reasons for “resistance” to IRE are many and complex, many are likely technical or anatomical\nrelated to incomplete physical ablation. While mutations in apoptosis genes might contribute, this is an\nunnecessary over-simplification of the issue. It is certainly true that local recurrence rates are higher than\nwe would like, but the statement that “the rate of local recurrence is higher than other ablative therapies”\nis not supported by data. Two studies were referenced that had amongst the highest recurrence rates in\nthe literature but are not representative of the field, particularly open surgical IRE, and did not compare\nIRE to other forms of ablation, which have equally high, if not higher rates of local recurrence for pancreatic\ncancer. This statement needs to be amended/softened.\nAnswer:\nWe apologize for the confusion. “Resistance” to IRE was not attributed to mutations in apoptotic\npathways in the manuscript. In the first paragraph of introduction, the presence of residual cancer cells\npost-IRE was attributed to the uneven distribution of PEF intensity. PEF intensity at the certain area is\ninversely correlated with its distance to the electrode, and therefore can be affected by electrode\narrangement as well as the complexed anatomy of pancreatic tumors [19]. It could also be distorted by the\nheterogeneous components of the tumor microenvironment [20]. Therefore, we believe that our statement\nis in agreement with the reviewer’s comment that “the reasons for resistance to IRE are many and complex,\nmany are likely technical or anatomical related to incomplete physical ablation”.\nIn the second paragraph of the introduction, we summarized the contemporary approaches for the\nelimination of the residual cancer cells, including chemotherapy, radiotherapy, targeted therapy, and\nimmunotherapy [9, 21-24]. These therapies share a common mechanism: most of them kill cancer cells\nby inducing apoptosis. It was then postulated that cancer cells with mutations in apoptotic pathways could\nbe resistant to these therapies. Therefore, resistance to IRE was not attributed to mutations in apoptotic\nsignaling.\nWe thank the reviewer for clarifying that IRE is not inferior to other ablative techniques in terms of\nlocal recurrence. The incorrect statement that “the rate of local recurrence is higher than other ablative\ntherapies” is removed from the revised manuscript. The related paragraphs are shown below:\n…The resulting pulsed electric field (PEF) then eliminates cancer cells by inducing irreversible disruption of their\ncell membranes2, and promotes systemic anti-tumor immunity through the release of damage-associated molecular\npatterns3. However, the PEF intensity is unevenly distributed in the tumor4, 5. In regions where PEF intensity falls\nbelow the threshold for completion cell eradication, residual cancer cells may survive and ultimately drive tumor\nrecurrence.\nCurrent approaches to eliminating these residual cancer cells often rely on inducing apoptosis6-10. Therefore,\ntheir efficacy may be compromised when cancer cells harbor mutations in the apoptotic cascades. Ferroptosis is\nan iron-dependent, non-apoptotic form of regulated cell death that can overcome resistance to conventional\nchemotherapies11, 12.\nMinor thing is that there are too many non-standard, undefined abbreviations in graphic abstract.\nAnswer: We apologize for the confusion. Graphic abstract is removed because it is not allowed in Nature\nCommunications per the journal’s formatting instructions.\n33 / 47\nReviewer #6 (Remarks to the Author); expert in PROTAC:\nReview\nThis manuscript by Linrong Li, Shihao Su et al. presents a novel study investigating a strategy to enhance\nthe efficacy of irreversible electroporation (IRE) in primarily pancreatic cancer by sensitizing residual\ntumour cells to ferroptosis. During IRE, residual viable cancer cells can remain a significant cause of\ntumour recurrence. The authors demonstrate that iron-based metal-organic framework nanoparticles\n(MOF-Fe) can suppress recurrence by inducing iron overload and subsequently ferroptosis; however, this\neffect is attenuated by IRE-induced upregulation of ferritin heavy chain 1 (FTH1). To overcome this\nlimitation, the authors employ an arachidonic acid-derived PROTAC, C20U4V, to selectively degrade\nFTH1, thereby amplifying ferroptotic activity in both pancreatic cancer cell lines and organoids.\nEncapsulation of C20U4V within a ROS-responsive micelles (called M-C20U4V) enables in vivo delivery,\nand in combination with MOF-Fe, they show enhanced ferroptosis and therapeutic efficacy in murine\nmodels. This study is conceptually novel, supported by experimental data, and of clear translational\nrelevance. While a few minor points requiring clarification, the manuscript is of sound quality and in my\nopinion would be suited publication in Nature Communications.\nAnswer:\nWe appreciate the reviewer’s favorable evaluation. Detailed responses are listed as follows.\nComments\nSublethal PEF Induces Anti-Ferroptotic Gene Expression\nAs shown pulsed electric fields (PEF) upregulate anti-ferroptotic genes in pancreatic cancer cells. However,\nin Figure 1B, immunohistochemical staining of 4-Hydroxynonenal (4-HNE) in KPC tumours appears non-\nsignificant. It may be more appropriate to include this panel as a supplementary figure, especially since\nthe subsequent panels (PANC-1 data) clearly demonstrate significant effects.\nAnswer:\nWe apologize for the confusion. Fig. 1 is re-organized to improve logic flow. First, C11-BODIPY\noxidation (Fig. 1a) was re-analyzed. C11-BODIPY is a ratiometric fluorescence probe of lipid peroxidation.