Metal-Based Nanomedicines for Inducing Programmed Cell Death to Enhance the Efficacy of Cancer Immunotherapy

Introduction

Cancer is a major health concern worldwide and a leading cause of mortality, and decreases average life expectancy in all countries.¹ There were 18.5 million new cancer cases and 10.4 million cancer deaths estimated to occur globally in 2023, and there will be anticipated 30.5 million cases and 18.6 million deaths from cancer globally in 2050.² Currently, chemotherapy, surgery, radiotherapy, targeted therapy and immunotherapy are the mainly therapeutic tactics for cancer. In recent years programmed cell death (PCD) has emerged as a potent approach to induce cell suicide, and it has drawn widespread attention as a research hotspot for cancer treatment. PCD is provoked by endogenous and exogenous factors that disturb cellular homeostasis and trigger cell suicide under dedicated molecular pathways,^3,4 and multiple types of PCD such as ferroptosis, cuproptosis, calcicoptosis, apoptosis, pyroptosis, necroptosis, and PANoptosis have been elucidated and serve as targets for cancer therapy. Metal ions have essential effects on cell homeostasis, and increasing researches on the relationship of metal ions and tumor treatment have shown that several metal ions can induce PCD and provide novel insight for cancer therapy.^5,6

PCD has been demonstrated to be related to modulate the immunosuppressive tumor microenvironment (TME) and trigger an immunostimulatory response.^7,8 Cancer immunotherapy that harnesses the patient’s immune system to combat cancer cells has revolutionized cancer therapy. Over the past decades, cancer immunotherapy mainly including immune checkpoint inhibitors, vaccines, and immune cell therapies has been successfully implemented in the clinic and emerged as a novel therapeutic paradigm in solid and hematological malignancies.⁹ However, despite substantial achievements of cancer immunotherapy, patients experience low response, and a substantial proportion fail to achieve clinical benefit from cancer immunotherapy.^10,11 As a prominent immune checkpoint blockade therapy, anti-programmed cell death protein 1 (PD-1) and anti-programmed cell death ligand 1 (PD-L1) monoclonal antibodies have a low response rate of approximately 10–30%.^12,13 Therapeutic potential of the combination of PCD and immunotherapy was proposed as a promising approach to improve the efficacy of caner immunotherapy.^14,15 Metal ions, their targets and induced PCD, and mechanism of modulating immune response are summarized in Table 1. However, clinical utility of metal ions for eliciting PCD is prominently restricted by insufficient selectivity and targeting ability, toxicity, and dysregulation of systemic ion metabolism.¹⁶ Hence, the targeting delivery of metal ions to malignant cells is urgently needed to enhance therapeutic efficacy while minimizing adverse systemic effects.

Table 1 Summary of Metal Ions, Their Molecular Targets, Induced Types of PCD, and Mechanisms of Immune Modulation

The past few decades have witnessed significant advancements of nanotechnology, and multiple nanopharmaceuticals such as Doxil, Abraxane, Marqibo, and Onivyde are approved and marketed for carcinoma therapy.¹⁷ Nanoparticles can improve the solubility of hydrophobic drugs, stability of unstable agents, modulate the pharmacokinetics and biodistribution of loaded drug, realize passive targeting and active targeting via surface modification, reverse drug resistance, and minimize toxicity.^17–19 A nanoparticle-based delivery system can achieve targeted delivery of metal ions, strengthening their PCD potency and minimizing off-target toxicity. Metal ions can be engineered into therapeutic nanostructures either by directly forming nanoscale particles or by being strongly coordinated into fabricated nanocarriers. This nanoformulation strategy not only enables passive tumor accumulation via the enhanced permeability and retention (EPR) effect but also allows further surface modification for active targeting. Therefore, the nanoparticles function not merely as delivery vehicles but as integral components that modulate biodistribution, prolong systemic circulation, and reduce off-target toxicity, ultimately leading to enhanced therapeutic efficacy.

This review introduces the mechanism of PCD triggered by metal ions, the potency of PCD to modulate an immunosuppressive TME and induce an immunostimulatory response, and nanoformulations for integrating PCD and cancer immunotherapy. Relevant studies were retrieved from PubMed through title/abstract searches using combinations of “metal”, “nanoparticle”, and “immunotherapy”. As depicted in Scheme 1, the review focuses on ferroptosis, cuproptosis, calcicoptosis, and other PCD triggered by zinc and manganese. The interplay between PCD and cancer immunotherapy is discussed, and potential tactics to boost immunotherapy via PCD are presented. The review analyzes advantages and challenges in nanomedicine-loaded metal ions to trigger PCD and promote potency of immunotherapy, explores future research orientations, and proposes strategies to address existing challenges. Moreover, we expect to offer some rewarding suggestions and enlightenments for cancer immunotherapy.

Scheme 1 Metal ion-induced programmed cell death.

Nanoparticles for Ferroptosis and Cancer Immunotherapy

Ferroptosis was first identified in 2012 by Dixon et al as a PCD pattern and is characterized by iron-driven lipid peroxidation.²⁰ Ferroptosis has emerged as an important modulator in a scope of pathophysiological incidences encompassing oncology, ischemic organ injury, stroke, acute kidney injury, chronic kidney disease, cardiomyopathy, and neurodegenerative diseases.²¹ The importance of ferroptosis has piqued research interest of scholarly community, especially in cancer treatment. Despite the need to elucidate the physiological and pathological roles of ferroptosis, its mechanism of induction and function have been gradually revealed.

Regulation Mechanism of Ferroptosis

Ferroptosis is mediated by several primary pathways, encompassing iron metabolism, lipid peroxidation, and antioxidant mechanisms (Figure 1).²² It is crucial to determine if interactions among the aforementioned pathways affect cellular susceptibility to ferroptosis, posing profound implications for overall fitness.

Figure 1 Summary of the metabolic pathway of ferroptosis. The intricate modulation of ferroptosis is regulated via several primary pathways, encompassing iron metabolism, lipid peroxidation, and antioxidant mechanisms.

Iron is indispensable in physiological processes, is acquired by cells via intestinal absorption and degradation of erythrocytes, and plays a pivotal character in the pathway of ferroptosis. Extracellular ferric ion (Fe³⁺) preliminarily binds to transferrin (TF) to be internalized into cells via endocytosis by transferrin receptor 1 (TFR1).²³ Fe³⁺ is reduced to Fe²⁺ after being transferred into the endosome by six-transmembrane epithelial antigen of prostate 3 (STEAP3), and then Fe²⁺ is liberated into the cytoplasm by divalent metal transporter 1 (DMT1). If the Fe²⁺ is not utilized, it can be preserved in mitochondria and cytoplasm in the form of a labile iron pool (LIP) or sequestered within a ferritin complex.^24,25 Ferroportin is the sole protein responsible for exporting iron from the intracellular compartment to extracellular matrix, and it exerts a vital role in ferroptosis.²⁶ Under pathological conditions, dysregulation in genes encoding iron metabolism, particularly reduction in ferritin heavy chain 1 expression coupled with overexpression of TFR1, could trigger a substantial accumulation of intracellular iron to elicit a surge in reactive oxygen species (ROS) via the Fenton reaction.^25,27,28 The elevated oxidative microenvironment can ultimately result in susceptibility of vulnerable cells to ferroptosis.²⁵ Also, ferritinophagy which is regulated by nuclear receptor coactivator-4 (NCOA4) can activate ferroptosis by increasing intracellular iron density due to its involvement in ferritin breakdown.²⁹

In addition, polyunsaturated fatty acids (PUFA) are essential components for cell membranes, and they are perceived as key stimuli for lipid peroxidation, which is a crucial process responsible for the onset of ferroptosis.³⁰ PUFA undergoes esterification with long-chain acyl-coenzyme A (CoA), which is facilitated by acyl-CoA synthetase long-chain family member 4 (ACSL4), leading to the production of PUFA-CoAs. Lysophosphatidylcholine acyltransferase 3 (LPCAT3) which is responsible for delivery of PUFA-CoAs into cell membrane phospholipids, plays a pivotal role in the subsequent peroxidation.³¹ Once integrated into phospholipids, the PUFA-CoA-integrated phospholipids are subjected to enzymatic oxidation by lipoxygenases (LOX) or autoxidation, and a cascade of biological reactions is initiated to generate the iron-dependent lipid peroxides that are a hallmark of ferroptosis.^27,32 Once LOX, LPCAT3, and ACSL4 enzymes are overly active, lipid peroxidation occurs, resulting in oxidation of PUFA and accumulation of lipid peroxides. As displayed in Figure 1, eventually excessive lipid peroxides synergize with intracellular overloaded iron to cause a Fenton reaction, resulting in increased ROS levels, loss of structural integrity of the lipid bilayer, cytotoxicity, and death.²⁷

