Multifunctional Manganese‐Based Nanocomposites for Enhanced Immunotherapy by Regulating Lactate Metabolism and Activating the cGAS‐STING Pathway

ABSTRACT

The efficacy of tumor immunotherapy is frequently hindered by immunosuppressive tumor microenvironments (TME). Activating the cyclic guanosine monophosphate‐adenosine monophosphate synthase stimulator of interferon genes (cGAS‐STING) pathway and depleting the tumor metabolic byproduct lactate offer promising strategies to reverse the immunosuppressive TME and enhance immunotherapy efficacy. In this study, multifunctional honeycomb manganese oxide nanocomposites (AL@hM) loaded with banoxantrone (AQ4N) and lactate oxidase are designed to activate the cGAS‐STING pathway and deplete lactate for achieving potent cancer immunotherapy. Upon internalization by tumor cells, lactate oxidase catalyzes the oxidation of lactate, generating hydrogen peroxide and exacerbating hypoxia, thereby reducing lactate levels within both tumor cells and the TME. This lactate depletion promotes cGAS activation and immunosuppressive TME reprogramming. Concurrently, hypoxia‐mediated toxic 1,4‐diazabicyclo[2.2.2]octane‐1,4‐dione (AQ4) production and manganese ions‐driven reactive oxygen species generation facilitate the accumulation of cytosolic double‐stranded DNA (dsDNA). These processes collectively enhance the sensitivity of manganese ions‐dsDNA‐cGAS recognition, leading to robust activation of the cGAS‐STING pathway and strong immune response. This study presents an innovative strategy that combines activation of the cGAS‐STING pathway and lactate metabolism regulation to remodel the tumor immune microenvironment for potentiating immunotherapy outcomes.

The multifunctional honeycomb manganese oxide nanocomposite (AL@hM) synergistically activates the cGAS‐STING pathway by inducing cytoplasmic dsDNA accumulation, activating cGAS through lactate depletion, and enhancing cGAS‐dsDNA recognition with Mn²⁺. This synergistic effect significantly reverses the immunosuppressive tumor microenvironment and enhances immunotherapy efficacy.

1. Introduction

In recent decades, cancer immunotherapy has achieved significant progress [1, 2]. However, its efficacy is markedly impeded by the immunosuppressive tumor microenvironment (TME) and insufficient immune system stimulation [3, 4]. Recent studies demonstrated that the cyclic guanosine monophosphate‐adenosine monophosphate synthase stimulator of interferon genes (cGAS‐STING) pathway has emerged as a critical innate immune regulator and a promising target for overcoming immunoresistance in solid tumors [5, 6, 7]. The cGAS‐STING pathway is initiated by cGAS, a cytosolic DNA sensor that recognizes cytosolic double‐stranded DNA (dsDNA), leading to the synthesis of 2′, 3′‐Cyclic GMP‐AMP (cGAMP), which subsequently activates STING [8, 9]. STING recruits and activates tank‐binding kinase 1 (TBK1), leading to the phosphorylation of both STING and the transcription factor interferon regulatory factor 3 (IRF3). This cascade ultimately induces the secretion of type I interferons (IFN‐I) and other cytokines [10, 11, 12]. Although STING functions as a tumor suppressor within cells, it is primarily found in an inactive state in various cancers. Therefore, key strategies to activate the cGAS‐STING pathway include enhancing cGAS activation, increasing cytoplasmic dsDNA levels, and improving the binding affinity between cGAS and dsDNA. These approaches boost cGAMP production and potentiate its interaction with STING [13, 14, 15].

Manganese ions (Mn²⁺) serve as a critical agonist in the robust activation of the cGAS‐STING pathway. Mechanistically, Mn²⁺ markedly increases cGAS sensitivity to cytosolic dsDNA, encompassing cytosolic nuclear DNA and mitochondrial DNA (mtDNA). This interaction lowers the threshold for DNA recognition, triggers the conformational activation of cGAS, and subsequently amplifies downstream signaling cascades [16, 17, 18]. Manganese oxides (MnO₂) nanomaterials have garnered significant attention in biomedical applications and tumor immunotherapy due to their favorable properties, such as facile synthesis, non‐toxicity, excellent biocompatibility, and biodegradability [19, 20]. As a prototypical MnO₂ nanostructure, honeycomb manganese oxide (hMnO₂) nanospheres possess high loading capacity and unique TME‐responsiveness, enabling a Mn²⁺‐mediated Fenton‐like reaction that drives hydroxyl radical (•OH) accumulation. This •OH generation induces mitochondrial dysfunction and mtDNA damage, resulting in the cytosolic leakage of mtDNA fragments [21, 22, 23]. Nevertheless, the quantity of mtDNA produced solely through ROS‐mediated stress is frequently inadequate to elicit potent cGAS‐STING pathway activation. Although DNA‐damaging agents can enhance cytosolic dsDNA production and accumulation, standalone activation of the cGAS‐STING pathway is typically insufficient to reverse the immunosuppressive TME, which is further intricately regulated by metabolic networks [24, 25, 26].

