Bimetallic peroxide nanoparticles induce PANoptosis by disrupting ion homeostasis for enhanced immunotherapy

Abstract

PANoptosis has recently emerged as a potential approach to improve the immune microenvironment. However, current methods for inducing PANoptosis are limited. Herein, through biological screening, the rational use of the nutrient metal ions Cu²⁺ and Zn²⁺ had great potential to induce PANoptosis. Inspired by these findings, we successfully developed hydrazided hyaluronic acid–modified zinc copper oxide (HZCO) nanoparticles as a PANoptosis inducer to potentiate immunotherapy. Bioactive HZCO actively delivered Cu²⁺ and Zn²⁺ while disrupting the cellular intrinsic ion metabolism pathway, resulting in double-stranded DNA release and organelle damage in cancer cells. Simultaneously, this process triggered the formation of PANoptosome and the activation of PANoptosis. HZCO-induced PANoptosis inhibited tumor growth and activated potent antitumor immune response, thereby enhancing the effectiveness of anti–programmed cell death 1 therapy. Overall, our work provides an insight into the development of PANoptosis inducers and the design of synergistic immunotherapy strategies.

Zinc-copper bimetallic peroxide nanoparticles are developed to activate PANoptosis and enhance tumor immunotherapy.

INTRODUCTION

Immunotherapy has emerged as a crucial tool in cancer treatment, harnessing the patient’s own immune system to recognize, attack, and eliminate tumor cells (1). However, the inherent immunoediting process of tumors, including elimination, equilibrium, and escape stages, can alter tumor immunogenicity and hinder the immune system’s ability to recognize and eliminate tumor cells, leading to immune resistance and ultimately culminating in the failure of immunotherapy (2). Inducing tumor immunogenic cell death (ICD), which can occur through various processes, such as pyroptosis, ferroptosis, cuproptosis, and necroptosis, has been identified as one of the most effective strategies for promoting the release of tumor-associated antigens from tumor cells and eliciting antitumor immune responses (3). Nevertheless, tumor cells usually use various mechanisms such as cell repair processes, molecular regulatory mechanisms, and alterations in the cell cycle to evade or resist a single death pathway (4–7). This considerably affects the efficiency of ICD in activating antitumor immune responses. The optimal strategy for activating immune responses should be a combination of multiple nonredundant pathways of ICD to comprehensively enhance the immunogenicity and sensitization of cancer cells.

The ability of combining different modes of ICD to enhance the immune response against tumors has been investigated (8–11). However, the complex nature of different cell death mechanisms often hinders the synergistic activation of antitumor immunity using simple combinations (12). The latest concept of PANoptosis presents potential ideas for the development of more potent immune-enhancing strategies. PANoptosis is an inflammatory regulatory cell death pathway that involves the activation of caspases and receptor-interacting protein kinases (RIPKs) and is regulated by PANoptosome. This process incorporates key features of pyroptosis, apoptosis, and necroptosis but cannot be characterized by any single form of death (13–15). It has the potential to generate a robust and long-lasting tumor-specific immune response by disrupting the cell barrier and repeatedly releasing damage-associated molecular patterns (DAMPs) (16–18). However, research on PANoptosis is still in its early stages. Effective strategies to induce PANoptosis encompass only some specific pathogens, DAMPs, pathogen-associated molecular patterns, cytokines, and chemotherapeutic agents (19–22). Therefore, there is a need to explore and develop effective strategies for inducing PANoptosis, which could have substantial implications for synergistic immunotherapy.

The antitumor strategies based on nutrient metal ions are promising methods for cancer therapy with the advantages of broad-spectrum antitumor activity, high efficacy, and low drug resistance (23–25). Interfering with intracellular ion homeostasis can effectively kill tumor cells by triggering various biological processes such as biocatalysis, disrupting osmotic balance, interfering with metabolism and signal transduction, and causing DNA damage (26). Nutrient metal ions, such as Ca²⁺, Fe²⁺, Cu²⁺, Mn²⁺, and Zn²⁺, have been demonstrated to activate adaptive immune responses by inducing specific forms of ICD, including pyroptosis, ferroptosis, cuproptosis, and necroptosis (10, 27, 28). However, if nutrient metal ions can induce PANoptosis is largely unknown. Exploring the potential of metal ions to induce PANoptosis is important for developing highly effective PANoptosis inducers and expanding the applications of immunotherapy.

In this study, we explored the potential antitumor effects of various metal ions and found that the combination of Cu²⁺ and Zn²⁺ has the ability to trigger PANoptosis under specific conditions. Inspired by this, hydrazided hyaluronic acid (HHA)–modified Zn-CuO₂ (HZCO) nanoparticles were developed and explored for their potential as a PANoptosis inducer for enhanced immunotherapy (Fig. 1). The bioactive HZCO disrupted intracellular Cu²⁺ and Zn²⁺ homeostasis by actively supplying Cu²⁺/Zn²⁺ and interfering with ion metabolic pathways. This further led to abnormal accumulation of Cu²⁺ and Zn²⁺, resulting in mitochondrial dysfunction, double-stranded DNA (dsDNA) release, and endoplasmic reticulum (ER) stress. Unlike the apoptosis or pyroptosis induced by Cu²⁺ or Zn²⁺ alone, HZCO successfully triggered the formation of the absent in melanoma 2 (AIM2)–PANoptosome complex, leading to the activation of PANoptosis. PANoptosis induced by HZCO not only effectively inhibited tumor growth but also activated a strong antitumor immune response. HZCO combined with programmed cell death 1 (PD-1) therapy inhibited primary tumor growth and had a remarkable abscopal effect. Furthermore, motivated by the synergistic mechanism of Zn²⁺ and Cu²⁺, we examined the potential synergistic effects of the preclinically approved drug elesclomol (ES) and Zn²⁺. It was confirmed that, with the assistance of Zn²⁺, the antitumor effect and immune response of ES were substantially improved due to the occurrence of PANoptosis, indicating its potential for clinical application. This study highlights the effectiveness of nutritional metal ion–induced PANoptosis, providing a theoretical basis and important reference for the design of PANoptosis inducers and the development of synergistic immunotherapy strategies.

Fig. 1. Schematic diagram of HZCO to activate PANoptosis and enhance immunotherapy.

The bioactive nanoparticles HZCO activate the apoptotic signaling pathway while concurrently disrupting zinc homeostasis within tumor cells. This disruption sets off a domino effect of cellular death processes, culminating in PANoptosis. HZCO-induced PANoptosis not only eliminates tumor cells with high efficiency but also alters the immune profile of cold tumors, effectively converting them into hot tumors. This alteration increases the tumors’ susceptibility to immune detection and attack. Consequently, when HZCO is combined with PD-1 therapy, it markedly impedes the progression of primary tumors and produces a considerable abscopal effect.

RESULTS

Biological screening of PANoptosis

To investigate the potential of inducing nutritional metal ions via PANoptosis, the anticancer effects of various metal ions were initially explored. 4T1 cancer cells were used to investigate the killing effects of various metal ions (Fig. 2A). The cytotoxic effects of K⁺, Na⁺, Mg²⁺, Ca²⁺, Mo²⁺, Ga³⁺, and Fe²⁺ on cancer cells were minimal, and they were unable to inhibit cancer cell growth even at concentrations as high as 1280 μM (Fig. 2B). In contrast, Zn²⁺, Cu²⁺, Mn²⁺, Co²⁺, Ni²⁺, and V³⁺ exhibited noticeable cancer cell killing abilities. The different metal ions induced various forms of cell death (Fig. 2D). For example, Cu²⁺, Co²⁺, and V³⁺ induced obvious apoptosis, characterized by the formation of a substantial number of apoptotic bodies; Ni²⁺-treated cells exhibited cytoplasmic vacuolation without karyopyknosis, suggesting that paraptosis was induced by Ni²⁺; and Mn²⁺ caused necrosis, characterized by increased membrane permeability and cell swelling. Cancer cells treated with excess Zn²⁺ presented characteristic features of pyroptosis, including membrane vesiculation and cytoplasmic vacuolation. Among the aforementioned metal ions, Zn²⁺ exhibited unique characteristics. Owing to its strict regulation of cell cycle processes, Zn²⁺ has been shown to cause hypersensitive acute toxicity to cancer cells with an extremely low toxicity threshold, which vastly distinguishes it from other metal ions (Cu²⁺, Mn²⁺, Co²⁺, Ni²⁺, and V³⁺) that exhibit concentration-dependent cytotoxicity (Fig. 2, B and C). Within the concentration range of 40 μM, Zn²⁺ exhibited favorable cellular safety and enhanced cell proliferation to some extent. However, surpassing this threshold resulted in explosive cell death. Treatment with 60 μM Zn²⁺ nearly eradicated all the cell viability (Fig. 2C). After further research was conducted on Zn²⁺, the types of cell death exhibited sensitive concentration-dependent characteristics. At a concentration of 60 μM, Zn²⁺ tended to promote the necroptotic cell death. However, Zn²⁺ at a concentration of 80 μM evidently triggered pyroptosis (fig. S1). This meant that Zn²⁺ switched between different death signals at very low concentrations, suggesting that Zn²⁺ was a potential candidate for inducing PANoptosis. We hypothesized that zinc overload occurs simultaneously with the activation of apoptotic signals and that pyroptosis, apoptosis, and necroptosis are expected to occur rather than just additive effects. Therefore, the effects of other ions on zinc overload were further investigated. 4T1 cells were cotreated with a relatively safe concentration (40 μM) of Zn²⁺ in combination with other metal ions (Cu²⁺, Co²⁺, Ni²⁺, V³⁺, Mn²⁺, Ca²⁺, Mg²⁺, and Fe³⁺). Among these bioactive metal ions, only Cu²⁺ exhibited a synergistic therapeutic effect with Zn²⁺ (Fig. 2, E and F). When the cells were exposed to 125 μM Cu²⁺ combined with 40 μM Zn²⁺, their viability rapidly decreased to only ~4.1%, whereas Cu²⁺ alone had minimal cytotoxic effects (Fig. 2G). Notably, these two groups of cells exhibited distinct forms of cell death, with Cu²⁺ inducing typical apoptosis and Cu²⁺ + Zn²⁺ triggering lytic cell death, similar to zinc overload–induced pyroptosis (Fig. 2, F and H). Compared with the Cu²⁺ and Zn²⁺ treatments alone, the Cu²⁺ + Zn²⁺ treatment induced considerable release of lactate dehydrogenase (LDH) and high-mobility group box 1 protein (HMGB1) (Fig. 2, I and J), which are important markers of inflammatory cell death. We hypothesized that Cu²⁺ promoted zinc overload by disrupting zinc metabolic pathways in cancer cells. The detection of the intracellular free zinc concentration by a Zn-specific probe after Cu²⁺ treatment for 2 hours confirmed that Cu²⁺ effectively increased the intracellular free Zn concentration (fig. S2). Moreover, treatment with Cu²⁺ for 2 hours up-regulated the expression of metallothionein (MT) and metal-regulated transcription factor 1 (MTF-1) in cancer cells (fig. S3). MT and MTF-1 are crucial proteins that sustain intracellular zinc homeostasis, and their expression levels are positively correlated with the concentrations of free zinc ions within the cell (29, 30). Therefore, Cu²⁺ increased the intracellular content of free Zn²⁺, which was attributed to the substitution of Cu²⁺ for the bound zinc in MT, owing to the fact that Cu²⁺ has a stronger chelating affinity with MT (31).