System Xc⁻, a ubiquitous amino acid antiporter in phospholipid bilayers, regulates the exchange of cystine and glutamate at a stoichiometric ratio of 1:1 and is essential for cellular uptake of cystine.^33,34 Within cells, cystine is converted into cysteine, which is an indispensable precursor for biosynthesis of glutathione (GSH).³⁵ GSH is an endogenous antioxidant responsible for scavenging ROS via glutathione peroxidases (GPXs). Therefore, inhibition of this antiporter can lead to depletion of intracellular GSH and an increase of glutamate, which participates in ROS production after conversion into glutamine by glutaminase enzyme.^36–38 GPX4 possesses the potency to degrade a range of lipid peroxides and block the detrimental cascade of lipid peroxidation, exerting a pivotal role in ferroptosis. A dramatic decline in the activity of GPX4 results in overproduced ROS and elevated oxidative stress, inducing ferroptotic cell death by the active ROS and lipid peroxides (Figure 1).³⁹ Furthermore, p53 as a cancer suppressor protein can mediate cystine uptake by inhibiting the light chain SLC7A11 of the antiporter, influencing GPX4 activity and triggering ferroptosis.¹⁵

Interplay Between Ferroptosis and Cancer Immunotherapy

Ferroptosis has been identified as an important player in regulation of immunosuppressive microenvironments and differentiation of immune cells, and its role in immunotherapy is becoming increasingly evident.

Macrophages are important in tumor immunosuppression by supporting tumor development and progression and resistance to therapy. They can polarize into three phenotypes: unactivated M0, classically activated M1, and alternatively activated M2 macrophages. M1 macrophages show high expression of iron-sequestering proteins such as ferritin and low expression of iron-exporting proteins such as FPN, store iron, and are resistant to ferroptosis, helping to fight cancer cells. On the contrary, M2 macrophages promote tumor cell proliferation and immune evasion by releasing iron, and they are susceptible to ferroptosis owing to high FPN and low ferritin expression.⁴⁰ M2 macrophages can be repolarized into the M1 phenotype by ferroptosis inducers, and iron can promote M1 polarization under certain conditions.^41,42 Similarly, Treg cells and myeloid-derived suppressor cells have an immunosuppressive role and antagonize ferroptosis through high expression of GPX4 or other proteins. Induction of ferroptosis in these cells may induce cell death and reverse their immunosuppressive function.⁴²

Ferroptosis is accompanied by release of oxidation products, and damage-associated molecular patterns (DAMPs) such as high mobility group box 1 (HMGB1) can trigger inflammatory and immune responses during cell death.^43,44 HMGB1 is one of the key elements required for activation of the innate and adaptive immune systems by binding to pattern recognition receptors.⁴⁵ Recovery of CD8⁺ cytotoxic T cell function within the TME is an important factor determining response to cancer immunotherapy, and ferroptosis is a key metabolic regulator of CD8⁺ T cells activity.⁴⁶ Ferroptotic cells liberate lipid cytokines as “find me” signals, which recruit antigen-presenting cells and other immune cells to the site.⁴⁴ LOXs not only oxygenate esterified PUFAs as ferroptotic signals but also contribute to the release of oxidized lipid mediators as immunomodulatory signals from ferroptotic cancer cells, enhancing anti-tumor immunity.⁴⁴ Arachidonate 15-lipoxygenase-derived lipid mediators mediate dendritic cells (DCs) maturation and regulate adaptive immune responses.⁴⁷

Immune cells have an anti-tumor immunity function by releasing cytokines that trigger ferroptosis in cancer cells. For example, interferon γ released by CD8⁺ T cells and transforming growth factor-β released by macrophages decrease the expression of the antiporter system Xc⁻, followed by impaired uptake of cystine in cancer cells, promoting lipid peroxidation and ferroptosis in tumor cells.^48,49

Nanoparticles for Ferroptosis to Enhance Cancer Immunotherapy

Overload of iron can elicit tissue damage encompassing myocarditis that can progress to heart failure and neurological and neurodegenerative diseases.^50,51 Hence, the targeted delivery of iron is indispensable to protect healthy tissues. Ferroptosis has been broadly applied as a novel strategy to enhance the efficacy of cancer immunotherapy. PD-1 and its ligand PD-L1 have emerged as important immune checkpoints in tumor treatment. Although neutralization of the negative immune checkpoints with anti-PD-1 or anti-PD-L1 antibodies has generated impressive progress in treatment of several types of cancer, such therapy is limited by a low response rate.^12,13 As summarized in Table 2, various nanoparticles for delivery of iron ion have been designed and fabricated, aiming to augment the immune response against anti-PD-1 or anti-PD-L1 therapy by modulating activating immune cells.

Table 2 Iron-Based Nanomedicines for Triggering Ferroptosis to Enhance Cancer Immunotherapy

Cholesterol oxidase is responsible for catalysis of cholesterol to H₂O₂, and cholestenone enhances lipid peroxidation and ROS levels, promoting ferroptosis immune therapy.⁵² A novel nanozyme composed of iron metal-organic framework (MOF) for delivery of cholesterol oxidase and polyethylene glycosylation (Figure 2) ⁵² and catalytic hydrogel-loaded dimethyl maleic anhydride-modified cholesterol oxidase and a metalloporphyrin compound hemin with peroxidase-like activity⁵³ were developed for integrated ferroptosis and immunotherapy. In combination with PD-1 or PD-L1 inhibitor, the nanodrug delivery system exerted a synergistic therapeutic effect.

Figure 2 A novel nanozyme composed of iron metal-organic framework nanoparticles delivering cholesterol oxidase and polyethylene glycosylation for integrated ferroptosis and immunotherapy. The nanozyme depleted cholesterol, produced excessive H2O2, and enhanced ROS and lipid peroxides levels to promote ferroptosis. Concurrently, the nanozyme augmented immunogenic cell death by reducing PD-L1 expression, promoting DCs maturation and M1 macrophage polarization, revitalizing exhausted CD8+ T cells and priming CD8+ T cells. In combination with PD-1 inhibitor the nanozyme exerted a synergistic therapeutic effect.

Jingbo Ma et al synthesized a multifunctional nanocomposite of sonosensitizer HMME, Fe³⁺, and tannic acid, and the nanocomposite consolidated the function of HMME for producing ROS and Fe³⁺ for induction of ferroptosis and ROS.⁵⁴ More importantly, the nanocomposite could potentiate efficacy of immunotherapy by recruiting additional T cells and natural killer cells and promoting DCs maturation, and its combination with anti-PD-1 antibody could eradicate tumors. A hydrazide/Cu²⁺/Fe²⁺/indocyanine green coordinated nanoplatform was developed, the hydrazide-metal-sulfonate coordination significantly potentiated CD8⁺ T cell infiltration into tumor, and the nanoplatform and antibody against PD-1 synergistically eliminated the primary tumor and inhibited distant tumor metastasis and recurrence.⁵⁵ A metal-coordinated carrier-free nanodrug was prepared by co-assembly of a natural product of ursolic acid, sorafenib, Fe³⁺, low-molecular weight protamine, and epithelial cell adhesion molecule aptamer. The nanodrug induced immunogenic cell death (ICD) and augmented the immune response against PD-L1 via increasing infiltration of cytotoxic T cells to suppress tumor growth and distant metastasis.⁵⁶ A tannic acid-Fe³⁺-coated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG2000] (ammonium salt) micelle loaded with doxorubicin and anti-PD-L1 antibody enhanced anti-tumor immunity by activating CD4⁺ and CD8⁺ T cells and reducing the ratio of regulatory T cells to CD4⁺ T cells.⁵⁷

Postoperative recurrence and metastasis especially brain metastasis dramatically deteriorate the survival rates for breast cancer sufferers. A core-shell nanoparticle of Fe MOF-encapsulated hollow mesoporous organosilica nanoparticles was proposed to deliver doxorubicin to prevent recurrence and metastasis.⁵⁸ The nanosuspension synergistically improved doxorubicin chemotherapy, achieved remarkable ferroptosis by doxorubicin and iron ions, and significantly activated immune response including stimulating DCs, recruiting T cells, and facilitating antigen presentation. When used in conjunction with the PD-1 antibody, the nanosuspension could inhibit postoperative recurrence and brain metastasis of breast cancer. Zn-pyrophosphate core-shell nanoparticles for co-delivering a cholesterol derivative of dihydroartemisinin and pyropheophorbide-iron sensitized non-immunogenic colorectal tumor to anti-PD-L1 checkpoint blockade immunotherapy.⁵⁹

