Biomineralized bacterial outer membrane vesicles exert pleiotropic immunoferroptotic effects on immune-deserted liver cancer

Biomineralized bacterial outer membrane vesicles exert pleiotropic immunoferroptotic effects on immune-deserted liver cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biomineralized bacterial outer membrane vesicles exert pleiotropic immunoferroptotic effects on immune-deserted liver cancer Ying Luo, Zhongsheng Xu, Qianying Du, Lian Xu, Yi Wang, Jie Xu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4183359/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The immune landscape of late-stage liver cancer is featured by severe immunosuppression that is characterized by poor immunogenicity, T-cell exhaustion, and infiltration of a large number of immunosuppressive cells, leading to compromised therapeutic efficacy of mainstream immunotherapies. Herein, we developed a pleiotropic immunoferroptotic mobilization strategy to treat intractable advanced liver cancer. In this study, immunogenic bacteria-derived outer membrane vesicles (OMVs) were exploited as a vector to deposit Cu and Mn with mixed valence states via one-step biomineralization, followed by platelet membrane camouflage to enhance the circulation time and reduce systemic side effects of the metal biomineralized OMVs, which are denoted as OPCM. Mechanistically, the metal-deposited OPCM possesses POD, CAT, and GPX-like activities, thereby stimulating immunogenic ferroptosis, cGAS-STING activation, and tumor hypoxia alleviation, ultimately leading to pleiotropic immunoferroptotic mobilization to combat tumor growth, recurrence, and metastasis. Notably, the combination with typical αPD-L1 augmented the tumor suppression effect, since the administration of αPD-L1 not only rescued exhausted T cells, but also amplified the intensity of ferroptosis due to IFN-γ secretion by activated T cells. Overall, the metal biomineralized OPCMs in combination with αPD-L1 formed a closed-loop therapy that cycles from immunotherapy and ferroptosis therapy, providing new insights for treating immunosuppressive liver cancer. outer membrane vesicle (OMV) metal ions ferroptosis cCAS-STING immunotherapy liver cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction In clinical settings, late-stage liver cancer patients display low response rates to a variety of treatments, including chemotherapy, radiotherapy, and anti-angiogenetic therapy. [1] Immunotherapy, such as immune checkpoint therapy, is an evolving therapeutic paradigm for cancer treatment, however, its clinical response rate for liver cancer is only estimated to be around 15%, which entails further improvement. [2] The low response rate to mainstream immunotherapy is mainly attributed to the severe immunosuppression of liver cancer featured by poor immunogenicity, T-cell malfunction, infiltration of immunosuppressive cells owing to the insufficient release of tumor-associated antigens (TAAs), high expression of programmed cell death 1 ligand 1 (PD-L1), and a large number of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and tumor-associated macrophages (TAMs). [3] As a result, mono-immunotherapy, like immune checkpoint inhibitors (anti-PD-1/PD-L1 antibody) which mainly mobilize the body's CD8 + T cells to exert a tumoricidal effect, naturally showed sub-optimal effects on liver cancer treatment. Thus, it is imperative to seek a pleiotropic immune mobilization strategy that can induce highly immunogenic tumor cell death and mobilize both the innate and adaptive immune systems to suppress liver cancer growth, metastasis, and recurrence. Bacteria-derived outer membrane vesicle (OMV), secreted by Gram-negative bacteria, a neotype immune adjuvant or immune vaccine, holds great promise for boosting tumor immunotherapy. [4] OMV can initiate strong innate immune responses because OMV contains numerous pathogen-associated molecular patterns (PAMPs), such as peptidoglycan, lipopolysaccharide (LPS) and flagellin, and a growing body of evidence confirmed that OMV administration effectively mobilized multiple immune cells to resist tumor growth via various approaches, like promoting dendritic cell (DC) maturation and TAM re-polarization, inducing immunogenic pyroptosis, and even evoking trained anti-tumor immunity. [5] Nevertheless, the mere administration of OMV only moderately suppresses tumor growth in numerous studies and entails combination treatment to strengthen antitumor immunity since the OMV can only serve as an immune adjuvant to increase the immunogenicity of tumor cells and activate the innate immune system to inhibit tumor growth. Even worse, intravenous administration of pristine OMV can cause systemic inflammatory reactions, attacking major functional organs. Therefore, optimization of pristine OMVs and exploiting OMV as a vector to load synergistic therapeutic agents are needed to trigger a comprehensive immune attack on liver cancer cells by mobilizing multiple immune cells from both the innate and adaptive immune systems. In recent years, several lines of evidence confirmed that cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway plays a crucial role in awakening both the innate and adaptive immune systems by triggering the production of type 1 interferons (IFNs) and other inflammatory cytokines, leading to natural killer (NK) cell activation, TAMs repolarization, DC maturation, and cytotoxic T cell (CTL) infiltration in the tumor microenvironment (TME). [6] Numerous STING agonists have been proposed to activate this popular signaling pathway for antitumor purposes. [7] Other than that, therapeutic agents that can produce intracellular reactive oxygen species (ROS) have been employed to assist in cGAS-STING activation in synergy with STING agonists since ROS can attack cellular DNA and result in cytosol DNA exposure, which can be recognized by monomeric cGAS to produce cGAMP, thereby priming the cGAS-STING signaling pathway. [8] To exert the full potential of the cGAS-STING signaling pathway for antitumor purposes, multiple strategies have been applied to induce cytosolic DNA exposure and sensitize protein activities simultaneously. Among all STING agonists, Mn 2+ has received vast attention since it can not only catalyze Fenton-like reactions to produce intracellular ROS, leading to DNA damage and cytosol DNA exposure, but also independently activates cGAS, enhances dsDNA-binding capability of cGAS, accelerates the overall catalytic activity of dsDNA-bound cGAS, and induces the phosphorylation of both TBK1 and p65 in a STING-independent manner, leading to synergistic cGAS-STING activation and pleiotropic immune mobilization. [9] Nonetheless, the catalytic efficiency of Mn 2+ is relatively low as compared to the other transitional metals, such as Cu, Fe, and Co, thereby requiring further improvement. [10] Hence, we hypothesize that the addition of another transitional metal element with high catalytic efficiency, such as Cu, may help boost ROS production and cGAS-STING activation. In addition to the immunostimulatory effect induced by metal ions with univalent state, metal ions with mixed valence state that possess multiple enzyme-like activities were reported to exert greater tumoricidal effects. [11] For example, Mn 2+ and Cu + in a low valence state possess peroxidase (POD)-like activities while Mn 3+ , Mn 4+ , and Cu 2+ in a high valence state possess catalase (CAT)/glutathione peroxidase (GPX)-like activities. Thus, we hypothesized that Mn and Cu with mixed valence states possess ternary enzyme-like activities (POD, GPX, CAT), resulting in ROS production, GPX4 degradation, and O 2 generation, ultimately resulting in non-ferrous ferroptosis and hypoxia alleviation. Ferroptotic tumor cells can release abundant intracellular cytokines, damaged associated antigen molecules (DAMPs), and TAAs to promote DC maturation, while hypoxia alleviation can reduce intratumoral hypoxia-inducible factor 1α (HIF-1α) expression, leading to TAM polarization. Robust IFN-γ secreted by CTL and NK cells as a result of the activated STING pathway can inhibit the xc-system of tumor cells and further suppress the bio-activity of GPX4, thereby enhancing lipid peroxidation (LPO) accumulation and exacerbating ferroptosis of tumor cells. [12] Herein, a closed-loop therapeutic paradigm was formulated between ferroptosis and cGAS-STING activation. However, the introduction of abundant free metal ions into human bodies poses a serious health threat, which will lead to metal toxicity and organ dysfunction. Precise transportation of metal ions into the targeted tumor sites and preventing premature leakage of metal ions are crucial for developing a bio-safe and feasible technique for metallo-immunotherapy. Inspired by the fact that OMV is negatively charged and embedded with vast biomolecules and proteins just as cytomembrane, we hypothesized that OMV may serve as a favorable nanocarrier to deposit positively charged metal ions. Herein, in this study, we first harvested OMVs from Escherichia coli ( E. coli ), followed by Cu and Mn deposition. Afterward, to shield the pristine OMVs from