Fig. 2. Biological screening for ions that activate PANoptosis.

(A) Scheme of ion screening for PANoptosis. (B) Cytotoxicity of various metal ions at different concentrations. (C) Cytotoxicity of Zn²⁺ at different concentrations (n = 5). (D) Photographs of 4T1 cells treated with different metal ions for 24 hours. (E and F) Viabilities (E) and photographs (F) of 4T1 cells treated with different ions and Zn²⁺ (40 μM) (n = 5). (G) Viabilities of 4T1 cells treated with Zn²⁺ (40 μM) and Cu²⁺ (n = 5). (H) Schematic diagram of cell death after cotreatment with Cu²⁺ + Zn²⁺. (I and J) Release of LDH (I) and HMGB1 (J) from 4T1 cells treated with different formulations (n = 3 to 5). (K) Western blotting analysis of PANoptosis-associated proteins in 4T1 cells treated with different treatments. (L) Effect of VX-765 (pyroptosis inhibitor), Ac-DEVD-CHO (apoptosis inhibitor), and Nec-1 (necroptosis inhibitor) on the cytotoxicity of Cu²⁺ + Zn²⁺ [c(Cu²⁺) = 100 μM; c(Zn²⁺) = 40 μM] (n = 5). (M) Viabilities of HUVECs treated with Cu²⁺ and Zn²⁺ (40 μM) (n = 5). (N) Schematic diagram of the difference in the therapeutic efficacy of Cu²⁺ + Zn²⁺ treatment in normal and cancer cells. Data are presented as the means ± SD. Significant differences were performed in (L) using one-way ANOVA with Tukey multiple comparisons post hoc test. The statistical significance in (E), (G), and (M) was calculated via two-tailed Student’s t test. *P < 0.05; ***P < 0.001; n.s., not significant.

Notably, the morphological features of cell death induced by Cu²⁺ + Zn²⁺ did not appear to be exactly the same as those induced by pure zinc, as confirmed by the irregular morphology of the cytoplasmic vesicles (Fig. 2F). The specific apoptosis inhibitor Ac-DEVD-CHO, the pyroptosis inhibitor VX-765, and the necroptosis inhibitor Necrostatin-1 (Nec-1) significantly weakened the synergistic anticancer killing effect of Cu²⁺ + Zn²⁺, confirming that the combined treatment of Cu²⁺ + Zn²⁺ caused multiple cell death signals (Fig. 2L). Furthermore, we assessed the biochemical activation of cell death molecules via Western blotting analysis. Treatment with Cu²⁺ + Zn²⁺ not only concurrently cleaved the pyroptosis-related proteins caspase 1 and gasdermin D (GSDMD) as well as the apoptosis-associated proteins caspase 3/7/8 but also induced the phosphorylation of the necroptosis-related proteins RIPK1 and mixed lineage kinase domain-like protein (MLKL) (Fig. 2K). This suggested that the synergistic effect of Cu²⁺ + Zn²⁺ activated three distinct cell death pathways simultaneously: pyroptosis, apoptosis, and necroptosis. Owing to the faster and more intense cell death provoked by Cu²⁺ + Zn²⁺ treatment, Cu²⁺ alone did not cause changes in apoptosis-related signals at the same time point. Furthermore, in cells treated with Cu²⁺ + Zn²⁺, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck was observed, and RIPK1, caspase 8, and caspase 1 colocalized with the ASC speck (fig. S4). These findings indicated that RIPK1, caspase 8, and caspase 1 assembled with ASC in cells, indicating the formation of the PANoptosome. The PANoptosome formation is a crucial marker of cellular PANoptosis (32, 33). These results suggested that the synergistic action of Cu²⁺ + Zn²⁺ induced PANoptosis, thereby facilitating molecular cross-talk among pyroptosis, apoptosis, and necroptosis. We further explored the anticancer effects of Cu²⁺ + Zn²⁺ in other cell lines including CT26, MC38, B16F10, and PAN02 cells. Cu²⁺ + Zn²⁺ treatment also induced lytic cell death, which was closely associated with pyroptosis, apoptosis, and necroptosis in these additional cell lines, leading to the significant release of inflammatory markers including LDH and HMGB1 (figs. S5 to S8). This confirmed the universal ability of Cu²⁺ and Zn²⁺ to induce lytic cell death. It was noteworthy that the cell killing effect of Cu²⁺ + Zn²⁺ was not completely inhibited even after the cells were treated with three different inhibitors (VX-765 + Ac-DEVD-CHO + Nec-1) simultaneously (fig. S9). This might be attributed to the complex biological effects of ions, which could induce other forms of cell death, such as paraptosis and cuproptosis (34, 35). This observation further underscored the potential of Cu²⁺ and Zn²⁺ synergism in antitumor therapy. In addition, the addition of extra H₂O₂ enabled specific killing of cancer cells at a lower dose of Cu²⁺ + Zn²⁺ as the function of Cu²⁺ was dependent on H₂O₂ (fig. S10). These results confirmed that the combination of Cu²⁺ and Zn²⁺ had the potential to trigger PANoptosis by inducing apoptosis and disrupting zinc homeostasis.

The normal human umbilical cord endothelial cells (HUVECs) were much less sensitive to Cu²⁺ + Zn²⁺ treatment than were 4T1 cancer cells and remained healthy even after receiving a double dose of Cu²⁺ + Zn²⁺ (Fig. 2M). This was attributed to the fact that intracellular zinc and MT expression levels in normal cells were considerably lower than those in cancer cells with unlimited proliferation (36). As a result, the same dose of Cu²⁺ + Zn²⁺ treatment failed to trigger zinc overload in normal cells, thus preventing acute toxicity. Notably, the cytotoxicity of Cu²⁺ + Zn²⁺ treatment on HUVECs was lower than that of Cu²⁺ alone. This occurred because unoverloaded Zn²⁺ levels within normal cells counteracted the toxicity of Cu²⁺ by activating the Zn-MTF-MT metabolic axis (Fig. 2N). All these findings confirmed that the synergistic effect of Zn²⁺ and Cu²⁺ induced PANoptosis in tumor cells by disrupting zinc homeostasis, which had important implications for the design and development of PANoptosis inducers.

Preparation and characterization of HZCO

Inspired by the potential synergistic effects of Cu²⁺ and Zn²⁺ in inducing PANoptosis, a PANoptosis inducer capable of delivering both Cu²⁺ and Zn²⁺ simultaneously was further developed. Metal peroxides have gained considerable research attention as ideal donors of metal ions and H₂O₂. On the basis of the optimal ratio mentioned above, Zn-doped CuO₂ nanoparticles were synthesized via a chemical coprecipitation method (Fig. 3A). Zn-CuO₂ prepared with polyvinyl pyrrolidone (PVP) as a dispersant showed an irregular cluster structure containing ultrasmall nanodots (fig. S11), with an average hydrated particle size of ~100 nm and a surface potential of +20 mV (fig. S12). To enhance the physiological stability of Zn-CuO₂, surface modification was conducted by using a metal-hydrazide coordination method to attach HHA onto the surface of Zn-CuO₂, resulting in HZCO nanoparticles. The coordination of hydrazide groups to Cu²⁺/Zn²⁺ led to the reconfiguration of disordered Zn-CuO₂ nanoparticles into uniform nanoclusters (Fig. 3B). In addition, the hydrated size of HZCO slightly increased to ~130 nm, and the surface potential changed to −25 mV (fig. S12). These changes were attributed to the presence of carboxyl groups in HA, which facilitated the diffusion and penetration of the nanomaterials within tumor tissues. X-ray photoelectron spectroscopy (XPS) analysis was also conducted to further validate the successful synthesis of HZCO. The full XPS spectrum clearly displayed characteristic peaks corresponding to Zn, Cu, and O, whereas a prominent peak at 532 eV confirmed the presence of O-O chemical bonding (fig. S13). This substantiated the coexistence of Zn, Cu, and H₂O₂ within the HZCO. The ratio of Zn to Cu in the HZCO particles was ~1:3.6. The presence of peroxide radicals in the nanoparticles was further confirmed through the titanium sulfate method. The superoxide ions react with titanium sulfate to form a yellow peroxide-titanium complex, and the absorption peak at 412 nm increases with increasing concentrations of HZCO (fig. S14). This observation served as a solid confirmation of the substantial presence of O₂²⁻ within HZCO. The abundant O₂²⁻ was anticipated to be released under weakly acidic tumor conditions, resulting in H₂O₂ formation. Under Fenton-like catalysis mediated by Cu²⁺, H₂O₂ reacted to generate highly reactive ·OH species with potent oxidizing properties. Consequently, this process enhanced the efficient release of Zn²⁺ ions from MT, offering potential benefits in therapeutic applications. In addition, HZCO exhibited pH-dependent release (Fig. 3, C and D, and fig. S15). At pH 6.5 and pH 5.0, the release rates of Cu²⁺, Zn²⁺, and H₂O₂ were substantially higher than those in neutral environments. This pH-responsive behavior of HZCO enabled the precise release of Zn²⁺, Cu²⁺, and the ion effect enhancer H₂O₂ in weakly acidic tumor tissues, which held potential for metal ion–mediated cancer therapy.

Fig. 3. Preparation and in vitro biological effects of HZCO.

(A) Schematic diagram of the preparation process of HZCO. (B) Transmission electron microscopy image of HZCO. (C) Zn²⁺ release curve of HZCO in different pH solutions (n = 3). h, hours. (D) Cu²⁺ release curve of HZCO in different pH solutions (n = 3). (E) CLSM images of the uptake of Cy5.5-labeled HZCO by cells at different time points. (F) Cytotoxicity of HZCO at different concentrations in 4T1 cells and HUVECs (n = 6). (G) Viabilities of 4T1 cells after different treatments (n = 5). (H) LIVE/DEAD staining of cells treated with different formulations. (I) CLSM images of 4T1 cells stained with annexin V–FITC/PI after different treatments. (J and K) Western blotting analysis of PANoptosis-associated proteins in 4T1 cells treated with different treatments. (L) Immunofluorescence images of 4T1 cells after Cu²⁺ + Zn²⁺ treatment. Scale bars, 2 μm. (M) Effect of VX-765, Ac-DEVD-CHO, and Nec-1 on the cytotoxicity of HZCO (n = 5). (N to P) Release of LDH (N), IL-1β (O), and HMGB1 (P) from the cells treated with different formulations (n = 3 to 5). (Q) Schematic diagram of HZCO-activated PANoptosis. Data are presented as the means ± SD. Significant differences were performed in (M), (N), (O), and (P) using one-way ANOVA with Tukey multiple comparisons post hoc test. Statistical significance in (F) was calculated via two-tailed Student’s t test. *P < 0.05; ***P < 0.001.