A MOF was formed through the coordination between tannic acid and Fe³⁺ to deliver triptolide and was functionalized by folic acid-modified bovine serum albumin.⁶⁰ The nanoplatform triggered a potent systemic anti-tumor immune response by inducing ferroptosis and pyroptosis, and its combination with antibody against PD-L1 enhanced immunotherapy. Combined with aptamer-PD-L1 checkpoint blockade, iron-based MOF nanoparticles modified by MnO₂, glucose oxidase, and polyethylene glycol (PEG) strengthened the tumor treatment efficiency.⁶¹

Fe₃O₄/Gd₂O₃ hybrid nanoparticles conjugated to arginine-glycine-aspartic dimers for loading sorafenib and antibody against transforming growth factor-β achieved cumulative ferroptosis through Fe₃O₄/Gd₂O₃ hybrid nanoparticle-mediated Fenton reaction and sorafenib-mediated GSH synthesis blocking, and increased the potency of anti-PD1 therapy.⁶² Polydopamine, cyclic arginine glycyl aspartate, and anisamide-modified Fe₃O₄ nanoparticles enhanced cellular ferroptosis induced by Fe²⁺-mediated Fenton reaction via introducing glucose oxidase as a catalyzer for generation of H₂O₂, and in combination with antibody against PD-L1 the nanoparticles exhibited favorable synergistic effectiveness against colorectal cancer.⁶³ A biomimetic platelet membrane camouflaged with Fe₃O₄ nanoparticles for delivery of sulfasalazine triggered ferroptotic cell death, and the biomimetic nanoparticles induced an anticancer immune response and efficiently repolarized immunosuppressive M2 macrophages to anti-tumor M1 phenotype, drastically enhancing the efficacy of PD-1 inhibitor.⁶⁴

Ultrasmall body-centered cubic Fe nanoparticles with an Fe core approximately 2 nm in size and an iron oxide shell less than 0.7 nm were synthesized and further modified by the CRGDKGPD (iRGD) peptide. These nanoparticles could efficiently induce immunogenetic promotion of DCs maturation and adaptive T cell response. Combined with anti-PD-L1 antibody, the ultrasmall Fe nanoparticle-triggered ferroptosis significantly potentiated immune response and developed strong immune memory.⁶⁵ Ultrasmall iron nanoparticles were functionalized by fluorophenylboronic acid to generate nitrogen-boronate complex with bovine serum albumin and ¹³¹I-labeled antibody against PD-L1. The ultrasmall nanoparticles were responsive to an increase of adenosine triphosphate in tumor owing to a relatively stronger affinity of ribose structure in adenosine triphosphate to fluorophenylboronic acid. The ICD caused by radiopharmaceutical therapy and ferroptosis combined with antibody against PD-L1 exhibited a strong anti-tumor immunity.⁶⁶

A tetrapod spiky-like iron-palladium nanocrystal was engineered with decylamine as a coordinating ligand for co-reduction of Fe and Pd species, and the surface of nanocrystal was modified with polyvinylpyrrolidone to improve its biosafety and biocompatibility. The nanocrystal induced lipid peroxide accumulation, promoted ferroptosis, and effectively triggered the release of inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β) in macrophages, strengthening immunotherapy with antibody against PD-L1.⁶⁷

Researches have reported synergism of iron nanoparticle-mediated ferroptosis and A2 adenosine receptor blocker,⁶⁸ CD47 blocking antibody,⁶⁹ or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) check point inhibitor^70–72 for cancer immunotherapy.

Nanoparticles for Cuproptosis and Cancer Immunotherapy

In 2022, Tsvetkov et al discovered excessive copper-induced cell death as a distinct type of PCD from other modalities, and it was termed cuproptosis.⁷³ Cuproptosis is featured by excessive accumulation of copper in cells, followed by mitochondrial dysfunction and toxic protein stress, ultimately leading to cell death. Tumor cells demonstrate increased metabolism processes and energy consumption, which are intricately associated with mitochondrial function. This indicates opportunities for cuproptosis as a novel target for eradication of tumor cells.

Mechanism of Cuproptosis

Copper is indispensable for regulation of redox-active enzymes, which are involved in various metabolism processes, signaling pathways, and biological functions.⁷⁴ Intracellular copper homeostasis is tightly mediated by transporters, efflux protein, and enzymes responsible for conversion of Cu²⁺ to Cu⁺, and their coordinated processes maintain a precise balance of copper in cells. Disruption of copper homeostasis can lead to detrimental dysfunction of cells. Inadequate copper blunts normal metabolic activity, while excess copper produces cytotoxicity and ultimately results in cell death.

The copper ionophore elesclomol translocates Cu²⁺ into cells dependent on mitochondrial respiration and disrupts copper homeostasis mediated by solute carrier family 31 member 1 (SLC31A1) which is previously called copper transporter protein 1 (CTR1) as copper import protein, and ATPase copper transporting α/β (ATP7A/B) as copper export protein (Figure 3).⁷⁵ Accumulated copper in cells can directly combine with dihydrolipoamide S-acetyltransferase (DLAT), a lipoylated protein involved in the tricarboxylic acid cycle, to generate protein oligomers. Copper ions can undermine the synthesis of Fe-S cluster proteins, which are a crucial family of functional mitochondrial proteins involved in multiple process including cell energy metabolism, electron transfer, and substrate synthesis.⁷⁶ These steps synergistically result in proteotoxic stress response and ultimately lead to cuproptosis.

Figure 3 Pathways mediating cuproptosis. Elesclomol binds to Cu2 + and transports it into intracellular compartments. Cu+ is primarily transported into cells via solute SLC31A1 previously called CTR1, while ATP7A/B are responsible for exporting copper ion. Excess intracellular copper primarily leads to cuproptosis through mitochondrial protein toxicity stress mediated by FDX1. This FDX1 reduces Cu2+ to Cu+, promotes lipidation and aggregation of enzymes involved in the mitochondrial tricarboxylic acid cycle particularly DLAT, and degrades iron-sulfur cluster proteins. (reproduced with permission from Xiaojie Zhang et al (2024). Copyright 2024 Springer Nature).

Additional metabolic pathways mediating cuproptosis have been identified, among which ferredoxin 1 plays a pivotal role in protein lipoylation through integration into lipoic acid synthase and facilitation of its functional interplay with glycine cleavage system protein H.⁷⁷ On the other hand, the ferredoxin 1 gene is responsible for encoding a small iron-sulfur protein that converts Cu²⁺ to more toxic Cu⁺. However, Cu⁺ ions undermine the structure and function of iron-sulfur clusters, resulting in their degradation (Figure 3). GSH, a cellular protector, can reduce the concentration of free Cu²⁺ and inhibit cuproptosis by binding to Cu²⁺ and generating a complex. The combination of GSH and Cu²⁺ is beneficial for maintenance of intracellular copper ion balance, and ferredoxin 1-mediated Cu²⁺ reduction reduces the detrimental influence of Cu⁺ on iron-sulfur cluster proteins, defending cells against copper-induced injury.⁷⁸ Recent studies demonstrate that tumor suppressor p53 is also involved in modulation of cuproptosis. P53 is responsible for regulating transcription of GSH reductase to modulate biosynthesis and recycle GSH, and also is responsible for mediating the expression of genes linked to iron-sulfur proteins, regulating cellular GSH and iron-sulfur levels.⁷⁹ Pathways mediating cuproptosis are illustrated in Figure 3.

Relationship Between Cuproptosis and Cancer Immunotherapy

Cuproptosis is believed to be closely associated with immune cell infiltration and has an important role in reprogramming the immunosuppressive microenvironment. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) is demonstrated as a critical regulator of cancer immunity and facilitates various immune effector responses, and a cGAS-STING-mediated immune supportive microenvironment can hamper malignancy occurrence.^80,81 Cuproptosis has been reported to enhance anticancer immunity through the cGAS-STING pathway, activating tumor antigen-presentation. The cGAS-STING signaling in DCs is triggered by cuproptosis-stimulated cancer cells, followed by release of inflammatory factors. In addition, combined cuproptosis inducers and PD-1 inhibitor synergistically increases the circulating levels of CD45⁺CD8⁺ T cells, enhancing immunotherapy efficacy.⁸² Cuproptosis also significantly stimulates mitochondrial DNA release to activate innate immunity via cGAS-STING signaling in vivo. Subsequently, secretion of type I interferon and expression of interferon-β are upregulated and DCs maturation is promoted. In addition, cuproptosis-associated innate immunity activates T-cell immunity.⁸³ In vivo cuproptosis enhances infiltration of CD8⁺ T cells into tumor tissue, and cuproptosis potentiates the cytotoxicity of CD8⁺ T cells, which is realized by downregulating the WNT signaling pathway and PD-L1 expression.⁸⁴ Cuproptosis triggered by a mitochondria-targeted copper dithiocarbamate induces immunogenic death of cancer cells, leading to the release of DAMPs. The released DAMPs effectively elicit macrophages to M1 polarization and their migration towards target cell antigens, and secretion of relevant cytokines. The copper dithiocarbamate promotes antigen processing and presentation in cancer cells through the major histocompatibility complex-I pathway, activating CD8⁺ T cells and natural killer cells.⁸⁵

Nanoparticles for Cuproptosis to Enhance Cancer Immunotherapy

Copper overload in tissues leads to damages, including hepatotoxicity, nephrotoxicity, neurotoxicity, hemolysis, and cytotoxicity.⁸⁶ Nanotechnology provides a potent approach to enhance anti-tumor immune response by regulating cuproptosis, overcoming the obstacles of current cancer immunotherapy and minimizing off-target toxicity. Nanodrugs for inducing cuproptosis to boost cancer immunotherapy are presented in Table 3.

