Mn-based cGAS-STING activation for tumor therapy

Abstract

Immunotherapy has efficiently revolutionized the treatment of human neoplastic diseases. However, the overall responsive rate of current immunotherapy is still unsatisfactory, benefiting only a small proportion of patients. Therefore, significant attention has been paid to the modulation of tumor microenvironment (TME) for the enhancement of immunotherapy. Interestingly, recent studies have shown that cyclic GMP-AMP synthase-stimulator of interferon gene (cGAS-STING) was initially found as an innate immune sensor to recognize cytoplasmic DNA (such as bacterial, viral, micronuclei, and mitochondrial). It is a promising signaling pathway to activate antitumor immune responses via type I interferon production. Notably, Mn²⁺ was found to be a critical molecule to sensitize the activation of the cGAS-STING pathway for better immunotherapy. This activation led to the development of Mn²⁺-based strategies for tumor immunotherapy via the activation of the cGAS-STING pathway. In this critical review, we aimed to summarize the recent progress of this field, focusing on the following three aspects. First, we briefly introduced the signaling pathway of cGAS-STING activation, and its regulation effect on the antitumor immunity cycle has been discussed. Along with this, several agonists of the cGAS-STING pathway were introduced with their potential as immunotherapeutic drugs. Then, the basic biological functions of Mn²⁺ have been illustrated, focusing on its critical roles in the cGAS-STING pathway activation. Next, we systematically reviewed the Mn²⁺-based strategies for tumor immunotherapy, which can be classified by the methods based on Mn²⁺ alone or Mn²⁺ combined with other therapeutic modalities. We finally speculated the future perspectives of the field and provided rational suggestions to develop better Mn²⁺-based therapeutics.

Introduction

Immunotherapy is a therapeutic tumor modality that trains or stimulates the innate immune system. This stimulation identifies and then harnesses immune cells to attack and eliminate cancer cells, which has attracted tremendous research interest in recent years (1). Several immunotherapies, such as chimeric antigen receptor T-cell (CAR-T cell) immunotherapy, immune checkpoint, and vaccines, have been applied in the clinic, and revolutionized cancer therapies (2). However, only a small percentage of patients benefited from most current immunotherapy techniques. In contrast, the majority cannot be successfully treated because of some limitations. These limitations include high costs, treatment resistance, auto-immune effects, and low response rate of patients. Among these factors, the low response of tumor is the most critical one (3). The tumor variability and low immunity lead to an imbalance between the control of immunological and oncological signals, and then tumors evade immunoediting surveillance (4).

Appropriate activation of certain innate-sensing mechanisms within tumor cells or intrinsic immune cells may trigger an enhanced T cell response to combat the tumor (5). Recently, it was found that the activation of the cyclic GMP-AMP synthase-stimulator of interferon gene (cGAS-STING) pathway has remarkable potential to turn immunologically cold tumors into hot ones by various signaling pathways (6). The cGAS-STING pathway, an ancient intracellular DNA sensing mechanism, will be bound by cGAS and catalyze the production of the next signaling molecule, cyclic dinucleotide (CDN), when DNA is present in the cell that should not be present in the first place (7). The CDN will then go on to bind and activate the downstream signaling molecule, the stimulator of interferon genes (STING), which catalyzes the phosphorylation of the associated kinase into the nucleus, increasing the production of a variety of immune-related factors. Among them, type I interferons (IFNs) are the most representative (8). The produced type I IFN then induces additional cytokine production by dendritic cells (DCs) in a paracrine or autocrine manner to promote antigen presentation by T helper lymphocytes, thereby further enhancing the antitumor T cell response (9). Cancer cells differ from normal cells in that they are usually filled with cytoplasmic double-stranded DNA (dsDNA). Activation of the cGAS-STING pathway in cancer cells may hinder early tumor progression by upregulating a series of inflammatory genes (e.g., type I IFN) (10). Tumor-specific adaptive immune responses, including the activation of cytolytic CD8⁺ T cells, are dependent on type I IFN signaling in antigen-presenting cell (APC) types, the cGAS-STING pathway links both innate and adaptive mechanisms to promote antitumor immunity (11).

Interestingly, more recent studies demonstrated that the activation of cGAS-STING pathway was sensitized in the presence of Mn²⁺ (12). 1) Mn²⁺ enhanced the sensitivity of cGAS to dsDNA and its enzymatic activity; thus cGAS was able to produce the secondary messenger cyclic GMP-AMP (cGAMP) (one of the CNDs) even when lower concentrations of dsDNA were present in the cytoplasm; and 2) Mn²⁺ also enhances STING activity by augmenting cGAMP/STING binding affinity (13). Importantly, Mn²⁺ itself may serve as a potent cGAS activator, directly activating cGAS in the absence of dsDNA and inducing cells to produce type I IFNs and cytokines without any infection (13). Thus, Mn²⁺ can facilitate the activation of cGAS and STING in all aspects, from the production of cGAMP to the binding affinity of cGAMP/STING.

With the multiple functions of Mn²⁺ for enhancing the cGAS-STING pathway, Mn²⁺-mediated therapies have attracted great research attention as potent antitumor agents or adjuvants (14). Recently, we have witnessed a surge in the development of Mn²⁺-based strategies for tumor immunotherapy. Along with such significant advancement, it would be a good chance to summarize the recent progress in the field, which could give useful information for future research direction. In this critical review, we introduced the roles of cGAS-STING pathway in cancer immunotherapy and discussed the critical participation of Mn²⁺ in cGAS-STING pathway. Furthermore, we systematically reviewed the Mn²⁺-based antitumor immunotherapy, including its combination with various therapeutic modalities (Figure 1). Finally, future perspectives have been speculated for further development of the field.

Figure 1.

Schematic representation of Mn²⁺-mediated activation of the cGAS-STING pathway alone and its use in combination with multiple therapeutic modalities for enhanced antitumor treatment modalities. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon gene; TAA, tumor-associated antigen; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand 1. The image was created at BioRende.com.

Roles of cGAS-STING pathway in cancer and cancer immunotherapy

cGAS-STING pathway in cancer immunity cycle

Nucleic acid sensing is an essential component of the innate immune system that can stimulate an immune response to pathogens and diseased cells (15). cGAS is an innate immune sensor that identifies a variety of cytoplasmic dsDNA, including DNA with bacterial, viral, micronuclei, and mitochondrial (Figure 2) (16). The cGAS does not rearrange the catalytic structural domain, although other nucleic acids (ssDNA, ssRNA and dsRNA) also bind to cGAS. As a result, the cGAMP may not be synthesized (17). The activation of cGAS is sequence independent of dsDNA. cGAS binds to dsDNA to form a 2:2 complex with a conformational shift in the active site, catalyzing the production of cGAMP with cytoplasmic ATP and GTP as substrates (16). Longer DNA is more effective in activating cGAS and encouraging the production of liquid-like droplet formation, which increases cGAS’ sensitivity to dsDNA. cGAS is activated only when a certain level of cytosolic dsDNA exists (16,17). After that, the newly created cGAMP serves as a second messenger by binding to and activating the adaptor protein STING (18), which is a cytosolic receptor that senses both exogenous and endogenous CDNs and is located on the endoplasmic reticulum (ER) membrane (19). STING transitions from the ER to the ER-Golgi intermediate chamber and then to the Golgi (20). Upon activation, STING simultaneously recruits kinases in parallel, such TANK-binding kinase 1 (TBK1) and IKB kinase (IKK), which phosphorylate nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) (21). Phosphorylated IRF3 polymerizes to form a dimer which is then transferred to the nucleus and activates transcription of genes encoding IFNs such as interferon-β (IFN-β) (21). NF-κB enters the nucleus and enhances the expression of inflammatory and immune response genes in conjunction with other transcription factors (22). The activation of the cGAS-STING pathway, which can trigger a potent and antigen-specific immune response with a positive feedback loop to drive anticancer immunity, is essential for antitumor immunity (23).