On the other hand, considering the excellent Fenton-like catalytic effect of Cu²⁺, it was expected to catalyze the generation of highly oxidative ·OH from overexpressed H₂O₂ within tumor cells, thereby attacking antioxidant systems represented by MT. Therefore, the Fenton-like catalytic effect of HZCO was further investigated via methylene blue (MB) bleaching. In the presence of H₂O₂, HZCO successfully caused the discoloration of MB, confirming its good Fenton-like catalytic effect (fig. S16). Furthermore, considering the pH-responsive degradation behavior of HZCO and its inherent ability to self-supply H₂O₂, the self-catalytic ·OH generation behavior of HZCO under acidic conditions was subsequently investigated via a 3,3′,5,5′-tetramethylbenzidine (TMB) probe (fig. S17). Under neutral pH conditions, HZCO did not cause TMB coloration. However, a notable blue coloration of TMB was clearly observed under weakly acidic conditions, and the spectrum of the mixed solution exhibited a prominent absorption peak at ~650 nm, which was the characteristic of oxidized TMB. These results confirmed the acid-triggered self-catalytic ability of HZCO, providing considerable potential for disrupting zinc homeostasis and inducing PANoptosis.

In vitro cell assay

Afterward, in vitro cell experiments were conducted to validate the biological effects of HZCO. First, the internalization of Cy5.5-labeled HZCO by cancer cells was verified. The intracellular red fluorescence reached its peak at 4 hours and then gradually decreased (Fig. 3E), suggesting that HZCO was efficiently taken up by the cells due to surface modification with HA. HA binds to CD44 receptors that are overexpressed on the surface of cancer cells, thereby initiating a receptor-mediated endocytosis mechanism (37). The colocalization of HZCO with lysosomes confirmed that HZCO was internalized by cells through the endocytosis pathway. In addition, the separation of red and green fluorescence at 4 hours indicated successful escape/release of HZCO from lysosomes, which facilitated their biological effects of these compounds inside the cells. The cell toxicity of HZCO was subsequently tested via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Consistent with this hypothesis, HZCO exhibited substantially higher toxicity toward cancer cells than toward normal cells (Fig. 3F). This difference might be due to the heightened sensitivity of cancer cells to reactive oxygen species (ROS) and Zn²⁺ caused by the overexpression of H₂O₂ and MT. Furthermore, the synergistic mechanism between CuO₂ and Zn²⁺ in HZCO was validated to confirm the superior cytotoxic effect of zinc overload (Fig. 3G). At the concentrations of Zn²⁺ below 10 μM, the cytotoxicity of HZCO was attributed primarily to the toxicity of CuO₂ due to a less pronounced zinc overload effect mediated by insufficient exogenous Zn²⁺. However, when the concentration of Zn²⁺ reached 20 μM, the free Zn²⁺ remained nontoxic, while the cytotoxicity of HZCO significantly increased compared with that of CuO₂. The maximum difference in viability between the HZCO and CuO₂ groups was observed at a concentration of Zn²⁺ of 40 μM. The LIVE/DEAD fluorescence assay further provided evidence of the superior cytotoxic effect of HZCO, highlighting the synergistic killing effect of Zn and CuO₂ (Fig. 3H). Notably, the cell death induced by HZCO was markedly different from that induced by CuO₂ (Fig. 3I). The CuO₂ treatment group exhibited typical apoptotic features, whereas the HZCO-treated cells presented relatively intact nuclei, cell swelling, and cell membrane blistering, which were characteristics of lytic cell death. We subsequently assessed the expression of cell death–related molecules in cancer cells following HZCO treatment via Western blotting analysis (Fig. 3, J and K). In HZCO-treated cells, the precursor of caspase 1 was cleaved to activate cleaved caspase 1, which was characterized by a decrease in caspase 1 expression and an increase in active cleaved caspase 1 expression. The activated cleaved caspase 1 further cleaves GSDMD and releases the N-terminal domain of GSDMD (N-GSDMD) (38). Therefore, the expression of GSDMD decreased in the HZCO-treated group, whereas the expression of N-GSDMD increased. In addition, there was an increase in the expression of markers associated with apoptosis (cleaved caspases 3, 7, and 8) and necroptosis (pMLKL) in HZCO-treated cells (Fig. 3K), suggesting that the cell death induced by HZCO involved multiple forms of cell death, including pyroptosis, apoptosis, and necroptosis. Owing to the more rapid and intense cell death caused by HZCO, equivalent concentrations of Zn²⁺ and CuO₂ had negligible effects on the aforementioned proteins. Treatment of cells with VX-765, Ac-DEVD-CHO, or Nec-1 effectively alleviated the cytotoxicity of HZCO, suggesting that the anticancer effects of HZCO could be attributed to coactivation of pyroptosis, apoptosis, and necroptosis (Fig. 3M). We further detected the formation of PANoptosome by immunoprecipitation and fluorescence colocalization, which are the key markers of PANoptosis activation (14, 19). After HZCO treatment, RIPK1, caspase 1, caspase 8, and ASC interacted with each other (fig. S18), indicating the formation of the PANoptosome. Furthermore, ASC specks were observed in HZCO-treated cells, with RIPK1, caspase 1, and caspase 8 colocalizing with ASC specks (Fig. 3L), thus visually confirming the assembly and formation of the PANoptosome. Thus, HZCO successfully led to the formation of PANoptosome through a synergy between its components Cu²⁺, Zn²⁺, and H₂O₂, which in turn triggered caspase 1–dependent pyroptosis, caspase 3/7–dependent apoptosis, and MLKL-dependent necroptosis. All these death signals were intricately intertwined with each other, ultimately resulting in PANoptosis (Fig. 3Q). As a potent form of inflammatory cell death, PANoptosis can result in the leakage of inflammatory contents from the cell (39). Through the regulation and activation of caspase 1 by PANoptosome, HZCO treatment led to the cleavage of the precursors of the intracellular pro-inflammatory cytokines, pro–interleukin-1β (IL-1β) and pro–IL-18, which is manifested as the decreased expression of pro–IL-1β and pro–IL-18 (fig. S19). As a result, the intracellular contents, including LDH, HMGB1, IL-18, and IL-1β, were released into the cells after HZCO treatment, resulting in a notable increase in their extracellular concentrations (Fig. 3, N to P, and fig. S19). Therefore, HZCO induced a strong inflammatory response and thus activated an antitumor immune response. In addition, HZCO also effectively induced lytic cell death in other cell lines, including CT26, PAN02, B16F10, and MC38. This lytic cell death was closely associated with pyroptosis, apoptosis, and necroptosis, which caused a considerable release of LDH and HMGB1 (figs. S20 to S23). These findings confirmed the broad-spectrum ability of HZCO to induce lytic cell death. It was noteworthy that, similar to the effects observed with the combined treatment of Cu²⁺ + Zn²⁺, the simultaneous use of VX-765, Ac-DEVD-CHO, and Nec-1 further mitigated the cancer cell inhibition induced by HZCO, but it did not completely restore cell viability (fig. S24). This suggested that, owing to the diversity and complexity of the anticancer mechanisms of metal ions, HZCO may also induce other forms of cell death, such as cuproptosis or paraptosis, in addition to causing PANoptosis (34, 35).

To investigate the mechanism of HZCO-induced PANoptosis, tetrathiomolybdate (TM), a copper chelator, was used to eliminate the impact of Cu²⁺ on cells. Compared with that in the control group, the toxicity of HZCO toward TM-pretreated cells was nearly eliminated (Fig. 4A), indicating that intracellular free Cu²⁺ was essential for initiating cell death. This further confirmed that the cytotoxicity of HZCO was a result of the synergistic effect of Cu²⁺ and Zn²⁺. Considering the slow toxicity of Cu²⁺ and the acute toxicity of Zn²⁺, it was speculated that the toxicity of HZCO was primarily due to the intracellular overload of free Zn²⁺. Subsequently, the effect of CuO₂ on the intracellular Zn²⁺ concentration was investigated via the use of Zinpyr-1, a specific Zn²⁺ probe (Fig. 4B). The green fluorescence intensity inside the cancer cells increased after CuO₂ treatment for 2 hours, indicating an increase in the amount of intracellular free Zn²⁺. This was attributed to the displacement and oxidation effects of CuO₂ on the MT overexpressed within cancer cells, leading to the release of endogenous Zn²⁺. In addition, the expression of MT increased when the cells were treated with Zn²⁺, CuO₂, or HZCO for 2 hours (fig. S25), strongly confirming that CuO₂ stimulated the release of endogenous Zn²⁺ from intracellular MT proteins. Higher intracellular Cu concentrations were detected at 8 hours in the HZCO group than in the CuO₂ group (Fig. 4C), suggesting that HZCO potentially induces abnormal Cu²⁺ accumulation through a cascade reaction triggered by zinc overload. This was attributed to the disruption of energy metabolism caused by HZCO-mediated zinc overload and the inhibition of copper-transporting adenosine triphosphatase (ATPase) (ATP7A) (Fig. 4D) (40). In addition, the levels of the MTF-1 and the zinc transporter SLC30A2 were also reduced after HZCO treatment for 8 hours (Fig. 4D). Therefore, HZCO not only directly delivered ions to induce cell overload but also disrupted ion metabolic pathways to further create a positive feedback loop, thereby further amplifying the anticancer effects of Cu²⁺ and Zn²⁺ (Fig. 4E).

Fig. 4. Potential mechanism of HZCO-induced PANoptosis.

(A) Effect of TM on the cytotoxicity of HZCO (n = 5). (B) CLSM images of cells stained with Zinpyr-1 after different treatments. 2.5 D, 2.5 dimensional. (C) Intracellular Cu²⁺ concentration in cells treated with CuO₂ or HZCO (n = 4). (D) Western blotting analysis of important proteins in the Cu²⁺ and Zn²⁺ metabolic pathways after the corresponding treatment. (E) Schematic diagram of HZCO-mediated ion metabolism disorders. (F and G) CLSM images (F) and flow cytometry (G) of the DCF probe for detecting intracellular ROS levels in cells after various treatments. (H) Effect of the ROS scavenger N-acetyl-l-cysteine (NAC) on the cytotoxicity of HZCO (n = 5). (I and J) CLSM images (I) and flow cytometry (J) of the JC-1 probe for detecting mitochondrial damage after various treatments. (K) Western blotting analysis of important proteins involved in mitochondrial damage and ER stress after the corresponding treatments. (L) Release of dsDNA from 4T1 cells treated with various formulations (n = 4). (M) Western blotting analysis of AIM2 and ZBP1 in cells after the corresponding treatment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (N) Contents of ATP in the cells treated with different formulations (n = 3). (O) Effect of 4-phenylbutyric acid (4-PBA) on the cytotoxicity of HZCO (n = 5). (P) Schematic of the potential mechanism of HZCO to induce PANoptosis. Data are presented as the means ± SD. Significant differences were performed in (L) and (N) using one-way ANOVA with Tukey multiple comparisons post hoc test. Statistical significance in (A), (C), (H), and (O) was calculated via two-tailed Student’s t test.