Reprogramming of energy metabolism is a well‐recognized hallmark of cancer. To meet their high metabolic demands, many cancer cells preferentially rely on aerobic glycolysis. This process, known as the Warburg effect, results in substantial lactate production. Lactate accumulation in the TME is a key contributor to its immunosuppressive phenotype [27, 28, 29]. The lactate‐enriched TME shaped by cancer cells impairs the antigen‐presenting function of dendritic cells (DCs), induces dysfunction in tumor‐infiltrating T cells, and polarizes tumor‐associated macrophages toward the M2 phenotype, thereby conferring a tumor‐promoting advantage in vivo [30, 31, 32]. Recent studies have established that lactate‐induced cGAS lactylation impairs its electrostatic binding to dsDNA, resulting in suppressed cGAMP generation and dampened downstream signaling [33, 34]. Therefore, depleting lactate in the TME represents a promising therapeutic strategy to reactivate the cGAS‐STING pathway and alleviate immunosuppression [35]. Lactate oxidase (LOx) specifically catalyzes the oxidation of lactate to pyruvate and hydrogen peroxide (H₂O₂), with concurrent consumption of oxygen (O₂). This process facilitates H₂O₂‐dependent Fenton‐like reactions and improves the efficacy of hypoxia‐activated prodrugs [36, 37, 38].

Herein, we designed a versatile nanosystem, denoted AL@hM, which acts as both a cGAS‐STING nanoagonist and an immunosuppressive TME regulator to enhance cancer immunotherapy. As illustrated in Scheme 1A, the hMnO₂ nanoparticles were synthesized using an oleic acid (OA)‐templated reduction method, enabling precise control of their morphology and porosity. Banoxantrone (AQ4N) was encapsulated into the hMnO₂ framework via electrostatic interaction. Polyvinylpyrrolidone (PVP)‐modified LOx was anchored to the hMnO₂ surface through stable hydrogen bonds formed between the oxygen atoms of PVP and the hydroxyl groups on hMnO₂ (Figure 1B). Upon internalization by tumor cells, AL@hM undergoes degradation in response to elevated glutathione (GSH) levels and the acidic TME (Scheme 1B). LOx‐mediated lactate depletion concomitantly induces H₂O₂ production and exacerbates tumor hypoxia. The combined effects of •OH generated via Mn‐based Fenton‐like reactions to release mitochondrial DNA (mtDNA) and hypoxia‐mediated activation of cytotoxic AQ4 induce severe DNA damage to release abundant cytosolic nuclear DNA. Concurrently, decreased lactate levels alleviate cGAS lactylation, thereby promoting cGAS activation. Furthermore, Mn²⁺ acts as a metallic agonist to enhance the binding affinity between cGAS and dsDNA. These synergistic effects activate the cGAS‐STING pathway and induce IFN‐β secretion, leading to the maturation of DCs, M1 polarization of macrophages, and infiltration of CD8⁺ T cells. Thus, this study presents a versatile and effective nanoplatform that harnesses the synergy between cGAS‐STING pathway activation and metabolic reprogramming to enhance anticancer immunotherapy efficacy.

SCHEME 1.

Schematic illustration of the AL@hM nanosystem for enhanced cancer immunotherapy. (A) Fabrication process of the AL@hM nanosystem. (B) Therapeutic mechanism of AL@hM through lactate metabolism‐amplified manganese ions‐dsDNA‐cGAS sensing axis to potentiate cGAS‐STING pathway activation for stimulating the immune response and enhancing the efficacy of immunotherapy.

FIGURE 1.

(A) TEM images of AL@hM. (B) Schematic diagram illustrating the mechanism of PVP‐modified LOx fastened on the surface of A@hM via a hydrogen bond to construct AL@hM. (C) STEM‐HAADF image of AL@hM and the corresponding elemental mapping image. (D) Full XPS spectra of AL@hM. (E) The high‐resolution Mn 2p XPS of AL@hM. (F) N₂ adsorption‐desorption isotherms of hMnO₂ and AL@hM. (G) The pore size distribution of hMnO₂ and AL@hM. (H) FT‐IR of hMnO₂, AQ4N, LOx, AL@hM. (I) XRD pattern of AL@hM. (J) Hydrodynamic diameter distribution of hMnO₂, A@hM, L@hM, and AL@hM. (K) The hydrodynamic size changes of AL@hM dispersed in PBS, H₂O, RPMI 1640 medium, and serum for 16 days. Data represent means ± SD (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

2. Results and Discussion

2.1. Characterization of AL@hM

To construct the AL@hM nanosystem, hMnO₂ nanocarriers were first prepared via a templated reduction method employing OA. Transmission electron microscopy (TEM) imaging confirmed the successful formation of hMnO₂ with a well‐defined honeycomb‐like architecture, which consisted of interconnected lamellar MnO₂ platelets (Figure S1A). The porous structure of hMnO₂ was then utilized to load AQ4N through electrostatic adsorption to obtain AQ4N‐loaded hMnO₂ (A@hM). Separately, lactate oxidase (LOx) was conjugated to the surface of the resulting A@hM to obtain AL@hM via hydrogen bonding, as supported by previous reports [39, 40, 41]. The morphologies of the intermediate products (A@hM (Figure S1B) and L@hM (Figure S1C) and the final AL@hM (Figure 1A) nanosystem were well preserved compared to the pristine hMnO₂. A critical challenge was to immobilize LOx onto the A@hM surface with high efficiency while preserving its enzymatic activity. To address this, we employed polyvinylpyrrolidone (PVP), a biocompatible polymer, as an adaptor molecule for two key reasons: (1) PVP acts as a capping agent capable of forming a stable PVP/protein complex [40, 41], and (2) its abundant carbonyl groups facilitate directional anchoring of LOx to the A@hM surface via hydrogen bonding (Figure 1B). The PVP modification did not compromise the catalytic activity of LOx toward lactate (Figure S2). High‐angle annular dark‐field scanning transmission electron microscopy (HAADF‐STEM) and corresponding energy‐dispersive X‐ray spectroscopy (EDS) elemental mapping confirmed the homogeneous distribution of C, N, O, and Mn throughout the AL@hM nanosystem (Figure 1C). The survey X‐ray photoelectron spectroscopy (XPS) spectrum of AL@hM also confirmed the presence of Mn, O, N, and C elements (Figure 1D). The high‐resolution Mn 2p spectrum displayed two distinct peaks at 653.07 and 641.35 eV, corresponding to the 2p_1/2 and 2p_3/2 of Mn⁴⁺, respectively (Figure 1E). Deconvolution of the N 1s spectrum showed a predominant peak at 399.7 eV (Figure S3), which is assigned to N─H bonds, thereby verifying the successful incorporation of AQ4N.