Table 3 Copper-Based Nanomedicines for Inducing Cuproptosis to Boost Cancer Immunotherapy

As depicted in Figure 4, a biomimetic cuproptosis amplifier was fabricated by Cu²⁺-regulated coordinative self-assembly of near-infrared II (1000–1700 nm) ultrasmall polymer dots and doxorubicin, followed by camouflaging of cancer cytomembrane. Overexpressed GSH in the TME reduced Cu²⁺ to Cu⁺, resulting in disassembly of the amplifier, photothermal therapy, chemotherapy, and cuproptosis. Cuproptosis elicited significant DCs maturation and infiltration of CD⁴⁺ and CD⁸⁺ T cells through ICD and reshaped the immunosuppressive TME via downregulated Tregs. The amplifier together with anti-PD-L1 antibody elicited a powerful anti-tumor immune response.⁸⁷

Figure 4 Schematic illustration of the biomimetic self-assembly of cuproptosis amplifier for synergistic tumor immunotherapy. (a) Preparation of the near-infrared II semiconducting polymer and the biomimetic cuproptosis amplifier (PCD@CM). (b) Near-infrared-II fluorescence/photoacoustic imaging-mediated chemotherapy and photothermal amplified cuproptosis, provoking anti-tumor immunotherapy combined with immune checkpoint blockade mediated by anti-PD-L1 antibody. , downregulation; , upregulation. (reproduced with permission from Yeneng Dai et al (2024). Copyright 2024 Elsevier).

A sodium alginate hydrogel incorporating elesclomol-Cu and galactose was developed to trigger persistent cuproptosis, and the hydrogel abrogated radiation-induced PD-L1 upregulation, significantly enhancing the sensitization of cancer to radiotherapy and immunotherapy.⁸⁸ A hydrogel composed of glycyrrhizic acid, copper ions, and celastrol was fabricated for synergistic cuproptosis and apoptosis, and the hydrogel repolarized tumor-associated macrophages (TAMs) into M1 phenotype, induced T cell proliferation and infiltration, activated antigen presentation, and upregulated PD-L1 expression. Upon co-administration with PD-L1 antibody, the hydrogel synergistically alleviated both primary and metastatic tumors.⁸⁹

Yiming Xu et al decorated lung cancer cytomembrane onto glucose oxidase-loaded copper-layered double hydroxide nanoparticles to generate an intelligent biomimetic nanodrug. The nanodrug significantly induced cuproptosis and PD-L1 upregulation in lung cancer cells and sensitized the therapeutic potency of antibody against PD-L1.⁹⁰ An Escherichia coli and Cu₂O nanoparticle microbial nanohybrid was fabricated by electrostatic interaction through simple mixing, and the nanohybrid reversed the immunosuppressive microenvironment by inducing DCs maturation and T cell activation. Upon synergism with PD-1 antibody, the nanohybrid inhibited relapse and metastasis of colon tumors.⁹¹

Tumor-targeting peptides RGD coated hollow mesoporous copper sulfide nanoparticle for delivery of the nitric oxide donor L-Arginine induced cuproptosis, promoted immune cell infiltration and activation, and converted “cold” tumors into “hot” ones. In addition, their combination with antibody against PD-L1 significantly enhanced the immunotherapy response rate in triple-negative breast cancer.⁹² A Cu²⁺-based MOF loaded with the copper ionophore elesclomol and surface modified with PEG polymer was developed for cuproptosis induction to increase anticancer immune response, and combing the MOF with PD-L1 antibody reshaped the immunosuppressive TME to an immunogenic milieu, significantly inhibiting tumor growth.⁹³

A ROS-sensitive polymer was designed and used to encapsulate elesclomol and copper. The micelle triggered cuproptosis, promoted DCs maturation and CD8⁺ cell infiltration, and reprogrammed the TME. Moreover, the micelle dramatically increased PD-L1 expression and effectively enhanced the response rate to anti-PD-L1 therapy.⁹⁴ Copper oxide nanoparticles were encapsulated into the PEG-modified copper ionophore elesclomol, and the nanodrug triggered an immune response and reshaped the immunosuppressive microenvironment by increasing the number of tumor-infiltrating lymphocytes and secretion of inflammatory cytokines. In addition, combining the nanoparticles and anti-PD-1 therapy substantially increased the anticancer potency.⁹⁵ A novel bifunctional nanoparticle comprising a core of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol, PEG-coated copper ions and peroxide, and an erastin shell was constructed for synergistic cuproptosis and ferroptosis. Erastin of anti-Warburg potency sensitized cancer cells to cuproptosis, resulting in ICD, enhanced antigen presentation, increased proliferation and infiltration of T cells, and upregulated PD-1 expression. When combined with PD-L1 antibody, the nanoparticles supported T cells to mediate cancer regression and prevent metastasis.⁹⁶

Self-amplified cuproptotic nanoparticles have been produced using the natural product celastrol as a versatile copper ionophore and scavenger for GSH to amplify cuproptosis, and the celastrol-copper complex was encapsulated by PEG. The self-amplified cuproptotic nanoparticles evoked ICD to trigger a potent immune response, and their combination with antibody against PD-L1 effectively eradicated metastatic tumors in an animal model.⁹⁷ Aloe emodin, a natural compound found in a plant, was chelated to copper ions and self-assembled into nanoparticles under modification with PEG and folic acid conjunction. The nanoparticles elicited maturation of DCs, infiltration of lymphocytes, transformation of “cold tumors” into “hot tumors”, and potently increased the efficacy of immune checkpoint blockade.⁹⁸

An inhalable poly (2-(N-oxide-N,N-diethylamino) ethyl methacrylate)-coated copper-based MOF loaded with pyruvate dehydrogenase kinase 1 siRNA, which blocks the copper efflux protein ATP7B, was fabricated to trigger cuproptosis and promote immunotherapy. The nanodrug triggered ICD and upregulated membrane-associated PD-L1 expression and soluble PD-L1 secretion, demonstrating synergism with the PD-L1 antibody.⁹⁹ Another inhalable nanoparticle was composed of a Cu²⁺-chitosan shell and a low-molecular-weight heparin-tocopherol succinate core and was loaded with disulfiram, which chelated with Cu²⁺ to suppress ATP generation and Cu⁺ transporter ATP7B expression. The inhalable nanoparticles enhanced cuproptosis and activated the cGAS-STING pathway to increase innate and adaptive immunity, and strong anticancer immunity was realized by combing with PD-L1 antibody.¹⁰⁰

Nanoparticles for Calcicoptosis and Cancer Immunotherapy

Calcium overload is generally featured by excessive accumulation of Ca²⁺ in cytoplasm or mitochondria. Under endoplasmic reticulum (ER) stress, the capability of cells to manipulate Ca²⁺ homeostasis is undermined. Ca²⁺ is sustainably released from the ER, which is the primary intracellular calcium ion pool, and cytosolic Ca²⁺ concentration is increased, followed by transport of Ca²⁺ into mitochondria, leading to mitochondrial Ca²⁺ overload.¹⁰¹ In some conditions, Ca²⁺ overload can cause cell death through a distinct mechanism defined as calcicoptosis, which offers a novel strategy for cancer treatment.

Mechanism of Calcicoptosis

ER Ca²⁺ channel protein transmembrane and coiled-coil domains 1 (TMCO1) exert an important function in regulating calcium overload and maintaining calcium homeostasis, and inhibiting TMCO1 expression disrupts intracellular calcium homeostasis.¹⁰² Participation of calcium in signaling among organelles determines the fate of cells and influences cell survival or programmed death.