the body's immune defense, platelet membrane (PM) was adsorbed on the surface of the OMVs by physical extrusion. Mechanistically, upon the stealth effect of PM, OPCM selectively accumulated at the tumor site, followed by phagocytosis by tumor cells. Once getting into the tumor cells, Cu and Mn ions were subsequently released from the OMVs into the cytoplasm. On the one hand, glutathione (GSH) depletion, ROS production, and O 2 generation can be achieved due to the mixed valence state of Cu and Mn, leading to down-regulation of HIF-1α, inactivation of GPX4, accumulation of lipid peroxidation, and cytosol DNA exposure, leading to the alleviation of tumor hypoxia, initiation of tumor ferroptosis, and activation of the cGAS-STING signaling pathway. On the other hand, Mn 2+ boosted the activation of the cGAS-STING signaling pathway by sensitizing the activities of various molecules in this molecular pathway. As a result, abundant immunogenic factors (CRT, ATP, HMGB1), TAAs, and type 1 IFNs can be produced and released due to broken cell membranes induced by LPO accumulation, and further awaken both the innate and adaptive immune systems by activating NK cells, re-polarizing M2 TAMs, promoting DC maturation, and recruiting cytotoxic T cells (CTLs) into the TME. Abundant IFN-γ secretion as a result of the massive immune stimulation further exacerbates tumor ferroptosis by down-regulating the GPX4 pathway, forming a closed-loop therapy. Specifically, Mn 2+ possesses magnetic resonance imaging (MRI) performance and can realize theranostic purposes in a TME-responsive manner. This paradigm was proved to significantly amplify the therapeutic effect of anti-PD-L1 antibody (αPD-L1) to treat hepatocellular carcinoma (HCC) in two mouse models. As a proof of concept, this is the first study that applied OMVs as metal ion transporters by one-step biomineralization and identified the critical role of both Cu and Mn in inducing ferroptosis and ferroptosis-enhanced cGAS-STING activation for tumor metallo-immunotherapy. 2. Results and discussion 2.1. Characterization of various OMV-based nanodrugs Firstly, pristine OMV from E. coli was extracted as previously reported, followed by one-step biomineralization of Cu and Mn ions, and coating of PM. [5c] Next, a range of input concentrations of CuCl 2 and MnCl 2 , ranging from 50 mM to 800 mM, were applied to assess the optimal loading capacity (LC) of OMVs for both Cu and Mn ions. The results of inductively coupled plasma-mass spectrometry (ICP-MS) indicated a dose-dependent increase of LC for both Cu and Mn. When the input concentration of Cu and Mn reached 800 mM, the LC reached ~6% for both metals (Figure S1) . The molar ratio of Cu and Mn in the OPCM sample was determined to be approximately 1.2:1 by ICP-MS. Based on this result, 800 mM input of Cu and Mn was chosen to synthesize OPCM for the following experiments. As shown in Figure 1a, the morphology of pristine OMVs was spherical under transmission electron microscopy (TEM). After the coating of PM, a double membrane structure was identified under TEM. Beyond that, sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) assay confirmed that protein bands from the original PM and OMV were reserved in the OPCM, indicating the co-existence of both PM and OMV (Figure 1c). In addition, TEM mapping was done to verify the elemental composition of the ultimate OPCM. As indicated in Figure 1d, the C, O, Cu, and Mn elements were shown around the OPCM. Dynamic light scattering (DLS) revealed that the hydrodynamic size of OMVs, OP, OCM, and OPCM was 228, 396.9, 412.3, and 506.6 nm, respectively (Figure S2). The results of DLS demonstrated that the coating of PM and deposition of Cu and Mn ions enlarged the diameter of OMVs, which might be ascribed to the aggregation of OPCM induced by metal ion deposition. The zeta potentials of OMV, PM, OP, OCM, and OPCM were all negative, among which OP displayed the most negative value, and OCM displayed the least negative value, indicating the successful loading of Cu and Mn ions in the OPCM (Figure 1b). The elemental composition of the OPCM was detected further by X-ray photoelectron spectroscopy (XPS), which revealed the co-existence of Cu and Mn elements in the OPCM (Figure 1e). Interestingly, after analysis of Cu2P and Mn2P, we identified two strong binding energy peaks (932.18 and 934.28 eV) corresponding to Cu + and Cu 2+ , and three strong binding energy peaks (640.88, 646.18, and 642.28 eV) that were assigned to Mn 2+ , Mn 3+ , and Mn 4+ (Figure 1f, g). The presence of Cu and Mn ions with mixed valence states was believed to be correlated with a series of redox reactions that took place in the process of metal ion biomineralization due to the presence of polysaccharide, protein molecules, and biomacromolecules anchored on the surface of pristine OMV. The X-ray powder diffraction (XRD) pattern showed the amorphous structure of OPCM (Figure S3). All the above results demonstrated that OPCM was successfully synthesized via one-step biomineralization. In addition, the electrochemically active area (ESCA) of biomineralized OMVs was measured through the double-layer capacitance method (Figure 1h, i, j, and Figure S4) . Compared with the mono-metallic biomineralized OMV, the dua-metallic biomineralized OMV obtained the highest value of ESCA (0.0481 mF/cm 2 ), indicating that the dual-metallic biomineralized OMV enabled the most abundant active sites, which was mainly attributed to the introduced Cu ions with high catalytic activity. Metal ions in a low valence state with POD-like activities decompose H 2 O 2 into hydroxyl radical (•OH) under an acidic environment, while metal ions in a high valence state with CAT/GPX-like activities, promote O 2 production and GSH degradation into oxidized glutathione disulfide (GSSG), thereby alleviating intratumoral hypoxia and preventing ROS elimination. [13] Owning to the co-existence of Cu + , Cu 2+ , Mn 2+ , Mn 3+ , and Mn 4+ , high content of H 2 O 2 and GSH in tumor cells and TME were supposed to be catalyzed into O 2 , ·OH, and GSSG. Based on the results of the electrochemical test, the factual catalytic activity of OPCM was validated via 3,3,5,5-tetramethylbenzidine (TMB), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and a dissolved oxygen monitor. TMB was applied to evaluate the POD-like activity of OPCM. The intermediate ·OH could oxidize TMB from colorless to blue with obvious characteristic light absorption at about 575 nm (Figure S5a). Absorption at this wavelength was enhanced in a concentration-dependent manner with the addition of H 2 O 2 , which indicated that OPCM had a favorable chemo-dynamic effect via the Fenton-like reaction. A dissolved oxygen monitor was employed to confirm the CAT-like activity of OPCM. Figure S5b showed a time-dependent change of O 2 generation in three groups with different concentrations of OPCM + H 2 O 2 , among which 400 μg/mL OPCM displayed the maximum O 2 increase, suggesting the ability of OPCM to catalyze O 2 generation for alleviating intratumoral hypoxia status. To evaluate the GPX-like activity of OPCM, DTNB was chosen as an indicator, since DTNB can measure GSH content due to the weakening of its characteristic UV–Vis absorption peak at 412 nm when reacting with GSH. As revealed by Figure S5c, a time-dependent decrease pattern of GSH content was identified, which indicated that the as-synthesized OPCM possessed GPX-like activity and may potentiate ROS production and GPX4-dependent ferroptosis in tumor cells. Taken together, the obtained OPCM could not only decompose H 2 O 2 but also could deplete GSH, leading to simultaneous O 2 generation, and ROS production, implying great promise for anti-tumor application. Subsequently, the TME-responsive release behavior of OPCM was measured by ICP-MS. Figure S6 showed distinct acid-responsive release behavior of Cu and Mn, which may be attributed to the acid-responsive degradation of PM and OMV. The results proved that Cu and Mn ions could be released in tumor cells accurately and efficiently. Encouraged by the ternary enzyme-like activities, the biosafety and tumor-killing effect of OMV-based nanodrugs were further evaluated at the cellular level. M2 TAMs and JAWSII cells were chosen to test the biocompatibility of OMV-based nanodrugs on non-tumor cells. As revealed in Figure 1k and 1l , CCK-8 results showed that the pristine OMV, OCM, and OPCM had no obvious cytotoxicity on M2 TAMs and JAWSII cells even when the concentration reached 300 μg/mL. The cytotoxicity of OCM on RAW264.7 and JAWSII cells was determined to be lower than that of OPCM, and higher than that of OMV, which might be ascribed to PM-mediated intracellular uptake of metal-deposited OPCM. To test the antitumor effect of OPCM on Hepa1-6 cells, different concentrations of OMV, OCM, and OPCM were incubated with Hepa1-6 cells for 24 h. Figure 1m revealed that the pristine OMV at 300 μg/mL lowered the viability of Hepa1-6 cells to about 70%, OCM at 300 μg/mL lowered the viability to about 60%, OPCM at the same concentration considerably lowered the viability to around 45%, suggesting the effective