\nIt emits red fluorescence at reduced state, and shifts to green at oxidized state. According to the suggestion\nfrom Reviewer #2, oxidation index was calculated as:\n𝑂𝑥𝑖𝑑𝑎𝑡𝑖𝑜𝑛 𝐼𝑛𝑑𝑒𝑥 = 𝑀𝐹𝐼𝑠𝑎𝑚𝑝𝑙𝑒 (𝑜𝑥𝑖𝑑𝑖𝑧𝑒𝑑)−𝑀𝐹𝐼𝑏𝑙𝑎𝑛𝑘 (𝑜𝑥𝑖𝑑𝑖𝑧𝑒𝑑),𝑀𝐹𝐼 = 𝑚𝑒𝑎𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑖𝑛𝑑𝑒𝑥 [14]:\n𝑀𝐹𝐼𝑠𝑎𝑚𝑝𝑙𝑒 (𝑟𝑒𝑑𝑢𝑐𝑒𝑑)−𝑀𝐹𝐼𝑏𝑙𝑎𝑛𝑘 (𝑟𝑒𝑑𝑢𝑐𝑒𝑑)\nAfter re-analysis, the level of C11 oxidation was similar between the control and the PEF group (Fig.\n1a), which was consistent with the results of MDA concentration (Fig. 1b). Ferroptosis inhibitors (Lip-1,\nFerr-1) and a stronger anti-oxidant (tocopherol) was then added to PEF-treated cells, but did not rescue\nthe cell viability (Fig. 1c, d), suggesting that PEF probably did not cause ferroptotic cell death. Changes\nin the signaling pathway were then analyzed (Fig. 1e-h), and showed that ferroptosis suppressor genes\n34 / 47\nand NRF2 pathway were upregulated after treatment with PEF and cell debris. The findings from the in\nvitro studies were then verified in KPC-A719 tumor models. In tumor treated with IRE (referred to as PEF\nablation according to Reviewer #5’s suggestion), 4-HNE staining in the viable regions of PEF ablation-\ntreated tumors were similar to that of control (Fig. 1k). Separate tumor models were treated with PEF at\nFTH1 was significantly elevated on day 2 post-PEF. Taken together, we have demonstrated that low-\nintensity-PEF upregulated ferroptosis suppressor genes in pancreatic cancer cells instead of inducing\nferroptotic cell death.\nWe respectfully propose retaining Fig. 1k in the main figure, as it provided direct evidence that low-\nintensity PEF, when applied at the current setting, did not induce ferroptotic cell death.\nFig. 1 (a-j) | PEF upregulated ferroptosis suppressor genes in pancreatic cancer cells\n35 / 47\nFig. 1 (k, l) | PEF upregulated ferroptosis suppressor genes in pancreatic cancer cells (continued).\na, Flow cytometry histograms and quantification of C11-BODIPY oxidation. N = 7. b, MDA concentration. N = 3. c,\nExperiment scheme for panel d. d, Panc-1 viability after PEF and incubation with Lip-1, Ferr-1, or tocopherol. N =\n6. e, GO analysis of biological process (GO_BP) for genes upregulated in the PEF + cell debris group relative to\nthe control group. f, Volcano plots of transcriptome. Differentially expressed genes (DEGs) in the ferroptosis\npathway are labeled with red (upregulated) or blue (downregulated). g, GSEA on the WikiPathway gene set. h,\nVolcano plots of transcriptome. DEGs in the NRF2 pathway are labeled with red (upregulated) or blue\n(downregulated). Cells in panels e-f were treated with PEF and incubated with cell debris for 24 h. N = 3. i,\nImmunoblots of Panc-1 cells. Quantification is included in Supplementary Fig. 1f. N = 3 to 5. j, Hematoxylin-\neosin (H&E) staining of KPC-A719 tumors at 24 h after IRE. The border between the viable tumors and ablation\nzone is marked with a black dashed line. Residual cancer cells are indicated with black arrowheads. Scale bar =\n100 μm. k, 4-HNE staining of KPC-A719 tumors and quantification. Scale bar = 100 μm. N = 6 to 8. l, Immunoblots\nof KPC-A719 tumors. Quantification is included in Supplementary Fig. 1g. N = 9 to 12. PEF intensity = 600 V/cm\nin cell culture or as specified for in vivo studies. Data are presented as mean ± standard error of mean (SEM).\nStatistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison,\nStudent’s two-sided t-test, or ratio paired t-test. n.s. = not significant. *p < 0.05. **p < 0.01.\nFigure Labeling and Mis-referencing in the Text:\nThere are multiple inconsistencies between figure panels and their citations in the text:\nFigure 1C is incorrectly labelled as Figure 1B.\nFigures 1I–K are referred to in the text as Figures 1E–G.\nFigure 1D in the text corresponds to Figure 1H.\nFigure 1E in the text is Figure 1I.\nFigure 1H in the text refers to Figure 1L.\nFigures 1J and 1K are not referenced in the text at all. They appear to be extensions of earlier panels and\nmay benefit from reordering to align with their corresponding experiments and improve overall clarity.\nAnswer:\nWe thank the reviewer for the corrections. The revised manuscript has been carefully examined for\ntypos and grammatical errors.\n36 / 47\nAnti-Ferroptotic Gene Upregulation and Ferroptosis Resistance\nThe demonstration that PANC-1 cells upregulate anti-ferroptosis/antioxidative stress genes post-PEF is\nconvincing. The data showing that Ferrostatin-1 (Ferr-1) and Liproxstatin-1 (Lip-1) do not restore viability\nsuggests that PEF does not sensitize cells to ferroptosis. However, a useful addition could be to investigate\nwhether PEF-treated cells gain resistance to ferroptosis. For example, treating both PEF and non-PEF\ncells with erastin and comparing their sensitivities could yield informative insights about sensitization/de-\nsensitization of model systems to ferroptosis post electroporation.\nAnswer:\nWe thank the reviewer for the insightful suggestion. In a separate project, we have developed PEF-\nresistant Panc-1 cells by ten cycles of selection. For each cycle, cells were treated with PEF, seeded in\nflask, and cultured to confluency. After ten cycles, the cells were tested and indeed formed more colonies\nthan the parental cells after PEF treatment.\nFollowing the reviewer’s comments, both parental cells and PEF-resistant cells were treated with\nerastin or RSL3 for 24 h, and their viability measured (see figure below). Compared to the parental cells,\nthe PEF-resistant cells were less sensitive to erastin, but more sensitive to RSL3. These results suggested\nthat the acquired resistance to PEF may involve other mechanisms beyond ferroptosis. Unfortunately,\nfurther mechanistic studies would be out of scope for the current manuscript.