2.1 Absorption and transport of Cu in vivo

The source of Cu for the human body is mainly daily food and water. Radioisotope studies have shown that Cu begins to be absorbed in the stomach after entering the digestive tract, but the mainly in the duodenum and the front of the small intestine. Some Cu can also be absorbed in the distal part of the small intestine. Elemental Cu cannot be absorbed because it is insoluble in water. Most Cu in food is absorbed as soluble chelates rather than ions. The mechanisms of Cu absorption are saturated active transport and unsaturated diffusion. The transport of copper in the human body is influenced by the amount of copper present in the diet. When the dietary copper content is low, copper is primarily transported through the saturated active pathway. Conversely, when the dietary copper content is high, diffusion plays a dominant role in copper transport. Copper in the diet is transported to the intestinal mucosa with the help of various complexing agents, such as amino acids. Among the amino acids that bind to copper, L-histidine is the most predominant, followed by threonine, glutamic acid, cystine, and cysteine.⁸⁴ After being absorbed, copper is first transported into intestinal mucosal cells in the form of metallothionein bound to macromolecular proteins. It then enters the bloodstream through the basolateral membrane of intestinal mucosal cells, where it undergoes phosphorylated adenosine triphosphatase (p-ATPase)-mediated copper transport.

2.2 Intracellular Cu uptake

After entering the blood, most Cu binds with albumin, while a few combine with small molecular components such as amino acids to form loose compounds, which can benefit its further absorption and utilization in other tissues.^{85, 86} The main modes of cellular uptake of Cu are mainly ceruloplasmin transmitter and free cupric pathway. Plasma ceruloplasmin complexes contain approximately 75%–95% of the Cu present in the plasma. Each molecule of ceruloplasmin binds 6–7 copper ions at different sites, enabling it to transfer copper to cells and intracellular proteins via receptors.^{87, 88} Studies have shown that leukocytes have the highest number of receptors for Cu. Once plasma ceruloplasmin binds to the receptor on the cell membrane, the copper ions are reduced from Cu²⁺ to Cu⁺ and separated from ceruloplasmin. This process allows the copper ions to enter the cell and be utilized for various intracellular functions. The other is the free cupric pathway, in which Cu first enters the histidine-rich (N)-terminal Cu binding site in plasma albumin, combines with the free histidine, and transports to the metal ionophores in cell membranes of liver cells and fibroblasts.^{89, 90} The Cu-binding ionophores then transfer the Cu to intracellular Cu-binding protein. Cu from the free cupric pathway is also reduced to Cu⁺ before entering the cell.

2.3 Extracellular Cu efflux

The efflux of Cu is accomplished by a protein called P type of ATPase (P2-ATPase), which is a transmembrane single-chain polypeptide. The N-terminal metal binding site of the protein is composed of Cu binding sites, halogen transport sites, and phosphorylation sites. In the role of ATPase, an aspartate amino acid residue in the phosphorylated region of P2-ATPase is phosphorylated, which changes the conformation of the protein, and transfers Cu to the center of the enzyme. Then dephosphorylation of P2-ATPase results in the efflux of Cu to extracellular.^{91, 92}