tumor-killing effect of bimetallic biomineralized OPCM. To summarize, compared with the cytotoxicity on RAW264.7 and JAWSII cells, OMV, OCM, and OPCM exerted stronger cytotoxicity on Hepa1-6 cells, which might be attributed to excessive GSH and H 2 O 2 in tumor cells. Furthermore, the existence of PM on the outer shell of OPCM facilitated intracellular uptake of OPCM due to the ligand-receptor interaction, leading to more metal ion intake compared with non-tumor cells. Based on the results of CCK-8, 300 μg/mL of OMV, OP, OCM, and OPCM were used to evaluate other cellular performance. Effective intracellular uptake of OPCM in tumor cells is a prerequisite for achieving desirable antitumor efficacy. Therefore, the intracellular uptake of DiI-labeled OPCM was assessed by confocal laser scanning microscopy (CLSM). Figure S7a displayed a time-dependent increase of red fluorescence (FL) signals around the DAPI-stained nuclei, suggesting that the DiI-labeled OPCM was effectively engulfed by Hepa1-6 cells. This phenomenon was also verified by flow cytometry (FCM) as demonstrated in Figure S7b and S7c, suggesting that tumor cells could effectively engulf OPCM. To further evaluate the inhibitory effect of various OMV-based nanomaterials on tumor cell growth, Calcein-AM (green)/propidium iodide (PI) (red) kit and Annexin V-FITC/PI assay were conducted and observed by both FL microscopy and FCM. According to the results of live/dead cell staining under FL microscopy, the control, OMV, and OP groups did not exhibit obvious cell damage, indicating that OMV and OP without metal ions were biocompatible (Figure S8) . Notably, the OCM group displayed a decreased number of live cells and a significantly increased number of dead cells, suggesting that the biomineralization of Cu and Mn on the OMVs increased cytotoxicity on cancer cells. Compared with the above four groups, the last OPCM group exhibited the least Calcein-AM-stained live cells and the most prevalent PI-stained dead cells, indicating that OPCM possessed the strongest anticancer efficacy. The results of FCM were in line with the result of live/dead cell staining. (Figure 1n, and o). Taken together, the as-synthesized OPCM was successfully synthesized, which possessed a typical membrane structure with vast bacterial biomacromolecules and platelet proteins, ternary enzyme-like activities (POD-like, CAT-like, and GPX-like activities), pH responsiveness, and considerable anti-tumor effect, which might be due to the metal ion-induced tumoricidal activity and the PM-mediated intracellular uptake. 2.2. OPCM-initiated immunogenic ferroptosis and cGAS-STING activation Given the favorable ternary enzyme-like activities, distinct cellular uptake behavior, and effective tumoricidal effect of OPCM validated in the above studies, the antitumor therapeutic mechanism of OPCM was further evaluated at the cellular level. First, ·OH generation was detected using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe since non-fluorescent DCFH-DA can be converted into fluorescent DCF by ROS oxidization. Specifically, a small-molecule lipophilic antioxidant, Fer, was added to the OPCM group to antagonize ferroptosis induction. The result of CLSM revealed that OMV group displayed negligible DCFH-FITC FL signals, while the OCM and OPCM groups exhibited greater DCFH-FITC FL signals, with the OPCM showing the strongest FL signals which were reduced by the addition of Fer, suggesting that the biomineralization of both Cu and Mn potentiated ROS production in Hepa1-6 cells and the initiation of ferroptosis (Figure 2a) . Accordingly, a similar result was observed via FCM (Figure S9). It is reported that the overproduction of ROS results in LPO and mitochondrial membrane damage, leading to ferroptosis. Therefore, OPCM-induced LPO was assessed by a BODIPY-C11 fluorescent probe (581/591 nm) which is a lipid-soluble fluorescent indicator of LPO. During the LPO, the BODIPY-C11 dye changes from the aggregate state with red FL to the monomer state emitting green FL. The pristine OMVs had little effect on the LPO of Hepa1-6 cells, while cells cultured with the OCM and OPCM emitted more intense green FL signals, suggesting that the biomineralization of Cu and Mn ions on the OMVs induced LPO and played a crucial role in initiating tumor ferroptosis (Figure 2b). Similar to the result of ROS detection, the intensity of LPO was reduced by the addition of Fer. These results collectively demonstrated that OPCM successfully induced cancer cell ferroptosis as shown by ROS production and LPO. Experiments in vitro showed that the OPCM was capable of consuming GSH which is an endogenous antioxidant. [14] Depletion of GSH in tumor cells inactivates GPX4, resulting in an imbalance of redox reactions and then the accumulation of LPO, and subsequent ferroptosis. [15] Therefore, we further determined whether the OPCM activated the GPX4-related ferroptotic pathway. As shown in Figure S10 , the OCM and OPCM groups exhibited distinct reduced levels of intracellular GSH content compared to the control and OMV groups, demonstrating the GPX-like activity of OPCM. To test whether GSH consumption induced by OPCM leads to GPX4 degradation, a western blot (WB) was conducted. The result showed that the expression of GPX4 protein was declined in the OCM and OPCM groups compared to the control and OMV group, suggesting that OPCM contributed to ferroptosis via GPX4 degradation in addition to ROS production (Figure 2c and Figure S11). It was reported that overexpression of intracellular ROS broke the integrity of the mitochondrial membrane, leading to the decrease of mitochondrial membrane potential (MMP) and the release of mitochondrial DNA (mtDNA). [16] To further assess OPCM-induced mitochondrial damage, the mitochondrial function was evaluated by JC-1 dye that can monitor MMP change. When the positive charge of cell membrane potential declines, the JC-1 dye changes from the aggregate state (red color fluorescence) to the scattered state (green color fluorescence). According to the results of CLSM, compared with the control and OMV group, cells cultured with OCM and OPCM displayed a distinct increase in green FL signals and a decrease in red FL signals, suggesting that the JC-1 dye changed from the aggregate state to the scattered state (Figure S12) . The result of FCM was in accordance with the CLSM results as shown in Figure S13, illustrating that OPCM treatment broke the mitochondrial membrane integrity and declined the MMP of Hepa1-6 cells, which might result in the release of mtDNA into the cytosol. To confirm DNA damage, γ-H2AX was used as a DNA damage marker. According to the immunofluorescence (IF) results of γ-H2AX, OCM and OPCM exhibited greatly enhanced green γ-H2AX FL signals compared to the control, OMV, and OP groups, hinting that the bimetallic biomineralized OCM and OPCM effectively induced DNA damage which may lead to cytosol dsDNA exposure (Figure 2d, Figure S14). Interestingly, cytosol dsDNA exposure is not only a result of intracellular ROS attack but also an activator of the cGAS-STING signaling pathway which plays a prominent role in the cancer immunity cycle by producing pro-inflammatory cytokines, such as type 1 IFN. Hypothetically, the cGAS-STING signaling pathway could also be synergistically activated by Mn 2+ anchored on the OPCM as reported by previous studies. Hence, the expression levels of STING, p-STING, TBK1, p-TBK1, IRF3, and p-IRF3 were measured and corresponding statistical analyses were conducted. The schematic diagram of cGAS-STING activation is displayed in Figure 2e . As demonstrated in Figure 2f and 2g, the OMV treatment had little impact on the expression level of all the above six proteins just as the control group. The bimetallic biomineralized OCM and OPCM showed insignificant influence on the expression of non-phosphorylated proteins (STING, TBK-1, IRF3), but augmented the expression of phosphorylated proteins (p-STING, p-TBK1, and p-IRF3). Through statistical analysis, the expression ratio of phosphorylated proteins versus non-phosphorylated proteins was significantly increased in the OCM and OPCM groups compared with the control and OMV groups, suggesting that the bimetallic biomineralized OCM and OPCM successfully activated the cGAS-STING signaling pathway (Figure 2h). To summarize, GSH depletion and ROS generation could be achieved due to the mixed valence states of Cu and Mn in OCM or OPCM, thus enabling tumor cells to undergo non-ferrous ferroptosis via inactivation of GPX4 and accumulation of LPO. In this process, the mitochondrial membrane was broken and the intranuclear DNA and mitochondrial DNA were damaged, leading to the cytosol mtDNA and dsDNA exposure, which might be conducive to activating the cGAS-STING signaling pathway. Notably, ferroptotic cancer cells are known to be immunogenic as the affected cells could release TAAs, and damage-associated molecular patterns (DAMPs), ultimately leading to antitumor immune activation. Therefore, the immunostimulatory effect of OPCM will be assessed and discussed in the following sections. 