The nitrogen adsorption‐desorption isotherms of hMnO₂ and AL@hM are presented in Figure 1F. The Brunauer–Emmett–Teller (BET) surface area was determined to be 169.54 m²/g for hMnO₂ and 89.92 m²/g for AL@hM. The corresponding pore volume decreased from 0.51 cm³/g (hMnO₂) to 0.12 cm³/g (AL@hM) (Figure 1G). This substantial reduction in surface area and pore size confirms the successful encapsulation of AQ4N and LOx within the porous structure. Fourier transform infrared (FT‐IR) spectroscopy confirmed the presence of key functional groups from the encapsulated components in AL@hM (Figure 1H). Specifically, the amide I band at 1620–1650 cm⁻¹, corresponding to C═O stretching vibrations in LOx, was clearly observed. The distinct phenolic C─O stretching and benzene ring vibrations of AQ4N were presented between 1180–1360 cm⁻¹, while the broad O─H stretching mode of phenolic hydroxyl groups appeared in the 3090–3320 cm⁻¹ range. Significantly, the preservation of these vibrational signatures of the C═O, phenolic O─H, and aromatic ring vibrations was also presented in the AL@hM spectrum, providing conclusive evidence for the successful encapsulation of both LOx and AQ4N within the hMnO₂ nanostructure. The crystallographic structure of AL@hM was confirmed by powder X‐ray diffraction (XRD). The XRD pattern exhibited characteristic diffraction peaks at 2θ values of 12.16°, 24.57°, 36.52°, and 65.82°, correspond to the (001), (002), (100), and (110) crystallographic planes (Figure 1I), respectively, consistent with the standard pattern of MnO₂ [20, 41].

Dynamic light scattering (DLS) analysis confirmed that AL@hM possessed a hydrodynamic diameter of 151.7 ± 2.14 nm (Figure 1J). The surface zeta potential of the nanomaterials became progressively more negative following the stepwise loading of the therapeutic agents (Figure S4). The loading capacities of LOx and AQ4N in AL@hM were quantitatively determined using UV–vis spectroscopy and corresponding standard curves (Figure S5). The encapsulation efficiency was determined to be 10.6 ± 0.14 wt.% for LOx and 3.26 ± 0.05 wt.% for AQ4N. The Mn content in the AL@hM was determined to be 42.3% by inductively coupled plasma mass spectrometry (ICP‐MS). In addition, AL@hM demonstrated superior colloidal stability in physiological media, with the hydrodynamic diameter varying within ± 10% throughout a 16‐day incubation period (Figure 1K). This sustained nanostructural integrity underscores its potential for intravenous administration and targeted drug delivery.

2.2. The Performance Evaluation of AL@hM

To systematically investigate the biodegradation behavior of AL@hM in response to the TME, physiological and TME‐like conditions were simulated (Figure 2A). AL@hM maintained its structural integrity under pH 7.4 without GSH, whereas its honeycomb‐like architecture underwent significant degradation under mildly acidic conditions following 12 h of incubation (Figure 2A). Importantly, the combined effect of mildly acidic conditions and GSH resulted in complete structural disintegration of AL@hM. This pH and GSH dual‐responsive degradation mechanism is consistent with the requirements for tumor‐specific drug release. Notably, under TME‐mimetic conditions, the liberation of Mn ions exhibited a time‐dependent profile, with 82.1% release efficiency achieved within 24 h (Figure 2B). Under physiological conditions, AL@hM demonstrated sustained drug retention, with cumulative releases of merely 9.8% (Figure 2C) and 8.6% (Figure S6) over 24 h. In contrast, the release of AQ4N and LOx reached 76.4% and 70.8%, respectively, within 24 h under mildly acidic conditions with 10 mM GSH, representing a 7.8‐fold increase in release efficiency compared to physiological conditions. This significant enhancement confirms that AL@hM maintains circulatory stability while enabling tumor‐specific activation of therapeutic agents. It was reported that LOx catalyzes intracellular lactate and O₂ into H₂O₂ and pyruvate. Co‐incubation of lactate solution with AL@hM resulted in a 61.85% reduction in lactate levels compared to the control group after 30 min (Figure 2D), indicating its lactate‐depleting capacity. LOx activity was assessed by incubating AL@hM with lactate solution for varying durations. AL@hM has been observed to produce more H₂O₂ with higher lactate concentrations (Figure 2E) and longer incubation times (Figure S7). This enhanced H₂O₂ generation suggests potential to improve chemodynamic therapy (CDT) efficacy and promote a hypoxic tumor microenvironment.