Figure 2.

The cGAS-STING signaling pathway in cancer immunity cycle and Mn²⁺ plays a vital role in this pathway. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon gene; IFN, interferon; TNF, tumor necrosis factor; IL, interleukin. The image was created at BioRende.com.

Thus, the presence of cytoplasmic DNA is a warning sign that can prompt a potent innate immune response (23). It is worth mentioning that cGAS recognition of cytoplasmic dsDNA is irrelevant to a nucleotide sequence (24). And this DNA sensing route can therefore be utilized to identify self-dsDNA leakage brought on by cellular malfunction, a characteristic of many precancerous cells, as well as a variety of microbial diseases (25). In intracellular homeostasis, DNA is largely segregated in the cytoplasm within the nucleus and mitochondria (26). DNA damage or microbial infection may be the two main causes of DNA being found in the cytoplasm (23). Cancer cells are often accumulated by cytosolic dsDNA. This accumulation may include nucleus or mitochondrial DNA (mtDNA) spillage due to chromosomal destabilization, DNA damage, functional defects of DNA repair proteins and tumor suppressor genes mutations (23,27).

However, the mechanism by which tumor cells cause host immune cells to activate cGAS-STING pathway is a basic issue that is still poorly understood (28). In early preneoplastic cells, the cGAS-STING pathway functions as a tumor suppressor to inhibit DNA damage-induced tumorigenesis. The cGAS within the tumor senses cytoplasmic DNA produced by DNA damage from multiple sources. Then, cGAS activates STING to upregulate gene expression of type I IFN, IFN-stimulated genes (ISGs) and senescence-associated secretory phenotype (SASP) genes, which would suppress or retard cancer progression. Additionally, the cross-talk between tumors and nearby immune cells that controls antitumor immunity is made possible by the cGAS-STING pathway (16). DCs, macrophages, and APCs are believed to eliminate necrotic malignant cells (29). There are two main hypotheses about how tumor cells activate APCs (Figure 2): 1) Tumor cells are phagocytosed by DCs, and then DNA fragments from the tumor enter the cytoplasm to activate the cGAS-STING pathway, which secretes a series of cytokines to re-circulate and activate IFN signaling to enhance the presentation of tumor antigens for antitumor immunity (30); and 2) Tumor-derived cGAMP is transferred to neighboring cells via gap junctions, promoting direct activation of the STING pathway. Alternatively, tumor cells secrete cGAMP into the extracellular space and gain access to immune cells via the folate transporter protein SLC19A1 (31). Tumor DNA and tumor-derived cGAMP regulate the activation of the cGAS-STING signaling pathway by APCs, triggering the clearance of tumor cells by immune cells.

Agonists for the cGAS-STING pathway are crucial activators because the pathway’s hyperactivity plays a role in tumor regression, increased survival, and improved immunity. Therefore, investing a lot of time and effort in developing medicines targeting the cGAS-STING pathway is more worthwhile. It is important to note that most STING pathway agonists currently undergoing clinical trials are direct activators of the STING protein.

STING agonist

STING agonists have been explored for cancer immunotherapy, given their ability to trigger a robust innate and adaptive immune response. STING agonists are subdivided into various categories, which include natural CDNs, CDN derivatives, and other novel and unique non-nucleotide compounds (8). CDNs are a group of cyclic dinucleotide family members, consisting of cyclic di-GMP (c-di-GMP), cyclic di-AMP (c-di-AMP), and cyclic AMP-GMP (cGAMP). cGAMP also contains the subtypes of 3’,3’-cGAMP and 2’,3’-cGAMP (17). Bacteria naturally create the canonical 3’,3’-cGAMP with the typical 3’,5’-phosphodiester bond (32). The only known naturally occurring mammalian CDN with a noncanonical 2’,5’-phosphodiester linkage is noncanonical 2’,3’-cGAMP (Figure 3) (9,33). CDNs are susceptible to degradation by phosphodiesterase and nucleases since it contains two phosphodiester bonds (34). Moreover, the intrinsic negative charges, hydrophilicity, and tiny size of CDNs pose challenges for in vivo delivery, such as rapid clearance, weak cellular targeting, inefficient cytoplasmic transport and systemic inflammatory toxicity (35). As the molecular interactions between natural CDN and STING have been studied in depth, the increasing understanding of this pathway has stimulated a number of synthetic CDN derivatives that possess greater potency, stability and binding affinity (8). ADU-V19 is a newly modified CDN that is resistant to phosphodiesterase by chemical modification and functions in human STING (17). ADU-S100 is similar to ADU-V19 (17), which is the first one to enter clinical trials. In comparison to cGAMP and other prokaryotic-derived CDNs, it exhibits superior stability, lipophilicity, and dramatically increased STING activation (33).

Figure 3.

Structure of naturally occurring CDNs. The c-di-GMP, c-di-AMP, and 3’,3’-cGAMP are bacteria CDNs, while 2’,3’-cGAMP is a mammalian CDN. CDN, cyclic dinucleotide; cGAMP, cyclic GMP-AMP.

Non-nucleotide small molecule STING agonists have grown into a vast system. For example, recent studies have demonstrated that the flavonoid flavone-8-acetic acid (FAA) initiates immunity through contact with STING pathway and was one of the initial synthetic STING agonists to be identified (36). The xanthone 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a synthetic anti-vascular drug that dramatically reduces tumor load by disrupting the tumor vascular system (37). In 2012, DMXAA was discovered to target STING activation and trigger the production of type I INF (38). Interestingly, DMXAA only increases murine cGAS-STING signals but not of humans, which would explain why cancer patients in clinical trials did not respond to treatment (39). Therefore, species specificity is an essential concern for new drug development. A potent method for finding new inhibitors and agonists for many therapeutic targets is high-throughput drug screening (40), based on which more and more STING agonists have been explored, such as dispiro diketopiperzine (DSDP) (41), 6-bromo-N-(naphthalen-1-yl) benzo dioxole-5-carboxamide (BNBC) (42), G10 (43), and C11 (44).

Besides new compound discovery, another solution to address the delivery issue is to employ nanoparticles. Nanoparticles are nano-size particles that can deliver a drug with high selectivity, bioavailability, and low cytotoxicity (45). In the past few years, various types of STING agonist nanoparticle formulations, such as liposomes (46,47), polymeric nanoparticles (48), as well as inorganic materials (49,50), have been successfully developed. They have shown satisfactory antitumor effects by modulating immune cells to increase their anticancer activity. Additionally, drug delivery methods utilizing biomaterials on various scales (from nanocarriers and microparticles to hydrogels) have been created to cross cell and tissue barriers, enhance therapeutic efficacy, and lessen side effects (9). Several excellent reviews have described STING agonists delivery systems involving biomaterials at various scales, so we will not be discussed them in detail here (8,9,17).