Excessive accumulation of ions within the cell may hinder the function of the electron transport chain, compromise the respiratory chain, and eventually result in abnormal accumulation of oxygen free radicals in the mitochondria (41). The 2,7-dichlorofluorescein diacetate (DCF) ROS probe was used to examine the ability of HZCO to induce oxidative stress. An equivalent concentration of Zn²⁺ did not induce oxidative stress, and CuO₂ led to only a mild increase in the levels of ROS. In contrast, the HZCO group presented substantial enhancement of intracellular green fluorescence, indicating an abnormal accumulation of intracellular ROS (Fig. 4, F and G). This was attributed to the impairment of mitochondrial function caused by HZCO-mediated zinc overload and the enhancement of Fenton-like catalysis resulting from the disruption of Cu²⁺ metabolism during HZCO treatment. The cell viability was significantly improved after treatment with the ROS inhibitor (Fig. 4H), further confirming that oxidative stress was one of the important factors involved in HZCO-induced cell death. Owing to the abnormal accumulation of ROS, the mitochondrial membrane potential of the HZCO group was considerably reduced (Fig. 4, I and J). Moreover, the mitochondrial damage caused by HZCO affected the synthesis of adenosine 5′-triphosphate (ATP) inside the cells (Fig. 4N), and insufficient ATP further led to a reduction in the activity of Na⁺- and K⁺-dependent ATPase (Na⁺,K⁺-ATPase) and Ca²⁺-dependent ATPase (Ca²⁺-ATPase) (fig. S26), resulting in a substantial increase in oncotic pressure in cancer cells. Moreover, the pro-apoptotic protein Bcl2-associated X (BAX) was up-regulated, the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) was down-regulated, and cytochrome c was released from the mitochondria (Fig. 4K). Furthermore, HZCO-mediated ion overload further led to the release of mitochondrial DNA (mtDNA; a circular dsDNA) from the mitochondria (Fig. 4L). dsDNA has been proven to be a DAMP that can induce PANoptosome assembly and PANoptosis activation under certain conditions (32, 42). We used Western blotting analysis to detect the effect of HZCO on the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway and AIM2 in cancer cells, both of which are crucial intracellular DNA sensing pathways. As expected, HZCO treatment successfully activated the cGAS-STING pathway in cancer cells, as characterized by the phosphorylation of STING, interferon regulatory factor 3 (IRF3), and TANK-binding kinase 1 (TBK1) (fig. S27). In addition, HZCO treatment up-regulated the expression of AIM2 and the innate immune sensor Z-DNA binding protein 1 (ZBP1), which is closely associated with AIM2 (Fig. 4M), suggesting that HZCO was capable of simultaneously activating the signaling pathways related to AIM2. Moreover, immunoprecipitation confirmed interactions between AIM2 and RIPK1, ZBP1, caspase 8, caspase 1, and ASC (fig. S18). The fluorescence colocalization results further verified that AIM2 and ZBP1 colocalized with ASC specks (fig. S28), confirming that HZCO mediated the formation of AIM2-PANoptosomes. Furthermore, the ability of HZCO to induce cell death in AIM2-silenced cells was substantially weaker than that in normal cells (fig. S29). In AIM2-silenced cells, the expression of the PANoptosis-related proteins cleaved caspases 1, 3, 7, and 8, N-GSDMD, and pRIPK1/pMLKL was obviously reduced, as well as the release of LDH and HMGB1 (fig. S30). These findings further confirmed the critical role of AIM2 in HZCO-mediated PANoptosis.

On the other hand, when cells are exposed to oxidative stress or calcium imbalance as well as other stressors, destabilization, abnormal folding, and aggregation of ER proteins can consequently trigger an ER stress response (43). In addition, as the principal organelle for zinc ion storage, the function of ER is highly sensitive to the intracellular free zinc ion levels. Excessive or insufficient zinc levels can both result in an ER stress response (44). To verify the effect of HZCO on ER stress, the cells were treated with HZCO combined with an ER stress inhibitor, and their activity was detected via the MTT assay. The ER stress inhibitor considerably alleviated the cytotoxicity of HZCO, providing initial evidence that ER stress is a potential pathway for HZCO-induced cell death (Fig. 4O). In addition, HZCO treatment caused the phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK), a kinase associated with ER stress, and subsequently led to the phosphorylation of eukaryotic initiation factor 2α (eIF2α) and activation of the activating transcription factor 4 (ATF4) andC/EBP homology protein (CHOP) (Fig. 4K). The activation of the PERK-eIF2α-ATF4-CHOP signaling pathway strongly confirmed the ability of HZCO to induce ER stress, which assumes an extremely vital role in the activation of the inflammasome, the regulation of the expression of AIM2 and caspase 8 and the generation of PANoptosome (45, 46). Therefore, the bioactive HZCO disrupted the metabolic pathways of Cu²⁺ and Zn²⁺ within cancer cells, causing ion disorder and oxidative stress. This further led to mitochondrial damage, dsDNA release, and ER stress, ultimately promoting the formation of AIM2-PANoptosome complexes and triggering the activation of PANoptosis (Fig. 4P).

Transcriptomic analysis

To further explore the molecular mechanism of HZCO treatment, a transcriptomic analysis was conducted on 4T1 cells treated with HZCO. This analysis included differential gene expression (DEG) analysis, Gene ontology (GO) enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. A volcano plot revealed that there were 6385 DEGs between the HZCO-treated group and the control group (Fig. 5A). Among these genes, 3923 genes were up-regulated, while 2462 genes were down-regulated. The GO and KEGG databases currently do not include pathways specifically related to PANoptosis. Therefore, the GO and KEGG analysis did not reveal any enrichment of PANoptosis-related pathways. The GO enrichment analysis revealed that the DEGs were primarily localized to the nucleus and mitochondria regions (Fig. 5E). These DEGs were mainly involved in apoptosis pathways, such as the regulation of the apoptotic signaling pathway, intrinsic apoptotic signaling pathway, DNA repair, negative regulation of the cell cycle, and regulation of cysteine-type endopeptidase activity, and others. The GO analysis also revealed that HZCO treatment affected cell growth and several fundamental cellular functions, including ubiquitin-like protein transferase activity, ubiquitin protein ligase activity, catalytic activity acting on DNA, ubiquitin-like protein ligase activity, protein serine/threonine kinase activity, iron-sulfur cluster binding, and ubiquitin protein ligase activity.

Fig. 5. Transcriptomic analysis of cells treated with HZCO.

(A and B) Volcano plot (A) and heatmap (B) of DEGs between the control group and the HZCO group. (C to E) KEGG enrichment [(C) and (D)] and GO enrichment (E) analysis of DEGs between the control group and the HZCO group.

KEGG analysis revealed notable enrichment of several pathways, including herpes simplex virus 1 (HSV-1) infection, nuclear factor κB (NF-κB) signaling pathway, tumor necrosis factor (TNF) signaling pathway, apoptosis, p53 signaling pathway, platinum drug resistance, forkhead box O (FoxO) signaling pathway, apoptosis-multiple species, mitogen-activated protein kinase signaling pathway, and estrogen signaling pathway (Fig. 5, C and D). These findings were consistent with previous reports on PANoptosis (47–50). Of particular interest was the considerable enrichment of HSV-1 infection, which was attributed to the close association between HSV-1 and PANoptosis. Recent studies have confirmed that HSV-1 induces AIM2-dependent inflammasome activation and PANoptosome formation, resulting in PANoptosis, and the release of cytokines such as IL-1β and IL-18 (13, 47, 51). The heatmap analysis further supported these findings, revealing considerable up-regulation of PANoptosis-related genes, including Zbp1, Aim2, Il-1b, and Il-18rap, in the HZCO group (Fig. 5B). This strongly confirmed the effective activation of PANoptosis by HZCO treatment. In addition, the up-regulation of the MT2 gene indicated changes in intracellular zinc homeostasis caused by HZCO. The observed changes in genes related to the ER and mitochondria (such as Hspa5, Bcl2l11, Bnip3l, Cpt1c, Hmox1, Eif2s3x-ps1, Atf4, Bik, and Hspa8) further supported the notion that HZCO damages key organelles (Fig. 5B), which was an important factor in triggering PANoptosis. Together, these findings confirmed that HZCO effectively triggered PANoptosis in cancer cells by inducing mitochondrial damage, dsDNA release, and ER stress, consequently affecting fundamental cellular functions.

In vivo antitumor effect

Inspired by the ability of HZCO to effectively induce PANoptosis, it is anticipated that HZCO would exhibit remarkable in vivo antitumor effects. To investigate the antitumor effect of HZCO, mice bearing a 4T1 breast cancer model were subsequently established (Fig. 6A). The tumor-bearing mice were randomly divided into four groups: (i) phosphate-buffered saline (PBS), (ii) Zn²⁺, (iii) CuO₂, and (iv) HZCO. The formulations were administered via intratumoral injection, with an equivalent HZCO dose of 10 mg/kg. Zn²⁺ treatment alone had a minimal effect on tumor restriction (Fig. 6, B to E, and fig. S31). However, CuO₂ nanoparticles demonstrated a moderate ability to inhibit tumor growth by effectively inducing apoptosis. Because of their exceptional ability to interfere with zinc homeostasis, the HZCO nanoparticles substantially suppressed tumor growth. To further validate the antitumor effects, various histological analyses, such as hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, and Ki-67 staining, were conducted (Fig. 6F). Compared with those in the control groups, the tumors treated with HZCO exhibited more severe nuclear dissolution and fragmentation as well as noticeable infiltration of lymphocytes. The fluorescence signal of the apoptotic marker TUNEL considerably increased, indicating widespread tumor cell death induced by HZCO. In addition, the expression of Ki-67, a classic marker of cell proliferation, was obviously decreased in the presence of HZCO, suggesting that HZCO has inhibitory effects on cell proliferation and migration. The up-regulation of proteins associated with PANoptosis in tumors, including cleaved caspase 3, N-GSDMD, MLKL, and RIPK1, further substantiated the ability of HZCO to achieve in vivo antitumor effects through the PANoptosis mechanism (Fig. 6G). Furthermore, to validate the broad applicability of the antitumor effects of HZCO, we established other subcutaneous tumor models using the CT26 and B16F10 cell lines. Owing to the potent synergistic action of Cu²⁺ and Zn²⁺, HZCO demonstrated remarkable tumor growth inhibition in both the CT26 and B16F10 models. These results demonstrated the broad-spectrum antitumor properties of HZCO (fig. S32).

Fig. 6. Evaluation of the antitumor effect of HZCO.

(A) Scheme of the subcutaneous (s.c.) mouse 4T1 tumor model for HZCO treatment. All formulations were administered by intratumoral (i.t.) injection, and the equivalent injection dose of HZCO was 10 mg/kg. (B) Tumor growth curves of tumor-bearing mice that received with different treatments. D, days. (C) Average volume change curve of tumors in different groups (n = 6). (D) Average weight of tumors in different groups (n = 6). (E) Photographs of the mice that received various treatments at different time points. (F) TUNEL, H&E, and Ki-67 staining images of tumor sections in different groups. (G) Immunofluorescence staining images of related proteins (cleaved caspase 3, N-GSDMD, RIPK1, and MLKL) in the different groups. (H) Scheme of the subcutaneous mouse 4T1 tumor model for HZCO treatment. Different doses of HZCO (2.5, 5.0, and 10.0 mg/kg) were administered by intravenous (i.v.) injection every 2 days, with two replicates. (I) Tumor growth curves of tumor-bearing mice treated with different doses of HZCO. (J) Average volume change curves of tumors in different groups (n = 8). (K) Kaplan-Meier survival curves of tumor-bearing mice in different treatment groups (n = 8). Data are presented as the means ± SD. Significant differences were performed in (C), (D), and (J) using one-way ANOVA with Tukey multiple comparisons post hoc test.