2.3. Exploration of ICD and immune cell regulation in vitro Ferroptosis was reported to be immunogenic as numerous studies proved that abundant DAMPs (ATP, CRT, and HMGB1) are released from cell membrane rupture due to LPO accumulation, which promotes immune stimulation, such as DC maturation and TAM re-polarization. [17] In addition, the end-product of the cGAS-STING signaling pathway, type 1 IFN, was reported to mobilize multiple immune cells to exert tumoricidal effects. Furthermore, OMVs were also confirmed to be a strong immune adjuvant for immune stimulation. Therefore, the immune stimulatory effects of OPCM were examined on DCs and TAMs. Figure 3a and 3b elucidated the co-culture system of Hepa1-6 cells and DCs or TAMs respectively. The Hepa1-6 cells were placed on the upper chamber, while DCs or TAMs were seeded on the bottom of the six-well plate. As demonstrated in the FCM results, compared to the control group, the proportion of matured DCs was increased by 3.1-, 2.6-, 5.4-, and 6.6-fold in the OMV, OP, OCM, and OPCM groups, respectively, which showed that all the OMV-based nanodrugs were capable of inducing ICD and therefore promoting DC maturation and activating cellular antitumor immunity (Figure 3c, 3d). Notably, the population of CD80 + CD86 + cells in the OPCM group was the largest and 6.6-fold higher than that of the control group, suggesting that metal ions with mixed valence states played a dominant role in DC maturation. The cytokine secretion level was also measured to confirm the ICD effect and DC maturation. Firstly, the ICD effect of OPCM on CRT expression was examined by IF staining. As revealed by Figure S15 , the OCM and OPCM groups displayed more green FL signals in comparison with the control group, OMV group, and OP group, suggesting the exposure of CRT induced by OCM and OPCM. Subsequently, we decided to further measure the secretion level of another two DAMPs, ATP and HMGB1, via enzyme-linked immunosorbent assay (ELISA). Type 1 IFN, and typical pro-inflammatory cytokines secreted by matured DCs, IL-6 and TNF-α were also tested via ELISA. The secretion levels of ATP and HMGB1 were distinctly higher in the OCM and OPCM groups than other groups, confirming the ICD effect of metal-deposited OMVs (Figure 3e, 3f). The secretion level of type 1 IFN was consistent with the results of ATP and HMGB1 (Figure 3g). There was a little variation of type 1 IFN among the control, OMV, and OP group. The OCM and OPCM groups remarkably enhanced the secretion level of type 1 IFN, suggesting the activation of the cGAS-STING signaling pathway. IL-6 and TNF-α were also higher in the OCM and OPCM groups than other groups, illustrating the effective DC maturation induced by bimetallic biomineralized OMVs (Figure 3h, 3i). Notably, the secretion levels of IL-6 and TNF-α were slightly higher in the OMV and OP groups than those in the control group, which might be ascribed to the intrinsic immunostimulatory effect of OMV on immature DCs. Next, the immunostimulatory effect of OMV-based nanodrugs on M2 TAMs was assessed. The OMV, OP, OCM, and OPCM groups all displayed higher CD80 + cells than the control group, among which the OPCM displayed the highest proportion of CD80 + cells among which the OPCM displayed the highest proportion of CD80+ cells (74.2%) which indicated the enhancement of M1 TAMs (Figure 3j, 3k) . Conversely, the number of CD206 + cells was significantly lowered in all four intervention groups, among which the OPCM group displayed the minimum number of CD206 + cells, suggesting that all the OMV-based nanodrugs were capable of decreasing the number of M2 TAMs (Figure 3l, 3m). The secretion levels of ATP, HMGB1, and type 1 IFN in the coculture system of Hepa1-6 and TAMs followed the same pattern just as that of Hepa1-6 and DCs. The bimetallic biomineralized OCM and OPCM significantly enhanced the secretion of ATP, HMGB1, and type 1 IFN (Figure 3n, 3o, 3p). In addition, we measured classical anti-inflammatory cytokine, IL-10, secreted by M2 TAMs, and classical pro-inflammatory cytokine, IL-12, secreted by M1 TAMs, to further verify the TAM re-polarization effect induced by OCM and OPCM. As expected, the secretion of IL-10 was significantly reduced in the OCM and OPCM groups, while the secretion of IL-12 was remarkably enhanced in these two groups, suggesting the successful re-polarization of M2 TAMs into M1 TAMs (Figure 3q, 3r). In short, the as-synthesized OMV, OP, OCM, and OPCM could all promote DC maturation and TAM re-polarization with the OPCM exerting the greatest immune activation effect. The potent immunostimulatory effect of OPCM might be ascribed to the collective effort induced by PAMPs anchored on OMVs and Cu and Mn-induced cGAS-STING activation and immunogenic ferroptosis. Inspired by the results of the in vitro experiments, we hypothesized that the OPCM might serve as a potent immune adjuvant to exert antitumor immunity in combination with typical immunotherapy in the following in vivo studies. 2.4. Exploration of anti-tumor effect in vivo Based on the satisfactory inhibitory effect of OMV-based nanodrugs on tumor cells in vitro , the in-depth anti-tumor effect in vivo was further investigated in two mouse models, including C57BL/6J and BALB/c mice. To be specific, Hepa1-6 cells were inoculated into the right flank of C57BL/6J mice, while H22 cells were inoculated into the right flank of BALB/c mice. [18] The treatment protocol is depicted in Figure 4a . As a crucial immunosuppressive factor in the TME, high expression of PD-L1 could trigger malfunction of CTL and compromise T cell-related tumoricidal effect. Encouraged by the efficient tumor-killing effect as demonstrated in the above in vitro studies, a commonly used immunotherapeutic agent, αPD-L1, was applied to synergize with OPCM to boost the pleiotropic immune stimulation effect of OPCM and concurrently induce tumoricidal effects on liver cancer models. The mice were divided into five groups, including the control, OMV, αPD-L1, OPCM, and the combination of αPD-L1 and OPCM. Firstly, we analyzed the antitumor effect of the four intervention groups on C57BL/6J mice. The body weight of mice in all groups showed no abnormalities, suggesting that all the treatments exhibited no obvious systemic toxicity (Figure 4b). As revealed by the growth of tumor relative volume, the combination group (αPD-L1 + OPCM) presented a higher tumor inhibiting capacity compared with the other groups (Figure 4c). The results of the individual tumor growth curve, the representative photos of mice during treatment, the photograph and average weight of extracted tumors in each group, and the corresponding tumor inhibition rates were consistent with the outcome of relative tumor volume as shown in Figure 4d, 4e, S16, and S17. Afterward, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), proliferating cell nuclear antigen (PCNA), and hematoxylin and eosin (H&E) staining assays were conducted to assess the extent of tumor apoptosis, tumor proliferation, and tumor necrosis. As demonstrated in Figure 4f, 4g, 4h, prevalent red PCNA FL signals were shown in the control group. The PCNA FL signals were slightly diminished in the OMV and αPD-L1 groups, while the FL signals were largely reduced in the OPCM and the combination groups, suggesting a lower cell proliferation in the two groups. Conversely, the results of TUNEL staining showed the opposite outcomes, with the combination group showing the greatest green TUNEL FL signals and the control group showing the least FL signals, suggesting that the combination group presented the highest cell death ratio. The H&E staining results were consistent with the TUNEL and PCNA staining assays, in which excessive apoptosis and necrosis were found in the OPCM and combination groups, implying the effective antitumor effect induced by the combination of OPCM and αPD-L1. The survival time was significantly prolonged in all intervention groups compared with the control group, suggesting the long-term antitumor protection of the OMV-based nanodrugs and/or αPD-L1 (Figure S18). Notably, there was only one mouse dead in the combination group, suggesting the effective therapeutic capacity and the long-term protective effect of the combination therapeutic paradigm. In summary, the above data suggested that the therapeutic effect of pristine OMVs was moderate, while the Cu and Mn deposited on the OMVs augmented the therapeutic efficacy of pristine OMVs for liver cancer, which was further strengthened by the combination with αPD-L1. Since the TME of liver cancer is complex and heterogeneous, another hepatocellular carcinoma (HCC) cell line model, H22 cells, was chosen to establish BALB/c tumor-bearing mouse models to confirm the therapeutic potential of the OMV-based nanodrugs for HCC treatment. Similar to the therapeutic outcomes of the C57BL/6J tumor-bearing mouse model, all intervention groups displayed obvious tumor inhibition, with the combination group showing the greatest tumor suppression effect (Figure S19) . The body weight was stable in different groups, showing no unusual changes during the treatment processes. The representative digital