FIGURE 2.

(A) TEM images of AL@hM after various treatments in different time intervals. (B) Accumulated releasing profiles of Mn ions from AL@hM under different conditions. (C) Accumulated releasing profiles of AQ4N from AL@hM under different conditions. (D) Lactate consumption ability of different nanomaterials. (E) Changes of H₂O₂ concentration after various treatments with various concentrations of lactate. (F) MB degradation after AL@hM incubation with different concentrations of GSH. (G) MB degradation after AL@hM incubation with different concentrations of lactate. (H) ESR spectra of hydroxyl radical trapped by DMPO under different GSH concentrations in the presence of 30 mM lactate using AL@hM. (I) The depletion of GSH (10 mM) after treatment with different concentrations of AL@hM. Data represent means ± SD (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

The mildly acidic environment and elevated GSH levels synergistically facilitated the disintegration of AL@hM nanoparticles, releasing Mn ions to initiate Fenton‐like catalytic reactions. As shown in Figure 2F, the methylene blue (MB) absorption peak at 653 nm gradually decreased with increasing GSH concentration. This phenomenon is attributed to the Mn²⁺ in solution also gradually increased, which was able to react with H₂O₂ to produce •OH. Moreover, MB degradation was dependent on lactate concentration (Figure 2G). Electron spin resonance (ESR) spectroscopy employed 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) as •OH spin trap, revealing characteristic 1:2:2:1 quartet signals, showing remarkable consistency with the MB degradation profiles (Figure 2H). These results demonstrate the significant Mn²⁺ release and •OH production of AL@hM under mildly acidic conditions with elevated lactate and GSH levels. To quantitatively assess the GSH‐depleting capacity of hMnO₂, DTNB was employed as a colorimetric probe. As demonstrated in Figure 2I, TNB absorbance at 412 nm was decreased with increasing concentrations of AL@hM, demonstrating the consumption of GSH through redox interaction.

2.3. In Vitro Antitumor Efficacy Evaluation

Building on the enhanced catalytic performance and drug release capabilities, the intracellular behavior of AL@hM was further investigated. Initially, the cellular uptake of Cy5.5‐labeled AL@hM was assessed. As shown in Figure 3A, the red fluorescence of Cy5.5 was prominently observed in 4T1 cells within 2 h of incubation. With increasing incubation time, the fluorescence intensity of Cy5.5 was concomitantly increased, revealing a time‐dependent cellular uptake profile. MTT assays were conducted to evaluate the ability of AL@hM for suppressing tumor cell proliferation. Compared to normoxic conditions, the viability of 4T1 cells was significantly reduced after treatment with AL@hM under hypoxic conditions (Figure 3B). Increasing AL@hM concentrations resulted in progressive decreases in cell viability under hypoxic conditions, indicating that the toxicity of AQ4 gradually increases in a concentration‐dependent manner (Figure S8). Notably, AL@hM exhibited minimal cytotoxicity toward L929 cells under physiological conditions, demonstrating the biological safety of nanoparticles (Figure S9). When different nanomaterials were co‐incubated with 4T1 cells for 12 or 24 h, the AL@hM‐treated group demonstrated significantly greater lactate depletion than other groups (Figure 3C). Furthermore, significant lactate levels were detected in the culture supernatant of 4T1 cells (control group). In contrast, LOx, L@hM, and AL@hM‐treated groups exhibited significantly reduced extracellular lactate levels (Figure 3D). Rescue experiments demonstrated that exogenous lactate supplementation reversed the antiproliferative effects of AL@hM (Figure S10). The restoration of intracellular lactate levels correlated with recovered cell viability, confirming that the nanoparticles inhibit tumor growth via lactate depletion. Collectively, these results indicate that AL@hM accelerates apoptosis by depleting intracellular lactate.

FIGURE 3.

(A) Cellular uptake of AL@hM following incubation for 2 h and 6 h. (B) Cell viability in 4T1 cells after different treatments, hMnO₂ (70 µg mL⁻¹), LOx (6.5 µg mL⁻¹) + AQ4N (0.2 µg mL⁻¹), A@hM (70 µg mL⁻¹), L@hM (70 µg mL⁻¹), or AL@hM (70 µg mL⁻¹). (C) The changes of lactate content in the supernatant of the cell culture medium after various treatments. (D) The extracellular concentration of lactate after 4T1 cells treatment with different nanomaterials under hypoxic conditions. (E) CLSM images of hypoxia levels in 4T1 cells after different treatments under a hypoxic environment. (F) CLSM images of ROS content after different treatments. (G) Mitochondrial transmembrane potential analysis in 4T1 cells under different treatment conditions. (H) Bio‐TEM characterization of mitochondrial structural alterations in 4T1 cells treated with PBS or AL@hM. (I) Immunofluorescence detection of oxidative DNA damage (8‐OHdG, red) in 4T1 cells treated with different nanoagonists. Mitochondria and nuclei were labeled with TOMM20 (green) and DAPI (blue), respectively. Data represent means ± SD (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Subsequently, LOx‐induced hypoxia exacerbation was assessed using hypoxia probes to monitor intracellular responses. The results demonstrated that 4T1 cells treated with LOx, LOx@hMnO₂, and AL@hM exhibited pronounced red fluorescence, indicating significant intracellular hypoxia (Figure 3E). LOx‐mediated oxygen consumption intensified hypoxia, facilitating the conversion of non‐toxic AQ4N to toxic AQ4 and enhancing chemotherapeutic effects. Intracellular ROS accumulation after different treatments was evaluated using the 2’,7’‐dichlorodihydrofluorescein diacetate (DCFH‐DA) probe. As shown in Figure 3F, 4T1 cells incubated with LOx@hMnO₂ and AL@hM exhibited stronger DCF fluorescence, suggesting that intracellularly generated hydrogen peroxide is further converted into hydroxyl radicals. Dynamic changes in GSH levels accompanied the continuous accumulation of intracellular ROS. Notably, the GSH level in the AL@hM‐treated group was significantly depleted compared to other groups, confirming the catalytic activity of Mn²⁺ released from the nanosystem for efficient Fenton‐like reactions (Figure S11).