To further evaluate the antitumor effect of HZCO, it was administered via tail vein injection with different doses (Fig. 6H). HZCO still exhibited a considerable tumor-suppressive effect, and the tumor growth rate in mice was negatively correlated with the concentration of HZCO (Fig. 6, I and J). At a dose of 10 mg/kg, HZCO effectively suppressed tumor growth in the mice, leading to prolonged survival (Fig. 6K). These results provide further confirmation of the outstanding antitumor efficacy of HZCO. Throughout the treatment period, the body weights of the mice in each group remained stable (fig. S33), and there were no pathological changes in major organs following the various treatments, indicating minimal toxic side effects of HZCO (fig. S34). All these data suggested the biologically safe HZCO. Its ability to interfere with zinc homeostasis and induce PANoptosis resulted in notable inhibition of tumor progression and prolonged the survival period of the mice without causing any substantial toxicity.

In vivo evaluation of the immune response

PANoptosis is a cell death process that results in immunogenic properties. When cells undergo PANoptosis, they release a variety of immune-related signaling molecules, intracellular antigens, heat shock proteins, and other cellular contents (14, 17). These substances are recognized by the immune system, thereby initiating an immune response. Inspired by this, the ability of HZCO to induce ICD was further investigated. First, HZCO induced calreticulin (CRT) up-regulation and translocation to the cancer cell membrane (Fig. 7A and fig. S35). CRT translocation releases an “eat me” signal to the surrounding microenvironment, which promotes the activation and phagocytosis of immune cells. In addition, HMGB1 and ATP were actively released from cancer cells treated with HZCO, which serve as “find me” signals that facilitate inflammation, immune cell infiltration, and activation (Fig. 7, B and C, and fig. S35). In contrast, treatment with Zn²⁺ or CuO₂ had minimal effects on CRT exposure and the release of HMGB1 and ATP, confirming that HZCO effectively induced ICD through PANoptosis. The immature dendritic cells (DCs) tend to mature upon stimulation by these DAMPs. As expected, the maturity of DCs cocultured with HZCO-pretreated 4T1 cells significantly surpassed that of the other groups due to HZCO-mediated ICD through triggering PANoptosis (Fig. 7, D and E). This process facilitated the ability of DCs to effectively present antigens and activate T cells, promoting the occurrence of an immune response (Fig. 7F).

Fig. 7. Analysis the antitumor immune response induced by HZCO.

(A and B) Expression of CRT (A) and HMGB1 (B) in the cells after different treatments. (C) Content of ATP released from the cells after different treatments. (D and E) Representative flow cytometry analysis (D) and quantification (E) of DC maturation in different groups. (F) Schematic diagram of HZCO-mediated ICD effect and DC maturation. (G) Scheme of the in vivo immune evaluation procedure. (H and I) Flow cytometry analysis (H) and quantification (I) of DC maturation in TDLNs in different groups. (J) Proportion of CD3⁺ T cells in tumors after different treatments. (K and L) Flow cytometry analysis (K) and quantification (L) of CD8⁺ T cell proportion among CD3⁺ T cells in different groups. (M and N) Flow cytometry analysis (M) and quantification (N) of CD4⁺ T cell proportion among CD3⁺ T cells. (O) Immunofluorescence staining images of CD4⁺ and CD8⁺ T cells in different groups. (P and Q) Flow cytometry analysis (P) and quantification (Q) of IFN-γ⁺ cell proportion in CD8⁺ T cells. (R and S) Flow cytometry analysis (R) and quantification (S) of M1CD80⁺ cell proportion among CD11b⁺F4/80⁺ cells. (T and U) Flow cytometry analysis (T) and quantification (U) of CD206⁺ cell proportion in CD11b⁺F4/80⁺ cells. (V to X) Levels of IL-1β (V), TNF-α (W), and IFN-γ (X) in the different groups. Data are presented as the means ± SD. Significant differences were performed using one-way ANOVA with Tukey multiple comparisons post hoc test.

Because of the remarkable ability of HZCO to trigger ICD and stimulate DC maturation in vitro, it was expected that HZCO would also trigger effective in vivo immune responses (Fig. 7G). Immunofluorescence staining and Western blotting for CRT and HMGB1 in tumor tissues further confirmed that HZCO resulted in a pronounced increase in CRT levels and HMGB1 release in tumor tissues, indicating its effective capacity to induce ICD (fig. S36). In addition, tumor-draining lymph nodes (TDLNs) and tumor cells were collected to evaluate tumor-specific immune activation by preparing single-cell suspensions. Consistent with the in vitro experiments, mice treated with HZCO showed substantially improved DC maturation in TDLNs (Fig. 7, H and I), confirming that HZCO-mediated ICD stimulates in vivo DC maturation. Owing to the powerful ability of HZCO to induce ICD, the percentage of CD3⁺ T cells in the tumors of the mice treated with HZCO reached ~0.194%, which was much greater than that in the PBS (~0.054%), Zn²⁺ (~0.063%), and CuO₂ (~0.095%) groups (Fig. 7J). These findings confirmed the potential of HZCO to activate the body’s adaptive T cell antitumor immune response by inducing PANoptosis. Among CD3⁺ T cells, CD3⁺CD4⁺ T cells are critical for activating CD3⁺CD8⁺ T cells and potentiating antitumor immune responses through the secretion of various immune-modulatory factors. Once CD3⁺CD8⁺ T cells recognize tumors, they induce apoptosis via the release of perforin and granzymes. After HZCO treatment, the increased proportions of CD8⁺ and CD4⁺ T cells among the total CD3⁺ T cells indicated that cytotoxic T cells capable of killing tumors infiltrated the tumor after receiving tumor-associated antigens presented by DCs (Fig. 7, K to O). Moreover, the increased presence of activated CD8⁺ T cells in the HZCO group suggested that the immune system generated a specific immune response against tumor cells (Fig. 7, P and Q). The immunosuppressive tumor microenvironment (TME) poses a considerable challenge to tumor immunotherapy. However, treatment with HZCO did not exacerbate immune suppression reactions, as evidenced by the fact that the proportions of myeloid-derived suppressor cells (MDSCs) did not significantly increase (fig. S37). In addition, HZCO treatment effectively reduced the percentage of M2-polarized macrophages while simultaneously increasing the proportion of M1 macrophages (Fig. 7, R to U). This was attributed to the ability of HZCO to activate the NF-κB pathway through the induction of oxidative stress (52). M2-polarized macrophages typically dampen T cell–mediated immune responses by releasing immunosuppressive factors, whereas M1 macrophages exhibit effective pro-inflammatory effects. Therefore, the capacity of HZCO to promote the polarization of macrophages from the M2 phenotype to M1 phenotype contributes to reversing immunosuppression in the TME, thereby increasing the effectiveness of immunotherapy. Cytokines play a crucial role in immune responses among lymphocytes, sustaining the activation of antitumor immunity. The levels of various cytokines were measured via an enzyme-linked immunosorbent assay (ELISA). As expected, high levels of pro-inflammatory cytokines, such as IL-1β, TNF-α, and interferon-γ (IFN-γ), were exclusively observed in the HZCO treatment group (Fig. 7, V to X). In contrast, the Zn²⁺ and CuO₂ alone groups exhibited relatively weak inflammatory responses, thus confirming that HZCO triggered a robust inflammatory response in the TME through the synergistic effect of Cu²⁺ and Zn²⁺. Overall, HZCO effectively converted the immunosuppressive “cold” TME into an immune-infiltrated “hot” TME, thus enhancing the efficacy of cancer immunotherapy.

Synergistic immunotherapy

Tumor cells have various strategies to evade the immune system, one of which involves the PD-1/programmed death ligand 1 (PD-L1) pathway (53). Recently, various soluble factors produced by immune cells, such as TNF-α, IL-17, and IFN-γ, have been identified as potent inducers of PD-L1 (54). The strong inflammatory response induced by HZCO in tumors triggered the up-regulation of PD-L1 expression in tumor cells (fig. S38). This potentially enhanced the efficacy of PD-L1 blockers and promoted a more robust antitumor immune response. To investigate the potential of HZCO in combination with PD-L1/PD-1 therapy, bilateral murine tumor models were established, and the tumor-bearing mice were randomly divided into four groups: (i) PBS; (ii) HZCO (intratumoral injection, 10 mg/kg), (iii) αPD-1 (intravenous injection, 2.5 mg/kg), and (iv) HZCO (intratumoral injection, 10 mg/kg) + αPD-1 (intravenous injection, 2.5 mg/kg) (Fig. 8A). The tumor growth curves revealed that the PBS treatment led to a rapid tumor growth, whereas αPD-1 therapy alone partially inhibited the growth of both the primary and distant tumors, possibly because of inadequate infiltration of T cells into the tumor sites (Fig. 8, B to E). Conversely, HZCO treatment efficiently suppressed the growth of primary tumors but only slightly inhibited the growth of the distant tumors, and this limited inhibition of the distant tumors was attributed to the innate immune evasion mechanisms used by cancer cells. HZCO combined with αPD-1 demonstrated the most substantial suppression of tumor growth, resulting in marked suppression of both primary and distant tumor growth. This combined therapy led to a substantial extension of survival (Fig. 8F), indicating a remarkable abscopal effect induced by this combination treatment. This abscopal effect was attributed to the PANoptosis mediated by HZCO, which effectively eliminated the primary tumor and activated systemic antitumor immune responses, particularly those involving CD8⁺ T cells. The αPD-1 functioned by blocking the PD-1/PD-L1 pathway, counteracting tumor immune suppression, and enhancing the antitumor immune response mediated by CD8⁺ T cells, thus leading to enhanced tumor inhibition via this synergistic effect. To validate this, immune cell infiltration and the expression levels of related cytokines in distant tumors were examined (Fig. 8G). Compared with those in the PBS treatment group, both the αPD-1 and HZCO treatment groups exhibited enhanced DC maturity in the draining lymph nodes of distant tumors (Fig. 8, H and I), which could be attributed to the alleviation of immunosuppression through αPD-1 treatment and the stimulation of an adaptive immune response by HZCO. Notably, the combination of HZCO and αPD-1 had the highest level of DC maturation, suggesting that the combination of HZCO and αPD-1 has the potential for a synergistic antitumor immune effect. Moreover, the combination treatment of HZCO + αPD-1 significantly increased the overall proportion of T cells in the distant tumors, with the CD3⁺ T cell ratio increasing to ~0.41%, which was much higher than that in the PBS (~0.05%), HZCO (~0.08%), and αPD-1 (~0.04%) groups (Fig. 8J). Correspondingly, there was an increase in the proportion of CD8⁺ T cells and activated CD8⁺ T cells (Fig. 8, K to N), while the number of immunosuppressive regulatory T cells (T_regs) within the CD4⁺ T cell population did not significantly change (Fig. 8, P and Q), confirming the effective antitumor immune effects. In addition, M1 macrophages with potent pro-inflammatory effects considerably infiltrated distal tumors after HZCO + αPD-1 treatment (Fig. 8O). Elevated levels of pro-inflammatory cytokines, including IL-12, IL-1β, TNF-α, and IFN-γ, were also observed, further supporting the occurrence of an inflammatory response in the distant tumors caused by HZCO + αPD-1 treatment (Fig. 8, R to U). These results demonstrated that the combination of HZCO and αPD-1 activated a potent systemic antitumor immune response, effectively inhibiting both primary and distant tumors. During the treatment period, the mice that received HZCO + αPD-1 treatment did not exhibit any abnormal weight loss throughout the whole experiment (fig. S39). In addition, H&E staining of the major organs revealed no apparent damage (fig. S40), confirming the favorable safety profile of the combination therapy. Overall, the combination of HZCO and αPD-1 demonstrated an impressive synergistic effect on eliciting antitumor immune effects and achieving effective tumor suppression in both primary and distant tumors (Fig. 8V).

