The immune system is composed of innate and adaptive immunity that cooperate to recognize and defend the body against foreign/non-self-substances and infect-ed/malignant cells to maintain homeostasis [11,12]^[4][5]. The innate immune system is the first line of defense against infection, responding to and eliminating harmful immunogens in a rapid and nonspecific manner [13,14]^[6][7]. Short-lived innate immunity subsequently bridges the activation of the adaptive immune system for specific and durable immune responses. Adaptive immunity can provide robust long-term protection against repeated encounters with pre-exposed antigens by the immunological memory effect, which is a cardinal feature of immunotherapy for durable and effective disease control [13,15]^[6][8].

2.1. Antigen-Presenting Cells

DCs and macrophages not only play central roles in innate immune responses but they are also primary antigen-presenting cells (APCs) that induce antigen-specific T cell immunity by presenting cancer antigens in the context of major histocompatibility complex (MHC) molecules [22]^[9]. They require maturation signals such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) for immune activation [23]^[10]. These signals can be recognized by pattern recognition receptors (PRR), including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and stimulator of interferon genes (STING) [22]^[9]. Upon maturation, DCs and macrophages exhibit an elevated expression of MHC molecules and costimulatory molecules (B7-1/B7-2) for the priming of T cells, and they secrete cytokines and chemokines that further activate T cells and enhance their migration to peripheral lymphoid organs or to the target site [24,25]^[11][12].

2.2. Effector Immune Cells

CD8⁺ T cells or cytotoxic T lymphocytes (CTLs) are the key effector cells for the adaptive immune response, which can directly recognize and eliminate cancer cells in an antigen-specific manner [29,32]^[13][14]. The multistep immune cell–cancer cell and immune cell–immune cell interactions that are involved in the killing of cancer cells via CTL are referred to as the ‘cancer-immunity cycle’ (Figure 2) [33]^[15]. In the first step of the cycle, cancer cell antigens released by spontaneous cell death are captured by professional APCs such as DCs (possibly accompanied by the co-release and uptake of DAMP-based maturation signals). APCs then process antigen epitopes and present them to CD8⁺ T cells in the context of MHC I molecules in peripheral lymphoid organs [34]^[16]. Naïve T cells receive an antigenic signal via the T-cell receptor (TCR) and are primed and activated along with the co-stimulatory signals provided by APC maturation [18]^[17]. The activated T cells then migrate and infiltrate the tumor bed, recognize cancer cells via their cognate antigens bound to MHC I molecules, and exert cytotoxic activity for the induction of apoptosis [18,33]^[15][17]. NK cells can also directly engage and eliminate cancer cells [35,36]^[18][19]. Unlike CTLs, NK cells are innate immune cells and therefore do not require antigenic signals; their function is dependent on the repertoire and balance of activating and inhibitory receptor signaling [37]^[20]. For example, the inhibitory killer cell immunoglobulin-like receptor (KIR) can suppress NK cell activity after the recognition of MHC I molecules, which prevents the immune response and the subsequent collateral damage to healthy cells expressing MHC I [36,37]^[19][20]. Conversely, activating receptors can engage their ligands to promote NK cell activity. These include NK group 2 member D (NKG2D), such as MHC class I chain-related A and B (MICA/MICB) and several UL-16 binding proteins (ULBPs), natural cytotoxicity receptors (NCRs) like NKp30 and NKp46, and DNAX-activating molecule (DNAM1) [18,37,38]^[17][20][21].