Meanwhile, JC‐1 staining was employed to evaluate AL@hM‐induced mitochondrial destabilization by monitoring changes in mitochondrial membrane potential (Figure 3G). Cells treated with PBS exhibited negligible green fluorescence and strong red fluorescence. In contrast, AL@hM treatment induced significant mitochondrial depolarization, characterized by strong green fluorescence and attenuated red aggregates. Biological transmission electron microscopy (bio‐TEM) was employed to assess mitochondrial integrity. As depicted in Figure 3H, control cells displayed intact mitochondrial structures with distinct cristae. In contrast, AL@hM treatment induced severe structural damage, characterized by shrunken mitochondria, diminished cristae, and disrupted outer membranes, indicating substantial mitochondrial impairment. To further characterize mitochondrial stress, mtDNA damage was evaluated using 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG) immunofluorescence. CLSM images revealed that the AL@hM group exhibited the strongest 8‐OHdG signal colocalized with mitochondria, substantiating substantial oxidative mtDNA damage (Figure 3I). Considering that compromised mitochondrial integrity facilitates mtDNA translocation to the cytosol through permeability transition pores, cytosolic mtDNA levels were quantified via RT‐qPCR (Figure S12). Notably, the AL@hM group displayed a pronounced elevation in cytosolic mtDNA, consistent with its superior ROS generation capacity compared to other nanoagonists. The apoptotic effect was further investigated via Annexin V‐FITC/PI staining and flow cytometry. The live/dead staining assay confirmed the cytotoxicity of AL@hM. In CLSM images, both PBS‐ and hMnO₂‐treated 4T1 cells displayed strong green fluorescence, with negligible red fluorescence, suggesting minimal apoptosis in either group (Figure S13A). In contrast, red fluorescence was observed in AQ4N+LOx‐treated cells due to hypoxia exacerbated by LOx‐lactate reactions that promoted the generation of toxic AQ4. AL@hM exhibited the strongest cytotoxicity, driven by the synergistic effects of toxic AQ4 and ROS. Compared to other groups, the AL@hM‐treated group showed the lowest survival rate and highest apoptosis ratio, consistent with MTT and live/dead staining results (Figure S13B). The minimal catalytic effect of AL@hM under physiological conditions minimizes systemic toxicity, highlighting its potential as a promising therapeutic approach for cancer treatment.

2.4. In Vitro cGAS‐STING Pathway Activation and DC Maturation

The ability of AQ4 to induce cellular DNA damage has been well‐established, with recent studies highlighting the critical role of cGAS in sensing such damage [42, 43, 44]. Mn²⁺ accumulation and lactate consumption have been shown to activate the cGAS‐STING pathway [45, 46]. These findings support the hypothesis that AL@hM synergistically activates the cGAS‐STING pathway in tumor cells, upregulating IFN‐β secretion and promoting DC maturation (Figure 4A). To confirm AQ4 activation and Mn‐related ROS cause nuclear DNA fragmentation, the nuclear DNA damage was evaluated. AL@hM treatment caused significant nuclear DNA damage (Figure 4B). We further quantified cytosolic dsDNA release using PicoGreen. AL@hM‐treated cells resulted in higher dsDNA release compared to other treatment groups (Figure S14). This direct increase in dsDNA release confirms the damaging effects of AQ4 and ROS, thereby promoting cytosolic DNA release and subsequent activation of the cGAS‐STING pathway.

FIGURE 4.