images of H22 tumor-bearing mice, the digital photograph of dissected tumors in different groups 20 d post-treatment, the extracted tumor weight, the individual tumor growth curve, and the tumor inhibition rates elucidated the same outcomes. Apart from eliciting the high therapeutic efficacy, OPCM is also able to achieve TME-responsive T 1 weighted MR imaging due to the presence of Mn 2+ . We first demonstrated the MRI performance of OPCM in vitro . As revealed by Figure S20a , Mn concentration-dependent 1/T 1 increase was found in all PBS systems containing 500 μg/mL OPCM. The T 1 relaxation rate was 0.7854 mM -1 s -1 in pH 7.4 PBS system, 2.210 mM -1 s -1 in pH 6.0 PBS system, 2.434 mM -1 s -1 in pH 5.0 PBS system, and 2.636 mM -1 s -1 in pH 5.0 + 10 mM GSH PBS system, among which the PBS system with pH 5.0 + 10 mM GSH displayed the strongest T1 signal and the largest T 1 relaxation rate (Figure S20b). The above data indicated that OPCM released more Mn 2+ in acidic environment with high GSH content, suggesting that the OPCM might release abundant Mn 2+ in the TME. Then, we further detected its imaging capability in vivo . As shown in Figure 4i and Figure S21, the OPCM displayed favorable T 1 -weighted MRI performance. After i.v. injection of OPCM, the T 1 signal at the tumor site displayed a time-dependent increase until the 4 th hour and then started to decrease until the 24 th hour. To summarize, OPCM exhibited a strong antitumor effect on liver cancer models, and the effect was further amplified by αPD-L1, which might be attributed to the synergistic effect of immunogenic ferroptosis, cGAS-STING activation, and salvation of T-cell. The mechanism and the alternation of the tumor immune microenvironment will be discussed in detail in the following parts. 2.5. Exploration of immunogenic ferroptosis and cGAS-STING activation in vivo To further clarify the underlying mechanism of OPCM-based treatments on the TME of liver cancer, the tumor tissues in each group were collected and some important proteins and markers were investigated in vivo . In the in vitro study, the OPCM was demonstrated to possess POD-like, CAT-like, and GPX-like activities. Moreover, in cellular studies, OPCM was proven to induce ferroptosis and activate the cGAS-STING signaling pathway, leading to the release of DAMPs and type 1 IFN. Hence, we conducted IF staining to verify the expression of GPX4, DHE, HIF-1α, p-STING, p-TBK1, p-IRF3, CRT, and HMGB1. As expected, the red GPX4 signals were largely reduced in the OPCM and combination group compared with the control group, while the intervention of OMV and αPD-L1 had no significant influence on the expression of GPX4, suggesting that Cu and Mn ions in the OPCM played the key role in degrading GPX4 (Figure 7a, 7d). Contrary to the result of GPX4, the dihydroethidium (DHE) frozen staining revealed that the OPCM and the combination groups greatly enhanced the ROS generation, while OMV had little impact on the ROS level, indicating the POD-like activity of OPCM (Figure 5a, 5e) . Since the OPCM was capable of catalyzing H 2 O 2 to generate O 2 , a cellular hypoxia indicator, HIF-1α, was chosen to detect the status of tumor hypoxia. According to the IF results (Figure 5a, 5f), the tumors in the control group displayed high expression of HIF-1α. The intervention of OMV and αPD-L1 showed negligible effects on reducing tumor hypoxia, while the bimetallic biomineralized OPCM and the combination treatments significantly reduced tumor HIF-1α signals, illustrating the intratumoral O 2 generation capability of OPCM. Previous cellular experiments demonstrated that DNA damage induced by OPCM could activate the cGAS-STING signaling pathway, therefore we further evaluated the influence of OPCM on the cGAS-STING pathway in vivo . As expected, the treatment with OMV or αPD-L1 exhibited negligible effect on the expression of p-STING, p-TBK1, and p-IRF3, while the bimetallic biomineralized OPCM group and the combination group markedly enhanced the expression of the three phosphorylated proteins, corroborating that Cu and Mn deposited on the OPCM activated the cGAS-STING signaling pathway (Figure 5b, 5g, 5h, 5i) . As reported in previous studies, CTLs and NK cells are capable of secreting IFN-γ which exacerbates ferroptosis by inhibiting system xc - and downregulating the expression of intracellular GPX4. [12] In our study, the administration of αPD-L1 was believed to activate CTLs, while OPCM was demonstrated to activate the cGAS-STING signaling pathway which further promoted DC maturation, CTL, and NK activation, resulting in abundant release of IFN-γ. As revealed by the IF staining of IFN-γ in the tumor tissues (Figure S22), the administration of OMV slightly enhanced the expression of IFN-γ which may be attributed to the immunostimulatory effect of OMV, while the αPD-L1, OPCM, αPD-L1 + OPCM groups displayed significantly enhanced expression of IFN-γ, and the combination group showed the greatest expression, suggesting that the co-administration of αPD-L1 and OPCM activated the immunoferroptosis pathway to the greatest level. As observed in the above IF staining of GPX4, the administration of αPD-L1 down-regulated GPX4 expression. Moreover, the co-administration of both OPCM and αPD-L1 reduced the expression of GPX4 to the least as compared with other groups, confirming the immunoferroptosis pathway activated by the synergistic immunoferroptotic effect of αPD-L1 and OPCM. To confirm the immunogenic effect of OPCM-induced ferroptosis, the expression levels of CRT and HMGB1 were measured by IF (Figure 5c, 5j, 5k) . As shown by the IF results, the OMV and αPD-L1 slightly enhanced CRT expression and reduced the expression of intracellular HMGB1 in comparison with the control group, while the OPCM and the combination group drastically increased the expression of CRT and downregulated intracellular HMGB1, elucidating that OPCM induced immunogenic ferroptosis. To summarize, the OPCM exerted obvious POD-like, GPX-like, and CAT-like activities in vivo , resulting in GPX4/ROS-dependent immunogenic ferroptosis, tumor hypoxia alleviation, and cGAS-STING activation. Moreover, OPCM and αPD-L1-induced immune activation further amplified the intensity of ferroptosis by IFN-γ, which forms a closed-loop therapy cycle. The alteration of tumor cell signaling pathways and transformation of tumor metabolism may explain the mechanism of the potent antitumor effects of OPCM. 2.6. Immune cell evaluation in vivo To delineate the immune landscape of HCC after various treatments, the infiltration level of various antitumor immune cells, including DCs, CTLs, M1 TAMs, M2 TAMs, and NK cells were analyzed by FCM. The matured DCs (CD11c + CD80 + CD86 + ) are crucial competent antigen professional cells (APCs) and regulate the adaptive antitumor immunity, thereby cross-priming CTLs. To investigate DC maturation, tumor tissues, tumor-draining lymph nodes (TDLNs), and spleens were harvested post-treatment for FCM analysis (Figure 6a, 6c) . Compared with the control group with ~12.8% intratumoral CD80 + CD86 + cells, the proportion of matured DCs showed a remarkable increase in the OPCM and combination groups, particularly in the latter group with 3.9-fold increase. Similarly, the combination group displayed the highest frequency of CD80 + CD86 + cells in the TDLNs (~41.3%) and spleen (~33.9%). Notably, the population of matured DCs in the OMV groups from the tumor (~28.7%), TDLNs (~24.9%), and spleen (~27.6%), was also obviously higher than that in the control group, which might be ascribed to the intrinsic immunostimulatory of OMVs on DCs as reported by previous research. [19] The FCM results of DC analysis in different organs clarified that OPCM was capable of promoting DC maturation which was necessary to initiate cellular immunity, and the enhancement was more pronounced in co-administration of OPCM and αPD-L1, which might be ascribed to the concerted immunostimulatory efforts of the PAMPs anchored on the OMV, abundant DAMPs outflow induced by ferroptosis, and cGAS-STING activation. Matured DCs are professional APCs that can process tumor antigens and present them to naive T cells, thereby activating T-cell function and differentiating them into CTLs. Since the OMV-based nanodrugs were proven to promote DC maturation successfully, therefore we decided to analyze the proportion of tumoricidal CTLs within tumors, TDLNs, and spleens. As revealed by the FCM outcomes (Figure 6b, 6d) , compared to the control group with 6.86% CD3 + CD8 + T cells in the tumors, 14.1% CD3 + CD8 + T cells in the TDLNs, and 6.84% CD3 + CD8 + T cells in the spleens, the treatment of OMV and αPD-L1 slightly increased the proportion of CTLs, while the combination group displayed the remarkable increase of CTLs with 3.3-fold increase in the tumor, 3.2-fold increase in the TDLNs, and 2.8-fold increase in the spleen, suggesting that the as-synthesized OPCM in combination with αPD-L1 could effectively mobilize a large number of intratumoral and systematic T cells, and prompt them differentiating into CTLs to exert tumoricidal effects. Owing to the O 2 generation