During ER stress such as excessive misfolded protein, DNA damage, oxidative stress, and pro-apoptotic signals, Ca²⁺ is liberated into cytoplasm to activate dependent proteases approaching the ER. The activated proteases can trigger and release Caspase-12 into cytoplasm and activate the calcium/calmodulin-dependent protein phosphatase, which is responsible for dephosphorylation of the pro-apoptotic protein Bad, followed by release of cytochrome C to induce apoptosis.¹⁰³ A previous study has uncovered a synergistic effect between cellular oxidative stress and calcium overload, ultimately leading to cell death.¹⁰⁴ Figure 5 schematically illustrates the mechanism of calcicoptosis.

Figure 5 Schematic illustration of the mechanism of calcicoptosis. Under endoplasmic reticulum stress, Ca2+ is sustainably released from the endoplasmic reticulum, which is the primary intracellular calcium ion pool, and cytosolic Ca2+ concentration is increased and transported into mitochondria, leading to mitochondrial Ca2+ overload. In some conditions, Ca2+ overload can cause cell death through a distinct mechanism defined as calcicoptosis, which offers a novel strategy for cancer treatment. (reproduced with permission from Jie Gu et al (2024). Copyright 2024 Springer Nature).

Necroptosis, which is responsive to death stimuli such as tumor necrosis factor α and Fas ligand, also depends on calcium homeostasis. Upon reorganization of the ligand by the receptor on cell membrane, receptor-interacting protein kinase (RIPK)-1 forms a necrosome complex with RIPK3 and mixed lineage kinase domain-like protein (MLKL). The necrosome triggers mitochondria to produce ROS, leading to cell death.¹⁰⁵ The formation of a necrosome complex results in increased cytosolic calcium, followed by trimerization of MLKL and its translocation to the cell membrane. The MLKL enables influx of calcium into the cell and intensifies necroptosis in a feedback manner through interaction with the transient receptor potential melastatin 7 channel.¹⁰⁶ Calcium acts as a modulator of necrosome complex proteins and cell death.

Necrosis which is featured by rapid disintegration of cytoarchitecture, release of cellular contents, and an inflammatory reaction is also triggered by calcium overload.^107,108 Increased intracellular calcium concentration is induced by activation of transient receptor potential cation channel subfamily V member 1 to trigger cell death mainly through a necrotic pathway.¹⁰⁹ The release of Ca²⁺ from ER into the cytoplasm and their accumulation in mitochondria lead to Ca²⁺ overload and opening of mitochondrial permeability transition pores, resulting in swelling, mitochondrial rupture, and release of their contents to induce necrosis.¹¹⁰

Calcium ions also exert an important character in regulation of other PCD such as ferroptosis, pyroptosis, autophagy, and paraptosis.¹¹⁰ Precise regulation of calcium signaling in cells is a powerful tool in cancer therapy.

Relationship Between Calcicoptosis and Cancer Immunotherapy

The regulation of intracellular calcium ions plays an essential role in immune cell activation, and targeting increased intracellular calcium ions can significantly stimulate the proliferation of cytotoxic lymphocytes.¹¹¹ Moreover, activation of T lymphocyte-associated transcription factors, for example, nuclear factor of activated T cells, nuclear factor kappa-B, and c-Jun N-terminal kinase, are heavily dependent on excessive accumulation of intracellular calcium ions.^112,113 Necroptosis is dependent on calcium homeostasis and triggers cells to release DAMPs that promote an anti-tumor immune response.¹¹⁴ HMGB1 is liberated from cells undergoing necrosis triggered by calcium overload, acting as a DAMP to activate macrophages and DCs in the TME.¹¹⁵ Further, calcium overload facilitates the exposure of calreticulin localized in ER to deliver intensive pro-phagocytic signals to myeloid cells.^116,117 Previous researches have demonstrated that the concentration of calcium ion in macrophages might be closely associated with their phenotype.

Increase in cytoplasmic calcium ions can activate p38 and nuclear factor kappa-B for repolarizing TAMs to the M1 phenotype and stimulate transcription factor EB for reprograming the metabolism of TAMs.¹¹⁸ Excessive ROS and lipid peroxidation triggered by calcium overload through the ROS/p38-MAPK/diacylglycerol-O-acyltransferase 1 pathway are also speculated to inhibit M2 macrophage polarization.¹¹⁹

Nanoparticles for Calcicoptosis to Enhance Cancer Immunotherapy

Hypercalcemia results in clinical manifestations such as nausea, renal dysfunction, nephrocalcinosis, vascular calcification, and cardiac arrhythmias.¹²⁰ Therefore, the targeted delivery with nanomedicine can enhance efficacy and minimize toxicity. Nanoparticles for delivering Ca²⁺ or calcium-based nanovehicles to strengthen cancer immunotherapy are illustrated in Table 4.

Table 4 Calcium-Based Nanomedicines for Inducing Calcicoptosis to Strengthen Cancer Immunotherapy

Calcium carbonate-based nanoparticles are the most widely used vehicle for calcium overload therapy. pH-responsive and catalase-delivered calcium carbonate nanoparticles have been constructed to reshape the TME for enhanced immune checkpoint blockade. CaCO₃ nanoparticles reacted with protons in an acidic TME to reprogram it, and the released Ca²⁺ led to overload in cancer cells, followed by liberation of DAMP signals and repolarization of M2 TAMs to the M1 phenotype, enhancing tumor antigen presentation by DCs. Consequently, the CaCO₃ nanoparticles triggered a T cell-regulated immune response that can combine with antibody against PD-1 to stimulate local and systemic immune responses, suppressing growth of both primary and metastatic tumors.¹²¹ A calcium carbonate nanoparticle hydrogel was loaded with the anticancer drug bufalin as an inhibitor of Na⁺/K⁺-ATPase to increase intracellular Ca²⁺ level. The resulting pyroptosis enhanced the efficacy of PD-1 antibody to induce an inflammatory TME, achieving synergistic potency in stimulating an immune response.¹²² A core-shell Cu₂O and CaCO₃ nanocomposite that was responsive to acidic pH and H₂S sulfuration was used for photothermal, photodynamic, chemodynamic, and calcium-overload-mediated therapy, reprogrammed TAMs of the M2 phenotype to that of M1, and initiated a T cell-regulated immune response. Combined CD47 blockade and the nanocomposite induced a strong immune response, effective ablation of the primary tumor, and inhibition of cancer recurrence and metastasis.¹²³ Zheng et al used a modified double emulsion method to encapsulate doxorubicin and erianin into CaCO₃ nanoparticles. The multifunctional nanoparticles effectively elicited calcium overload and oxidative stress damage to activate hybrid ferroptosis and apoptosis pathways and led to prominent ICD. Additionally, the CaCO₃ nanoparticles synergistically amplified the potency of anti-PD-L1 antibody.¹²⁴ CaCO₃ nanoparticles were utilized to deliver a carbonic anhydrase inhibitor that improved the sensitivity of cancer to radiotherapy and further were modified with liposomes. The nanoparticles induced cellular calcium overload, strengthened ICD triggered by radiotherapy and DCs maturation, and repolarized macrophages from pro-tumor M2 to anti-tumor M1 phenotype, amplifying systemic anti-adaptive immunity. With PD-L1 antibody, the efficacy of CaCO₃ nanoparticles plus radiotherapy was increased, resulting in longer survival time.¹²⁵ A pH-sensitive nanoparticle for co-delivery of curcumin as a Ca²⁺ enhancer, CaCO₃ and MnO₂ were encapsulated by a cancer cell membrane, and the released Ca²⁺ triggered calcium overload and ROS production in mitochondria and ER, leading to ICD. In addition, the nanoparticle repolarized macrophages and induced DCs maturation via antigen presentation and enhanced immune responses of the anti-PD1 antibody.¹²⁶ Calcium carbonate nanoparticles encapsulating catalase assembled colloidosomes could activate strong anticancer immunity to significantly amplify the efficacy of co-loaded antibody against PD-1 and dramatically reinforce the therapeutic outcome of epidermal growth factor receptor-expressing CAR-T cells.¹²⁷

Hyaluronic acid-catechol, PEG-polyphenol, and PEG-IR780 self-assembled into nanoparticles for carrying Ca²⁺ and lactate dehydrogenase A inhibitor GSK2837808A. Satisfying glucose nutrition required by CD8⁺ tumor-infiltrating lymphocytes and destabilizing regulatory T were realized by inhibiting lactate dehydrogenase A, and further CD8⁺ T cell activation and tumor infiltration were promoted by the released DAMPs triggered by Ca²⁺ overload in mitochondrion and amplified mitochondrial dysfunction. Cooperating with CTLA-4 antibodies further enhanced therapeutic efficacy.¹²⁸