\nCytotoxicity of erastin and RSL3 in PEF-resistant Panc-1 cells (for review only)\nParental or PEF-resistant Panc-1 cells were treated with erastin or RSL3 for 24 h. Cell viability was measured using\nCCK-8 assay. N = 6.\n37 / 47\nArachidonic Acid-Derived PROTACs Targeting FTH1\nC20U4V is the chosen molecule for FTH1 degradation. However, C20U5V also appears to have very\nsimilar activity. What was the rationale for selecting C20U4V over C20U5V? Furthermore, why was\nC20U5V excluded from the CCK-8 assay? I imagine that the EPA will have a different interactome to AA\nand therefore it would be interesting to perform some of the later experiments with MOF-FE and PEF to\ndetermine what difference are present between compounds.\nAnswer:\nWe agree with the reviewer that CCK-8 assay alone was insufficient for compound screening. A\nsecond round of immunoblot screening was performed on chimeras with IC values close to 10 μM (Fig.\n50\n4d, Supplementary Fig. 7a). Panc-1 cells were incubated with MOF-Fe and each chimera (10 μM) for 12\nh, and their FTH1 expression was compared to that treated with MOF-Fe only. C20U4V exhibited the\nlowest IC values (5.38 ± 1.42 μM for Panc-1 and 3.65 ± 0.98 μM for MIAPaCa-2, Fig. 4c, Supplementary\npresence of MOF-Fe. Therefore, C20U4V was selected for further investigations.\nSummary of screening for C20U4V\nFig. 4c, Scattered plots of the IC values of the PROTAC library against Panc-1 and MIAPaCa-2 cells. Cells were\n50\ntreated with each chimera for 72 h. N = 6. d, Scatter plots of the change of FTH1 expression in Panc-1 cells after\ntreatment with MOF-Fe (3 μg Fe/mL) and the PROTAC library for 12 h. N = 3.\nSupplementary Fig. 7a, Representative immunoblots of FTH1 after treatment with MOF-Fe and chimeras (10 μM).\nC20U5V exhibited an IC value of 12.64 ± 0.52 μM for Panc-1 and 11.70 ± 0.54 μM for MIAPaCa-2.\nMOF-Fe. Therefore, it was less potent than C20U4V. According to the reviewer’s suggestion, additional\nexperiments were performed on the sensitizing effects of C20U5V. As shown in Supplementary Fig. 11,\nPanc-1 cells treated with the MOF-Fe + C20U5V dual-treatment or MOF-Fe alone showed similar levels\nof C11-BODIPY oxidation. MOF-Fe and C20U5V synergistically inhibited colony formation in Panc-1 cells.\nThe PEF + MOF-Fe + C20U5V triple-treatment also resulted in fewer colonies than PEF or the dual-\ntreatment. Therefore, our results support that the multiple PUFA-derived PROTACs could degrade FTH1\nand potentiate MOF-Fe against pancreatic cancer cells.\n38 / 47\nSupplementary Fig. 11 | Effects of MOF-Fe and C20U5V on pancreatic cancer cells.\na, Histograms and quantification of C11-BODIPY oxidation. N = 3. b-d, Colony quantification (b), HSA synergy\nscores (c), and representative images (d) in Panc-1 cells. N = 3. e, Representative images and quantification of\ncolonies by Panc-1 cells after treatment with PEF and/or MOF-Fe + C20U5V. N= 3 to 6. Data are presented as\nmean ± SEM. Statistical significance was determined using one-away ANOVA followed by Dunnett’s multiple\ncomparison or ratio-paired t-test. n.s. = not significant. ***p < 0.001. ****p < 0.0001.\n39 / 47\nAlthough the degradation is shown to be VHL-dependent, arachidonic acid (AA) is not likely to be inherently\nselective for FTH1. It would therefore strengthen the conclusion to demonstrate that FTH1 degradation is\na result of specific binding of VHL and FTH1 rather than a by-product of a loss of stability of FTH1 through\nnon-specific targeting of C20U4V. A suggested approach is a competition assay, where FTH1 is pre-\nsaturated with (S,S,S)-C20U4V prior to treatment with C20U4V to confirm target specificity.\nAnswer:\nWe agree that pre-saturation with (S,S,S)-C20U4V provides direct evidence that FTH1 degradation is\ndue to the specific binding of VHL and FTH1. Since Panc-1 cells would encounter excessive stress when\nMOF-Fe, C20U4V, and the blocking (S,S,S)-C20U4V were simultaneously added, we switched to FTH1-\nOE Panc-1 cells for the competition assay. Cycloheximide (CHX) was added to FTH1-OE cells to inhibit\nthe de novo protein synthesis, and the cells were incubated with C20U4V (10, 20 μM) and (S,S,S)-C20U4V\n(20 μM) for 12 h. No degradation of FTH1 was observed (Supplementary Fig. 9f). In contrast, 20 μM of\nC20U4V successfully degraded FTH1 in the same setting when (S,S,S)-C20U4V was absent\n(Supplementary Fig. 9e). These results supported that FTH1 degradation was not due to the non-specific\nloss of stability.\nCHX is commonly used to study protein stability. A similar assay to study protein degradation by\nPROTACs has been reported [2].\nSupplementary Fig. 9 | Evaluation of PUFA-derived PROTACs (additional data 1).\ne, Immunoblots and quantification of FTH1 in FTH1-OE Panc-1 cells after treatment with C20U4V in the presence\nof CHX (e) or (S,S,S)-C20U4V + CHX (f). N = 3. Data are presented as mean ± SEM. Statistical significance was\ndetermined using one-away ANOVA followed by Dunnett’s multiple comparison. n.s. = not significant. ****p <\n0.0001.\n40 / 47\nA pull-down assay combined with Mass spectrometry could establish to us that C20U4V is binding to FTH1\nand also could help define the interactome of the arachidonic acid and further support target engagement\nof C20U4V.\nAnswer:\nWe thank the reviewer for this excellent suggestion. A biotin-conjugated derivative of arachidonic acid\n(Biotin-PEG -AA, BPAA) was first synthesized and its chemical structure confirmed (Supplementary Fig.