cGAS agonist

As a drug target for cancer immunotherapy, the cGAS agonist has received less attention than the STING agonist (51). Despite cGAS agonists more naturally mimic the endogenous STING signal being stimulated. Promoting antitumor immunity by activating the cGAS requires the accumulation of dsDNA in the cytosol, which may be endogenous or exogenous (25). Many traditional cancer treatments (such as radiation, some chemotherapies, etc.) may result in endogenous DNA damage with the generation of a significant amount of dsDNA, activating the STING pathway (52). Due to oxidative stress and mitochondrial dysfunction, mtDNA is released into the cytoplasm as one of the sources of endogenous DNA to stimulate the dsDNA-sensitive pathway in the cytoplasm in cancer, apart from the nuclear compartment (52). Therefore, some drugs are proposed to promote cGAS-STING-mediated antitumor responses by increasing the leakage of mtDNA in the cytoplasm (16). Under optimal conditions, the STING signaling pathway directly or indirectly mediates cancer cell death via bolstering cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses (53). Besides, it is an attractive immunotherapeutic strategy for the treatment of cancer to import the exogenous DNA called interferon stimulatory DNA (ISD). The use of ISDs to activate cGAS will present a number of dosing challenges. Garland et al. designed a nucleic acid immunotherapeutic with NanoISD as a cGAS agonist (25). NanoISD is a nucleic acid nanoformulation engineered to be deoxyribonuclease resistant, which is taken up by cells and released into the cytoplasm via endosomal escape, thereby stimulating the cGAS responder to effectively activate the STING pathway. Intra-tumoral administration of NanoISD resulted in an increased local production of pro-inflammatory cytokines, enriching NK cells and T lymphocytes in mouse tumors. In preclinical tumor models, NanoISD’s shown a better response to ICB therapy and lowers tumor burden (25). Thus, NanoISD is a new immunostimulatory agent for the treatment of immunocompromised cancers.

Mn²⁺: A crucial player in cGAS-STING pathway

Manganese in physiological system

Mn, the most common and abundant metallic element on Earth, is a silver-gray, easily oxidized metal naturally present in the positive oxidation states (+2, +3, +4, +6, and +7) (54). Mn is a transition element located in group VIIA of the periodic table. Despite its 11 oxidation states ranging from −3 to +7, only two are of known significance in biological systems, i.e., Mn²⁺ and Mn³⁺ (55). The most stable form of manganese is Mn²⁺, but Mn³⁺ is a potent oxidant that typically exists in disproportionate amounts to Mn²⁺ and Mn⁴⁺ or forms complexes with proteins like transferrin (Tf) (56). Manganese is one of the most abundant metals in the tissues of mammals and is required for various physiological processes, including development, reproduction, neuronal function, immune regulation, and antioxidant defenses (57). Manganese acts in the body partly as a component of metalloenzymes and partly as an activator of enzymes.

Manganese levels in human blood typically range from 4−15 mg/L (58). Manganese concentration was higher in bone, liver, pancreas and kidney. The manganese concentration in the brain, heart, lungs and muscles is less than 20 nmol/g (59,60). In addition to intravenous injection, manganese can be absorbed through cutaneous permeation, ingestion, and inhalation. It reaches cells through passive diffusion or active transport in the gastrointestinal tract and the lung, where it is quickly absorbed. From there, it distributes to various tissues through blood circulation. Mn can transport in a biphasic pattern in human intestinal cells using a saturable mechanism, much like other divalent cations like calcium and iron (61). Transporters on the cell surface that control Mn influx from the cell matrix include the divalent metal transporter 1 (DMT1), Tf, two Zinc transporters (ZIP8 and ZIP14), dopamine transporter, calcium channels, citrate transporter, choline transporter and ceruloplasmin. Cytosolic Mn efflux is also mediated by SLC30A10, ferroportin (FPN) and sodium-calcium exchanger (NCX) (54). The Mn/Tf/TfR complex can be endocytosed intracellularly, packaged in endosomes for release, and then finally released into the cytosol by endosomal DMT1. On the other hand, the ATP13A2 gene (PARK9) produces a lysosomal type 5 P-type ATPase that shuttles cytosolic Mn into lysosomes. Mn can be stored in the Golgi apparatus by the secretory route Ca²⁺/Mn²⁺ ATPase isoform 1 (SPCA1), Mg transporter HIP14, SLC30A10 and calcium channels.

The organelles with the largest amounts of Mn are the mitochondrion and the nucleus, which function as Mn storage pools (54). With an average retention of 10 d, ingested Mn turns over quite quickly. The primary method of elimination is by feces and hepatobiliary excretion. The liver conjugates the majority of extra Mn to bile, which is then excreted in the feces (62). Most diets contain a sufficient quantity of Mn. Mn is closely regulated for intake, transport, and excretion to preserve homeostasis. Mn could cross the blood-brain barrier and predominantly accumulate in the globus pallidus and the brain’s striatum when exceeding the liver elimination capability, causing Mn intoxication (63). Importantly, Mn and iron (Fe) share cellular transporters. Iron deficiency permits increased transport of Mn and is a risk factor for the accumulation of manganese toxins in the brain. Early symptoms of Mn poisoning include sleep disturbance, fatigue, anorexia, psychological disturbance, and emotional lability (64). The nonspecific distribution and neurotoxicity of manganese raise the limitation for its clinical translation.

Role of Mn in cGAS-STING pathway

Some metal ions, such as Ca²⁺ and Zn²⁺, are involved in intracellular communication as second messengers in immune cells. So far, It is conceivable that the use of Mn²⁺ for innate antiviral activity has gone unnoticed (65). An almost 30-year-old study found that intraperitoneal injection of MnCl₂ increased the activity of murine NK cells, which was likely mediated through the release of type I IFNs (66). Recently, Mn²⁺ has been reported to promote cGAS and STING activation comprehensively (14). Storing Mn²⁺ was released from the mitochondria and/or Golgi apparatus after DNA virus infection. Additionally, Mn-binding proteins like metallothionein and calprotectin can provide free Mn²⁺ (67). The two ways to released Mn²⁺ altogether leads to the increase of cytosolic free Mn²⁺ to reach approximately 20 μmol/L for sufficient cGAS-STING pathway activation. The released Mn²⁺ is highly important for two main reasons (Figure 2). First, Mn²⁺ increases Cgas’s enzymatic activity and sensitivity to dsDNA, allowing it to create the secondary messenger cGAMP in the presence of low levels of dsDNA. Second, through increasing the binding affinity between cGAMP and STING, Mn²⁺ also improves STING activity. Thus, the freed cytosolic Mn²⁺ significantly decreases the host cells’ detection threshold for dsDNA and viruses. When Mn²⁺ is present, the cGAS-STING pathway is hyperactivated, causing even the smallest DNA stimulation or disruption to trigger the generation of IFNs (67).

Notably, the cGAS-STING pathway not only senses danger from viral and bacterial infections but also the dsDNA from damaged mitochondria and endocytosed debris of dead cells. It functions in DCs to prepare cytotoxic T cells for an antitumor response in tumor immunology (22). For instance, Lv et al. showed that Mn²⁺ greatly enhanced CD8⁺ T cell and NK cell activation in a cGAS-STING-dependent manner, as well as macrophage maturation and antigen presentation (68). Additionally, the liberated Mn²⁺ finally converted the immunosuppressive milieu to support anticancer immunity, polarized tumor-associated macrophages from M2 to M1 phenotypes, and promoted CTL infiltration on tumor cells (69).

Importantly, Mn²⁺ is a potent activator of cGAS. Recent research has discovered that Mn²⁺ directly activates cGAS independent of DNA and causes a specific catalytic synthesis of 2’,3’-Cgamp (12,70). Compared to other STING agonists, manganese is uniquely positioned to activate the cGAS-STING pathway. Moreover, manganese is a powerful and versatile metal for antitumor therapy. First, manganese is an essential metal ion for cell growth, biocompatible and effective in enhancing catalytic activity, especially in various oxidation reactions. Secondly, it is an ideal MRI contrast agent in its own right, significantly enhancing the T1-weighted MRI signal. Finally, manganese has large reserves, is inexpensive, and is very easy to transport and store. These advantages make manganese-mediated antitumor immunotherapy easy to apply and can significantly reduce the cost of tumor treatment.