4.1 Chemotherapy based on Cu nanoparticles

The latest research found that the traditional abstinence drug disulfiram (DSF), which was metabolized to dithiocarbamate (DTC) by the stomach and blood in the human body, exhibited a certain antitumor effect. DTC could form a complex with Cu in the form of DTC-Cu²⁺ chelation (CuET) in vivo. CuET interferes with ubiquitination protein degradation system, which makes tumor cells unable to timely clean up “garbage” proteins, resulting in metabolic disorders of tumor cells, and eventually death. Herein, Shi et al. reported a tumor microenvironment (TME) responsive PEGylated Cu²⁺ doped with hollow mesoporous silica nanoparticles loaded with DSF (PEG/Cu-HMSNs).⁹⁶ Once internalized into tumor cells, the weak acid environment can lead to its rapid biodegradation and accelerated DSF release. Then the toxic CuETs and Fenton-like reaction synergistically enhanced DSF-based chemotherapy (Figure 4A). In another work, Shi et al. reported a pyridine (Pyr)-functionalized generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers complexed with Cu(II) for MRI-guided radio-chemotherapy (Figure 4B).⁹⁷ Results demonstrated that the G5.NHAc-Pyr/Cu(II) complexes effectively inhibited the proliferation of different tumor cells, promoted the production of intracellular reactive oxygen species (ROS), blocked the cell cycle, and ultimately led to apoptosis. In this study, dendritic polymer monodisperse nanostructure and modified surface were utilized to construct dendritic macromolecule Cu complex as an integrated diagnosis and treatment nanoplatform for tumor MRI and chemotherapy. The research thus provides a new design for the construction of novel diagnostic and treatment integrated nanomaterials. At the same time, enhanced diagnosis and treatment effect is achieved through combined radiotherapy or ultrasound technology, providing theoretical guidance and direction Cu-assisted combination therapy without systemic toxicity.

4.2 Cu and radiotherapy (RT)

Cu has 29 isotopes, including ⁶⁴Cu,⁶⁷Cu,⁶²Cu,⁶¹Cu, and ⁶⁰Cu. Cu isotopes can easily accumulate in cancer cells and release β-rays and γ-rays for cancer diagnosis and treatment. Moreover, the long half-life of Cu isotopes allows their clinical application. However, the small difference between the sensitivity of tumor tissue and normal tissue to radiation limits the antitumor effect of RT. To maximize the radiosensitivity, high atomic number nanomaterials like gold, bismuth, tungsten, and platinum are frequently employed in RT. Through the enhanced permeability and retention (EPR) effect, they can be augmented at the tumor sites and become diagnostic and therapeutic probes integrating multimodal imaging (CTI, PAI, and MRI) and multimodal therapy (PTT and RT). Recently, Li et al. reported that the PEGylated Cu_2−xSe nanoparticles can be used as a highly effective radiosensitizer.⁹⁸ They also found that W-GA-PEG CPN can greatly improve the effect of RT.⁹⁹ These studies propose the application of Cu in tumor RT, which is expected to be further developed in clinical practice. However, the optimal Cu isotopes in RT is needed.

On the contrary, the disturbances of copper metabolism can also lead to radiotherapy resistance. For example, liver cancer cells are often accompanied by the loss of endogenous copper metabolisation-related protein COMMD10, which intensifies the accumulation of Cu in cells, thus further aggravating the radiotherapy resistance of liver cancer. Copper chelating agent can reverse the above effect of radiotherapy resistance. It was found that the accumulation of Cu caused by COMMD10 low expression could inhibit the ubiquitination degradation of hypoxia inducible factor-1α (HIF-1α), promote the nuclear translocation of HIF-1α and increase the transcription and translation of ceruloplasmin, thus forming a positive feedback regulatory loop of HIF-1α/Ceruloplasmin and inhibiting the cell ferroptosis, resulting in the radiotherapy resistance.

4.3 Phototherapy based on Cu nanoparticles

With d-d electron transition, copper sulfide (CuS) nanoparticles show significant near-infrared absorption regardless of size or form, with good photothermal properties. Additionally, the simple preparation of CuS nanoparticles and adjustable size promise their application in photothermal therapy (PTT). Li et al. first modified CuS with mercaptoacetic acid for PTT. To enhance the photothermal conversion efficiency of CuS, Tian et al. prepared a flower-like CuS with a superlattice structure.¹⁰⁰ However, due to the large size, its application in vivo was limited (Figure 5A). Dai et al. prepared a disk mounted Cu₉S₅ for PTT. Surprisingly, Liu et al. found that the better significant killing efficiency of CuS than that of precious metal nanomaterials, such as gold nanoparticles. Due to its low cost and high photothermal conversion efficiency, CuS nanoparticles had attracted extensive attention in the field of PTT. Besides PTT, the researchers observed that these CuS nanoparticles also had intrinsic near-infrared photodynamic properties, which produced high concentrations of ROS to kill tumor cells in one collaborative study. They found that under near-infrared light irradiation, Cu⁺ released from the CuS nanocrystals interacted with the surrounding biological environment to generate redox reactions, resulting in electron transfer, which reflected the characteristics of photodynamic therapy (PDT) (Figure 5B).¹⁰¹ Moreover, Lin et al. found that Cu⁺ directly activated hydroxyl radical (•OH), through the hydrogen or electron transfer process, realizing PDT (Figure 5C).¹⁰² All the results revealed the dual antitumor mechanism of Cu-based nanoparticles-PDT and PTT.