\n3\n74, 75, 110). Panc-1 cells were incubated with MOF-Fe and different concentrations of BPAA (0, 5, 10,\n20\nμM) for 24 h. The cell lysate was incubated with streptavidin-coated magnetic beads. The beads were\nwashed and subjected to immunoblots and qualitative proteomics.\nThe pulldown assay confirmed the interaction between FTH1 and BPAA (Fig. 4g, Supplementary\nFig. 8a).\nSummary of pulldown assay\nFig. 4g, Chemical structure of BPAA and immunoblots of FTH1 pulled down by BPAA. N = 3. Supplementary Fig.\n8a, Quantification of FTH1 pulled down by BPAA. N = 3. Data are presented as mean ± SEM. Statistical significance\nwas determined using one-away ANOVA followed by Dunnett’s multiple comparison. n.s. = not significant. *p <\n0.05.\nQualitative proteomics revealed 648 proteins, including FTH1, that might have interacted with BPAA\n(Supplementary Fig. 8b). GO analyses of the interactome on biological process (BP), molecular function\n(MF), and cellular components (CC) yielded the enrichment of a broad span of GO terms, e.g., “DNA\nreplication initiation”, “DNA helicase activity”, and “podosome” (Supplementary Fig. 8c-d). This\nobservation was consistent with the ubiquitous roles of AA in cellular physiology [25]. Seven ferroptosis-\nrelated and eight NRF2-related proteins were identified in the interactome, among which NQO1 was also\neffectively degraded by C20U4V (Supplementary Fig. 8f-h).\nThese results suggested that arachidonic acid (AA) may have interacted with other proteins in addition\nto FTH1. However, given the lack of highly-specific ligands for FTH1, C20U4V still provided a unique\nopportunity to degrade FTH1 in a PROTAC manner. We believe that the main conclusion of this manuscript,\ni.e., disruption of iron homeostasis is a feasible approach to lowering the risk of tumor recurrence after\nIRE, is not affected.\n41 / 47\nSupplementary Fig. 8 | Analysis of proteins pulled down by BPAA.\na, Quantification of FTH1 pulled down by BPAA. N = 3. b, Venn plots of proteins in the control group and those\npulled down by BPAA. Panc-1 cells were incubated with MOF-Fe (3 μg Fe/mL) (control) or MOF-Fe + 20 μM BPAA.\nTop-five ranked proteins and FTH1 are listed. c-e, GO analyses of biological process (BP, c), molecular process\n(MF, d), and cellular components (CC, e) for the 648 proteins pulled down by BPAA. f, g, Venn plots of proteins in\nthe BPAA-pull down group and those in the ferroptosis (f) or NRF2 (g) pathway. h, Immunoblots and quantification\nof SLC7A11 and NQO1 after treatment with MOF-Fe and C20U4V. N = 4. Data are presented as mean ± SEM.\nStatistical significance was determined using one-away ANOVA followed by Dunnett’s multiple comparison. n.s. =\nnot significant. ****p < 0.0001.\n42 / 47\nA DC titration assay for C20U4V would also be beneficial to quantify compound potency and the optimal\n50\ntherapeutic window of C20U4V.\nAnswer:\nWe agree. Panc-1 cells were incubated with MOF-Fe and up to 50 μM of C20U4V for 12 h. Substantial\ndegradation of FTH1 was observed at 20 and 50 μM (Supplementary Fig. 7f).\nSupplementary Fig. 7f, Immunoblots and quantification of FTH1 treated with MOF-Fe and C20U4V. N = 4. .\nMicelle-Loaded C20U4V (M-C20U4V)\nIn Figure 5, degradation kinetics for C20U4V are measured at 12 hours, whereas M-C20U4V is measured\nat 48 hours. For proper comparison, these should be performed at the same timepoint.\nAnswer:\nWe agree. Degradation kinetics for C20U4V was performed in Panc-1 (Fig. 7e), MIAPaCa-2\n(Supplementary Fig. 12c), and KPC-A719 (Supplementary Fig. 12d) cells. These results suggested that\nM-C20U4V exhibited degradation ability similar to that of C20U4V.\nSummary of M-C20U4V degradation kinetics\nImmunoblots and quantification of FTH1 in Panc-1 (Fig. 7e), MIAPaCa-2 (Supplementary Fig. 12c), and KPC-\nA719 (Supplementary Fig. 12d) cells after treatment with MOF-Fe and C20U4V for 12 to 48 h. N = 3 to 4.\n43 / 47\nIn the supplementary file they do perform a 48 Hour titration of C20U4V and show that it is no longer\ncapable of degradation, is this due to stimulation of FTH1 expression of FTH1 or that they’re compound\nisn’t potent?\nAnswer:\nIn our opinion, C20U4V alone did not reduce FTH1 expression in Panc-1 cells (Supplementary Fig.\n7d) because it also elevated the level of FTH1 mRNA (Supplementary Fig. 7c). This observation is in line\nwith the upregulation of NRF2, which is a central mediator of anti-oxidative responses and is known to\nstimulate the transcription of FTH1 mRNA. The oxidative stress induced by C20U4V is consistent with a\nprevious report that exogenous arachidonic acid promoted RSL3-induced ferroptosis [16].\nFTH1-OE Panc-1 cells were used to evaluate the standalone capacity of C20U4V for FTH1\ndegradation. Cycloheximide (CHX), an inhibitor of protein synthesis, was added to stop the de novo\nsynthesis of FTH1. C20U4V was added at 10 and 20 μM and incubated with the cells for 12 h. As shown\nin Supplementary 9e, around 70% of FTH1 was degraded by 20 μM C20U4V. CHX is commonly used to\nstudy protein stability. A similar assay was reported to study protein degradation by PROTACs [2]. We did\nnot apply this experiment setting to the parental Panc-1 cells, because their FTH1 expression would be\nfurthered lowered by CHX, making the results unreliable.