Yu et al constructed a mineralized porphyrin MOF encapsulating calcium phosphate for amplified cell damage caused by calcium overload and photodynamic therapy. The MOF could induce cell immunogenic death to liberate tumor-associated antigen, promote DCs maturation and enhance anticancer activity of CD8⁺ T cells, and co-administration with PD-1 antibody demonstrated prominent elimination of primary tumor and obvious inhibition for metastasis.¹²⁹ A transformable TiO₂ core and CaP shell nanosonosensitizer combining ROS generation and intracellular calcium overload substantially strengthened ICD, T cell recruitment, and infiltration into the immunogenic cold tumor. In combination with PD-1 antibody, the nanosonosensitizer regulated sonodynamic therapy-triggered systemic anticancer immunity.¹³⁰

Calcium hydroxide nanoparticles coated with a layer of silica and further conjugated with anti-CD205 antibody were designed for targeted delivery to DCs. The elevated cytosolic calcium triggered nuclear factor of activated T cells and the nuclear factor kappa-B signaling pathway, followed by enhanced anticancer immune response and increased efficacy of anti-PD-L1 antibody.¹³¹

The mitochondrial photosensitizer N770 conjugated mesoporous silica nanoparticles for delivery of CaO₂ achieved phototherapy, and calcium overload triggered ER stress and mitochondrial damage, and relieved the immunosuppressive microenvironment. Moreover, mesoporous silica nanoparticles together with antibody against PD-L1 significantly potentiated systemic anti-tumor immunity.¹³² Bovine serum albumin-templated ultrasmall CuS-MnO₂ nanoparticles were adhered to the surface of CaO₂ nanoparticles via surface electrostatic interaction, and the nanoparticles were wrapped with hyaluronate acid. A large mass of O₂ and Ca²⁺ produced by CaO₂ nanoparticles strengthened photodynamic therapy and Ca²⁺ overload separately, amplifying ICD. Combing the nanoparticles with antibody against PD-L1 achieved enhanced immunotherapy efficacy and long-term protection.¹³³

PEG-decorated manganese-doped calcium sulfide nanoparticles rapidly liberated Ca²⁺, Mn²⁺, and H₂S responding to a TME, and the released H₂S was a crucial synergist for Ca²⁺ in pyroptosis-triggered calcium overload by disrupting intracellular calcium homeostasis and interfering with oxidative phosphorylation pathways (Figure 6). Via activation of calcium overload-regulated pyroptosis and the cGAS-STING pathway, manganese-doped calcium sulfide nanoparticles stimulated both innate and adaptive anticancer immune response and boosted the efficacy of anti-PD-1 therapy.¹³⁴ A porous poly(acrylic acid) stabilized CaS nanoparticles delivering zinc protoporphyrin as a messenger amplifier resulted in Ca²⁺-dependent tumor immunogenic death and triggered release of tumor-associated antigens as an in situ vaccine to activate the immune response. Integration of CaS nanoparticles and anti-PD-1 antibody fabricated immune memory to produce long-term immunity against tumor metastasis and recurrence.¹³⁵

Figure 6 Schematic illustration of the underlying therapeutic mechanism of manganese-doped calcium sulfide nanoparticles for cancer cell pyroptosis and cGAS-STING pathway activation. (a) The mechanism of pyroptosis and activation of the cGAS-STING signaling pathway triggered by manganese-doped calcium sulfide nanoparticles. (b) H2S strengthened the adaptive immune response. (c) H2S amplified the innate immune response. (d) Reshaping of the immunosuppressive tumor microenvironment.134 , downregulation; , upregulation; , inhibition. (Reprinted with permission from Lin Liu et al (2024). Copyright (2024) American Chemical Society).

Zinc-Based Nanoparticles and Cancer Immunotherapy

Zinc ions exert an essential character in a vast array of physiological and cellular processes, including activation of matrix metalloproteinases, cell proliferation, development, metabolism, DNA biosynthesis and transcription, and PCD. Zinc ions also are involved in protection from oxidation stress, inflammation response, and immune regulation.⁵ Transportation of zinc ions across cytomembranes is regulated by the cooperation of zinc transporter proteins, known as zinc transporter family or Zrt/Irt-like proteins (ZIPs). ZIPs are responsible for influx of zinc ions from the extracellular compartment or intracellular organelles into cytoplasm, while zinc transporters take charge of zinc ion efflux from cytoplasm to extracellular compartment or intracellular organelles.¹³⁶ Given their essential characters in physiological and cellular processes, it necessitates stringent control of zinc ions homeostasis.

Zinc Triggered PCD

Zinc ion-elicited apoptosis is regulated by promotion of B-cell lymphoma-2 associated X protein (Bax) expression which triggers the liberation of cytochrome C and activation of caspase 3, eventually leading to apoptosis.¹³⁷ An increase in the ratio of Bax to B-cell lymphoma-2 contributes to deterioration of hypoxia inducible factor-1α followed by decreased expression of the inhibitor of survivin, ultimately triggering apoptosis.¹³⁸ Also zinc ions can strengthen the expression of Smad2 and PIAS1 as transcription activator 1, followed by activation of P21 gene expression and promotion of apoptosis.¹³⁹ ZIP9, a zinc transporter protein and membrane androgen-binding receptor, is revealed to be associated with apoptosis via activating G protein.¹⁴⁰

In cancer development, lysosomal function is usually upregulated to satisfy the enhanced energy requirement for rapidly proliferating tumor cells.¹⁴¹ Transient receptor potential mucolipin 1, a cation channel with dual permeability to calcium and zinc ions, is upregulated in certain cancer cells and can be activated to trigger lysozincrosis via release of zinc ions from lysosomes, mitochondrial swelling and impairment, and energy depletion.¹⁴² Moreover, normal cells expressing low level of transient receptor potential mucolipin 1 are not susceptible to lysozincrosis, indicating its possibility for cancer therapy.

Autophagy, a conserved catabolic process, is triggered in response to stress such as energy deprivation, hypoxia, infection, and ER stress and results in degradation of intracellular components.¹⁴³ Multiple studies consistently indicated that zinc triggers autophagy; however, the mechanisms are still poorly clarified. Extracellular-signal-regulated kinase 1/2 (ERK1/2), metal responsive transcription factor-1 (MTF1), and calcium/calmodulin-dependent protein kinase kinase-B/AMP-activated protein kinase (CaMKKb/AMPK) are involved in induction of autophagy by zinc.¹⁴³ Zinc ions are also responsible for triggering necroptosis,¹⁴⁴ ferroptosis¹⁴⁵ and pyroptosis.¹⁴⁶ The mechanism for Zn²⁺ induced PCD is summarized in Figure 7.

Figure 7 Schematic illustration of a representative signaling pathway for zinc ion-induced PCD. (reproduced with permission from Yawen You et al (2025). , upregulation. Copyright 2025 Royal Society of Chemistry).

Zinc Triggered PCD and Cancer Immunotherapy

Zinc ions can enhance the activity of an array of immune cells, including T cells, B cells, and natural killer cells; can polarize macrophages into the M1 phenotype; and alleviate the immunosuppressive state of microenvironment through inhibiting release of inflammatory molecular and inflammatory response. Zinc ions can enhance tumor antigen presentation and recognition of antigen by immune cells and suppress immune checkpoint protein expression to reinforce an anticancer immune response.¹⁴⁷

Zinc ions have emerged as an immunologic adjuvant to activate the cGAS-STING signaling pathway in initiation of anticancer immunity and transformation of a “cold” tumor into a “hot” one.¹⁴⁸ Zinc ion-modulated ferroptosis increases the potency of immune cells such as T cells or macrophages, which is related to the release of DAMPs and interferon γ.¹⁴⁵ Excess zinc ion-triggered tumor cell pyroptosis also resulted in liberation of mass DAMPs.¹⁴⁷ Autophagy and necroptosis also lead to the liberation of DAMPs, activation of immune cells, and antigen presentation.¹⁴⁹ Therefore, the combination of zinc ions and cancer immunotherapy can enhance the response rate.

Zinc-Based Nanoparticles Enhance Cancer Immunotherapy

Overexposure of zinc can damage the nervous system,¹⁵⁰ and the targeted delivery of zinc is highlighted to reduce its toxicity. Nanoparticles for delivering Zn ions or zinc-based nanovehicles to boost cancer immunotherapy are shown in Table 5.