4.4 CDT based on Cu nanoparticles

The important progress of CDT is Fenton or Fenton-like reactions to produce highly cytotoxic hydroxyl radicals. Some Fenton catalysts have been used in tumor CDT, mainly by converting the endogenous hydrogen peroxide of cancer cells into free radicals. Cu⁺-based Fenton-like reactions can occur effectively even under weak acidic and neutral conditions, reaching 160 times fold in comparison to Fe²⁺-based Fenton reactions. But because of the low redox potential of Cu²⁺/Cu⁺ (~0.16 V), Cu⁺ is very easy to be oxidized to Cu²⁺, which makes the Fenton-like reaction difficult to occur in vivo. Therefore, using specific molecules in the TME to control the production of Cu⁺ in tumor tissue for the Fenton-like reaction can be ingenious and crucial. For example, Xu et al. developed multifunctional copper doped hydrotalcite nanoparticles with artificial surface defects (d-Cu LDH) to improve its photothermal conversion efficiency (PTT) and singlet oxygen (¹O₂) generation (PDT). At the same time, hyperthermia led to the dissolution and release of Cu⁺ (CDT).¹⁰³ Similarly, GSH can also convert Cu²⁺ to Cu⁺ for further CDT. Li et al. designed a self-assembled copper-amino acid nanoparticle (Cu-Cys NP) for in situ GSH “AND” H₂O₂ sequentially triggered CDT.¹⁰⁴ Chen et al. demonstrated Janus-type nanoarchitectures, Cu-Fe-MSNs for CDT.¹⁰⁵ Moreover, Peng et al. constructed an organic nanotherapeutic agent based on cyanine and Cu²⁺ for combined PTT and CDT.¹⁰⁶ However, the endogenous hydrogen peroxide of tumor cells is not sufficient to achieve satisfactory chemokinetic effect. Therefore, it is necessary to develop chemical kinetic reagents that can increase the function of intracellular hydrogen peroxide. Metal peroxide, which is composed of peroxy group and metal ion, is an excellent hydrogen peroxide supply material. Since iron, copper, manganese, cobalt, and other metal ions have been proved to have good Fenton catalytic activity, Fenton metal peroxide will be a potential selffeeding chemical dynamic anticancer agent of hydrogen peroxide. Chen et al. successfully synthesized copper peroxide nanoparticles and realized selfsustaining chemokinetic treatment of hydrogen peroxide. The team prepared copper peroxide nanoparticles using the complexation of copper ions and hydrogen peroxide assisted by hydroxide ions, which could be reversed under acidic conditions. Therefore, copper peroxide nanoparticles had the characteristics of acid response, and decomposed and released hydrogen peroxide and copper ions with Fenton-like catalytic activity under weak acidic conditions. The accompanying Fenton-like reaction effectively generated hydroxyl free radicals, so as to kill cells efficiently. Both cell and animal experiments showed that copper peroxide nanoparticles had good chemical kinetic effect on tumor. As hydrogen peroxide self-contained enhanced chemical kinetic intelligent anticancer drugs, copper peroxide nanoparticles had a good application prospect in cancer therapy.