\nWe acknowledge that the degradation potency of C20U4V may be not as good as we wished. After\nall, the binding affinity of AA to ferritin was around 60 μM [18], which is much higher than the typical ligands\nfor protein-of-interest in PROTACs. Unfortunately, to our best knowledge, there still lacks a highly specific\nligand for FTH1. We believe that the data obtained with C20U4V supports our hypothesis: disruption of\niron homeostasis could sensitize pancreatic cancer cells to IRE.\n44 / 47\nSupplementary Fig. 7c, mRNA expression of NRF2, HMOX1, and FTH1 after C20U4V treatment. N = 3.\nSupplementary Fig. 7d, Immunoblots and quantification of NRF2, HMOX1, and FTH1 after C20U4V treatment.\nN= 3 to 5.\nSupplementary Fig. 9e, Immunoblots and quantification of FTH1 in FTH1-OE Panc-1 cells after treatment with\nC20U4V in the presence of CHX. N = 3.\nCould the authors clarify this\nAre they authors able to treat at even higher concentrations given the low level of activity or are they\nconstrained by solubility of the molecule?\nAnswer:\nC20U4V was used at maximal 50 μM in cell culture and degraded FTH1 in the presence of MOF-Fe\n(Supplementary Fig. 7f). Encapsulation in polymeric micelles substantially increased the water-solubility\nof C20U4V. In a typical animal experiment, M-C20U4V was injected at a concentration of about 2 mg\nC20U4V/mL, equivalent to 2.8 mM of C20U4V.\nSupplementary Fig. 7f, Immunoblots and quantification of FTH1 treated with MOF-Fe and C20U4V. N = 4. .\nDual Treatment with MOF-Fe and M-C20U4V in vivo\nThere are some referencing issues in this section:\nFigure 8 is mistakenly cited as Figure 6 in the text.\nFigure 8K should say M-C20U4V, not C20U4V.\nAnswer:\nWe thank the reviewer for the corrections. The revised manuscript has been carefully examined for\ntypos and grammatical errors.\nConclusion\nThis study presents a promising strategy for enhancing ferroptotic cancer therapy following IRE via\ntargeted degradation of FTH1 using a PUFA-based PROTAC. The results are compelling, but improving\nfigure organization, correcting some references, and clarifying compound selection and specificity would\nbe advisable.\nAnswer:\nWe thank the reviewer for the favorable evaluation. We appreciate this opportunity to further improve\nour manuscript.\n45 / 47\nReferences\n1. Ma, H.T. and R.Y. Poon, Gene down-regulation with short hairpin RNAs and validation of specificity\nby inducible rescue in mammalian cells. Curr Protoc Cell Biol, 2010. Chapter 27: p. Unit 27 2.\n2. Adhikari, B., et al., PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A\nkinase. Nat Chem Biol, 2020. 16(11): p. 1179-1188.\n3. Markelc, B., M. Cemazar, and G. Sersa, Effects of Reversible and Irreversible Electroporation on\nEndothelial Cells and Tissue Blood Flow, in Handbook of Electroporation, D. Miklavcic, Editor. 2016,\nSpringer International Publishing: Cham. p. 1-14.\n4. Zhang, Z., et al., Rapid dramatic alterations to the tumor microstructure in pancreatic cancer\nfollowing irreversible electroporation ablation. Nanomedicine (Lond), 2014. 9(8): p. 1181-92.\n5. Sharabi, S., et al., A statistical model describing combined irreversible electroporation and\nelectroporation-induced blood-brain barrier disruption. Radiol Oncol, 2016. 50(1): p. 28-38.\n6. Gehl, J., T. Skovsgaard, and L.M. Mir, Vascular reactions to in vivo electroporation:\ncharacterization and consequences for drug and gene delivery. Biochim Biophys Acta, 2002.\n1569(1-3): p. 51-8.\n7. Bhutiani, N., et al., Irreversible electroporation enhances delivery of gemcitabine to pancreatic\nadenocarcinoma. J Surg Oncol, 2016. 114(2): p. 181-6.\n8. Peng, W., et al., Optimal Irreversible Electroporation Combined with Nano-Enabled\nImmunomodulatory to Boost Systemic Antitumor Immunity. Adv Healthc Mater, 2024. 13(7): p.\ne2302549.\n9. Zhao, J., et al., Irreversible electroporation reverses resistance to immune checkpoint blockade in\npancreatic cancer. Nat Commun, 2019. 10(1): p. 899.\n10. Xie, Y., et al., Ferroptosis: process and function. Cell Death Differ, 2016. 23(3): p. 369-79.\n11. Mercadal, B., et al., Dynamics of Cell Death After Conventional IRE and H-FIRE Treatments. Ann\nBiomed Eng, 2020. 48(5): p. 1451-1462.\n12. Batista Napotnik, T., T. Polajzer, and D. Miklavcic, Cell death due to electroporation - A review.\nBioelectrochemistry, 2021. 141: p. 107871.\n13. Zhou, N., et al., FerrDb V2: update of the manually curated database of ferroptosis regulators and\nferroptosis-disease associations. Nucleic Acids Res, 2023. 51(D1): p. D571-D582.\n14. Nakamura, T., et al., Phase separation of FSP1 promotes ferroptosis. Nature, 2023. 619(7969): p.\n371-377.\n15. Bock, F.J. and S.W.G. Tait, Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell\nBiol, 2020. 21(2): p. 85-100.\n16. Kagan, V.E., et al., Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat Chem\nBiol, 2017. 13(1): p. 81-90.\n17. Chen, Y., et al., Small-molecule ferritin degrader as a pyroptosis inducer. J Am Chem Soc, 2023.\n145(17): p. 9815-9824.\n18. Bu, W., et al., Ferritin couples iron and fatty acid metabolism. FASEB J, 2012. 26(6): p. 2394-400.\n19. Edd, J.F. and R.V. Davalos, Mathematical modeling of irreversible electroporation for treatment\nplanning. Technol Cancer Res Treat, 2007. 6(4): p. 275-86.\n20. Golberg, A., et al., Tissue heterogeneity in structure and conductivity contribute to cell survival\nduring irreversible electroporation ablation by \"electric field sinks\". Sci Rep, 2015. 5: p. 8485.\n21. Lin, M., et al., Irreversible electroporation plus allogenic Vgamma9Vdelta2 T cells enhances\n46 / 47\nantitumor effect for locally advanced pancreatic cancer patients. Signal Transduct Target Ther,\n2020. 5(1): p. 215.\n22. Yu, B., et al., Electric pulse responsive magnetic nanoclusters loaded with indoleamine 2,3-\ndioxygenase inhibitor for synergistic immuno-ablation cancer therapy. ACS Appl Mater Inter, 2020.