Manganese-based therapy for cancer treatment

Based on the unique properties of Mn for activating cGAS-STING pathways, it is valuable to explore the antitumor function of Mn itself and to use it to develop a variety of nanoformulations. More importantly, this therapeutic modality might be combined with other effective antitumor therapeutic modalities to address each other’s disadvantages (Table 1). For efficient tumor immunotherapy, some manganese-containing formulations are available. Still, the function of manganese does not involve activation of the cGAS-STING pathway, so such examples are not discussed in this article.

Table 1. Summary of Mn-mediated activation of cGAS-STING pathway for cancer treatment.

Category	Formulations	Cell line	Administration	Therapeutic effect	Ref.
IFN, interferon; cGAMP, cyclic GMP-AMP; PD-1, programmed cell death protein 1.
Mn alone	MnCl2	B16F10/MC38	Intranasally or intravenously injected	More resistance to tumors with significantly suppressed tumor growth and greatly increased survival	(68)
	MnCl2	Hepa1-6	Intravenously injected	The number of tumor nodes in the liver was significantly decreased	(71)
	MnCl2	MC38	Nose drop	Encourage the creation of ROS and mitochondrial lipid peroxidation, that decrease DHODH function and cause ferroptosis in tumors	(72)
	MnP-PEG	B16-F10	Intratumorally injected	Leading to 57.3- and 13.3-fold higher production of IFN-β and interleukin-6 than free cGAMP, combination with a checkpoint inhibitor leads to significant tumor regression	(73)
	Mn-MOF@PEG	Panc02	Intravenously injected	The volume and weight of tumors in mice decreased, increased the percentage of CD8+ T cells but decreased the percentage of CD4+ T cells in the tumor tissue	(74)
	MnJ	B16-F10	Intramuscularly injected	Tumor growth was greatly suppressed and greatly blocked lung metastases	(75)
Mn plus chemotherapy	PL/APMP-DOX NPs	4T1	Intravenously injected	Tumor volume reduced 66.3%, prolonged lifespan of the tumor-bearing mice, inhibited tumor relapse and metastasis	(14)
Mn plus radiotherapy	Alg-Mn	CT26 and B16-F10	Peritumorally injected	Significantly delayed the growth of primary tumors and abscopal tumors, and increased the survival time	(76)
	NaGdF4:Nd@ NaLuF4@PEG-polyphenol/ Mn (DSPM)	4T1	Intravenously injected	Anticancer therapeutics in primary tumors, accompanied by robust systemic immune therapeutic performance against metastatic tumors	(77)
Mn plus photothermal therapy	CMM-DiR	B16-F10	Intravenously injected	The robust antitumor immunity of nanoplatform was observed in primary tumors, recurrent tumors, metastatic tumors and multinodular tumors	(78)
	ONc-Mn-A-malF127	B16-F10	Intravenously injected	Elevated levels of IFN-β in vivo, achieved eradication of primary and distant tumors	(79)
Mn plus chemodynamic therapy	NanoMn-GOx-PTX	4T1	Intravenous injection	Tumor suppression rate was 66.56%	(80)
	MnO@mSiO2-iRGD NPs	B16-F10	Intravenously injected	Synergized with α-PD-1 blocking antibody to highly elicit cytotoxic T lymphocyte infiltration and restrict melanoma progression and metastasis	(81)
Mn plus sonodynamic therapy	PIMS NPs	4T1	Intravenously injected	Efficiently inhibiting the growth of distant tumors and restraining lung metastasis	(82)
Mn plus immunotherapy	CMPCDA	CT26, B16-F10 and tobacco carcinogen-associated syngeneic squamous cell	Intravenously injected	Remarkable therapeutic efficacy in multiple difficult-to-treat murine tumor models	(83)
	Mn-cGAMP NVs	B16-F10	Intratumoral injection	Obviously delayed the primary tumor growth, prolong survival, effectively delayed and prevented distal tumor growth	(84)
	Mn2+ + YM101	CT26, EMT-6, H22, and B16-F10	Mn2+ intranasally or intratumorally For Ab treatment, hIgG, α-PD-L1, or YM101 by intraperitoneal injection	Successfully overcame the weak immunogenicity-caused treatment resistance and effectively reinvigorated adaptive antitumor immunity by stimulating innate immunity	(85)
Mn plus TME regulation	Mn/CaCO3@ PL/SLC	B16-F10	Intravenous injection	Suppressed tumor growth and prolonged survival time of tumor-bearing mice effectively and prevented tumor metastasis	(86)
	TMA-NPs	4T1	Intratumorally	Inhibited the primary large tumor progression and retarded distant tumor growth	(87)

Antitumor treatment based on manganese alone

Antitumor strategies in the form of manganese ions

Manganese is involved in various physiological activities such as reproduction, growth, energy metabolism and antioxidant defense, and is an indispensable metal for maintaining human life activities (57). Mn²⁺ has been recently discovered to activate the cGAS and augment cGAMP-STING binding affinity directly (73). Mn²⁺ functions as DAMPs and plays an essential role in cGAS-STING signal transduction. Type I IFNs and other proinflammatory cytokines are triggered by the moderate concentration (0.05−0.5 mmol/L) of Mn²⁺ signal. A lower concentration of Mn²⁺ stimulation (2−50 μmol/L) leads to a lower threshold of the cGAS-STING activation. Hence, Mn²⁺ has the great potential for STING activation (88). The antitumor function of Mn²⁺ depends on CD8⁺ T cells and type I IFN signaling. In the murine hepatocellular carcinoma (HCC) model, the mice were treated with MnCl₂ intravenously every other day. The number of tumor nodes in the liver significantly decreased compared to the control group. In addition, an uptrend in the percentage of immune cells CD8⁺ T cells in the liver and spleen as well as upregulation of macrophage MHC-II expression were found in the MnCl₂-treated group, and the level of IFN-β expression was significantly increased in serum, liver and tumor tissue. These findings suggest that Mn²⁺ can stimulate myeloid cells to express more costimulatory and MHC molecules, which may help them perform their roles as APCs (71).

In another work, Lv et al. demonstrated that multiple modes of Mn²⁺ administration (intranasally, intravenously or intratumorally) induced cGAS-STING pathway activation and exerted potent systemic antitumor effects in different mouse models. In experimental mice, NK cell function, DCs and macrophage maturation/activation, CD8⁺ T cell differentiation/activation and memory T cells were found to be promoted in the tumor region after cGAS-STING pathway activation. As a result, tumor growth and metastasis were significantly inhibited (68). However, although Mn²⁺ is identified as a natural STING agonist, direct administration of free Mn²⁺ could not sufficiently accumulate into the TME with effective concentration. Due to the abundant PO₄³⁻ in the physiological conditions, the in-situ formation of manganese phosphate nanoparticles upon MnCl₂ injection may lead to potential toxicity. Consequently, manganese-based nanomedicine is a promising immunotherapy platform targeting the cGAS-STING pathway (77).

A recent study has shown a fascinating connection between the cGAS-STING pathway activated by Mn²⁺ and ferroptosis. Ferroptosis an iron-dependent and non-apoptotic form of cell death. Its hallmark is the excessive accumulation of lipid peroxides and reactive oxygen species (ROS) (89). Zhang et al. investigated at how MnCl₂ boosted mitochondrial production of ROS and lipid peroxidation to cause ferroptosis by suppressing the expression of the essential ferroptosis molecule, dihydroorotate dehydrogenase (DHODH), in tumor cells (72). The intrinsic association is that MnCl₂ upregulates the production of type I IFN expression by the cGAS-STING signaling pathway. IFN enables DHODH function, promotes mitochondrial lipid peroxidation and ROS production, and induces ferroptosis in tumor cells (Figure 4). Type I IFN, or the cGAS-STING signaling pathway inhibition restored DHODH expression and prevented MnCl₂-induced ferroptosis. Manganese exerts antitumor effects through activation of the cGAS-STING pathway and ferroptosis induction, indicating that Mn²⁺ has excellent potential in antitumor therapy and can help develop new therapeutic strategies for antitumor treatment.