4.5 SDT based on Cu nanoparticles

SDT can achieve inexpensive, safe and effective tumor therapy by utilizing the sonosensitive agents at tumor sites and the strong tissue penetration capacity of ultrasound. US radiation causes the transition from Cu²⁺ to Cu⁺, accelerating the formation of ·OH from H₂O₂ for SDT. As electron-hole pairs separate due to the transition, ROS creation for SDT is encouraged.¹⁰⁷ The Cu²⁺ contains 27 electrons. After TCPP chelates the Cu²⁺, an unpaired electron will appear in the middle of the overall molecular orbital. This state is also called open shell with the ground state of Cu(II)NS being a double state D0. After this nanoparticle entered tumor cells, the hyperexpressed GSH will reduce Cu(II)NS to univalent Cu(I)NS, and GSH will give an electron to pair with the unpaired electron.¹⁰⁸ The ground state of the Cu(I)NS is restored to the singlet state S0 for SDT.

However, the antitumor activity of SDT is usually based on the oxygen, which limits its application in solid tumors with hypoxic TME, such as pancreatic cancer. To overcome this limitation, more and more studies had been conducted to explore a variety of new sound-sensitive systems. At the same time, PTT, chemotherapy, PDT, CDT and immunotherapy also showed effective synergistic effect. Huang et al. developed a kind of Cu-metal-organic framework (Cu-MOF) nanoparticles loaded with acoustic free radical initiators for hypoxic-adaptive chemosonodynamic therapy of pancreatic cancer in situ. In anoxic TME of in situ pancreatic cancer, structural degradation of Cu-MOF results in the release of Cu²⁺ and AIPH, and further dissociation of Cu-MOF and decomposition of AIPH to produce cytotoxic alkyl radicals (R·) and N₂ bubbles after US irradiation at the tumor site, which does not require oxygen supply. In turn, Cu²⁺ consumed high levels of GSH in tumor cells and was reduced to Cu⁺, which catalyzed Fenton-like reactions to produce additional ROS. On the other hand, N₂ bubbles enhanced the deep penetration of nanoparticles through ultrasonic cavitation effect under the action of ultrasound. Therefore, this acoustic-chemokinetic synergy of hypoxic TME response provided an ideal means to achieve nonoxygen-dependent free radical generation and enhancement of the antitumor effect of SDT.

4.6 MDT based on Cu nanoparticles

Under microwave (MW) irradiation, Cu-based nanoparticles can also enhance the production of ROS. Meng at al. constructed a biomimetic multifunctional red blood cell membrane (RBC) coated nanoparticle (RBC-Zr@APC/C NPs).¹⁰⁹ Through the targeting effect caused by biomimetic alteration, the nanoparticles aggregated at the tumor site after i.v. administration and continued to convert O₂ to ROS for MDT. Copper-cysteamine was discovered by Chen et al. to be a new group of sensitizers that could produce ROS when activated by light, X-rays, microwaves, or ultrasound.¹¹⁰

4.7 Catalytic therapy based on Cu nanoparticles

Nanocatalysis medicine is a cutting-edge academic idea proposed by a scientific research team led by Shi et al. It aims to use nontoxic/low-toxic nanomaterials to initiate in situ catalytic reactions in tumor by responding to specific internal field microenvironment or exogenous laser and ultrasonic fields of action at tumor sites. It can effectively realize oxidative damage and cell death of tumor cells. This catalytic tumor treatment method does not use highly toxic chemotherapy drugs, has the characteristics of high efficiency, strong specificity, high safety, has been widely recognized by the peer, and has been rapidly developed in recent years. The redox reaction between Cu²⁺ and GSH can not only remove GSH and improve the oxidative stress of tumor cells, but also the reduction product Cu⁺ can transform H₂O₂ into more toxic •OH through Fenton-like reaction, enhancing the efficacy of catalytic therapy.