\n12(49): p. 54415-54425.\n23. Han, J.-H., et al., An electro-ferroptotic nanoammunition enables image-guided, spatiotemporally\ncontrolled cancer ferroptosis induction via irreversible electroporation. Chem Eng J, 2024. 487: p.\n150366.\n24. Long, X., et al., Simultaneous Delivery of Dual Inhibitors of DNA Damage Repair Sensitizes\nPancreatic Cancer Response to Irreversible Electroporation. ACS Nano, 2023. 17(13): p. 12915-\n12932.\n25. Brash, A.R., Arachidonic acid as a bioactive molecule. J Clin Invest, 2001. 107(11): p. 1339-45.\n47 / 47\nPoint-by-point Responses to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nThe manuscript has been substantially improved through revisions that appropriately address the\nreviewers' comments, and it aligns well with the scope and readership of Nature Communications.\nAnswer：\nWe appreciate the reviewer’s positive comments.\nReviewer #2 (Remarks to the Author):\nThe authors carefully considered and addressed all the comments raised by this reviewer in the\nprevious round of reviews. They also added substantial new data throughout that strengthens the\nmanuscript. The additional lipidomics results are impressive and this reviewer appreciates the extra work\nand care taken with this aspect. Also, the improved analysis of the RNA-seq data are convincing. This\nreviewer has no further reservations and recommends publication.\nAnswer：\nWe appreciate the reviewer’s positive comments.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nAnswer：\nWe appreciate the reviewer’s effort in reviewing the manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nAnswer：\nWe appreciate the reviewer’s effort in reviewing the manuscript.\nReviewer #5 (Remarks to the Author):\nThis revised manuscript describes a highly innovative approach to stimulate ferroptosis and improve\nthe efficacy of pulsed electrical fields (PEF) in pancreatic cancer cells in vitro and in vivo. The agent itself\nis effective in vivo, but more generally the study identifies iron homeostasis as an important therapeutic\ntarget in combination with ablation of PDAC. The revised manuscript is highly responsive to the critiques,\nincluding new data.\n1 / 6\nAnswer：\nWe appreciate the reviewer’s positive comments.\nIf anything, the response to one of my critiques (that IRE was described as have higher recurrence\nrates than other methods of ablation) was perhaps a little excessive. In the discussion, the statement is\nnow made that \"Tumor recurrence after IRE has been reported\". This wording implies that recurrence after\nIRE is rare, which of course it is not. There are many ways this statement could be improved to convey\nthat recurrence after IRE IS a problem (without getting into the messy and complicated issue of how it\ncompares to other methods of ablation).\nAnswer:\nWe agree that our previous revision on the statement of post-IRE recurrence was over-simplified. An\nupdated literature search was performed in PubMed using the key word “irreversible electroporation tumor\nrecurrence”. Related clinical studies from 2020 to 2025 are briefed in the following table. Base on this\nliterature analysis, we would like to change our statement to:\n“The tumor recurrence rate following IRE varies across different tumor types. It remains a clinical\nchallenge, particularly in the context of pancreatic cancer. Twenty-three out of 50 patients with pancreatic\ncancer patients developed local recurrence after IRE in the PANFIRE-2 phase II study. In a recent analysis\non a multi-institutional database, 32 (17%) out of 187 patients with pancreatic cancer patients had local recurrence\nfollowing IRE”.\nThe original reference from PLOS one (2013) is replaced with an updated report (Woeste et al,\nOptimizing Patient Selection for Irreversible Electroporation of Locally Advanced Pancreatic Cancer:\nAnalyses of Survival, Front Oncol 2021; 11: Vol. 11 Pages 817220).\nAuthor [Ref] Tumor Type #Patients/Lesions Outcome\nHendrikx [1] Pancreatic cancer 35 patients Local recurrence was seen in 24.2% of patients after a\nmedian time of 7.5 months\nGordon [2] Pancreatic cancer 50 patients Twenty-five patients of the studied cohort of 50\nexhibited tumor recurrence within 12 months\nWall [3] Pancreatic cancer 20 patients One patient local progression 18 months after IRE\nYang [4] Locally advanced 74 patients 9 patients (12.2%) developed local recurrence, and 30\npancreatic cancer patients (40.%) developed distant metastasis over a\nmedian follow-up of 46.9 months (IQR: 30.8-63.0).\nWoeste [5] Pancreatic cancer 187 patients 32 patients (17%) had local recurrence; 49 (26%)\npatients had distant recurrence\nRuarus [6] Pancreatic cancer 50 patients 23 patients developed local recurrence\n2 / 6\nXu [7] Hepatocellular 180 patients 50.6% of patients developed recurrence over a mean\ncarcinoma follow-up of 40.9 ± 12.6 months\nNarayanan [8] Hepatic epithelioid 14 patients with 47 Over a median follow-up of 15 months, residual tumors\nhemangioendothelioma lesions were observed in 9 tumors without indications of\nprogressive disease\nNarayanan [9] Colorectal liver 26 patients with 46 The 2-year local tumor progression-free survival rate\nmetastasis in proximity lesions was 51.3%\nto critical structures\nZhang [10] Solid liver tumor 78 patients Recurrence rate at 1, 3, and 6 months after IRE were 0\n(0%), 10 (13.9%), and 10 (13.3%), respectively.\nLi [11] Hepatic tumors at the 21 patients with 38 Local recurrence was observed in 1 (4.8%) patient on\nhepatocaval confluence lesions day 176, and distant recurrence in 8 (38.1%) patients.