Table 5 Zinc-Based Nanomedicines to Boost Cancer Immunotherapy

Sun et al fabricated an erythrocyte membrane-decorated zinc-phenolic nanocapsule for delivery of mitoxantrone and antibody against PD-L1 to treat triple-negative breast cancer with limit immune response. The zinc-phenolic nanocapsule triggered cancer cell pyroptosis, activated the cGAS-STING pathway, and amplified the efficacy of anti-PD-L1 antibody, achieving sustained immune response.¹⁵¹ Zhu et al synthesized ferritin heavy chain siRNA and hyaluronic acid warped arginine-stabilized zinc peroxide to induce Fe²⁺ overload and ferroptosis, and the liberated Zn²⁺ induced mitochondrial dysfunction and oxidative stress to further boost ferroptosis. The nanoagent-regulated ferroptosis exerted a potent immunogenic response for T-cell activation and infiltration, and integration of the nanoagent with anti-PD-1 antibody resulted in prominent anticancer efficacy in vivo.¹⁵² A polydopamine-coated zinc-copper bimetallic nanoplatform was introduced to spontaneously liberate Cu²⁺, Zn²⁺, and H₂O₂ in the acidic TME, leading to irreversible cuproptosis and cGAS-STING pathway activation. The zinc-copper bimetallic nanoplatform induced DCs maturation, T cell activation, and PD-L1 expression, sensitizing triple-negative breast cancer to antibody against PD-L1 therapy.¹⁵³ Lu et al fabricated hyaluronic acid-modified zinc peroxide-iron nanocomposites to reshape the TME and simultaneously trigger pyroptosis and ferroptosis, significantly enhancing the anticancer immune response to anti-PD-1 antibody.¹⁵⁴ Bioactive zinc-nickel hydroxide nanosheets initiated zinc overload-regulated pyroptosis, and the released Ni²⁺ amplified pyroptosis through concurrently inducing paraptosis, inhibiting autophagic flux, and triggering release of endogenous zinc ions. The nanosheets triggered liberation of DAMPs from cancer cells, followed by stimulation of DCs maturation, increase in CD8⁺ T cell infiltration, and transformation of macrophages to M1 phenotype, strengthening the therapeutic potency of the antibody against PD-1.¹⁵⁵

A bovine serum albumin nanocluster was constructed via an ion diffusion approach for co-delivery of zinc and sulfur to enhance cancer immunotherapy as demonstrated in Figure 8. The released zinc ions in a low pH TME prominently enhanced the cGAS-STING pathway. H₂S produced by the nanocluster further facilitated production of ROS by zinc ions via specifically inhibiting catalase in hepatocellular carcinoma cells, leading to further activation of cGAS-STING by the accumulated ROS. The nanocluster promoted infiltration of CD8⁺ T cells into the tumor and cross-presentation of DCs, and integration of the nanocluster and PD-L1 antibody resulted in a significant inhibiting effect on tumor growth and potent immune response.¹⁵⁶

Figure 8 Schematic and characteristics of a bovine serum albumin nanocluster for co-delivery of zinc and sulfur. (a) The therapeutic process of the nanoclusters. (b) The synthesis routine of the nanoclusters. (c) Scanning electron microscopy image of the nanoclusters. Scale bar: 1 µm. Inset: High-resolution transmission electron microscopy image. Scale bar: 20 nm. (d) Hydrodynamic size of the nanoclusters in different solutions. (e) X-ray diffraction pattern of the nanoclusters. (f) Element mapping of the nanoclusters. Scale bar: 200 nm. (g) Release profile of H2S from nanoclusters in solutions with different pH of 7.4, 6.5, and 6.0 (n = 3, mean ± standard deviation). , upregulation; , the following step. (reproduced with permission from Xiujun Cai et al (2021). Copyright 2021 John Wiley and Sons).

Manganese-Based Nanoparticles and Cancer Immunotherapy

Manganese, an indispensable trace element in the human body, participates in a vast array of physiologic processes such as serving as a cofactor for various enzymes, development, metabolism, hematopoiesis, protein and vitamin synthesis, endocrine regulation, protection from ROS, and redox homeostasis.^5,157 In recent decades, manganese has been used as an inducer for PCD and enhancer for immune function.

Manganese-Dependent Cell Death

Manganese ions downregulate system Xc⁻ and excitatory amino acid transporter, and cystine as a precursor for GSH biosynthesis is reduced, leading to depletion of GSH and its synthesis.^158,159 Blocking the biosynthesis of GSH resulted in excessive accumulation of ROS followed by lipid peroxidation overproduction, and ultimate cell death.^160,161 Manganese ions catalyze Fenton-like and Haber-Weiss reactions that produce ROS and exhaust GSH, similar to the iron-triggered Fenton reaction.¹⁵⁷ Manganese ions catalyze a Fenton-like reaction in the presence of H₂O₂ to generate ·OH. Manganese ions also increase mitochondrial H₂O₂ by fostering superoxide dismutase 2 activity, and releasing oxidoreductases from the Krebs cycle through triggering permeability transition.^162,163

Cellular uptake of manganese and iron ions is competitive, and several transporters responsible for iron ions transportation, such as DMT1, TFR, ferroportin, and ferritin, also deliver manganese ions.^164–166 Manganese ions disturb iron homeostasis through over-expression of TfR and upregulation of iron uptake in the brain, leading to significantly increased cellular levels of labile iron rather than total cellular iron levels.^167–169

Manganese ions trigger ferroptosis in dopaminergic neurons by regulating the hypoxia-inducible factor-1 α /p53/SLC7A11 signaling pathway.¹⁷⁰ Manganese ions drive ferroptosis in oral squamous cell carcinoma cells via nuclear translocation of Yes-associated protein/transcriptional co-activator with PDZ-binding motif and subsequent ACSL4 activation.¹⁷¹

Manganese ions at high concentration trigger cytochrome C liberation from mitochondria and caspase-8 regulated apoptosis in B cells. In neuronal cells, manganese ion-induced apoptosis is facilitated by transcriptional activation of caspase 3, which is induced by phosphorylation of zinc finger transcription factor SP-1.¹⁷² Manganese ions can inhibit the acetylation of histone H3 and H4 by augmenting the activity of histone deacetylase and decreasing that of histone acetyltransferase, which eventually triggers apoptosis.¹⁷³ Manganese ions induce apoptosis through p53- and p38-mitogen-activated protein kinases and the mitogen and stress response kinase-1 signaling pathway.^174,175

Manganese ions trigger necrosis through ROS-induced lysosomal membrane permeabilization and release of cathepsin D into the cytosol, and also via ROS-triggered DNA damage, translocation of apoptosis-inducing factor from mitochondria to the nucleus, and parthanatos.¹⁷⁶ Manganese ions also induce necroptosis in macrophages infected with Mycobacterium tuberculosis through the STING-tumor necrosis factor signaling pathway.¹⁷⁷

Manganese-Regulated Cancer Immunotherapy

Manganese ions exert vital roles in activation and functional regulation of immune cells including T cells, macrophages, and natural killer cells, and they play a role in cancer immunotherapy.¹⁴⁷ Manganese ions potently enhance the affinity of cGAS to its agonist DNA substrates, and cGAS is activated by combination with DNA, leading to enzymatic production of cGAMP and activation of STING downstream signaling.^178,179 The activated cGAS-STING pathway leads to generation of type I interferons and a vast array of pro-inflammatory cytokines, resulting in enhanced immune surveillance, cytotoxicity of natural killer cells and macrophages, and activation and proliferation of T cells.^80,180 Manganese ions modulate the function and upregulate the expression of costimulatory molecules on the membrane of antigen-presenting cells, boost T cell infiltration and survival in the TME, and enhance the strength of natural killer cells and their release of cytokines.¹⁴⁷

Although the mechanisms of manganese ions regulated tumor immunotherapy are not fully elucidated, previous studies have explored their clinical value. In a Phase I clinical trial, patients were administered different dose of manganese chloride intranasally or by inhalation in combination with PD-1 antibodies and chemotherapy. After a median follow-up of 11.8 months, the combined protocol exhibited manageable side effects and prominent potency across various tumor types including ovarian, breast, and pancreatic cancers, achieving a disease control rate of 90.9%.⁸⁰

Manganese-Based Nanoparticles Enhance Cancer Immunotherapy

Clinical application of manganese is significantly hindered by its neurotoxicity and non-specific distribution,¹⁸¹ and targeted delivery is of great importance to minimize neurotoxicity. As presented in Table 6, an array of nanoagents delivering manganese ions or manganese-based nanovehicles have been introduced to amplify the immune response and have been integrated with immunotherapy for cancer treatment.