2.3. Immunosuppressive Cells

Tumors in the escape phase often harbor various immunosuppressive cells that aid in immune evasion and tumor progression [41]^[22]. Among the best-characterized immunosuppressive cells are TAMs, MDSCs, and Tregs [30]^[23]. Consisting mainly of M2 macrophages, TAMs originate from monocytes that are recruited by tumor-derived cytokines, chemokines, and growth factors and then differentiate in response to IL-4, IL-10, and IL-13 [30]^[23]. They express CD68, CD163, CD204, and CD206 and produce a high level of IL-10 and arginase 1 (ARG-1), which are associated with a poor prognosis of solid tumors [42]^[24]. TAMs can negatively affect DCs and CTLs with cytokine regulation, express inhibitory ligands such as PD-L1 and B1-H4, and recruit Tregs by secreting chemokines [41]^[22]. MDSCs are immature myeloid cells that secrete various immunosuppressive substances, including ARG-1 and anti-inflammatory cytokines such as IL-10 and transforming growth factor-β (TGF-β), which upregulate PD-L1 expression and expand Tregs [10,30,43]^[3][23][25]. As a result, MDSCs disrupt T cell activation, DC antigen presentation, M1 macrophage polarization, and NK cell cytotoxicity [30]^[23]. The presence of MDSCs in the TME has been associated with drug resistance and metastatic progression, underscoring their pro-tumor effects [44,45,46]^[26][27][28]. Tregs are a subset of CD4⁺ T cells with critical immunosuppressive functions for maintaining normal immune homeostasis and self-tolerance [47]^[29]. They are attracted to hypoxic environments intrinsic to tumors, where they suppress anticancer immune cells; therefore, they are considered a significant barrier to cancer immunotherapy [48,49]^[30][31]. Tregs produce immunosuppressive cytokines such as IL-10, IL-35, and TGF-β; deplete IL-2; and express negative co-stimulatory molecules that suppress T cells [30,41]^[22][23].

3. Metal Ions in the Host Response and Immune Modulation

Metal ions play an important role in the regulation of host homeostasis [52,53]^[32][33]. Some transition metal ions such as Fe²⁺, Mn²⁺, and Zn²⁺ are also essential elements in bacteria that serve as a cofactor to catalyze, stabilize, and activate many proteins and enzymes involved in vital physiological processes such as DNA replication/transcription and central metabolism [54]^[34]. Intriguingly, they can trigger host responses that are potentially associated with the inhibition of bacterial pathogens; the innate defense system not only actively exocytose these ions and thus sequester from intracellular pathogens but also accumulate them in the phagolysosomes to kill the bacteria, possibly by overdose toxicity or reactive oxygen species (ROS) production via a redox reaction [54,55]^[34][35]. The impact of metal ions on the activation of innate immunity has been demonstrated with inflammasome-mediated responses. The inflammasome is an oligomeric protein complex that activates caspase-1 for the maturation of IL-1β and IL-18β and subsequent pro-inflammatory responses [56]^[36]. In particular, inflammasomes that contain the nod-like receptor (NLR) family member NLRP3 can respond to several metal ions such as Ca²⁺, Na⁺, and K⁺. An increase in extracellular Ca²⁺ levels has been observed at sites of infection and ischemic necrosis to activate the NLRP3 inflammasome, suggesting that Ca²⁺ is a DAMP signal released by damaged cells [57]^[37]. Na⁺ and K⁺ can also trigger the formation of the NLRP3 inflammasome by inducing an ionic imbalance within cells [56,58]^[36][38]. Intracellular K⁺ depletion has been shown to be necessary and sufficient to activate NLRP3 inflammasomes, which can be caused by active K⁺ efflux and the formation of membrane pores permeable to K⁺ [56]^[36]. Conversely, Na⁺ influx can dysregulate the Na⁺/K⁺ gradient and promote K⁺ efflux, thus leading to the activation of the NLRP3 inflammasome [58]^[38].  Metal ions can also regulate the pivotal immune cells in the adaptive immune system. Zn²⁺ is essential for the development and function of T cells and B cells, where its deficiency causes thymic atrophy and subsequent T cell lymphopenia, disrupts T cell subtype differentiation potentially causing immune dysfunction, impairs T cell activation and expansion, and reduces B cell maturation and antibody production [55]^[35]. In addition, Zn²⁺ exhibits various immunological functions related to the priming of adaptive immunity, including polymorphonuclear cell recruitment and phagocytic activation, the maturation and differentiation of DCs and NK cells, and pro-inflammatory cytokine production [55]^[35]. For the activity of CTLs, Mg²⁺ promotes the up-regulation of lymphocyte function-associated antigen 1 (LFA-1) and costimulatory T cell receptors to enhance their cytotoxicity [4]^[39]. TCR signaling is a key step in initiating adaptive immune responses by T cells, whereby Ca²⁺ plays a critical role by manipulating phospholipids and signaling proteins [61]^[40]. Specifically, Ca²⁺ can neutralize anionic phospholipids and subsequently increase the accessibility of immunoreceptor tyrosine-based activation motif (ITAM), leading to enhanced TCR sensitivity to antigenic signals by the amplification of downstream signaling [61]^[40].