In addition to Cu²⁺/Cu⁺, Cu atom also plays a vital role in tumor therapy, such as the Cu single-atom nanozyme, a peroxidase (POD) that catalysis H₂O₂ to •OH. Cu single-atom nanozyme has a unique metal active center and good microenvironment regulation ability, which can overcome the shortcomings of conventional nanozyme like element heterogeneity and low density of active sites. Wang et al. designed a biomimetic copper single-atom nanozyme system for self-enhanced nanocatalytic tumor therapy.¹¹¹ Likelywise, Shi at al. reported a bioinspired hollow N-doped carbon sphere doped with a single-atom copper species (Cu-HNCS) for effective antitumor therapy.¹¹²

No matter it is CDT, SDT, MDT, or catalytic therapy, all the therapies are relied on the electron rearrangement on the hybridized orbital of Cu to generate energy conversion, trigger reactions in tumor cells, transform nontoxic or low-toxic chemodrugs into more toxic chemodrugs, and effectively realize oxidative damage and cell death. These methods have the characteristics of high efficiency, strong specificity, high safety, and have been widely recognized by the scientists. They are helpful to solve some bottlenecks of chemotherapy, phototherapy, acoustic therapy, and other applications in clinical.

4.8 Cu and ferroptosis

Cu-based nanoparticles can catalyze not only Fenton-like reaction, but also artemisinin (ART) to produce ROS, which further aggravates the oxidative damage and lipid peroxidation buildup in cells, and thus kill cancer cells. Moreover, ART can also be used as a powerful autophagy inducer to increase the iron level in cells by degrading ferritin, promoting ferroptosis, and producing significant antitumor effects.¹¹³ The study demonstrates the interaction between metal ions in cells, expanding the application of ferroptosis in cancer treatment.

In addition to copper-mediated catalytic reactions, copper ions are also directly involved in the regulation of ferroptosis. The chelating agents TM and TEPA of copper ions can inhibit ferroptosis, while the addition of additional copper ions can promote ferroptosis, suggesting that copper ions may have the function of promoting ferroptosis. Moreover, copper ions did not affect the intracellular level of iron ions during the process of promoting ferroptosis, suggesting that copper ions may be a new type of Fe-promoting death metal ions besides iron ions. The results showed that the chelating agent reversed erastin induced glutathione peroxidase 4 (GPX4) protein decline, while copper ions promoted GPX4 protein decline. At the same time, copper ion did not affect GPX4 gene expression during ferroptosis. In addition, erastin combined with copper ions increased ubiquitination of GPX4 protein, and the autophagy inhibitor chloroquine (CQ) prevented GPX4 protein degradation. Therefore, the copper ion might promote GPX4 protein degradation via autophagy. Researchers found that the combination of copper ions and erastin significantly activated autophagy dependent ferroptosis, while knockdown of autophagy related gene autophagy protein 5 (ATG5) significantly inhibited the combination of copper ions and erastin induced ferroptosis. Then they found that erastin and copper ions selectively increased GPX4 binding to the autophagy receptor tax1 binding protein 1 (TAX1BP1). At the same time, TAX1BP1 knockdown did indeed inhibit GPX4 protein degradation and reduce cell sensitivity to ferroptosis, suggesting that TAX1BP1 was a key autophagy receptor in GPX4 protein degradation. This was the first ruled out erastin-induced GPX4 aggregation due to copper ion mediated ROS production. Based on a fluorescence conformational change on recombinant GPX4 protein, researchers found a significant spectral shift under copper ion treatment, indicating a direct interaction between copper and GPX4. Finally, they found that copper ions selectively bind to GPX4 cysteine residues C107 and C148. Similar results were obtained in vivo studies that copper promoted ferroptosis.

4.9 Vascular therapy based on Cu nanoparticles

Despite widespread clinical success, traditional cancer cell focused therapies have shortcomings that allow patients to develop drug resistance and suffer serious side effects. Tumor vascular targeting therapy is a promising approach to halt tumor progression by interfering with the vascular system instead of cutting off the supply of nutrients. This strategy has gained significant attention in recent years. Currently, there are two main strategies for tumor vascular targeting therapy. The first strategy involves blocking angiogenic pathways, such as VEGF and the copper signaling pathway, to prevent the formation of neovascularization. The second strategy involves obstructing or destroying established tumor vasculature. For instance, DNA nanorobots can be constructed to mediate the thrombin delivery system, which induces tumor blood vessel thrombosis and obstruction. Recently, Yu et al. reported a nanochelating agent (Imi-OSi, <6 nm) that effectively inhibited tumor by chelating with Cu,¹¹⁴ suppressing tumor angiogenesis. Moreover, under the stimulation of the TME, the Imi-OSi can change from dispersion to aggregation, forming secondary particles and blocking blood vessel functions (Figure 6). The rational design of functional nanomaterials provides a conceptual framework for vascular-targeted therapy. Furthermore, in the current era of rapidly advancing tumor therapies, combining immunotherapy with chemotherapy has led to unexpected tumor suppression. Antiangiogenic therapy has also shown benefits at a higher level. Multiple studies have demonstrated the synergistic effect of combining immunization with Cu-antiangiogenic therapy.