\nHitpass [12] Recurrent colorectal 23 patients Local tumor-progression free rate at 36 months was\nliver metastases receiving IRE as 57.4%.\nsalvage treatment\nBlazevski [13] Prostate cancer 134 patients In-field recurrence was present in 2.7-9.8% of patients.\nFailure-free survival at 3-years was 96.75%,\nJung [14] Prostate cancer 50 patients 13 patients showed local recurrence within 6 months\nWang [15] Renal cell carcinoma 8 patients with 9 Recurrence or metastasis occurred in two patients\nrenal cell within one year\ncarcinomas in a\nsolitary kidney\nDai [16] Small renal mass 47 patients with 48 5-year local recurrence-free survival was 93.3% in\ntumors confirmed renal cell carcinoma cases.\nGong [17] Tumors adjacent to 32 patients with 39 Recurrence in situ was observed in 5 of 32 patients,\nperihepatic important lesions and the median progression-free survival was 384 days\nstructures\nI recognize that this is not a clinical paper, but my only other suggestion to improve the manuscript\nwould be at least describe briefly the feasibility of translation of this agent to humans.\nAnswer:\nWe agree with the reviewer’s suggestion. C20U4V and its analogues exhibit potential for clinical\ntranslation since they are small-molecular agents. The MOF-Fe nanoparticles may require more time but\nremain possible because superparamagnetic nanoparticles (SPIOs) have been approved for clinical use.\nIn our opinion, our study indicated that a simultaneous delivery of iron and a FTH1 degrader would be\ncritical for the disruption of iron homeostasis. Given the current success of lipid nanoparticles and\nliposomes in clinical translation, a potential solution may reside in similar lipid-based formulations. For\nexample, while hydrophilic ferric ions could be encapsulated into the hydrophilic interior, the PUFA-derived\n3 / 6\nFTH1 degraders could be readily integrated into the lipid membranes. In addition, more specific FTH1\ndegraders would also be an interesting field to explore. This paragraph is rephrased and added to the\nlimitation section of the manuscript.\nReviewer #6 (Remarks to the Author):\nThis manuscript by Linrong Li, Shihao Su et al. presents an interesting and novel study investigating\na strategy to enhance the efficacy of irreversible electroporation (IRE) in pancreatic cancer through\nsensitisation of residual tumour cells to ferroptosis using a PROTAC-based approach. During IRE, residual\nviable tumour cells can remain, representing a significant cause of tumour recurrence. The authors\ndemonstrate that iron-based metal–organic framework nanoparticles (MOF-Fe) can suppress recurrence\nby inducing iron overload and subsequently ferroptosis; however, this effect is attenuated by IRE-induced\nupregulation of ferritin heavy chain 1 (FTH1). To overcome this limitation, the authors employ an\narachidonic acid-derived PROTAC, C20U4V, to selectively degrade FTH1, thereby amplifying ferroptosis\nactivity in both pancreatic cancer cell lines and organoids. Encapsulation of C20U4V within ROS-\nresponsive micelles (termed M-C20U4V) enables in vivo delivery, and in combination with MOF-Fe, they\ndemonstrate enhanced ferroptosis and therapeutic efficacy in murine models. This study is novel, well\nsupported by experimental data, and of clear translational relevance. Upon re-evaluation of this paper, I\nam satisfied with the corrections and additional experiments performed in response to my previous\nconcerns and therefore believe that this manuscript is suitable for publication in Nature Communications.\nAnswer：\nWe appreciate the reviewer’s positive comments.\nResponse to previous review:\nMy previous comment on Figure 1 regarding immunohistochemical staining of 4-hydroxynonenal (4-\nHNE) being superfluous was, in hindsight, a misunderstanding of the data being presented. The authors’\nre-adjustment of this figure now makes it flow more logically, and I agree that they should retain the data\nin the main figure. They have also corrected all misreferencing of this figure in the main text.\nAnswer：\nWe appreciate the reviewer’s positive comments.\nRegarding ferroptosis sensitivity in a PEF-resistant cell model, thank you for providing this additional\ndata for review. It is fascinating that you observe specific sensitivity to one but not both ferroptosis inducers,\nand I am sure that elucidating that mechanism would make for an exciting future paper. I am satisfied with\nthe additional data provided for the PROTAC evaluation of C20U4V and C20U5V, which now clearly show\nthat C20U4V performs appreciably better, as supported by the IC₅₀ and log fold-change data. Incidentally,\nit is common practice in the PROTAC field that if a PROTAC degrades a target, its IC₅₀ is typically referred\nto as its ‘DC₅₀’ value. These terms are often used interchangeably in the literature, so I will leave it to\nthe authors to decide which terminology they consider best practice.\n4 / 6\nAnswer：\nWe thank the reviewer for the suggestion. We would like to continue using IC50 for the following\nreasons. First, the IC50 term is also used in Fig. 1/Supplementary Fig. 1 for Lip-1, Ferr-1, and tocopherol.\nSwitching to DC50 might cause confusion for the audience. Second, the cell-inhibitory effect of PUFA-\nderived PROTAC may not be solely attributed to FTH1 degradation. PUFA by themselves may promote\nferroptosis. We thank the reviewer for the understanding.\nThe inclusion of the competition assay clearly demonstrates that C20U4V degradation activity\ndepends on the binding of the active PROTAC to FTH1, and that this can be sequestered by competition.