capability of Mn and Cu ions with high valence state and immunostimulatory capacity (DAMPs and inflammatory cytokines induced by OPCM, and PAMPs carried by OMVs) of OPCM, protumoral M2 TAMs can be polarized into antitumoral M1 phenotype to awaken innate antitumor immunity. To test this hypothesis, the phenotype and proportion of M1 TAMs and M2 TAMs were verified (Figure 6e, 6f, and 6g) . As demonstrated by FCM results, the frequency of M2 TAMs, F4/80 + CD11b + CD206 + cells, was largely decreased in the OPCM and combination groups, while the proportion of M1 TAMs, F4/80 + CD11b + CD80 + cells, was markedly promoted in these two groups as compared with the control group, confirming the TAM re-polarization effect of OPCM. In addition to DCs, CTLs, and TAMs, the proportion of another innate tumoricidal immune cell, NK cell was also detected since numerous studies proved that the activation of the cGAS-STING signaling pathway could increase the population of intratumoral NK cells via the production of type 1 IFN. As expected, the OPCM and combination groups significantly increased the frequency of intratumoral NK cells by 4.3-fold and 5.7-fold as compared with the control group, indicating that the as-synthesized OPCM were able to mobilize NK cells to exert antitumor immunity (Figure 6h and 6i) . Apart from FCM analysis of the immune cell population, some crucial cytokines and immunogenic factors were detected to corroborate the alteration of the tumor immune microenvironment by ELISA. On the one hand, we collected mouse serums post-therapeutic treatment to detect the secretion level of IL-6, TNF-α, and IL-12. Figure 6j showed that the serum content of pro-inflammatory cytokines, IL-6, TNF-α, and IL-12 were enhanced in all intervention groups as compared to the control group with the combination group showing the greatest enhancement, while the serum content of anti-inflammatory cytokines, IL-10 and IL-12, were downregulated in the intervention groups as compared to the control group, with the combination group displaying the lowest level of IL-10 and IL-12, indicating the effective DC maturation and TAM re-polarization effects induced by OPCM. On the other hand, we extracted tumor tissues post-treatment to detect the intratumoral content of ATP, HMGB1, and type 1 IFNs (Figure 6j, 6k). As expected, the expression levels of these three factors were markedly increased in the treatment groups with the combination group displaying the highest promotion, suggesting the induction of ICD and activation of cGAS-STING signaling pathway. Taken together, all the above results demonstrated that the OPCM could serve as a potent immunoadjuvant to stimulate the immunosuppressive TME by mobilizing and activating multiple immune cells from the innate and adaptive immune systems to inhibit tumor growth in combination with αPD-L1. 2.7. Long-term antitumor immune memory effect in vivo The activation of the tumor immune microenvironment is not only conducive to killing primary tumors but also helps the body build up long-term immune memory against tumor recurrence and metastasis. Encouraged by the effective tumor suppression effect and the immunostimulatory effect of the combinational therapeutic paradigm (αPD-L1 and OPCM) achieved on the primary tumor models, tumor-re-challenged mouse and lung metastasis models were established to verify the long-term antitumor immune memory. The schematic diagrams of the establishment of both tumor models are shown in Figure 7a and 7j . The body weight of re-challenged mouse models was recorded for 2 weeks since the 37 th day. The tumor weight of all re-challenged mouse models remained stable during the observation process (Figure 7b). Regarding the re-challenged tumor growth, the average tumor volume in the four intervention groups was smaller than that in the control group with 250 mm 3 (Figure 7c). Notably, the combination group showed the slowest tumor growth with an average tumor volume of around 70 mm 3 , indicating that the combination treatment possessed long-term resistance to tumor recurrence. The results of extracted tumor weight, the photograph of extracted tumors, and the representative digital photos of re-challenged mice were consistent with the outcomes of tumor volume growth, indicating that the OMV-based nanodrugs and/or αPD-L1 established long-term antitumor immune memory and thus provided long-term protection against tumor recurrence (Figure 7d, 7e, S23). Subsequently, the immune mechanism was further studied. TDLNs and spleens were extracted and digested to obtain a single-cell suspension, followed by FCM detection, to quantify the population of effector memory T cells (T em ). As revealed by the FCM results of the TDLNs, compared with only 19.1% of CD3 + CD8 + CD44 + CD62L - cells, the other four intervention groups displayed enhanced proportion of T em , particularly in the OPCM and combination groups with 1.9-fold, and 2.2-fold increase, respectively (Figure 7f, 7g) . Similarly, the FCM results of the spleens displayed the same outcome, with the combination group showing the highest proportion of T em (Figure 7h, 7i). These results indicated that the OMV-based nanodrugs combined with αPD-L1 could induce long-lasting antitumor immune memory and systemic immunity, which was beneficial for suppressing tumor recurrence via recognition of “old antigens”. To further verify that the combination paradigm of OPCM and αPD-L1 can suppress tumor metastasis, we established lung metastasis models by injecting Hepa1-6 tumor cells into the mice after various treatments through the tail vein. One week after tumor cells injection, the lung tissues of all mice were obtained, photographed, and sent for H&E staining to observe lung metastasis nodules. As revealed by Figure 7k and 7l , as compared with the four intervention groups, the H&E staining of lung sections in the control group showed a higher degree of canceration in which numerous metastasis nodules were found in-homogeneously, indicating the effective suppression of metastasis induced by OMV-based nanodrugs and/or αPD-L1 due to the activation of antitumor immune response. In short, we demonstrated that the OMV-based nanodrugs could not only suppress the primary tumor growth by ferroptosis induction and pleiotropic immune cell mobilization but also induced long-term antitumor immune protection against tumor recurrence and metastasis via evoking T em locally and systemically. 2.8. Evaluation of biodistribution and biosafety Effective accumulation of OPCM in tumor sites is a prerequisite for achieving a favorable anti-tumor effect. Hence, the biodistribution of OPCM was analyzed using an in vivo FL imaging system. After i.v. DiR-labeled OPCM administration, a clear and gradually increasing FL signal was observed at the tumor site from 2 h to 24 h, and the FL signal reached a maximum at 24-h post-injection and reduced at 48-h post-injection as shown in Figures S24a and S24b , verifying the efficient tumor accumulation of OPCM owing to the enhanced permeability and retention effect. Moreover, the mice were sacrificed at 48-h post-injection, followed by extraction of tumor tissues and major organs, including the spleen, heart, liver, lung, and kidney. Ex vivo FL imaging was done to capture the FL images of these tissues. The results showed that the liver displayed the highest FL signals, followed by the lung, tumor, spleen, kidney, and heart, illustrating the in vivo biodistribution of DiR-labeled OPCM (Figure S24c, S24d). To further detect the in vivo accumulation and distribution of Cu and Mn ions in tumor tissues and major organs, ICP-MS was conducted to measure the Cu and Mn content of each tissue at different time intervals (Figure S24f, S24g, S24h, S24i, S24j, S24k) . The results showed that Cu and Mn ions preferentially accumulate in the lung, heart, and liver, which might be due to the rapid blood supply and the capture by the reticuloendothelial system, followed by metabolization and elimination of the kidney over time. Notably, the maximum contents of Mn and Cu were found in the tumor at the 4 th hour, which was in accordance with the result of the MRI with the 4 th hour showing the maximum imaging intensity. To evaluate biological safety, 6-8-week-old C57BL/6J mice were treated by i.v. injection of OPCM at the 0 th d, 1 th d, 3 th d, 7 th d, 14 th d, 21 th d. At the 21st d, all mice were sacrificed and the whole blood samples, blood serums, and major organs were obtained to detect hematology parameters, liver function, and renal function markers, as well as histology observation. Figure S24k and S24l displayed that all hematology parameters and biochemical indexes were within the normal range, suggesting negligible toxicity to the liver and kidney with good safety. As demonstrated by Figure S24m the H&E staining of major organs showed no morphological abnormalities, implying no acute and chronic pathological toxicities and adverse events. The above results illustrated that OPCM was bio-compatible and posed no obvious threat to major organs, making it a feasible alternative for future clinical translation. 