Fig. 8. Evaluation of the antitumor effect and immune response to the combination of HZCO and αPD-1 therapy.

(A) Treatment schedule for in vivo tumor therapy with HZCO combined with αPD-1. (B and C) Growth curves (B) and average volume change curves (C) of primary tumors in different groups. (D and E) Growth curves (D) and average volume change curves (E) of distant tumors in different groups (n = 6). (F) Kaplan-Meier survival curves of tumor-bearing mice in different groups (n = 6). (G) Scheme of the in vivo immune evaluation procedure. (H and I) Flow cytometry analysis (H) and quantification (I) of DC maturation in TDLNs. (J) Proportion of CD3⁺ T cells in the tumors after different treatments. (K and L) Flow cytometry analysis (K) and quantification (L) of CD8⁺ T cell proportion in CD3⁺ T cells. (M and N) Flow cytometry analysis (M) and quantification (N) of IFN-γ⁺ cell proportion in CD8⁺ T cells. (O) Proportion of CD80⁺ cells in CD11b⁺F4/80⁺ cells in mice tumors after different treatments. (P and Q) Flow cytometry analysis (P) and quantification (Q) of proportion of Foxp3⁺ T cells in CD4⁺ T cells. (R to U) Levels of IFN-γ (R), TNF-α (S), IL-12 (T), and IL-1β (U) in different groups. (V) Schematic diagram of the synergistic anticancer mechanism of HZCO combined with αPD-1 therapy. Data are presented as the means ± SD. Significant differences were calculated via using one-way ANOVA with Tukey multiple comparisons post hoc test.

Combined antitumor effect of ES- and zinc-based therapy

HZCO exhibited a remarkable antitumor effect and activated the immune response by inducing PANoptosis, thus indicating its feasibility as a PANoptosis inducer for synergistic immunotherapy. Although HZCO has shown ideal biosafety in mouse tumor models, substantial challenges remain in clinical translation due to numerous uncertainties. Inspired by the effective induction of PANoptosis by Cu²⁺ + Zn²⁺ treatment and HZCO, we further explored the combined antitumor effect of the preclinically approved drugs ES (a copper ionophobe) and Zn²⁺ therapy in a 4T1 mouse tumor model (Fig. 9, A and B). The tumor-bearing mice were randomly divided into four groups: (i) PBS, (ii) ES (intravenous injection, 5 mg/kg), (iii) Zn²⁺ (intratumoral injection, 2 mg/kg), and (iv) ES (intravenous injection, 5 mg/kg) + Zn²⁺ (intratumoral injection, 2 mg/kg). Zn²⁺ treatment alone had a minimal effect on tumor growth inhibition, while ES treatment had a limited antitumor efficacy and the relative tumor growth rate was ~57.51% on day 10. Notably, a remarkable increase in the antitumor effect was observed when Zn²⁺ was combined with ES, with a relative tumor growth rate of ~33.45% on day 10 (Fig. 9, C and D, and fig. S41), confirming the synergistic effect between ES and Zn²⁺. The tumors of the mice were subsequently subjected to immunofluorescence staining to assess the potential of Zn²⁺ + ES treatment to induce PANoptosis.

Fig. 9. Evaluation of the synergistic antitumor effect of ES- and zinc-based therapy.

(A) Schematic diagram of the synergistic antitumor effect of the combination of ES and Zn²⁺. (B) Treatment schedule for in vivo tumor therapy by ES combined with Zn²⁺. ES was administered by intravenous injection at a dose of 5 mg/kg, and Zn²⁺ was administered by intratumoral injection at a dose of 2 mg/kg. (C and D) Growth curves (C) and average volume change curves (D) of tumors in different groups (n = 6). (E) Proportions of CD80⁺CD86⁺ cells among CD11c⁺ cells in different groups. (F) Proportion of CD3⁺ T cells in the mice tumors after different treatments. (G) Proportion of CD8⁺ T cells in CD3⁺ T cells in different groups. (H) Proportions of IFN-γ⁺ cells among CD8⁺ T cells in different groups. (I) Proportions of CD4⁺ T cells among CD3⁺ T cells in different groups. (J) Proportions of Foxp3⁺ cells among CD4⁺ T cells in different groups. (K and L) Proportions of CD206⁺ cells (K) and CD80⁺ cells (L) among CD11b⁺ F4/80⁺ cells in different groups. (M to P) Levels of IL-1β (M), IL-12 (N), IFN-γ (O), and TNF-α (P) in different groups. (Q) Schematic diagram of potential induction strategies for PANoptosis. Data are presented as the means ± SD. Significant differences were calculated via using one-way ANOVA with Tukey multiple comparisons post hoc test.

Compared with those in the Zn²⁺ or ES alone treatment group, the expression levels of PANoptosis-associated proteins including cleaved caspase 3, N-GSDMD, and MLKL were markedly increased after treatment with Zn²⁺ + ES, strongly confirming the potential of Zn²⁺ + ES to induce PANoptosis (fig. S42). The effect of the combined therapy on tumor immunity was further verified. As anticipated, treatment with ES or Zn²⁺ alone did not result in an increased proportion of T cells within the tumor tissue. However, when ES and Zn²⁺ were combined, there was a noteworthy increase in the proportion of both CD3⁺ and CD3⁺CD8⁺ T cells in the tumor tissue (Fig. 9, F and G). Furthermore, compared with those in the ES group, the proportions of activated killer T cells in the Zn²⁺ + ES group were significantly greater, but there was no substantial increase in the number of immunosuppressive T_regs (Fig. 9, H to J). These findings suggested that the combination of ES and Zn²⁺ successfully activated adaptive antitumor immunity mediated by T cells. Furthermore, there was a tendency for macrophages to polarize toward the M1 phenotype, and the levels of pro-inflammatory cytokines such as IL-12p70, IL-6, IL-1β, and TNF-α were significantly increased in the Zn²⁺ + ES group (Fig. 9, K to P). Furthermore, the proportion of DCs in the TDLN significantly increased in the Zn²⁺ + ES group (Fig. 9E). This clearly suggested that more DCs matured as a result of the homing effect of lymphocytes, which could further activate the systemic antitumor immune response. These findings confirmed that the presence of Zn²⁺ enhanced the antitumor effect and immune activation ability of ES. The underlying mechanism was explained by the fact that ES facilitated the intracellular delivery of Cu²⁺, thereby increasing the intracellular Cu²⁺ concentration and oxidative stress levels in tumor cells. This process induced damage to cancer cells while simultaneously triggering the release of intracellular Zn²⁺, which, in combination with exogenous Zn²⁺, leads to zinc overload and PANoptosis, thereby stimulating a potent antitumor immune response. This finding further supported the role of zinc overload in facilitating PANoptosis.

DISCUSSION

PANoptosis is a newly identified form of programmed cell death that involves the activation of multiple molecules from three distinct cell death pathways: pyroptosis, apoptosis, and necroptosis (13–15). As an innate immune pathway, PANoptosis holds substantial promise for tumor immunotherapy. However, owing to the nascent stage of PANoptosis research, effective induction strategies are still limited. Nutrient metal ions can induce programmed cell death by triggering biological processes such as biocatalysis, disrupting osmotic balance, interfering with metabolism and signal transduction, and causing DNA damage (26). In this study, the anticancer effects of various metal ions were preliminarily investigated, focusing on their potential to induce PANoptosis. We found that Cu²⁺ and Zn²⁺ had a notable synergistic anticancer effect. A PANoptosome complex was formed in Cu²⁺- and Zn²⁺-treated cancer cells, activating various cell death signals, including pyroptosis, apoptosis, and necroptosis. This suggests that the synergistic action of Cu²⁺ and Zn²⁺ can induce PANoptosis. Notably, at the same dose, Cu²⁺ and Zn²⁺ are much less toxic to normal cells than to cancer cells. This specific synergistic anticancer effect of Cu²⁺ and Zn²⁺ may be attributed primarily to the disruption of inherent zinc homeostasis within the cells. Inside the cell, zinc ions are mainly stored in MT in the form of bound zinc, with each MT molecule capable of selectively binding seven zinc atoms to form Zn-MT (55). MT can bind with various metals in a preferred order. Compared with that of Zn²⁺, the binding constant between Cu²⁺ and MT is substantially greater (56). Therefore, free Cu²⁺ in the cell can replace zinc ions in Zn-MT, leading to an increase in the level of free zinc ions within the cell. Rapidly proliferating cancer cells have a greater demand for Zn²⁺ than normal cells do, leading to higher zinc levels (36), similar to the high ROS levels observed in cancer cells. Consequently, cancer cells have a lower Zn²⁺ toxicity threshold than normal cells do. To maintain intracellular zinc homeostasis, free zinc ions are chelated by the six zinc fingers of MTF-1, a transcription factor specifically activated by zinc ions. Upon activation, MTF-1 induces MT gene expression by binding DNA and other transcription factors (29, 57). When the level of free zinc in the cell exceeds the adjustable range, zinc ions interfere with normal physiological processes, blocking the regulation of intracellular zinc homeostasis and causing ion overload and cell death. Owing to the low level of Zn²⁺ in normal cells, the combination of Zn²⁺ replaced by Cu²⁺ and exogenous Zn²⁺ does not reach the Zn²⁺ toxicity threshold in normal cells. This results in the continuous activation of the Zn²⁺–MTF-1–MT signaling axis, effectively eliminating abnormal Zn²⁺ and Cu²⁺. Therefore, a specific combination of Cu²⁺ and Zn²⁺ can selectively induce PANoptosis in cancer cells.