\nI think this is a strong experimental addition that helps strengthen the case for C20U4V. The pulldown\nassay is clean and clearly demonstrates FTH1 engagement, thereby addressing my previous suggestion\nof its inclusion. This addition substantially strengthens the use of arachidonic acid (AA) in this paper for\nPROTAC design. It is clear from the proteomics data that AA likely has several other targets; however, I\ndo not think this necessarily detracts from its use, as there are currently no other selective ligand\nalternatives for FTH1. The key takeaway from this work is that it establishes a viable method to counter\ntumour resurgence post-IRE, which the authors have successfully demonstrated.\nAnswer：\nWe appreciate the reviewer’s positive comments.\nOne possible suggestion for future work, likely beyond the scope of this paper, would be to employ a\ndegron-tagging system such as BromoTag, dTAG, or AiD to endogenously tag FTH1, allowing selective\ndegradation of FTH1 to compare responses between models treated with a selective degrader versus a\nmore promiscuous one.\nAnswer:\nWe agree that more advanced techniques will be helpful to develop FTH1-specific degraders. This is\nan intriguing field that deserves a vigorous study. This paragraph is re-phrased and added to the\nlimitation section.\nThe DC₅₀ titration data are clean and clearly show the treatment window for C20U4V at 12 hours. I\nam satisfied with its inclusion. Overall, I am pleased with all additional experimental changes that were\nsuggested in my previous review of this manuscript. It is clear that AA is not the ideal ligand for the\ndevelopment of C20U4V; however, I do not believe this detracts from the central message of the paper—\nthat this represents a viable strategy for preventing post-IRE tumour resurgence through ferroptosis. Upon\npublication, I anticipate that future studies will focus on refining the selectivity profile of this generation of\nFTH1 PROTACs, which is understandably beyond the scope of the current manuscript.\nAnswer：\nWe appreciate this opportunity to receive such valuable suggestions from an expert in the PROTAC\nfield.\n5 / 6\nReferences\n1. Hendrikx, B., et al., Use of Irreversible Electroporation in Pancreatic Cancer Patients: A Multi-\nCenter Experience. Curr Oncol, 2025. 32(10).\n2. Gordon, J.W.H., A. Goel, and R.C.G. Martin, 2nd, Evaluation of Recurrence Risk in Irreversible\nElectroporation-Treated Pancreatic Adenocarcinoma Patients Using Radiomics Signatures.\nCancers (Basel), 2025. 17(14).\n3. Wall, C., et al., Early Single-Center Experience With Irreversible Electroporation for Stage 2, 3, and\n4. Yang, P.C., et al., Prognostic factor analysis of irreversible electroporation for locally advanced\npancreatic cancer - A multi-institutional clinical study in Asia. Eur J Surg Oncol, 2020. 46(5): p.\n811-817.\n5. Woeste, M.R., et al., Optimizing Patient Selection for Irreversible Electroporation of Locally\nAdvanced Pancreatic Cancer: Analyses of Survival. Front Oncol, 2021. 11: p. 817220.\n6. Ruarus, A.H., et al., Percutaneous Irreversible Electroporation in Locally Advanced and Recurrent\nPancreatic Cancer (PANFIRE-2): A Multicenter, Prospective, Single-Arm, Phase II Study.\nRadiology, 2020. 294(1): p. 212-220.\n7. Xu, M., et al., Recurrence patterns and survival outcomes after irreversible electroporation for\nhepatocellular carcinoma: a 6-year multicenter experience. Eur Radiol, 2025.\n8. Narayanan, G., et al., Irreversible Electroporation as a Valid Treatment Option for Hepatic\nEpithelioid Hemangioendothelioma: An International Multicenter Experience. Cardiovasc Intervent\nRadiol, 2024. 47(7): p. 883-890.\n9. Narayanan, G., et al., Irreversible Electroporation in Treating Colorectal Liver Metastases in\nProximity to Critical Structures. J Vasc Interv Radiol, 2024. 35(12): p. 1806-1813.\n10. Zhang, X., et al., Novel irreversible electroporation ablation (Nano-knife) versus radiofrequency\nablation for the treatment of solid liver tumors: a comparative, randomized, multicenter clinical\nstudy. Front Oncol, 2022. 12: p. 945123.\n11. Li, T., et al., Percutaneous Ablation of Hepatic Tumors at the Hepatocaval Confluence Using\nIrreversible Electroporation: A Preliminary Study. Curr Oncol, 2022. 29(6): p. 3950-3961.\n12. Hitpass, L., et al., Recurrent Colorectal Liver Metastases in the Liver Remnant After Major Liver\nSurgery-IRE as a Salvage Local Treatment When Resection and Thermal Ablation are Unsuitable.\nCardiovasc Intervent Radiol, 2022. 45(2): p. 182-189.\n13. Blazevski, A., et al., Oncological and Quality-of-life Outcomes Following Focal Irreversible\nElectroporation as Primary Treatment for Localised Prostate Cancer: A Biopsy-monitored\nProspective Cohort. Eur Urol Oncol, 2020. 3(3): p. 283-290.\n14. Jung, E.M., et al., Contrast enhanced ultrasound (CEUS) with parametric imaging after irreversible\nelectroporation (IRE) of the prostate to assess the success of prostate cancer treatment. Clin\nHemorheol Microcirc, 2021. 77(3): p. 303-310.\n15. Wang, Z., et al., Percutaneous irreversible electroporation of renal cell carcinomas in an acquired\nsolitary kidney: A primary study. J Cancer Res Ther, 2024. 20(7): p. 1997-2003.\n16. Dai, J.C., et al., Irreversible Electroporation for the Treatment of Small Renal Masses: 5-Year\nOutcomes. J Endourol, 2021. 35(11): p. 1586-1592.\n17. Gong, J., et al., A retrospective study of irreversible electroporation for tumors adjacent to\nperihepatic important structure. Front Oncol, 2024. 14: p. 1387952.\n6 / 6"}]

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