(A) A schematic illustration depicting the activation of the cGAS‐STING pathway and the maturation of DCs induced by AL@hM. (B) Immunofluorescence visualization of nuclear DNA damage in 4T1 cells using γ‐H2AX (red) and DAPI (blue) staining after different treatments. CLSM images of (C) CRT exposure and (D) HMGB1 release in 4T1 cells following various treatments. Representative expression levels of cGAS‐STING pathway‐related proteins in (E) 4T1 cells and (F) BMDCs after various treatments. (G) Relative secretion levels of IFN‐β in 4T1 cells after different treatments. (H) Representative flow cytometry analysis of mature DCs (CD80⁺CD86⁺ gated on CD11c⁺) after treating BMDCs with various treatments for 24 h. Data represent means ± SD (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Previous studies have established that lactate inhibits cGAS via lactylation, thereby suppressing the cGAS‐STING signaling pathway [47, 48, 49]. Given the significant difference in lactate depletion between A@hM and AL@hM, the activation of the cGAS‐STING pathway in 4T1 cells was evaluated. Notably, AL@hM treatment resulted in a significant increase in intracellular cGAMP levels (Figure S15). This confirms that reducing lactate levels alleviates lactylation‐mediated inhibition, thereby restoring the capacity of cGAS to catalyze cGAMP generation. The ability of AL@hM to activate the cGAS‐STING pathway was evaluated in both cancer cells and mouse‐derived bone marrow‐derived dendritic cells (BMDCs). Western blot (WB) analysis demonstrated upregulated expression of cGAS, phospho‐STING (p‐STING), phospho‐TBK (p‐TBK1), and phospho‐IRF3 (p‐IRF3) in 4T1 cells and BMDCs after different treatments. Notably, AL@hM‐treated cells exhibited the most pronounced increase in protein expression, highlighting the synergistic effects of elevated cytosolic Mn²⁺ levels, cytosolic dsDNA accumulation, and lactate depletion in efficiently activating the cGAS‐STING signaling pathway. This effect was observed in both 4T1 cells (Figure 4E and Figure S16) and BMDCs (Figure 4F and Figure S17). Following cGAS‐STING pathway activation, the secretion of IFN‐β was quantified. IFN‐β levels in the culture supernatants of 4T1 cells and BMDCs were significantly higher in the AL@hM group compared to control and other treatment groups (Figure 4G and Figure S18). These results confirm that AL@hM effectively delivers Mn²⁺, AQ4N, and LOx to cells, thereby promoting cGAS‐STING pathway activation and which further triggers downstream anti‐tumor immune responses.

ROS and AQ4 trigger immunogenic cell death (ICD), resulting in the release of damage‐associated molecular patterns (DAMPs). These DAMPs stimulate cytotoxic T lymphocyte (CTL) infiltration, enhance antigen‐presenting cell (APC) maturation, and ultimately prime anti‐tumor immune responses through coordinated immunomodulatory cascades. To evaluate ICD induction, 4T1 cells were treated with PBS (control), hMnO₂, LOx+AQ4N, AQ4N@hMnO₂, LOx@hMnO₂, and AL@hM under acidic TME conditions containing lactate. The hallmark features of ICD, including calreticulin (CRT) surface exposure, high mobility group box 1 (HMGB1) release, and adenosine triphosphate (ATP) secretion, were analyzed. CRT exposure and HMGB1 release were visualized via CLSM (Figure 4C,D), while ATP levels were quantified by enzyme‐linked immunosorbent assay (ELISA) (Figure S19). Compared to other treatment groups, cells treated with AL@hM exhibited significantly elevated ICD markers. These results demonstrate that AL@hM effectively induces ICD and subsequently promotes immune activation.

To investigate this immunoregulatory potential, 4T1 cells were pre‐treated with various nanomaterials before co‐culture with BMDCs. Flow cytometric analysis revealed that BMDCs exposed to AL@hM exhibited a marked increase in the CD80⁺CD86⁺ population cells compared to control groups, demonstrating superior DC maturation capacity (Figure 4H and Figure S20). These experimental validation underscores that cGAS‐STING pathway activation drives the functional maturation of DCs, establishing critical prerequisites for initiating adaptive T‐cell mediated anti‐tumor immunity. To elucidate the mechanisms by which AL@hM modulates the TME, we investigated the polarization effects of AL@hM on RAW264.7 macrophages, given its abundance and critical role in tumor immunosurveillance. As shown in Figure S21, A@hM‐treated cells induced a modest upregulation of CD86. Notably, AL@hM‐treated cells elicited a significantly more pronounced upregulation of CD86, indicating a stronger M1‐polarizing effect. These findings demonstrate that AL@hM can promote macrophage repolarization toward an M1 phenotype, which attributable to its lactate‐depleting capability.

2.5. In Vivo Biodistribution, Biosafety, and Single‐Cell Sequencing Analysis

To evaluate hemocompatibility, hemolysis ratios were quantified at varying concentrations of AL@hM through a hemolysis assay. The results demonstrated observable red coloration in the supernatant even at high concentrations, indicating minimal hemolysis and superior hemocompatibility (Figure 5A). Next, the biodistribution of AL@hM in 4T1 tumor‐bearing mice was analyzed via fluorescence imaging of major organs and tumor sites following intravenous injection. Fluorescence intensity peaked at 6 h post‐injection and gradually declined over time, remaining detectable at 24 h, suggesting prolonged accumulation at the tumor site for sustained antitumor activity (Figure 5B and Figure S22A). After 24 h, the mice were sacrificed, major organs and tumor tissues were harvested, and fluorescence imaging was performed (Figure S22B,C). The results confirmed significant tumor‐specific enrichment of AL@hM. Following intravenous administration, the pharmacokinetic profile of AL@hM was systematically assessed, with comprehensive hematological and serum biochemical analyses conducted to evaluate systemic toxicity. Pharmacokinetic parameters were determined within 24 h of injection, revealing a blood circulation half‐life of 5.21 h (Figure 5C). Moreover, no significant toxic effects were observed in hematological and serum biochemical parameters after mice were treated with AL@hM at different time points, further confirming the safety profile of AL@hM (Table S1). Next, the in vivo lactate depletion efficacy of AL@hM was explored. After 24 h post‐intravenous injection, the tumor tissues were excised and homogenized for lactate quantification. As expected, the administration of free LOx did not induce lactate exhaustion in the tumor site, attributable to its lack of tumor‐targeting ability. Conversely, due to the intrinsic tumor‐targeting and enhanced retention characteristics of L@hM and AL@hM, lactate concentrations in mice treated with these nanocomposites were markedly reduced compared to control and LOx‐treated groups (Figure S23). Collectively, these results demonstrated that AL@hM effectively accumulates in tumor sites and exhausts intratumor lactate.