3. Conclusion In conclusion, we successfully developed Cu and Mn-deposited OPCM via one-step biomineralization, which possessed favorable biocompatibility, TME responsiveness, and POD/CAT/GPX-like activities, leading to the initiation of tumor ferroptosis and cGAS-STING activation for antitumor immunity. This study provided a state-of-art paradigm for treating immunosuppressive liver cancer, since the OPCM successively initiated ferroptosis and activated the cGAS-STING signaling pathway, leading to pleiotropic immune cell mobilization, including DCs, CTLs, M1 TAMs, M2 TAMs, NK cells, and T em . Moreover, OPCM and αPD-L1-induced immune activation further amplified the intensity of ferroptosis by IFN-γ secretion, which formed a closed-loop therapy cycle. As a result, the systematic immunostimulation led to both primary anti-tumor immunity and long-term anti-tumor immune memory, suppressing tumor recurrence and metastasis, thus providing an innovative combination therapeutic strategy for advanced liver cancer treatment. 4. Experimental section 4.1. Chemical and materials MnCl 2 ·4H 2 O and CuCl 2 ·2H 2 O were obtained from Sigma Aldrich (USA). BeyoPure™ LB Broth (premixed powder), protease inhibitor cocktail, and Ethylene Diamine Tetraacetic Acid (EDTA) were bought from Beyotime Biotechnology (Jiangsu, China). HEPES-NaOH was bought from Bio-sharp company. Hydrogen peroxide (H 2 O 2 , 30%), 5,5’-dithiobis-(2-nitrobenzoic acid) (C 14 H 8 N 2 O 8 S 2 , DTNB), and 3,3',5,5'-Tetramethylbenzidine (C 16 H 20 N 2 , TMB) were acquired from Aladdin (Shanghai, China). The Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Boster (Wuhan, China). Streptomycin-penicillin, trypsin, DAPI staining agent, Cell Counting Kit-8 (CCK8), DiI, DNA Damage Assay Kit by γ-H2AX Immunofluorescence, and JC-1 dye were obtained from Beyotime Biotechnology (Jiangsu, China). 1,1-dioctadecyl-3,3,3,3-tetramethylindo tricarbocyanine iodide (DiR) was bought from AAT Bioquest (CA, USA). Annexin V-FITC/PI Apoptosis Kit was bought from Elascience Biotechnology (Wuhan, China). Calcein-AM/PI Double Staing Kit and ROS assay kit were bought from Dojindo Laboratories (Kumamoto, Japan). BODIPY-C11 was obtained from ThermoFisher (USA). Anti-glutathione peroxidase 4 antibody was bought from Abcam (USA). Mouse-Reactive STING Pathway Antibody Sampler Kit was acquired from CST (USA). Calreticulin recombinant antibody was obtained from Proteintech (Wuhan, China). The enzyme-linked immunosorbent assays (ELISAs), including ATP, HMGB1, IL-6, TNF-α, Type 1 IFN, IL-10, and IL-12 were acquired from MEIMIAN (Jiangsu, China) or Bioswamp (Wuhan, China). αPD-L1 antibody was bought from Bio X cell (USA). FITC CD11c antibody, PE anti-mouse CD80 antibody, and APC anti-mouse CD86 antibody, and APC anti-mouse CD206, PE anti-mouse CD3, APC anti-mouse CD8a, FITC anti-mouse F4/80, PerCP/Cyanine5.5 anti-mouse/human CD11b, APC anti-mouse NK1.1, and APC anti-mouse CD8a, FITC anti-mouse CD44 and PerCP/Cyanine 5.5 anti-mouse CD62L were purchased from Biolegend (San Diego, CA, USA). All the chemicals were used as purchased without further purification. 4.2. Synthesis of OMV-based nanodrugs 4.2.1. Acquirement of OMVs OMVs was obtained according to a previously reported method. Briefly, Escherichia coli was inoculated into 250 mL LB medium and the medium was placed in a rotary shaker (180 rpm) with 37°C overnight. When OD600 of the LB medium reached 1.2, the medium was centrifuged at 5000 rpm for 10 min to collect bacteria. Afterwards, HM lysis buffer was prepared and the formula was as follows: 0.25 M sucrose, 1 mM Ethylene Diamine Tetraacetic Acid (EDTA), 20 mM HEPES-NaOH (pH 7.4), and 1х protease inhibitor cocktail. The collected bacteria were washed three times by PBS and then suspended in the HM lysis buffer. Then, the mixed solution was transferred to the ultrasonic cell disruptor in an ice bath for ultrasonic decomposition (power: 30%, time: 10 min). Subsequently, the whole solution was centrifuged at 3000 g for 5 min to eliminate the remaining bacteria and large bacterial fragments. After centrifugation, the supernatant was aspirated carefully and further underwent centrifugation at 15000 g for 30 min to collect the sediment. The sediment was resuspended in a cold HM buffer, passing through 0.8 μm and 0.45 μm filters at least 5 times in turn to get the OMVs. To quantify OMVs, the obtained OMVs solutions were dried at 60℃ and then weighed in a precision balance. 4.2.2. Preparation of platelet membrane (PM) First of all, whole blood was obtained from mice and then centrifuged at 200 g for 10 min at room temperature. Next, the upper platelet (PLT)-rich plasma was collected and further centrifuged at 800 g for 15 min at room temperature to collect the sediment. Cold PBS containing 1 mM EDTA and protease inhibitor was employed to suspend the PLT sediment. The mixed PLT solution was transferred thrice from room temperature to -80℃ refrigerator to obtain PLT fragments. Then, the PLT lysis was centrifuged at 4000 g for 30 min at RT to collect the sediment. Finally, the sediment was washed 3 times with PBS to obtain the pristine PM. 4.2.3. Preparation of OP, OP@Cu, OP@Mn, OCM, OPCM, and DiI or DiR-labeled OPCM To obtain PM-coated OMVs (OP), PM and OMV were dispersed in saline with a mass ratio of 1:1. Then, the mixed solutions were put into an ultrasonic bath under 20℃ for 5 minutes and physically extruded 11 times by an Avanti mini extruder through a porous polycarbonate membrane (400 nm). The mixture was centrifuged (4°C, 12 000 rpm for 30 min) to obtain the OP. To obtain Cu and Mn bio-mineralized OMVs (OCM), different input concentrations of Cu and Mn (50 mM, 100 mM, 200 mM, 400 mM, 800 mM) were separately mixed with 10 mg/mL OMVs to assess the optimal input amount of CuCl 2 and MnCl 2 by ICP-MS. After confirming the best input of CuCl 2 (800 mM) and MnCl 2 (800 mM), PM was mixed with the synthesized OCM to obtain OPCM. OPCM was dispersed and stored in saline for future use. OP@Cu and OP@Mn were prepared by mixing OMV, CuCl 2 or MnCl 2 , and PM similar to the preparation of OCM. DiI or DiR-labeled OPCM was prepared by mixing OPCM and DiI or DiR by physical stirring (250 rpm) for 1 h at 37℃. 4.3. Characterization of various OMV-based nanodrugs, enzyme-like activities, and pharmacokinetics of OPCM in vitro 4.3.1. Characterization of various OMV-based nanodrugs The morphology of various OMV-based nanodrugs was characterized by transmission electron microscopy (TEM). The elemental mapping of C, O, Cu, and Mn were also examined post TEM. The hydrodynamic diameters and zeta potentials of the OMV-based nanodrugs were analyzed by a surface zeta potential and particle size analyzer (Zetasizer Nano ZS90, Britain). Inductively coupled plasma-mass spectrometry (ICP-MS, Agilent 7700(MS), USA) was applied to quantify the content of Cu and Mn in OCM and OPCM. X-ray photoelectron spectroscopy (XPS) measurements of the valence states of Cu and Mn were obtained using the K-Alpha XPS System (Thermo Scientific K-Alpha, USA). The X-ray powder diffraction (XRD) pattern was obtained using an X-ray diffractometer (Rigaku Ultima IV, Japan). 4.3.2. Enzyme-like activities of OPCM in vitro To test the POD-like activity of OPCM, different concentrations of OPCM (50, 200, 300, 500, and 600 μg/mL) was incubated with 10 mM H 2 O 2 solution for 30 min at a 96-well plate. Then, 4 µL of TMB (80 × 10 -3 M) was added into the above solutions, followed by measurement of the wavelength absorption from 400 nm to 600 nm in a microplate reader (MultiSkan GO, Thermo scientific, USA). To track O 2 production, 0, 200, and 400 μg/mL OPCM were suspended in ultra-pure water and then sealed by paraffin. After 10 mM H 2 O 2 was injected into the airtight system, a dissolved oxygen meter (JPBJ-608, Shanghai Oustor Industrial Co.) was used to measure the dissolved oxygen content for 15 min. To detect the consumption of GSH induced by OPCM, the 5, 5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) solution was used. In brief, OPCM (500 µg/mL) were dissolved in PBS and reacted with GSH (5 mM). Afterward, the sediment was removed by centrifugation (13,000 rpm, 10 min) for various incubation durations (30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h) to collect the supernatant. Finally, DTNB solution (3 mg/mL, 10 µL) was added into the supernatant in a 96-well plate, the absorbance at 412 nm was measured by a microplate reader. 4.3.3. Pharmacokinetics of OPCM in vitro To detect the pH-responsive behavior of OPCM, OPCM was suspended in PBS solution with pH 5.0, pH 6.0, and pH 7.4. Then, the above PBS solutions containing OPCM were placed in a shaker (150 rpm, 37℃). 