Figure 4.

Manganese induces ferroptosis in tumor cells by activating a cascade reaction of the cGAS-STING pathway producing type-I IFN to inhibit mitochondrial DHODH (Reproduced with permission from Ref. 72, Elsevier). cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon gene; IFN, interferon; DHODH, dihydroorotate dehydrogenase.

Manganese-based nanomedicines

Manganese nanoformulations have been developed for cancer treatment. Several polyethylene glycols (PEGs)-modified Mn nanoparticles for specific DCs activation have been reported. Gao et al. developed PEG-modified manganese (II) phosphate nanocluster (MnP-PEG) with remarkably high biocompatibility and powerful ability to excite the cGAS-STING pathway (Figure 5) (73). Slightly negatively charged surface (about −11.0 mV), and the appropriate nano-size (about 150.0 nm) rendered the MnP-PEG nanoclusters allow for better pharmacokinetics and enhanced retention in the tumor. MnP-PEG nanoclusters displayed superior stability in buffers that simulated physiological circumstances and were resistant to enzymatic deterioration. When MnP-PEG nanoclusters were successfully ingested by DCs through endocytic and released Mn²⁺ in response to an acidic environment in the endo/lysosomes, STING was powerfully activated. Immature bone marrow-derived dendritic cells (BMDCs) were stimulated by MnP-PEG nanoclusters, which resulted in 57.3- and 13.3-fold greater production of IFN- and IL-6 than free cGAMP, respectively. In vivo administration of MnP-PEG encouraged DC and macrophage infiltration and maturation, and increased CD8⁺ T cell and NK cell activation and granzyme B production in the TME. The synthesis of MnP-PEG nanoclusters is a simple process with mild conditions, which facilitates the introduction of large-scale production to create greater clinical value. As a non-nucleotide nanoparticle stimulator, nanoclusters have a few advantages over natural CDNs to enhance their future clinical applications.

Figure 5.

Synthesis of MnP-PEG nanoclusters and schematic representation of its cGAS-STING activation mechanism (Reproduced with permission from Ref. 73, John Wiley and Sons). MnP-PEG, PEG-modified manganese (II) phosphate nanocluster; cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon gene; PEG-Ale, polyethylene glycol conjugated with a terminal alendronate; IFN, interferon; ER, endoplasmic reticulum; TBK1, TANK binding kinase 1; IRF3, interferon regulatory factor 3; NF-Κb, nuclear factor-κB.

Metal-organic frameworks (MOFs), which have found potential uses in various disciplines, are made up of metallic ions or ion ensembles as the nodes that interact with multidentate organic linkers (90). In particular, significant efforts have been invested in applying nanoscale MOFs (nMOFs) in cancer therapies or treating bacterial infections (91). Adding PEG to nMOFs to transport drugs, i.e., PEGylation, is a tried-and-true method for improving blood flow, cell absorption, and bioavailability (92). Liu et al. reported the preparation of Mn-based MOFs nanoparticles grafted with PEG (Mn-MOF@PEG) on its outer surface via conventional carbodiimide chemistry, which showed clear and regular shape and suitable size, strong colloidal stability, and pH-dependent Mn²⁺ releasing behaviors. Even after extensive incubations at pH 7.4, only around 10% of Mn²⁺ was liberated into the media. At pH 5.5, however, approximately 70% of Mn²⁺ was found in the media within the first 3 h. Such pH-dependent release behaviors are desired for nMOF as a metal ion reservoir because it can release metal ions locally inside cancer cells or at malignant locations with an acidic pH of less than 5.5 (93). Mn-MOF@PEG effectively inhibited pancreatic cancer cell proliferation by blocking the cell cycle in the G2-M phase, increasing the proportion of cells in G2-M, and inducing apoptosis. In the in situ pancreatic tumor model, Mn-MOF@PEG was observed to have a significant therapeutic effect (74,94). This study further demonstrated that the Mn²⁺ released from Mn-MOF could probably regulate innate immunity cells via activating the cGAS-STING pathway.

Only a few adjuvants have gained human use approval, with aluminum-containing adjuvants being the most common. T helper 2 (TH2) cell responses were primarily induced by aluminum-containing adjuvants, but T helper 1 (TH1) or CTL responses were not (95). As a result, aluminum-containing adjuvants are thought to be incapable of eliciting the cellular immune responses required for virus or tumor vaccinations. However, by activating the cGAS-STING and NLRP3-ASC pathways, Mn²⁺ stimulates immunological responses by promoting antigen absorption, antigen presentation, and germinal center formation (75). Zhang et al. generated various small Mn-containing jelly-like colloids (MnJ, <10 nm) with different Mn²⁺/OH⁻/PO₄³⁻ ratios and tested for their immunological activity. When the OH⁻/PO₄³⁻ ratio was between 1:3 and 2:1, MnJ could strongly activate both types I IFN responses and inflammasomes. The significant increase in the volume and weight of draining lymph nodes (dLNs), the increase in the number of mouse T follicular helper cells (Tfh) and germinal center B cells, the promotion of CD4⁺ and CD8⁺ T cell proliferation, and the promotion of BMDCs maturation after MnJ injection all demonstrate the powerful ability of MnJ to promote the migration and/or proliferation of immune cells, advantages that free Mn²⁺ cannot be matched. Accordingly, an in vivo cytotoxic assay revealed that mice inoculated with MnJ produced very potent CTL activities that killed ovalbumin (OVA)-bearing cells, but this did not occur in mice that had been immunized with aluminum. Therefore, MnJ promotes the immune response by modulating the immune system in a holistic manner (75). In lung metastasis models and melanoma models, immunotherapy with OVA-MnJ significantly inhibited tumor metastasis and growth and improved survival rates. MnJ vaccination increased the development of tumor antigen-specific CTLs, showing that cancer vaccines have a lot of potency. The MnJ simplicity and consistency of components, as well as Mn’s inexpensive cost and widespread availability, made this adjuvant even more appealing.

Manganese combination with other treatments

Manganese-based immunotherapies, combined with other therapeutic modalities, can improve immune conditions in the TME. Simultaneously escape the dilemmas faced by other treatment modalities. These combinations indicate a solid antitumor immune response for chemotherapy, radiotherapy, anti-PD-L1 immunotherapy, and photodynamic therapy.

Chemotherapy

Chemotherapy is a well-established treatment modality. A growing number of studies have shown that chemotherapy is also known to enhance anticancer immunity by inducing immunogenic cell death (ICD) (96). Most cytotoxic chemotherapeutics have two-side effects, on one side harming immune homeostasis (lymphopenia), and on the other side, helping to increase antitumor immunogenicity (97). In addition, cytotoxic drugs could stimulate effectors of the innate immune system, such as recruiting effector NK cells and DCs to the tumor site and potentiating tumor-associated macrophage cytotoxicity via proinflammatory cytokine production (98). Therefore, combining chemotherapy regimens with immunotherapy may lead to synergistic antitumor activity, but there are still many unknowns about the effectiveness of the combination.

The combination of chemotherapeutic drugs and Mn²⁺ can achieve synergistic antitumor effects by activating the cGAS-STING pathway (14,99). Hou et al. employed amorphous porous manganese phosphate (APMP) NPs to construct doxorubicin (DOX) loaded, and phospholipid (PL) coated hybrid nanoparticles (PL/APMP-DOX NPs) (Figure 6). Once the nanoparticles enter the acidic and phospholipase-rich TME, the nanoparticles will rapidly degrade and release DOX, which induces DNA damage. The intracellular accumulation of DNA and the release of Mn²⁺ are very beneficial to enhance the activation of cGAS-STING to produce type I IFN and promote DC maturation and infiltration. There was a significant difference between PL/APMP-DOX and DOX in tumor growth inhibition effects, in which the former achieved a much better survival rate. Their results indicated that the chemotherapy drugs in combination with Mn²⁺ offered the potential to amplify antitumor immunotherapeutic efficacy (14).