FIGURE 5.

(A) Hemolysis assays and corresponding photographs (inset) at varying concentrations of AL@hM. (B) Fluorescence imaging of mice after intravenous injection of AL@hM. (C) Blood‐circulation lifetime of AL@hM, where t_1/2 is the half‐life of Mn in the bloodstream. (D) Volcano plot of differentially expressed genes. (E) Clustering heatmap of gene expression after receiving AL@hM treatment (Red: up‐regulation; blue: down‐regulation). (F) Top 15 significantly enriched KEGG pathways that were up‐regulated. (G) GO enrichment analysis of tumor tissues following AL@hM treatment. (H) GSEA map of cytokine receptor binding comparing control and AL@hM treatment. (I) GSEA map of T cell receptor signal pathway comparing control and AL@hM treatment. Data represent means ± SD (n = 3).

Following confirmation of superior tumor targeting and biosafety, the RNA‐level biological responses and antitumor mechanisms of AL@hM were investigated. Transcriptome analysis of AL@hM‐treated tumor tissues was performed using RNA sequencing (RNA‐seq) to systematically evaluate the regulatory mechanisms underlying anti‐tumor immune responses. Volcano plots revealed significant alterations in gene expression between the control group and the AL@hM‐treated group after 24 h post‐treatment (Figure 5D). Notably, AL@hM induced marked changes in gene expression, including upregulation of interferon response genes (such as Ifngr2), pro‐inflammatory cytokine genes (such as Il24 and Il18r1), and leukocyte‐recruiting chemokine genes (such as Cxcr1, Cxcr2, Cxcl9, and Cxcl10), as well as downregulation of immunosuppressive genes (such as Havcr2, Atg12, and Ilrun) (Figure 5D,E). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of immunostimulatory pathways following AL@hM treatment, including the NF‐kappa B (NF‐κB), mTOR, TNF, and Toll‐like receptor signaling pathways, as well as the membrane DNA‐sensing pathway (Figure 5F). Notably, both upstream and downstream components of the STING signaling axis were significantly activated. Gene Ontology (GO) analysis further demonstrated significant enrichment of immunomodulatory processes, particularly those associated with immune system regulation and innate immune responses (Figure 5G). Gene Set Enrichment Analysis (GSEA) revealed significant upregulation of key immunomodulatory pathways and processes, such as the T cell receptor signaling pathway and cytokine receptor binding (Figure 5H,I).

2.6. Anti‐Cancer Effect and Immunotherapy of AL@hM In Vivo

After confirming the biosafety and tumor‐targeting capabilities, the antitumor performance of AL@hM was evaluated in 4T1 tumor‐bearing Balb/c mice following intravenous administration. The treatment schedule is outlined in Figure S24A, with body weight and tumor volume monitored every 2 days. The mice treated with hMnO₂, LOx+AQ4N, showed only slightly suppressed tumor growth. AL@hM displayed the most pronounced tumor inhibition effect (Figure S24C), with a tumor growth inhibition rate of 79.34% (Figure S24D), confirming its potent antitumor activity. Notably, no significant difference in body weight was observed (Figure S24B), demonstrating the excellent biocompatibility of AL@hM. These findings collectively indicate that AL@hM exhibits potent tumoricidal effects in 4T1 tumor‐bearing mice.

Inspired by the superior tumor cell‐killing and ICD induction ability of AL@hM, we further investigated its antitumor immunotherapy effects in bilateral 4T1 tumor‐bearing mice following intratumoral administration. Initially, 4T1 cells were inoculated subcutaneously into mice, which were randomly divided into six groups when the tumor volumes reached approximately 80 mm³. The right primary tumors were treated every three days, and growth of both primary and distant tumors was monitored (Figure 6A). Initially, no significant changes in body weight were observed across all treatment groups, indicating the excellent biosafety profile of AL@hM (Figure 6B). Growth of primary and distant tumors was monitored every two days following various treatments to assess therapeutic efficacy. The AL@hM‐treated group demonstrated significant inhibition of primary tumor growth compared to other groups, highlighting its superior antitumor activity (Figure 6C–E). Given the strong immunostimulatory capacity, AL@hM also significantly suppressed distant tumor growth (Figure 6F–H). H&E staining of tumor tissues showed structural disruption and necrotic features in the AL@hM group, indicating its potent antitumor effects (Figure 6I). TUNEL immunofluorescence staining revealed the strongest green fluorescent signals in the AL@hM‐treated group, confirming extensive apoptosis (Figure 6I). Ki‐67 immunohistochemical analysis further demonstrated that AL@hM significantly reduced tumor cell proliferation (Figure 6I). Additionally, H&E staining revealed no histopathological abnormalities in major organs of AL@hM‐treated mice compared to the control group (Figure S25). Given the excellent targeting and accumulation ability of AL@hM, tumor tissue hypoxia levels were measured following treatment with various nanomaterials. Immunofluorescence analysis results indicated that the strongest red fluorescence signals were observed in L@hM‐treated and AL@hM‐treated mice, demonstrating an extensive hypoxic status in the tumor (Figure S26). These findings indicate that AL@hM effectively inhibits tumor cell proliferation through chemotherapy, chemodynamic therapy (CDT), and immunotherapy.