4.10 Cuproptosis

The cell death is a normal life phenomenon, and its related research has been a point in the field of life science. According to the different mechanism, the cell death mode is also different, such as apoptosis, pyroptosis, necrosis, ferroptosis and so on. Among them, ferroptosis, an emerging cell death named in 2012, has become the focus of research in recent years. Similar to iron, intracellular copper ions exhibit cytotoxicity when concentrations exceed the threshold for maintaining homeostasis mechanisms. The researchers first performed experiments on different cell lines using copper ionophores with structures, demonstrating that copper ionophores could induce cell death, which was mainly dependent on the accumulation of copper ions in cells. To confirm the mechanism of this mode of cell death, they performed knockout of key apoptosis factors Bcl2 associated protein X (BAX) and Bcl2 antagonist/killer 1 (BAK1), and used caspase inhibitors for apoptosis, ferrostatin 1 for ferroptosis, necrostatin 1 for necrotic apoptosis and N-pancreatic cysteine for oxidative stress. It was found that the cell death induced by copper ionophores could not be eliminated. This suggested that copper ionophores induced cell death was a mechanism quite different from the known mode of cell death. Meanwhile, the researchers observed that cells that relied more on mitochondrial respiration were about 1000 times more sensitive to the copper inducer than cells that relied on glycolysis. Treatment with mitochondrial antioxidants, fatty acids and mitochondrial function inhibitors significantly changed the sensitivity of cells to copper ions. In addition, electron transport chain (ETC) complex inhibitors, as well as inhibition of mitochondrial pyruvate uptake, reduced cupric-induced cell death, while none of these inhibitors had an effect on ferroptosis. At the same time, it was found that the content of tricarboxylic acid cycle (TCA) related metabolites changed in copper ionophores treated cells, suggesting that copper ionophores induced cell death may act on the TCA stage. To further explore the metabolic pathway of cuproptosis, genome-wide CRISPR-Cas9 loss of function screening identified seven genes, including ferredoxin 1 (FDX1), associated with copper ionophores induced cell death. It had been confirmed that FDX1 and protein lipid acylation were the key factors of copper ionophores induced cell death. Excess copper promoted the aggregation and disfunction of fatty acylated proteins, triggering instability of ferrothiotubulin, leading to proteotoxic stress, and ultimately cell death. Overall, the team identified a new type of cell death that they named cuproptosis, to distinguish it from existing cell death methods. The main process of cuproptosis depended on the accumulation of copper ions in the cell. The identified mechanism of cuproptosis, which involves the direct binding of copper ions to fatty acylation components in the TCA cycle leading to protein aggregation and imbalance, is a novel form of cell death. The study revealed that FDX1 is a key regulator of cuproptosis and the upstream regulator of protein lipid acylation, which is highly correlated with various human tumors. The sensitivity of cell lines to cuproptosis is associated with the abundance of FDX1 and fatty acylated proteins, suggesting that copper ionophores may have therapeutic potential in targeting cancer cells with such metabolic characteristics (Figure 7A).¹¹⁵ However, further research is required to fully understand the specific mechanisms of cuproptosis.