4. Metal-Based Nanoparticles for Cancer Metalloimmunotherapy

4.1. Manganese- and Zinc-Based Nanoparticles for cGAS-STING Activation

Manganese-containing nanoparticles can stimulate innate and adaptive immunity through the cGAS-STING pathway [65,66]^[41][42]. Sun et al. showed that Mn²⁺ can potentiate the activity of the cyclic dinucleotide (CDN)-based STING agonist by an order of magnitude in multiple human STING haplotypes (Figure 3) [9]^[2]. The authors designed the self-assembled CDN-Mn²⁺ nanoparticle (CMP) to take advantage of a nanoparticle formulation to improve CDN and Mn²⁺ metabolic stability, cell permeability, intracellular activity, and in vivo performance. CMP was demonstrated to trigger anticancer immune responses through NF-kB and IRF3, the downstream signaling of the cGAS-STING pathway. It induced DC maturation, cytokine production, and M1 macrophage polarization, leading to the activation of CD8⁺ T cells and inhibition of immunosuppressive MDSCs for remarkable antitumor efficacy in multiple difficult-to-treat murine tumor models. Liu et al. developed biomineralized manganese oxide nanoparticles (Bio-MnO₂ NPs) in combination with radiotherapy (RT), showing that it could induce apoptosis and the DNA release of cancer cells to activate the cGAS-STING pathway [67]^[43]. Bio-MnO₂ NPs not only sensitized RT to stimulate cGAS-STING signaling but also relieved tumor hypoxia and generated ROS with the release of Mn²⁺ and oxidation of hydrogen peroxide into oxygen. As a result, RT plus Bio-MnO₂ NPs elicited robust anticancer immunity while alleviating the immunosuppressive TME, significantly inhibiting tumor growth in a murine non-small-cell lung cancer (NSCLC) model. Zinc ions can also directly and indirectly stimulate cGAS-STING activation and anticancer immune responses [69]^[44]. Wang et al. showed that zinc oxide nanoparticles (ZnO NPs) could promote cancer cell apoptosis with the production of ROS [70]^[45]. ZnO NPs enhanced cellular uptake and the tumor spheroid penetration of DOX by inhibiting P-glycoprotein (Pgp)-mediated drug efflux and three-dimensional (3D) spheroid architecture-induced drug resistance, leading to the efficient killing of cancer and cancer stem cells. In addition, ZnO NPs activated macrophages and boosted anticancer immunity while decreasing the toxicity of DOX against macrophages. Cen et al. synthesized pH-responsive ZnS@BSA (bovine serum albumin) nanoclusters using a diffusive self-assembly approach that can release zinc and sulfur ions in an acidic TME [69]^[44]. The released Zn²⁺ activated cGAS/STING signaling, whereas S²⁻ formed H₂S gas that inhibited catalase and cooperated with zinc ions to generate ROS in hepatocellular carcinoma (HCC) cells. The intravenous administration of ZnS@BSA significantly inhibited HCC tumor growth through anticancer immune responses that could be potentiated by the PD-L1 antibody. It also conferred long-term protection against tumor recurrence, indicating the induction of robust and durable anticancer immunity. 