Table 6 Manganese-Based Nanomedicines to Amplify Cancer Immunotherapy

A manganese-phenolic network platform was based on doxorubicin carrying PEG-poly(lactic-co-glycolic acid) nanoparticles and further modified by manganese-tannic acid. Manganese triggered a Fenton-like reaction and enhanced anti-tumor immunity by amplifying the cGAS-STING pathway, and integrated therapy of the nanodrug with CTLA-4 blocking antibody exerted superior treatment efficacy to monotherapy.¹⁸² Sun et al fabricated hollow mesoporous silica-coated MnO nanoparticles with conjugated iRGD peptide and applied them for cGAS-STING pathway-amplified immunotherapy, a Fenton-like reaction, and T₁-weighted magnetic resonance imaging. Integrated MnO nanoparticles and anti-PD-1 antibody enhanced tumor inhibition and activated the immune response.¹⁸³ PEG-coated manganese molybdate nanoparticles depleted highly accumulated GSH in a tumor and inhibited GPX4 expression, triggering ferroptosis. The manganese molybdate nanoparticles induced release of DAMPs, promoted DCs maturation and T cell infiltration, and reversed the immunosuppressive microenvironment, reprogramming “cold” tumors to “hot” ones. Integration with antibody against PD-L1 further enhanced anti-tumor efficacy and inhibited metastasis.¹⁸⁴ As illustrated in Figure 9, a hydrogen peroxide/ultrasound-propelled mesoporous manganese oxide nanomotor was fabricated to load mitochondrial sonosensitizers into mesoporous channels, and their surface was dual-functionalized with silk fibroin and chondroitin sulfate. Mn²⁺ ions regulated a Fenton-like reaction that decomposed excess H₂O₂ in the TME into oxygen and toxic hydroxyl radicals. The nanomotor effectively depleted intracellular GSH to downregulate GPX4, a vital regulator for ferroptosis, leading to accumulation of LPO as the hallmark for ferroptosis. The integrated Mn²⁺ and ultrasound promoted maturation of DCs and T cell-mediated immune response. Integration of the manganese oxide nanomotor and PD-L1 checkpoint inhibitor potently restrained primary tumor growth and prevented tumor recurrence by potentiating systemic anticancer immunity and providing long-term immune memory.¹⁸⁵ Risedronate-manganese nanobelts were fabricated via coordination-driven self-assembly and exerted an outstanding Fenton-like catalytic property and amplified radiotherapy-regulated oxidative stress. The released Mn²⁺ further activated the cGAS-STING pathway, boosting the efficacy of anti-PD-L1 antibody against primary and metastatic tumors.¹⁸⁶ Ultrathin manganese-based layered double hydroxide nanosheets delivering cytokine interferon γ were synthesized to strengthen ferroptosis and systemic anti-tumor immunity. Manganese ion-induced ferroptosis was further boosted by the loaded interferon γ-triggered downregulation of SLC7A11, which is responsible for uptake of cystine into cells for GSH biosynthesis. The released manganese ions activated the cGAS-STING signaling pathway and stimulated DCs maturation and T cell infiltration. Endogenous interferon γ secreted by activated CD8⁺ T cells promoted a cascade of immunogenic ferroptosis, forming a closed-loop treatment. Integrated nanosheets and antibody against PD-L1 achieved a potent abscopal effect on inhibition of both primary and distant tumors.¹⁸⁷

Figure 9 Schematic illustration of a hydrogen peroxide/ultrasound-propelled mesoporous manganese oxide nanomotor to achieve efficient mucus-traversing ability, deep tumor penetration, high anti-tumor efficacy, and potentiation of anti-tumor immunity. , downregulation; , upregulation; , blockade. (reproduced with permission from Yingui Cao et al (2022). Copyright 2022 John Wiley and Sons).

A dendrobium polysaccharide hydrogel embedded with Mn²⁺-pectin microspheres induced apoptosis in cancer cells, activating the cGAS-STING signaling pathway and initiating a cascade of anti-tumor immune responses. The hydrogel generated a synergistic potency with anti-PD1 antibody to inhibit metastasis and abscopal brain tumor proliferation.¹⁸⁸ A PEG-modified Mn²⁺-based MOF delivering paclitaxel induced pronounced apoptosis and promoted maturation of DCs and infiltration of T lymphocytes by activating the cGAS-STING pathway, enhancing the potency of anti-PD-L1 antibody.¹⁸⁹ Alginate microspheres embedded in a Pluronic F-127 matrix as a vehicle for Mg²⁺ and Mn²⁺ formed a hybrid hydrogel that elicited apoptosis and converted a “cold tumor” to a “hot tumor”, augmenting the therapeutic efficacy of antibody against PD-L1.¹⁹⁰ Zhou et al developed manganese-enriched zinc peroxide nanoparticles for synergistic anticancer immunotherapy. The nanoparticles elicited apoptosis, activated the STING pathway, and decreased the immunosuppressive TME. Integrated with PD-1 checkpoint blockage, the nanoparticles demonstrated potent inhibition of tumor growth and metastasis.¹⁹¹ A tumor cell membrane containing multienzyme-mimicking manganese oxide nanozymes induced apoptosis in cancer cells, and the released Mn²⁺ ions promoted maturation of DCs and M1 repolarization of macrophages by activating the STING pathway. With the support of PD-1 checkpoint blockade, robust anti-tumor immune response and long-term immune memory were achieved, and the growth of primary tumor and metastasis was prominently inhibited.¹⁹²

An in situ vaccine was fabricated by intercalating peroxydisulfate, a precursor of SO₄^·-, into manganese layered double hydroxide nanoparticles. Mn²⁺ mediated peroxydisulfate degradation through Fenton-type advanced oxidation in the tumor to produce in situ SO₄^·-, which elicited necroptotic cell death and adaptive immunity. The in situ vaccine activated the STING pathway to further enhance anticancer immunity. When integrated with anti-PDL1 antibody, remarkable inhibition of distant tumors was realized.¹⁹³

Perspectives

Cancer is responsible for high morbidity and mortality rates and is a prominent health burden worldwide. In recent decades, immunotherapy has emerged as a vital treatment for cancer following chemotherapy, surgical treatment, radiotherapy, and targeted therapy. Patients can benefit from a range of immunotherapeutic approaches including immune checkpoint inhibitors, CAR-T cell therapy, antibody-drug conjugate, cytokine therapy, and vaccination. Despite its great potential, cancer immunotherapy is confronted with significant challenges, and low response rates are a considerable hurdle. Excessive intracellular accumulation of several metal ions, such as Fe²⁺, Cu²⁺, Ca²⁺, Zn²⁺, and Mn²⁺, is important for regulating PCD through various signaling pathways. PCD is capable of reprogramming the immunosuppressive TME and inducing immunostimulatory responses,^7,8 indicating it as a viable option to enhance the potency of cancer immunotherapy. With advancement of nanotechnology, metal ions are used more extensively in cancer therapy, and efficacy of immunotherapy has been increased. However, issues in academic investigation and clinical application remain to be solved.

The precise mechanisms and signaling pathway for metal ion-mediated PCD and enhanced immune response by PCD have not been fully elucidated, and in-depth molecular mechanism study should be performed. The metabolism and long-term safety of metal ions and metal-based nanomaterials are unknown, and further investigation is required. Moreover, optimization of material formulations and development of more biocompatible nanomaterials are needed. One key limitation of this review is insufficient assessment of metabolism and long-term safety of the proposed nanodrugs.

Tumor heterogeneity has emerged as a mediator for the efficacy of immunotherapy.¹⁹⁴ However, this characteristic spatially and temporally evolves, and it is relatively complex and insufficiently characterized.¹⁹⁵ The influential factors affecting response to immunotherapy are complicated rather other only induction of PCD. Integrated strategies to overcome or minimize the detrimental impacts of tumor heterogeneity on immunotherapy and triggers of PCD for achievement of personalized treatment are promising research directions.

Additionally, in design of nanomaterials delivering metal ions or metal-based nanovehicles, druggability should be considered. The pharmaceutical industry operates on the “keep it simple, stupid” principle, and complicated manufacturing processes and standardization issues restrict up-scaling of nanodrugs from laboratory to industry scale.

In 2025, Clinicaltrials.gov retrieved no ongoing or completed clinic trials about metal ions and cancer immunotherapy. This is likely due to challenges in large-scale manufacturing and concerns regarding the safety of nanoparticle-based systems. Comprehensive preclinical data on safety and efficacy in large-scale animal tumor xenograft models are essential for successful clinical translation. To improve the prospect of clinical translation, simple and biocompatible nanosystems for loading metal ions and metal-based nanodrugs should be fabricated. Controlled clinical trials are needed to define the limitations and effectiveness of nanomaterials for metal ions in cancer immunotherapy.

Conclusion

In conclusion, there are challenges for cancer immunotherapy, and leverage of PCD induced by metal ions to amplify efficacy of immunotherapy has demonstrated encouraging achievements in scientific research. Simpler and safer nanocarriers are expected for personal treatment based on research in PCD and tumor heterogeneity, and translation of such methods into clinic is expected to be realized in the near future, along with patients benefit from immunotherapy.