Figure 6.

Schematic diagram of the mechanism by the PL/APMP-DOX NPs to stimulate natural immune antitumor activity by potentiating the cGAS-STING pathway (Reproduced with permission from Ref. 14, American Chemical Society). AMPM, amorphous porous manganese phosphate; DOX, doxorubicin; PL, phospholipid; cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon gene.

Radiotherapy

In practical practice, radiotherapy (RT) is still one of the leading cancer treatment methods. However, the immunosuppressive TME significantly speeds up tumor radioresistance (100). Researchers recently discovered that activating cGAS-STING enhances therapeutic radiation benefits in anticancer therapy by inducing immune responses. Since the 1950s, researchers have found that RT could directly damage tumor cell DNA and induce cell death, thus shrinking tumors (101). When damaged DNA reaches a certain threshold, and cytosolic DNA digestion by hosts DNase becomes overwhelmed, free DNA in the cytoplasm can activate the cGAS-STING pathway. It means that DNA damage from RT combined with Mn²⁺ may co-initiate the cGAS-STING pathway to achieve a synergistic antitumor effect. However, the researchers found that RT-induced DNA damage began accumulating in the cytoplasm after 24−72 h. At the same time, the free Mn²⁺ intratumorally injected will rapidly spread out from the tumor and metabolize to the kidney. This means that the effective concentration of Mn²⁺ in the tumor has to be maintained at the right time to improve the recognition of accumulated cytoplasmic DNA damage by cGAS (76).

Wang et al. developed Alg-Mn to effectively address the above-mentioned temporal and spatial inconsistencies that exist during practical clinical applications (Figure 7A) (76). Alginate (Alg) is a commonly available tissue engineering material that chelates divalent cations specifically (102). By injecting Alg-Mn around the tumor after radiotherapy, Mn²⁺ can be effectively replaced by Ca²⁺ in vivo due to the ability of Alg to chelate Ca²⁺ 1,000 times more than Mn²⁺, which sustains a slow emission for about 24−72 h synchronized with the metabolic time course of accumulated DNA damage to achieve synergistic amplification of cGAS-STING pathway activation in the tumor (Figure 7B,C). Alg-Mn also achieved more than 90% inhibition and significantly prolonged survival time in experiments treating bilateral tumor models in the side of the tumor without radiotherapy. Alg-Mn also achieved more than 90% inhibition and significantly prolonged survival time in experiments treating bilateral tumor models in which only unilateral tumors were treated with radiotherapy (103). This novel method dramatically decreases the pain of many intratumorally Mn²⁺ injections, increases patient compliance, and eliminates the risk of multiple injection adverse effects. The metabolic time course of accumulated DNA damage can be synchronized with the sustained-release of Mn²⁺ to synergistically accelerate the activation of cGAS-STING in tumors, which induces potent antitumor immunity.

Figure 7.

Alg-Mn continuously activates the cGAS-STING pathway to combat tumors. (A) Schematic representation of the mechanism by which Alg-Mn combined with RT-induced cumulative DNA damage jointly promotes antitumor immunity; (B) The relationship between Mn²⁺ and Ca²⁺ concentrations in the tumor area with time after peritumoral injection of Alg-Mn; (C) The trend of intratumoral damage DNA accumulation after radiotherapy and intratumoral accumulation after intratumoral injection of Mn²⁺ at different time points (Reproduced with permission from Ref. 16, Elsevier). STING, stimulator of interferon gene; IFN, interferon.

Mn²⁺ combined with radio sensitization is a viable strategy to facilitate synergistic antitumor. Radiosensitizers, chemical medicines, or nanoparticles that make tumors more sensitive to X-ray irradiation allow for better tumor management while retaining organ tolerance (104). High-Z metals, such as hafnium, gold, and lanthanide elements, can interact with X-rays through various physical processes to produce Auger electrons, Compton electrons, and photo-electrons, making them effective radio-sensitizers (105). These electrons then combine with a water molecule to produce deadly ROS, which causes irreversible DNA damage. Yan et al. fabricated a novel lanthanide doped radiosensitizer-based metal-phenolic network (MPN), NaGdF4: Nd@NaLuF4@PEG-poyphenol/Mn (DSPM) with surface modification of amphiphilic PEG-polyphenol via metal-phenolic coordination. Following cell internalization, core-shell NaGdF4: Nd@NaLuF4 downshifting nanoparticles (DSNPs) and Mn²⁺ were released as a result of the pH-responsive breakdown of DSPM. DSNPs made cancer cells more sensitive to X-ray radiation and encouraged the release of cytosolic dsDNA. The STING pathway was activated in both cancer cells and DCs, including the phosphorylation of STING, TBK1, and IRF3, by the released Mn²⁺, which made it easier for cGAS to recognizes cytosolic dsDNA. IFN expression was subsequently increased, promoting DC maturation and evoking potent anticancer immunity. This strategy resulted in strong immune therapeutic performance in primary, distant, and lung metastatic cancers with treatments sufficient for advanced radiotherapy. It is a sophisticated radio sensitization method that works with STING pathway activation-based immune stimulation to optimize radiotherapy for clinical application (77).

Photothermal therapy (PTT)

PTT may induce antitumor immune responses by releasing tumor-associated antigens (TAAs), DAMPs, and proinflammatory cytokines (106). Promoting the release of primary tumor antigens using PTT is one of the optimum approaches to preparing in situ tumor vaccines for individualized tumor therapy. Nevertheless, PTT immune response is also limited by the immunosuppressive TME in the majority of patients (107). The development of Mn²⁺ nanoplatforms are good choice for alleviating immunosuppression and provoking efficient antitumor immune responses.

Yang et al. developed a MnO₂ NPs-based biomimetic nanoplatform (CMM-DiR) with surface cancer cell membrane coating to immobilize the photothermal agent of DiR (Figure 8) (78). This biomimetic nanoplatform exerted antitumor effects via different mechanisms. First, the burst release of Mn²⁺ from the nanoplatform in TME was made possible by the fast degradation of the MnO₂ NPs in the nanoplatform in the presence of glutathione (GSH) and H⁺. The catalase-mimic MnO₂ also produced large amounts of O₂, and its oxidation raised the TME’s pH level. This reduced tumor hypoxia and encouraged T cell invasion. Second, the homotypic adhesion feature of the CMM-DiR allowed the tumor cells to absorb it. Due to the photothermal impact, tumor cells exposed to laser light released a significant amount of TAAs and DAMPs. This strategy could convert the initial tumor toward the STING vaccine (108). Moreover, as a STING agonist, Mn²⁺ was released in TME to activate the cGAS-STING pathway in immune cells infiltrating the tumor, and increase the uptake of the released antigen, thus enhancing the effect of PTT. The nanoplatform has observed potent antitumor immune benefits in a variety of tumor models, including primary, recurrent, metastatic, and multinodular tumors. In order to start highly effective and personalized antitumor immune responses, this strategy transforms primary tumors into in situ therapeutic vaccines. It also modifies the systemic immune response to promote incredible therapeutic efficacy against immunogenically deficient tumors of different types and stages.

Figure 8.

Diagram showing the covert transformation of an immunosuppressive tumor by a biomimetic nanoplatform (CMM-DiR) into an in situ STING-activating vaccination (ISSAV) and induction of an immune response (Reproduced with permission from Ref. 78, Elsevier). TAA, tumor-associated antigen; STING, stimulator of interferon gene; DC, dendritic cell.