FIGURE 6.

(A) Schematic illustration of the treatment schedule for in vivo antitumor efficiency analysis. (B) Relative body weight changes in tumor‐bearing mice following different treatments. (C) Tumor volume growth curves of primary tumors in mice during different treatments. (D) Ex vivo photographs of primary tumors from tumor‐bearing mice treated with different formulations. (E) Weight changes of primary tumors. (F) Tumor volume growth curves of distant tumors. (G) Ex vivo photographs of distant tumors from tumor‐bearing mice treated with different formulations. (H) Weight changes of distant tumors. (I) H&E, TUNEL, and Ki67 staining of tumor tissue after 14 days of treatment. Data represent means ± SD (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

2.7. In Vivo Immune Activation Analysis

The superior antitumor efficacy of AL@hM primarily results from the synergy between chemotherapy, CDT, and immunotherapy. To elucidate the underlying immunomodulatory mechanisms, we analyzed immune cell populations and key cytokine secretion profiles using flow cytometry and ELISA assays. Initial analysis focused on DC maturation in tumor‐draining lymph nodes (TDLNs). Flow cytometry results revealed significantly higher populations of mature DCs (CD80⁺CD86⁺) in all treatment groups compared to the control, which was attributed to cGAS‐STING pathway activation by Mn²⁺, AQ4, and lactate depletion (Figure 7A and Figure S27). Notably, the AL@hM‐treated group exhibited the highest DC maturation rate, indicating that metabolic reprogramming and cytosolic dsDNA accumulation synergistically enhance immune activation. Flow cytometric analysis of tumor‐infiltrating T cells revealed significantly higher proportions of CD4⁺ and CD8⁺ T cells in the AL@hM‐treated group compared to other treatment groups, indicating enhanced T cell‐mediated adaptive immune responses (Figure 7B and Figure S28).

FIGURE 7.

(A) Representative flow cytometry analysis of mature DCs (CD80⁺CD86⁺ gated on CD11c⁺) in tumor‐draining lymph nodes. (B) Representative flow cytometry analysis of CD8⁺ T cells (CD8⁺CD4⁺ gated on CD45⁺CD3⁺) in primary tumors following various treatments. (C) Representative flow cytometry analysis of M1 macrophages (F4/80⁺CD86⁺) in primary tumors under different treatments. (D) Representative flow cytometry analysis of M2 macrophages (F4/80⁺CD206⁺) in primary tumors under various treatments. (E) IFN‐β, (F) IL‐6, (G) TNF‐α, and (H) IL‐10 levels after 14 days of treatment. Data represent means ± SD (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Further analysis of macrophage phenotypes demonstrated a marked increase in M1 macrophages following AL@hM treatment, reaching 21.0%, approximately 3.6‐fold higher than that of the control group (Figure 7C and Figure S29A). Conversely, the proportion of M2 macrophages decreased to 15.95% (Figure 7D and Figure S29B). The M1/M2 ratio in the AL@hM‐treated group was an eightfold increase compared to the control group (Figure S30). This shift in macrophage polarization reflects the functional reprogramming of tumor‐associated macrophages (TAMs) from immunosuppressive M2‐like to immunostimulatory M1‐like phenotypes. Activation of the cGAS‐STING signaling pathway by AL@hM triggered robust secretion of immunostimulatory cytokines, including IFN‐β, TNF‐α, and IL‐6 (Figure 7E–G). These cytokines play a pivotal role in reprogramming the tumor immune microenvironment and antagonizing the immunosuppressive functions of regulatory T cells (Tregs). Specifically, IFN‐β not only potentiates DC maturation and antigen presentation but also directly inhibits the proliferation and suppressive capacity of Tregs [50]. Conversely, the anti‐inflammatory cytokine IL‐10 was significantly downregulated (Figure 7H). Given that IL‐10 is a key mediator of immunosuppression that inhibits effector T cell function and promotes Treg activity, its reduction alleviates the immunosuppressive microenvironment [51]. Collectively, synergistic cGAS activation and metabolic reprogramming convert an immunologically inert TME to an immunologically active state, thereby enhancing systemic antitumor immunity.

3. Conclusions

In summary, this study presents a straightforward yet effective nanoparticle (AL@hM) for cancer immunotherapy. Within tumor cells, LOx‐mediated lactate depletion exacerbates tumor hypoxia to promote toxic AQ4 production, whereas Mn²⁺ release induces ROS accumulation. These effects collectively enhance chemotherapy and CDT efficacy while promoting dsDNA release. Lactate consumption within the TME, together with dsDNA and Mn²⁺ accumulation, significantly activates the cGAS‐STING signaling axis, leading to increased IFN‐β production and the initiation of innate immune responses. The immunoregulatory effect of AL@hM promoted DCs maturation through cGAS‐STING pathway activation. This process enhances antigen presentation, T cell priming, and M1 macrophage polarization, thereby establishing a favorable immunological landscape for antitumor immunity. In vitro and in vivo results demonstrate that AL@hM exhibits promising immunotherapeutic effects by integrating cGAS‐STING pathway activation and metabolic reprogramming to overcome tumor immunosuppression and improve therapeutic outcomes. Collectively, this study highlights the potential of multifunctional nanosystems to simultaneously modulate metabolic and immune signaling pathways, providing a novel strategy to advance precision cancer immunotherapy.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll73950‐sup‐0001‐SuppMat.pdf.

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article