Subsequently, Cu-based nanoparticles were blowout applied to cuproptosis. Pan at al. reported a nonporous copper(I) coordination polymer nanoplatform loaded glucose oxidase (GOx) (GOx@[Cu(tz)]) for cuproptosis-based dynergistic cancer therapy. In this paper, they combined GOx with [Cu(tz)] to simultaneously increase the level of Cu and reduce the level of glucose and GSH in tumor cells, thus improving the sensitivity of cancer cells to cuproptosis (Figure 7B).⁶⁵ This study demonstrates the application of nanocarriers in cuproptosis, but the relationship between Cu and cuproptosis is still unclear, such as the relationship of Cu valence (Cu⁰, Cu¹⁺, and Cu²⁺), the Cu containing nanocarriers (CuS, CuO, and CuP), other metal ions (Fe²⁺, Zn²⁺, and Ca²⁺) and cuproptosis.

4.11 Immunotherapy based on Cu nanoparticles

Over the past few decades, immunotherapy has emerged as one of the most significant treatment approaches. However, the lack of T-cells and the immunosuppressive milieu severely restrict the clinical use of immunotherapy. Copper plays a crucial role in regulating immune function, and its deficiency can result in a decline of immune function. In copper-deficient mice, the level of interleukin was found to be only 40%–50% of that in normal mice. Moreover, increased serum copper levels were observed in various infections, which stimulated liver synthesis and release of ceruloplasmin, thus promoting resistance to microbial invasion. The elevation of blood copper levels is primarily attributed to a leukocyte endogenous substance secreted during the activation of neutrophils and macrophages. This substance is transported to the relevant target cells via the bloodstream and plays a critical role in immune regulation and sterilization. Many studies have shown that copper deficiency can affect the development of immune organs and normal immune function. The growth and development of immune organs in mice with severe copper deficiency were observed by biochemical and morphological methods, and spleen enlargement and thymus shrinkage were found. Thymus and spleen atrophy and decreased thymus weight were reported in copper deficient rats. T cells are the main participants of cellular immunity in animals and can assist humoral immunity of B cells. Copper plays an important role in T cell proliferation, assisting T cells and B cells. Copper deficiency can alter the composition of lymphoid tissue, and T cells are more sensitive to copper deficiency than B cells. T cells mature in the thymus and gradually differentiate into different subgroups with complementary and cytotoxic functions. Studies have shown that the number and percentage of CD4⁺ and CD8⁺ cells in T lymphocytes of copper deficient male rats decreased. The amount of copper can be increased by adding copper. Adding copper to feed can improve the cellular immune function of animals and enhance the body resistance. As a growth promoting agent, copper is widely used in culture production. Proper amount of copper can maintain the immune function of the body and promote the cellular and humoral immunity of the body. Although copper can promote growth and contribute to the play of immune function, it should not be added to feed without limit.

Recently, immunogenic cell death (ICD) with damage-associated molecular patterns (DAMPs) released from dying tumor cells as robust antigenic and adjuvant signals attracted increasing attention in antitumor immunotherapy. Various therapies, such as chemotherapy, PDT, PTT, CDT, RT, and so on. have been reported to incite the ICD-related immune response. The corresponding Cu-based therapy can still stimulate ICD. Shi et al. reported a biomimetic polymeric nanoparticle coloaded with phosphorus containing dendrimer copper complex (¹G₃-Cu) and endoplasmic reticulum stress drug fengjiamycin (Toy) for chemotherapy enhanced tumor immunotherapy, and further combined with programmed cell death-ligand 1 (PD-L1) antibody to activate long-term and sustained antitumor immune response (Figure 8A).¹¹⁶ CuS nanoparticles mediated PTT synergistically ablated tumors and promoted ICD to amplify antitumor efficacy (Figure 8B).¹¹⁷ Intracellular redox imbalance can be brought up by Cu-mediated GSH consumption and oxidative stress, which will further encourage ICD response (Figure 8C).¹¹⁸ Moreover, the CAT-like activity of Cu was reported to alleviate tumor hypoxia, reduce the antioxidant level and improve oxidative stress, thus inducing the polarizing of M2-type to M1-type tumor associated macrophage (TAM), reversing tumor immunosuppressive microenvironment and enhancing immunotherapy (Figure 8D).¹¹⁹ These studies have shown the effect of Cu and immunotherapy via ICD, but whether Cu metabolism directly affecting the function of immune cells has not been studied.