Chemodynamic therapy (CDT)

CDT is a newly developed tumor treatment method whose potential advantages in the fight against cancer have been thoroughly investigated (109,110). A Fenton/Fenton-like reaction is used in the definition of CDT as an in-situ therapeutic technique to produce highly toxic hydroxyl radicals (·OH) at tumor locations. Compared to other Fenton agents, Mn demonstrates unparalleled advantages. In addition to causing the production of TAAs, it may also improve the presentation of antigens, encourage T cell activation and infiltration, and aid in killing tumor cells by activating the cGAS-STING pathway (111). As we all know, Mn²⁺ converts H₂O₂ into highly toxic ROS through a Fenton-like reaction. The therapeutic use of this therapy is hampered by the tumor tissue’s inadequate hydrogen peroxide concentration (112). This problem could be solved by glucose oxidase, which consumes glucose in tumor cells to generate a high level of H₂O₂ to enhance cooperative cancer treatment.

A multifunctional manganese nanoplatform called NanoMn-GOx-PTX was created by Zhu et al. (80) It has an inner core made of manganese and an outer shell made of phospholipid bilayers, co-loaded with glucose oxidase (GOx), paclitaxel (PTX), and a near-infrared fluorescent dye. The PEG-modified phospholipid bilayer shell allows the slow release of encapsulated Mn²⁺ and drugs in the acidic microenvironment of tumors. Among other things, GOx consumes glucose in tumor cells, generating large amounts of H₂O₂, which is converted to ROS to induce oxidative damage to tumor tissue via an Mn²⁺-mediated Fenton-like reaction. When used as chemotherapy, PTX causes a prometaphase arrest in the cell cycle, resulting in DNA damage and senescence. As a result of the aforementioned factors working in concert, DNA damage builds up, which will synergistically work with Mn²⁺ to activate the cGAS-STING pathway and successfully trigger the natural immune response by creating a lot of type I interferon and proinflammatory cytokines. Furthermore, the T1-weighted MRI pictures might be improved by the released Mn²⁺. This nanoplatform demonstrated a positive synergistic effect in the 4T1 tumor-bearing mouse model concerning the antitumor activity in vivo and considerably decreased the quantities of chemotherapeutic medications.

Sonodynamic therapy (SDT)

SDT converts the energy of ultrasonic waves into chemical energy to generate ROS in the presence of a sonosensitizer under the focused athermal mode of short-term repetitive ultrasound irradiation (113). Sonosensitizers and low-intensity ultrasound exposure are inherently harmless to humans, and only the temporal and spatial overlap of the two can cause cytotoxicity in vivo. Unlike PDT, which has a lower tissue penetration and is limited to treating cancers located in superficial tissues, SDT has a deeper tissue penetration depth and is a safer method of treating deep tumors (114).

By using phenolic metal coordination, Tian et al. created a phenolic nanoadjuvant (PIMS NPs) that self-assembled sonosensitizer polymer (PEG-b-IR), GSH inhibitor (sabutoclax), Mn²⁺ and TME acidity sensitive phenolic polymer (PEG-b-Pho) (Figure 9A) (82). In this specific nanoadjuvant, the sonosensitizer polymer (PEG-b-IR) triggered ROS generation by ultrasound stimulation (Figure 9B), Sabutoclax reducing intracellular GSH level, thereby suppressing ROS consumption to killing cancer cells prominently. The amphiphilic phenolic polymer (PEG-b-Pho) can guarantee to induce the polymer structure degradation for the sustained release of Mn²⁺ in the acidic TME. The stimulation of the cGAS-STING pathway by the combined effects of Mn²⁺ and SDT-mediated ICD impact dramatically increased DCs maturation (Figure 9C). Additionally, this phenolic nanoadjuvant dramatically increases the sensitivity of tumors to PD-L1 checkpoint inhibitor immunotherapy, successfully limiting distant tumor growth and preventing lung metastasis. The combination of SDT and Mn-mediated immunotherapy offers a novel avenue for developing nanoadjuvants to improve cancer immunotherapy.

Figure 9.

PIMS NPs combines sonodynamic therapy with cGAS-STING activation for cancer immunotherapy. (A) A diagram illustrating the preparation of PIMS NPs for self-assembly; (B) Schematic illustration for the anticancer mechanism of PIMS NPs; (C) Schematic diagram of the mechanism by which TAAs and DAMPs are released under ultrasound treatment at the primary tumor site, and PIMS NPs degrade in an acidic environment, activating the cGAS-STING pathway to initiate an adaptive immune response (Reproduced with permission from Ref. 82, Elsevier). TAA, tumor-associated antigen; cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon gene.

Dual agonism-Mn²⁺ combined immunotherapy

It is very attractive to eliminate tumors using the patients’ immunity. Leveraging the cGAS-STING pathway activation by Mn²⁺ can increase infiltration of immune effector cells to convert an immunosuppressive, TME to an immunogenic TME, and ultimately improve patient response rates to immune adjuvants and checkpoint blockade immunotherapy. The STING pathway is a promising strategy in combination with cancer immunotherapy. Recently, Mn²⁺ combined with immune checkpoint inhibitors (ICI) showed significant outcomes.

In cold tumors, the anti-TGF-β/PD-L1 bispecific antibody YM101 had some limitations even though it has a dual effect on antitumor immunity (85). The strategy containing Mn²⁺ and YM101 showed an extensive antitumor range and superior curative efficacy by concomitantly activating the STING pathway and suppressing TGF-β and PL-1/PD-L1 signals, overcoming multiple immunosuppressive. This new treatment paradigm demonstrated a long-lasting anticancer impact and prolonged survival. It had not been linked with treatment-related toxicity in several mouse tumor models, including CT26, EMT-6, H22, and B16-F10. The combined treatment dramatically increased the densities and functionality of tumor-infiltrating CD8⁺ T and NK cells, according to the flow cytometry and RNA-seq data. Mn²⁺ was the critical element that normalized TEM with bolstered antitumor immunity and plays a vital role in the antitumor spectrum, even in immune-desert tumors.

Moreover, an open-label, dose-escalation phase 1 clinical trial showed that the intervention of Mn²⁺ resulted in a turnaround in antitumor immunotherapy in patients with multidrug (immune)-resistant cancer. A study was conducted on patients with failure in standard anticancer treatments. They studied anti-PD-1 treatment to assess the security and primary efficacy of Mn²⁺ priming therapy (Clinical Tri als.gov, NCT03991559) started in November 2018 (68). The combined therapy was administered to 22 patients with advanced, metastatic solid tumors for at least two doses. MnCl₂ solution was administered intranasally or by inhalation. Blood Mn levels rose after administration but stayed within the usual range. The generation of type I IFNs and certain proinflammatory cytokines was facilitated by elevated blood Mn levels, and the significantly elevated levels of IFN-α, IL-6, IL-8, and TNF-α demonstrated positive clinical efficacy with 45.5% objective response and 90.9% disease control. It implies that in these immunologically nonresponsive patients, Mn²⁺ reinvigorated anticancer immunotherapy. This phase 1 trial documented a manageable safety profile. Although treatment-related adverse events occurred in 86% of patients, but they could be resolved with supportive care and without any treatment-related death. With a median follow up almost one year, no Mn overdose-related toxicity and accumulation of Mn in the basal ganglia was observed (115). This study demonstrates the safety and efficacy of Mn²⁺ in patients with advanced metastatic solid tumors, suggesting the substantial potential of manganese to enter into clinical antitumor effects.