The application of exogenous ions in vivo may be severely hampered by delivery barriers such as suboptimal pharmacokinetics, low tumor accumulation, and nonspecific cell targeting. Nanotechnology provides a promising direction for the development of metal ion transport and tumor therapy. In this study, zinc-doped CuO₂ nanoparticles modified with HHA (HZCO) were developed for the simultaneous delivery of Cu²⁺ and Zn²⁺ to achieve synergistic tumor immunotherapy. HZCO increased zinc overload by actively delivering Zn²⁺ and coordinating/oxidizing with overexpressed MT in cancer cells. Excessive Zn²⁺ disrupts cellular energy metabolism by interfering with mitochondrial function, leading to the blockage of Cu/Zn ion transport. This results in the abnormal accumulation of copper and zinc within the cell, further amplifying the anticancer effects of Cu²⁺ and Zn²⁺. As essential functional ions in cells, the abnormal accumulation of Cu²⁺ and Zn²⁺ destroys the MT-mediated antioxidant defense system of cancer cells and impedes the function of the mitochondrial electron transport chain. This ultimately leads to the abnormal accumulation of oxygen free radicals in mitochondria. Excess ROS oxidizes sulfhydryl groups on the mitochondrial membrane, damages the integrity and function of the membrane, and accelerates the release of mitochondria-associated proteins and mtDNA. It has been confirmed that the dsDNA virus HSV-1 can induce PANoptosis by activating the AIM2-PANoptosome (32). Transcriptomic analysis revealed that genes remarkably altered after HZCO treatment were enriched in the HSV-1 pathway. In addition, the DNA sensors cGAS-STING and AIM2-related pathways were simultaneously activated following HZCO treatment. A previous study has shown that free zinc ions can enhance the activity of the cGAS enzyme by promoting cGAS-DNA phase separation, thereby effectively improving the activation efficiency of the cGAS-STING pathway (58). Activation of the cGAS-STING-IFN1 pathway can amplify AIM2 protein levels (59). Therefore, the activation of the DNA-sensing pathway by HZCO may not only depend on the release of dsDNA induced by ion overload but may also be related to the disruption of zinc homeostasis leading to increased intracellular free Zn²⁺. AIM2 has been shown to be closely related to the formation of the PANoptosome. AIM2 regulates the innate immune sensors pyrin and ZBP1 and mediates the assembly of the AIM2-PANoptosome to drive inflammatory signals (32). The interaction between AIM2, RIPK1, ZBP1, caspase 8, caspase 1, and ASC in HZCO-treated cells confirmed that HZCO triggers PANoptosis by mediating the formation of the AIM2-PANoptosome.

Furthermore, HZCO disrupts the mitochondrial membrane, causing the release of molecular signals such as mtDNA, mitochondrial proteins, and ROS into the cytoplasm. These signals are likely detected by ER sensors, triggering ER stress responses. A previous study has shown that the ER stress–related protein ATF4 interacts with the AIM2 promoter, thereby promoting AIM2 expression (60). Another ER stress–related protein, CHOP, is a stress response factor involved in the cell death signaling cascade, which helps activate inflammasomes, promote the caspase 1–dependent pyroptosis pathway, and regulate the expression and activity of caspase 8, thus contributing to the formation of the PANoptosome (45, 46). Therefore, HZCO-mediated ER stress strongly promotes the formation of the AIM2-PANoptosome and the activation of PANoptosis.

Because of its ability to efficiently induce PANoptosis, HZCO has demonstrated remarkable anticancer effects and an enhanced anticancer immune response in both in vitro and in vivo tumor immunotherapy studies. However, major challenges remain in the clinical translation of HZCO. The lack of specific targets means that high concentrations of HZCO in normal cells can cause unnecessary oxidative stress, metabolic changes, macromolecular dysfunction, and cell death, limiting its clinical application. The toxic side effects of HZCO may depend on its structure, ion ratio and functional properties, delivery pathway, and cell type. Therefore, future research should prioritize the delicate balance between the toxicity and efficacy of metal-based drugs such as HZCO, as well as achieve precisely targeted delivery and selective release of these drugs.

Another promising strategy involves relying on clinically approved or preapproved drugs to achieve antitumor effects similar to those of HZCO. ES, as a copper ionophobe, has been shown to increase the level of oxidative stress within tumor cells and induce antitumor effects (61, 62). Unfortunately, the vast majority of patients in clinical trials are insensitive to ES; therefore, ES has not yet been approved for clinical oncology treatment (63). Our study confirmed that the presence of Zn²⁺ enhanced the antitumor effect and immune activation ability of ES. This finding further supported the role of zinc overload in facilitating PANoptosis. Inspired by this mechanism, antitumor strategies involving the use of ROS and reactive nitrogen species (RNS), such as radiotherapy, photodynamic therapy, sonodynamic therapy, and nitric oxide (NO) gas therapy as well as metal anticancer drugs such as platinum drugs, have the potential to synergize with zinc therapy for PANoptosis because of the sensitivity of highly expressed MT in cancer cells to metal ions and electrophiles (Fig. 9Q). These findings are important for expanding current clinical immunotherapy strategies for cancer treatment.

In summary, the combination of Cu²⁺ and Zn²⁺ was confirmed to trigger PANoptosis, and the PANoptosis inducer HZCO based on Zn²⁺ and Cu²⁺ was successfully developed to enhance the efficacy of immune therapy. HZCO disrupted the balance of Cu²⁺ and Zn²⁺ in cells through the unique synergistic effects of its components, Cu²⁺, Zn²⁺, and H₂O₂, leading to the abnormal accumulation of Cu²⁺ and Zn²⁺ within the cells. Excessive Cu²⁺ and Zn²⁺ induced mitochondrial damage, dsDNA release, and ER stress, thereby triggering the formation of the AIM2-PANoptosome and the activation of PANoptosis. In vitro and in vivo results demonstrated that HZCO-induced PANoptosis effectively reshaped the immunosuppressive microenvironment of tumors and suppressed tumor growth. Furthermore, the combination of HZCO with αPD-1 therapy activated potent T cell–specific immune responses, effectively inhibiting the growth of primary and distant tumors. Inspired by these findings, ES- and zinc-based therapies were tested for their ability to suppress tumor growth and induce adaptive antitumor immune responses. Our work highlights the valuable insights into the rational design of PANoptosis inducers and the enhancement of immunotherapy efficacy.

MATERIALS AND METHODS

Materials

PVP [M_w (weight-average molecular weight ~10,000], copper(II) chloride dihydrate (CuCl₂·2H₂O), zinc chloride (ZnCl₂), sodium hydroxide (NaOH), and hydrogen peroxide aqueous solution (H₂O₂; 30%) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). HHA was prepared according to our previous experiments. MB and TMB were purchased from Macklin Biochemical Co. Ltd. (Shanghai, China). A LIVE/DEAD cell double staining kit [calcein AM/propidium iodide (PI)], an annexin V–fluorescein isothiocyanate (FITC)/PI apoptosis detection kit, MTT, Alexa Fluor 488 secondary antibody, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). VX-765, Ac-DEVD-CHO, and Nec-1 were purchased from MedChemExpress (United States).

Anti-CHOP (ab11419), anti-eIF2α (ab169528), anti-phospho-eIF2α (ab32157), anti-RIPK1 (ab300617), anti-PERK (ab229912), anti-ATP7A (ab308524), anti-caspase 1 (ab207802), anti-GSDMD (ab219800), anti-TMS1/ASC (ab309497), anti-HMGB1 (ab18256), anti-IL-18 (ab223293), and anti-IL-1β (ab254360) antibodies were obtained from Abcam. Anti-cleaved caspase 1 (#89332), anti-cleaved caspase 3 (#9661), anti-caspase 8 (#4790), anti-cleaved caspase 8 (#8592), anti-cleaved GSDMD (#10137), anti-ASC-Alexa Fluor 488 (#17507), anti-ZBP1 (#33402), anti-cGAS (#31659), anti-STING (#13647), anti-phospho-STING (#72971), anti-TBK1 (#3504), anti-phospho-TBK1 (#5483), anti-phospho-IRF3 (#4302), anti-IRF3 (#4947), anti-CHOP (#2895), anti-Bcl-2 (#3498), anti-BAX (#2772), and anti-cytochrome c (#4272) antibodies were obtained from CST. Anti-AIM2 (20590-1-AP), anti-caspase 3/p17/p19 (19677-1-AP), anti-MLKL (21066-1-AP), anti-CRT (10292-1-AP), anti-caspase 7/p20 (27155-1-AP), anti-phospho-MLKL (ab196436), anti-phospho-RIPK1 (66854-1-Ig), anti-phospho-PERK/EIF2AK3 (29546-1-AP), anti-SLC30A2 (67993-1-Ig), and anti-MTF1 (25383-1-AP) antibodies were obtained from Proteintech Group.

All flow cytometry antibodies were purchased from BioLegend. All ELISA kits were purchased from Invitrogen.

Cell lines

4T1 cells, PAN02 cells, B16F10 cells, CT26 cells, MC38 cells, and HUVECs were obtained from the American Type Culture Collection. The cells were cultured in a standard cell culture medium at 37°C with 5% CO₂ and regularly subcultured.

Animals

Female BALB/c mice (5 to 6 weeks) and C57BL/6J (5 to 6 weeks) were sourced from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) and housed in a specific pathogen–free animal facility maintained at a constant room temperature of 21° ± 1°C and a relative humidity of 40 to 70%, with ad libitum access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of Soochow University (no. 202309A0250).

To establish the tumor model, 50 μl of a solution containing cancer cells (2 × 10⁶ cells) was subcutaneously injected into the back of each mouse. The tumor model was considered successfully established when the tumor volume reached ~100 mm³.

Ion screening

To investigate the cytotoxic effects of metal ions on cancer cells, 4T1 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and cultured overnight. Subsequently, the cells were exposed to various concentrations of different metal ions (K⁺, Na⁺, Mg²⁺, Ca²⁺, Mo⁴⁺, Ga³⁺, Fe²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Co²⁺, Ni²⁺, Gd²⁺, and V³⁺). The cellular morphology was examined at predetermined time points via optical microscopy. After 24 hours of treatment, the toxicity of the metal ions to the cells was assessed via an MTT assay.

To investigate the synergistic cytotoxicity of different metal ions in combination with Zn²⁺ on tumor cells, 4T1 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and cultured overnight. Following this, a combination of Zn²⁺ and other metal ions was applied to treat the cells. After a 24-hour incubation period, the cell morphology and viability were observed and assessed via an optical microscope and an MTT assay, respectively.

To evaluate the impact of copper ions on zinc overload, the cells were initially exposed to varying concentrations of Cu²⁺ in the presence of 40 μM Zn²⁺. After a 24-hour incubation period, the cytotoxicity was assessed via the MTT method. In addition, a zinc ion probe was used to monitor the alterations in free zinc ions within the cells. Specifically, 4T1 cells were seeded onto 12-well plates and treated separately with Zn²⁺ and Cu²⁺ for 4 hours. After treating the cells with the zinc ion probe according to the manufacturer’s instructions, changes in the intracellular Zn²⁺ levels were subsequently observed via confocal laser scanning microscopy (CLSM; ZEISS LSM 800).