4.2. Iron- and Copper-Based Nanoparticles for M1 Macrophage Polarization

Iron-based nanoparticles are mainly oxidation products that include magnetite (Fe₃O₄), maghemite (γ-Fe₂O₃), and mixed ferrites (MFe₂O₄ where M = Co, Mn, Ni, or Zn) [72]^[46]. Such iron oxide nanoparticles have proved effective for drug delivery and cancer theranostics due to their biological and electromagnetic properties [73,74,75]^[47][48][49]. Recently, it has also been demonstrated that iron oxide nanoparticles can elicit anticancer immune responses. Ferumoxytol, an FDA-approved iron oxide nanoparticle for the treatment of anemia, has been shown to promote the polarization of macrophages into the M1 phenotype [76]^[50]. The polarization of M1 is characterized by an increase in pro-M1 genes (TNFα, CD86), a decrease in pro-M2 genes (IL10, CD206) in vitro, and an up-regulation of CD80 expression in vivo by macrophages. Furthermore, ferumoxytol could catalyze ROS generation through the Fenton reaction with hydrogen peroxide secreted by M1 macrophages, leading to the apoptosis of cancer cells accompanied by the expression of caspase 3 [77]^[51]. In vivo mice models have demonstrated that ferumoxytol suppresses the tumor growth of early mammary cancers and inhibits the liver and lung metastasis of small-cell lung cancer. In another study, Wu et al. used hollow Fe₃O₄ nanoparticles for the loading of _L-arginine (_L-Arg), which was followed by poly(acrylic acid) (PAA) sealing to provide pH-responsive release in the acidic TME [78]^[52]. M1 macrophages can efficiently convert _L-Arg into NO with elevated levels and catalytic activity of inducible nitric oxide synthase (iNOS), allowing for NO-based gas therapy. Therefore, _L-Arg could synergistically enhance the anticancer efficacy of iron oxide nanoparticles by utilizing their M2-to-M1 reprogramming capability. Chen et al. developed iron oxide nanoparticles to boost cancer vaccine activity [75]^[49]. They synthesized iron oxide-mesoporous organosilica core–shell nanospheres and then loaded protein antigen in the large-pore organosilica shell layer for vaccine application. The study showed that the hybrid nanoparticles could induce M2-to-M1 macrophage polarization and antigen-specific T cell immunity, leading to strong anticancer immune responses and the complete prevention of tumor development. Recent studies have also demonstrated that copper ions can induce M1 macrophage polarization [79,80]^[53][54].

4.3. Calcium- and Sodium-Based Nanoparticles for Ion Overloading Effect

Calcium-based nanoparticles can exhibit various anticancer and immunomodulatory effects caused by Ca²⁺ overloading. An et al. developed honeycomb OVA@CaCO₃ nanoparticles (HOCNs) that can be decomposed in an acidic TME and exert several functionalities associated with the release of Ca²⁺; they alleviate tumor acidity, support autophagy, and promote DAMP secretion from cancer cells [83]^[55]. When employed with the chemotherapeutic drug mitoxantrone, HOCN induced the maturation and antigen presentation of DCs and subsequently activated CTLs and effector memory T cells, leading to robust antitumor immunity for the regression of both primary and distant tumors. Recently, Zheng et al. reported curcumin-loaded CaCO₃ nanoparticles for pyroptosis-based cancer immunotherapy [84]^[56]. Curcumin could stimulate the release of Ca²⁺ from the endoplasmic reticulum into the cytoplasm, which could later accumulate and overload in the mitochondria. The surges of Ca²⁺ could break the dynamic Ca²⁺ equilibrium in the mitochondria, leading to ROS induction, cytochrome C release, caspase 3 activation, gasdermin E cleavage, and eventually cell blebbing and pyroptosis. Ca²⁺ overloading-mediated pyroptosis elicited robust anticancer immune responses (marked by DC maturation and T cell activation), thereby efficiently inhibiting tumor growth. Calcium-based nanoparticles can also re-educate TAM via “Ca²⁺ interference”, allowing for M2-to-M1 phenotype switching.

5. Conclusions

Metalloimmunotherapy offers a promising novel cancer treatment that involves utilizing metal ions to increase anticancer immune responses and anticancer efficacy. Metal-based nanoparticles can improve the pharmacological and drug-like properties of metal ions along with versatile nanoparticle engineering for the desired in vivo activity and functionality. The clinical development of metal-based nanoparticles should also consider cancer immunotherapy barriers and nanomedicine design principles to fulfil the full therapeutic potential of metalloimmunotherapy. In addition, combination therapy could be a promising approach to increse the utility of metal-based nanoparticles and improve their therapeutic outcomes with the rational selection of the combination counterparts.