A promising new platform for metalloimmunotherapy is a coordination nanomedicine made of bioactive metal ions and a STING agonist. For example, Sun et al. demonstrated that Mn²⁺ self-assembles with CDN (c-di-AMP, CDA) to form a coordination nanoparticle (CDN-Mn²⁺ particle, CMP_CDA), which produces potent antitumor immunity after local or systemic administration (83). CMP_CDA effectively delivered Mn²⁺ and STING agonist, to amplify STING activation with minimal side effects. Moreover, systemic treatment with CMP_CDA showed significant efficacy in murine tumor models. In another study, researchers utilized polymerized guanidine-containing disulfides (Cu⁺ unit) to assemble with cGAMP and then paired with Mn²⁺ ion to construct a robust Mn-cGAMP nano vaccine (Mn-cGAMP NVs) (Figure 10A) (84). The fixation cGAMP with Mn²⁺ not only improved the stability of Mn-cGAMP NVs but also potentiated the activation of STING. The presence of polysulfides on the NVs surface allows direct cytosolic delivery via a thiol-mediated endocytosis-independent pathway while avoiding endo-/lysosomes degradation. After inoculation of Mn-cGAMP NVs in poorly immunogenic B16-F10 melanoma mice, abundant tumor-infiltrating lymphocytes (TIL) infiltration in tumors was observed via the STING pathway activation. The NVs significantly increased the survival of B16-F10 melanoma mice by successfully inhibiting primary growth and inducing immunological memory to present distant tumors.

Figure 10.

A schematic diagram of the preparation process of Mn-cGAMP NVs, NVs entry into the cytosol mediated by thiol-exchange, and the synergistic amplification of intracellular STING pathway activation by Mn²⁺ and cGAMP (Reproduced with permission from Ref. 84, John Wiley and Sons). IFN, interferon; TNF, tumor necrosis factor; IL, interleukin; cGAMP, cyclic GMP-AMP.

TME regulation nanoplatforms

Tumor growth and progression are often accompanied by a microenvironment with abnormal characteristics such as lack of oxygen, low pH, increased oxidative stress, high glutathione concentration and overexpressed enzymes (101). These factors affect or limit the treatment of tumors, while providing corresponding therapeutic strategies for cancer-specific treatment.

Low extracellular pH may constantly induce T cells into an anergic state due to the ongoing generation of acidic metabolites (116). By converting carbon dioxide into protons, carbonic anhydrase IX (CAIX) is frequently regarded as a contributor to the acidification of the TME. It has been suggested that blocking CAIX is an efficient strategy to reverse local immunosuppression caused by tumor acidity (117). Zheng et al. developed a nanoplatform MnCO₃@PL/SLC, which is based on palmitoyl ascorbate (PA)-liposome (PL) loaded with Mn-doped CaCO₃ nanoparticles (Mn/CaCO₃ NPs) and CAIX inhibitor SLC-0111 (Figure 11) (86). The pH-sensitive nanoplatform is disassembled in an acidic compartment at the tumor site. Then, the simultaneous release of Mn, Ca, SLC-0111 and PA perform multiple antitumor immunotherapeutic modulation phases. In more detail, TAAs are released and subsequently activated by T cells to start the immune cycle when Mn initiates lipid peroxidation. Additionally, SLC- 0111 and PA enhance this effector T cells’ ability to invade and kill tumors. The former converts formerly cold tumors into hot tumors, and the latter provides a favorable environment for enhanced tumor-infiltrating T cells. Importantly, Mn enhances the all-immunity cycle by encouraging activation of the cGAS-STING pathway. Mn/CaCO3@PL/SLC exhibited excellent efficacy in tumor growth and metastasis with negligible adverse effects in the melanoma tumor model. Consequently, the nanoparticles enable a comprehensive intervention in all stages of cancer immunotherapy, allowing a continuous entry into a new immune cycle, which can undoubtedly improve the effectiveness of immunotherapy for tumor regression.

Figure 11.

Schematic illustration of the construction of Mn/CaCO₃@PL/SLC, which strengthens the all-immunity-cycle by causing ICD of tumor cells, regulating the immunosuppressive TME and enhancing the activity of tumor-infiltrating T lymphocytes (Reproduced with permission from Ref. 86, Elsevier). ICD, immunogenic cell death; TME, tumor microenvironment; TAA, tumor-associated antigen; DC, dendritic cell.

A significant predictor of a poor clinical outcome in solid tumors, hypoxia has also been linked to increased chemo- and radiation resistance. The DNA damage brought on by IR is more significant when oxygen is present than when it is not. This damage can be caused by IR radiation directly destroying macromolecules or by interacting with water to form hydroxyl (·OH) and hydrogen (·H) radicals (118). Therefore, overcoming hypoxia is among the most important attempts to enhance the antitumor effects of radiotherapy. In one case, a TA-based nanoplatform (TMA-NPs) was by chelating Mn²⁺ and loading STING agonist for the combined paradigm of immunotherapy and RT in treating tumors (87). Mn²⁺ antagonizes hypoxia in TME by catalyzing the decomposition of hydrogen peroxide to oxygen. While exposed to X-rays, a sufficient oxygen level can raise the quantity of ROS and enhance the antitumor efficacy with X-ray irradiation. Furthermore, the combination of RT and TMA-NPs significantly activates the cGAS-STING pathway. Thus, both localized and systematic antitumor immunity is significantly launched.

Summary and future perspectives

The cGAS-STING pathway is critical in the anticancer immune process as a natural bridge between innate and adaptive immunity (8). However, its dual role in tumorigenesis and immunity, makes it necessary to avoid high doses or sustained action of STING agonists in therapy, avoiding the risk of promoting inflammation-driven tumorigenesis due to uncontrolled activation of STING (119). Until now, most STING agonist studies have not encountered this problem, as treatment uses a single or small dose, resulting in a flare-up of IFN that favors immune activation (28). The main role of Mn²⁺ in the cGAS-STING pathway is to increase the sensitivity of the cGAS-STING signal to reduce the dosage of combined STING agonists or antitumor immunotherapy drugs, thus reducing the side effects brought by these drugs. For the application of the cGAS-STING pathway in brain tumors, the accumulation of excessive manganese ions in the brain may cause toxicity to the central nervous system. Therefore, the balance between manganese’s therapeutic and toxic effects is open to question. So, before clinical use of manganese-based STING-agonist in anticancer immunotherapy, it is necessary to conduct a thorough investigation of this pathway’s function in many types of tissues to achieve a good balance between obtaining an optimal antitumor effect and minimizing the side-effects. Various manganese-based STING-agonists have been explored concerning antitumor immunity and their therapeutic effects in combination therapy (23).

The World Health Organization recommends a concentration of manganese in drinking water <400 mg/L. In addition, manganese is unlikely to pose a risk to healthy adults when the dietary intake of manganese is 9.2 mg/d (120). Since the concentration of manganese that leads to immune cell death is relatively high, often requires up to 0.5 mmol/L, and a very weak manganese ion signal (2 μmol/L) is sufficient to sensitize the cGAS-STING pathway. But a high manganese concentration can directly affect the cell survival rate, while affecting the cell-level experimental validation. In addition, high concentrations of manganese intake may bring the risk of metal accumulation in the body leading to Parkinson’s. Therefore, for experimental feasibility and safety reasons, the concentration of manganese ions in most current nanoplatforms usually does not reach such high concentrations. The Mn-based paradigms discussed in this article are considered safe since they could promote antitumor therapies with blood Mn levels within the physiological range. In addition, the simplicity, stability, affordability and abundance of manganese make it promising and powerfully attractive for cancer therapeutic applications (68). Mn may be used alone or in combinations and is non-immunogenic and safe in the nano-delivery system compared with other STING nanoagonists. Overall, its advantages for cGAS-STING activation cannot be replaced.

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.