The expression of MT and MTF-1 in Cu²⁺-treated cells was detected via immunofluorescence staining to determine the effects of Cu²⁺ on intracellular Zn homeostasis. Briefly, 4T1 cells were inoculated in a 12-well plate and exposed to Cu²⁺ for 4 hours. Then, the cells were washed, fixed, and fluorescently labeled with MT/MTF-1 antibodies and Alexa Fluor 488 secondary antibody for immunofluorescence staining. The labeled cells were observed via CLSM.

To investigate the mechanism of cell death induced by Cu²⁺ and Zn²⁺, cells were pretreated with specific inhibitors targeting different cell death pathways. These inhibitors included Ac-DEVD-CHO (apoptosis inhibitor), VX-765 (pyroptosis inhibitor), and Nec-1 (necroptosis inhibitor). The pretreatment with these inhibitors lasted for 1 hour, followed by treatment with Cu²⁺ (125 μM) and Zn²⁺ (40 μM). After 24 hours, cell viability was determined via the MTT assay.

Synthesis of HZCO

To synthesize PVP-Zn-CuO₂ nanoparticles, 1000 mg of PVP, 20 mg of CuCl₂, and 10 mg of ZnCl₂ were dissolved in 10 ml of ultrapure water. Then, a NaOH solution (500 μl, 40 mg/ml) and H₂O₂ (500 μl) were added to the mixture. After 10-min reaction, the resulting precipitate was collected through centrifugation and washed three times with ultrapure water to obtain PVP-Zn-CuO₂ nanoparticles.

To prepare HZCO, an HHA solution (5 ml, 10 mg/ml) was slowly added to a suspension of PVP-Zn-CuO₂ nanoparticles (10 ml, 1 mg/ml) under ultrasonic conditions. The mixture was mixed evenly and then slowly stirred at room temperature for 12 hours. The resulting precipitate was collected by centrifugation and washed to obtain HZCO.

Measurement of ion release

The ion release capacity of HZCO was determined via a centrifugal method. HZCO at a concentration of 1 mg/ml was dispersed in PBS buffers with various pH values (pH 7.4, pH 6.5, and pH 5.0) and then incubated in a constant temperature shaker. At predetermined time points, the supernatant of the HZCO suspension was collected via centrifugation. The concentrations of Zn²⁺ and Cu²⁺ in the supernatant were measured via inductively coupled plasma mass spectrometry (NexION 350D).

Measurement of ·OH generation

The production of ·OH was examined using MB as an indicator. Specifically, 100 μl of an HZCO suspension at a concentration of 500 μg/ml was mixed with 3 ml of an MB solution at a concentration of 10 μg/ml, along with 10 mM H₂O₂. The mixture was then incubated for 2 hours, after which the absorption spectrum of the solution was recorded using a UV-vis-NIR spectrometer (Lambda 750).

The autocatalytic ability of HZCO to generate ·OH was evaluated via a TMB probe. Specifically, 100 μl of the HZCO suspension (500 μg/ml) was introduced to 3.0 ml of a TMB solution (50 μg/ml) at various pH values (5.0, 6.5, and 7.4). After 30 min of thorough mixing, the absorption spectrum of the resulting solution was measured via UV-vis spectroscopy.

Cellular experiments

HZCO-triggered PANoptosis

To investigate HZCO-triggered PANoptosis, the cells were plated in 96-well plates at a density of 1 × 10⁴ cells per well and then pretreated with three different inhibitors: Ac-DEVD-CHO (an apoptosis inhibitor), VX-765 (a pyroptosis inhibitor), or Nec-1 (a necroptosis inhibitor). The pretreatment with these inhibitors lasted for 1 hour, followed by treatment with HZCO. After 24 hours, cell viability was assessed via the MTT assay.

To visualize HZCO-triggered PANoptosis, 4T1 cells were seeded into six-well plates at a density of 1 × 10⁵ cells per well and exposed to HZCO for 24 hours. The cells were then washed three times with PBS. The cells were stained with annexin V–FITC for 15 min and PI for another 5 min, following the instructions provided in the manual. The cells were observed via CLSM and analyzed by flow cytometry. Moreover, the extracellular secretion of LDH and IL-1β was detected using an LDH assay kit and an ELISA assay kit, respectively, following the provided protocols.

In addition, Western blotting analysis was conducted to investigate the expression of PANoptosis-related proteins. 4T1 cells were seeded in culture dishes and incubated with different formulations (Zn²⁺, CuO₂, and HZCO) for 12 hours. Subsequently, the cells were collected and lysed, after which the proteins were extracted. The protein concentration was quantified using the bicinchoninic acid method. Then, Western blotting analysis was performed to determine the expression of marker proteins related to PANoptosis, including caspase 1/3/6/7/8, ASC, N-GSDMD, IL-1β, IL-18, MLKL/p-MLKL, and RIPK1/p-RIPK1.

In vivo tumor therapeutic efficiency of HZCO

4T1 tumor-bearing mice with an initial tumor volume of ~100 mm³ were randomly divided into four groups (n = 6): (i) PBS, (ii) Zn²⁺, (iii) CuO₂, and (iv) HZCO. Intratumoral injection of 50 μl of the respective preparations was administered to the mice, with an equivalent dose of HZCO at 10 mg/kg. Tumor volume and body weight were measured every 2 days. The tumor volume was calculated via the formula V = L × W²/2, where L represents the length of the tumor and W represents the width of the tumor. After 12 days, the mice were euthanized, and their main organs were stained with H&E.

Furthermore, H&E, Ki-67, and TUNEL assays were conducted to evaluate the antitumor effects of HZCO. Briefly, 24 hours after treatment, tumor samples from each group were collected and stained with H&E, Ki-67, and TUNEL using standard protocols. Images were captured via a fluorescence microscope.

Evaluation of HZCO-triggered immune responses

To investigate the ability of HZCO to induce ICD, 4T1 cells were cultured with HZCO (40 μM) as well as with equivalent concentrations of Zn²⁺ and CuO₂. After incubation for 24 hours, the culture media were collected, and ATP levels were measured via ATP ELISA kits. The cells were then washed, fixed, and fluorescently labeled with anti-CRT/anti-HMGB1 antibodies and Alexa Fluor 488 secondary antibody for immunofluorescence staining. The labeled cells were observed via CLSM.

To assess the in vivo ICD-inducing ability of HZCO, 50 μl of formulations (Zn²⁺, CuO₂, or HZCO) with an equivalent dosage of 10 mg/kg of HZCO was intratumorally injected into mice bearing 4T1 tumors. After 24 hours, the tumors were collected and labeled with anti-CRT and anti-HMGB1 antibodies via immunofluorescence histochemistry. This allows the evaluation of the expression of CRT and HMGB1 in the tumor tissue.

To evaluate the HZCO-induced immune response in vivo, tumor-bearing mice were treated with HZCO (10 mg/kg). After 72 hours, the tumors and draining lymph nodes were excised to prepare single-cell suspensions. Then, the cells were stained with flow cytometry antibodies following the manufacturer’s instructions (for T cells, CD3-FITC, CD4-APC, and CD8-PE; for T_regs, CD3-FITC, CD4-APC, and Foxp3-PE; for tumor-associated macrophages (TAMs), CD11b-PE, F4/80-FITC, CD80-PE, and CD206-APC; and for MDSCs, CD45-FITC, CD11b-PE, and Gr-1-APC). The stained cells were subjected to flow cytometry analysis (BD Biosciences Accuri C6) and analyzed with the FlowJo software. In addition, the supernatant of the single-cell suspension was collected, and the levels of cytokines within the tumor were measured via ELISA.

In vivo systemic immunotherapy involving the combination of HZCO and αPD-1

The antitumor efficacy and abscopal effect of the combination of HZCO and αPD-1 were assessed in a bilateral tumor-bearing mouse model. To establish the model, 50 μl of a 4T1 cell suspension (1 × 10⁶ cells) was injected subcutaneously into the right flank of each mouse to create a primary tumor. After 5 days, 1 × 10⁶ 4T1 cells were injected subcutaneously into the left flank to create a distant tumor. Once the volume of the primary tumor reached 100 mm³ (designated as day 0), the mice were randomly divided into four groups (n = 6): (i) PBS, (ii) HZCO (intratumoral injection, 10 mg/kg), (iii) αPD-1 (intravenous injection, 2.5 mg/kg), and (iv) HZCO (intratumoral injection, 10 mg/kg) + αPD-1 (intravenous injection, 2.5 mg/kg). On days 0 and 2, the mice were treated with intratumoral injections of HZCO. In addition, the mice received intravenous injections of αPD-1 on days 1, 3, and 5. The sizes of the bilateral tumors and the body weights of the mice were monitored every 2 days. If the tumor volume exceeded 1500 mm³, then the mice were considered dead and were immediately euthanized.

In vivo evaluation of systemic immune responses induced by HZCO combined with αPD-1

Mice bearing 4T1 bilateral tumors were divided into four groups: (i) PBS, (ii) HZCO (intratumoral injection, 10 mg/kg), (iii) αPD-1 (intravenous injection, 2.5 mg/kg), and (iv) HZCO (intratumoral injection, 10 mg/kg) + αPD-1 (intravenous injection, 2.5 mg/kg). These mice were treated as described above. On day 7, the mice were euthanized, and their tumors were collected to prepare single-cell suspesions. Then, the cells were stained with flow cytometry antibodies following the manufacturer’s instructions (for T cells, CD3-FITC, CD4-APC, and CD8-PE; for T_regs, CD3-FITC, CD4-APC, and Foxp3-PE; for TAMs, CD11b-PE, F4/80-FITC, CD80-PE, and CD206-APC; and for MDSCs, CD45-FITC, CD11b-PE, and Gr-1-APC) following the manufacturer’s instructions. The stained cells were subjected to flow cytometry analysis (BD Biosciences Accuri C6) and analyzed with the FlowJo software.

In vivo tumor therapeutic efficiency of ES combined with Zn²⁺

4T1 tumor-bearing mice with an initial tumor volume of ~100 mm³ were randomly divided into four groups (n = 6): (i) PBS, (ii) ES (intravenous injection, 5 mg/kg), (iii) Zn²⁺ (intratumoral injection, 2 mg/kg), and (iv) ES (intravenous injection, 5 mg/kg) + Zn²⁺ (intratumoral injection, 2 mg/kg). The mice were treated every other day, and this treatment schedule was repeated twice. Tumor volume and body weight were measured every 2 days. The tumor volume was calculated via the formula V = L × W²/2. Furthermore, the systemic immune response triggered by the combination of ES and Zn²⁺ was also assessed. After receiving the various treatments mentioned above, the mice were euthanized, and their tumors were collected to produce single-cell suspensions. The proportion of lymphocytes in the tumors was analyzed by flow cytometry via the same protocol as previously described.

Statistical analysis

All the results in this work were presented as the mean values ± SD. All data were biologically replicated at least three times. Statistical analysis was conducted via the GraphPad Prism 9 software. Two-tailed Student’s t test was used for two-group comparison, and one-way analysis of variance (ANOVA) with a Tukey post hoc test was used for multiple comparisons. Statistical differences were calculated with two-tailed Student’s t test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

This PDF file includes:

Other experimental procedures

Figs. S1 to S42