200 uL solutions were taken out of the three solutions at each time point (15min, 30min, 1 h, 4 h, 8 h, 24 h, and 48 h). Afterward, the 200 µL solutions were centrifuged at 15000 rpm for 5min to obtain the supernatant for ICP-MS to detect the level of Cu and Mn. 4.4. Cellular experiments 4.4.1. Cell culture RAW264.7 cells, Hepa1-6 cells and H22 cells were purchased from Boster (Wuhan, China). JAWSII cell was obtained from MeisenCTCC (Zhejiang, China). RAW264.7 cells, Hepa1-6 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL) under a humidified atmosphere of 5% CO 2 at 37°C. H22 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C with 5% CO 2 . JAWSII cells were cultured in RPMI-1640 medium supplemented with 20% FBS, 1% penicillin– streptomycin, and 5 ng/mL GM-CSF at 37°C with 5% CO 2 . It is worth noting that RAW264.7 cells and JAWSII are half adherent and half suspended, H22 cells are fully suspended, while Hepa1-6 cells are fully adherent. The culture medium of Hepa1-6 cells, H22 cells, and RAW264.7 cells were sub-cultured every two days, while the JAWSII cells were sub-cultured every three days. M2 TAMs were obtained by culturing RAW264.7 cells with interleukin 4 (IL- 4) (40 ng/mL) overnight. 4.4.2. Cytotoxicity of various OMV-based nanodrugs JAWII and M2 TAMs were selected to verify the bio-compatibility of the OMV-based nanodrugs. Hepa1-6 cells were chosen to examine the anti-tumor effect of the OMV-based nanodrugs. All cell lines were seeded in 96-well plates at a density of 1 × 10 4 cells per well. M2 TAMs were obtained with the aforementioned method. After cell attachment, JAWSII, M2 TAMs, and Hepa1-6 cells were treated with gradient concentrations of OMV, OCM, and OPCM (50, 100, 150, 200, 250, 300 μg/mL) for 24 h. After repeated washing, the cell viability was measured by the CCK-8 assay following the manufacturer’s instructions. 4.4.3. The intracellular uptake of DiI-labeled OPCM The phagocytosis of DiI-labeled OPCM was explored in Hepa1-6 cells under confocal laser scanning microscopy (CLSM) (Nikon, Tokyo, Japan) and flow cytometry (FCM) (FACS Vantage SE, Becton Dickinson, San Jose, CA, USA). For CLSM characterization, Hepa1-6 cells (1 × 10 5 cells per well) were separately cultured in con-focal dishes under a humidified atmosphere of 5% CO 2 at 37℃, followed by cell attachment overnight. M2 TAMs were obtained by the aforementioned method. Next, the culture medium was discarded and replaced with fresh FBS-free DMEM containing DiI-labeled OPCM, followed by co-incubation for 30 min, and 1, 2, 3, and 4 h. Then, the confocal dishes were removed from the cell incubater and rinsed with PBS thrice. Afterwards, the cells were fixed with 1 mL 4% formalin for 15 min. Subsequently, the cells were washed with PBS three times and counter-stained with 100 μL DAPI staining solution for another 15 min. Finally, the cells were washed with PBS and then visualized by CLSM to take fluorescence (FL) images to observe the cellular uptake of DiI-labeled OPCM in Hepa1-6 cells. FCM characterization was also applied for quantitative analysis. Hepa1-6 cells (2 × 10 5 cells per well) were cultured in 6-well plates under a humidified atmosphere of 5% CO 2 at 37℃ respectively. Then, the medium was discarded and replaced with FBS-free DMEM containing DiI-labeled OPCM, followed by co-incubation for 30 min, and 1, 2, 3, and 4 h. Then, cells were collected by trypsin and suspended in 300 μL PBS solution to undergo FCM analysis. 4.4.4. Antitumor effect induced by various OMV-based nanodrugs To further detect the antitumor effect of OMV-based nanodrugs on Hepa1-6 cells, live/dead cell staining and FCM were conducted to confirm the viability of Hepa1-6 cells after various treatments. Hepa1-6 cells (1 × 10 5 cells per well) were cultured in six-well plate under a humidified atmosphere of 5% CO 2 at 37℃, followed by cell attachment overnight. Afterwards, 300 μg/mL of OMV, OP, OCM, and OPCM suspending in fresh FBS-free DMEM were added into the six-well plate respectively. After co-incubation for 24 h, the cells were sent for live/dead cell staining based on the manufacturer’s instructions, followed by observation under an FL microscope (Nikon Ti-S, Tokyo, Japan). Simultaneously, the cells were also collected by trypsin, followed by staining of Annexin V-FITC and PI in PBS solution based on the manufacturer’s instructions. Finally, FCM was performed to analyze the cell apoptotic rates of Hepa1-6 cells in various treatment groups. 4.4.5. The detection of cellular ROS generation, lipid peroxidation (LPO), and mitochondrial membrane potential (MMP) Hepa1-6 cells (1 × 10 5 cells per well) were separately cultured in con-focal dishes under a humidified atmosphere of 5% CO 2 at 37℃, followed by cell attachment overnight. Then, the Hepa1-6 cells were divided into 5 groups and incubated with PBS, OMV, OCM, OPCM, or OPCM + Fer for 24 h respectively. Afterwards, all cells were stained with DCFH-DA for detection of ROS. The cells were also stained with JC-1 dye and bodipy-C11 dye to observe variations in MMP and LPO respectively. All dishes were visualized by CLSM. In addition, cells were collected for FCM analysis of MMP. MMP was calculated as the ratio of the JC-1 aggregate/monomer. 4.4.6. Intracellular evaluation of GSH content and GPX4 expression Hepa1-6 cells (1 × 10 5 cells per well) were seeded in six-well plate under a humidified atmosphere of 5% CO 2 at 37℃, followed by cell attachment overnight. Afterwards, PBS, 300 μg/mL of OMV, OCM, and OPCM suspending in fresh FBS-free DMEM were added into the six-well plate respectively. After co-incubation for 24 h, cells were harvested to detect the concentration of GSH and GSSG via GSSG/GSH Quantification Kit on the one hand. On the other hand, proteins were extracted from cells to detect the expression level of GPX4 by western blot. 4.4.7. Intracellular detection of immunogenic cell death and cGAS-STING activation Hepa1-6 cells (1 × 10 5 cells per well) were plated in con-focal dishes under a humidified atmosphere of 5% CO 2 at 37℃, followed by cell attachment overnight. Afterwards, PBS, 300 μg/mL of OMV, OP, OCM, and OPCM suspending in fresh FBS-free DMEM were added into the con-focal dishes respectively. After co-incubation for 24 h, immunofluorescence staining of intracellular γ-H2AX and CRT was conducted to detect DNA damage and CRT exposure. Simultaneously, all cells were seeded in in six-well plate and then treated with PBS, 300 μg/mL of OMV, OCM, and OPCM suspending in fresh FBS-free DMEM for 24 h. Afterwards, proteins were extracted from cells to detect the expression level of STING, p-STING, TBK1, p-TBK1, IRF3, and p-IRF3 by western blot. 4.4.8. Immune stimulation experiments induced by various OMV-based nanodrugs A coculture transwell system (6-well plate, 0.4 μm-sized microporous membrane) was used to evaluate the immunostimulatory effect of OMV-based nanodrugs on immature DCs and M2 TAMs. JAWSII cells were seeded on the bottom of the coculture transwell system, while the Hepa1-6 cells were placed on the upper chamber. After cell attachment overnight, the old culture medium was replaced by FBS free DMEM medium containing 300 μg/mL OMV, OP, OCM, and OPCM. 24 h after co-incubation, the JAWSII cells were obtained and stained with FITC CD11c antibody, PE anti-mouse CD80 antibody, and APC anti-mouse CD86 antibody following the manufacturer’s instruction. Finally, the JAWSII cells underwent FCM analysis to determine the proportion of matured DCs. In addition, the supernatant of the coculture transwell system was collected and sent for ELISA to detect the secretion level of ATP, HMGB1, Type 1 IFN, IL-6, and TNF-α. Similar to the experiment of JAWSII cells, RAW264.7 cells were seeded on the bottom of the coculture transwell system, while the Hepa1-6 cells were placed on the upper chamber. Notably, the RAW264.7 cells were first stimulated to differentiate into M2 TAMs by 40 ng/mL IL4. After cell attachment overnight, the old culture medium was replaced by FBS free DMEM medium containing 300 μg/mL OMV, OP, OCM, and OPCM. 24 h after co-incubation, the JAWSII cells were obtained and stained with PE anti-mouse CD80 antibody, and APC anti-mouse CD206 antibody following the manufacturer’s instruction. Finally, the RAW264.7 cells underwent FCM analysis to determine the proportion of M1 TAMs and M2 TAMs. In addition, the supernatant of the coculture transwell system was collected and sent for ELISA to detect the secretion level of ATP, HMGB1, Type 1 IFN, IL-10, and IL-12. 4.5. Animal experiment 4.5.1. Establishment of syngeneic HCC mouse models All animal experiments were approved by the Ethics Committee of Chongqing Medical University and Institutional Animal Care. The permit number for the animal experiments is Research Ethics Review No. 223 (2023). C57BL/6J and BALB/c mice (6-8 weeks, male) were bought from the experimental animal center of Chongqing Medical University. Subcutaneous syngeneic C57BL/6J HCC mouse models were developed by injecting 5 × 10 6 Hepa1-6 cells suspended in 100 μL PBS into the right flanks of 6-8-week-old male C57BL/6J mice (5 mice per group). H22 tumor-bearing BALB/c mice were established by injecting 2×10 6 H22 cells suspended in 100 μL PBS into the right flanks of 6-8-week-old male BALB/c mice (3 mice per group). 4.5.2. Evaluation of antitumor effect in vivo The schematic diagram of therapeutic and observation process for subcutaneous syngeneic HCC mouse models was shown in Figure 6a. Subcutaneous tumors of C57BL/6J and BALB/c mice reached a measurable size 7 d post tumor cell inoculation. Then, the tumor-bearing C57BL/6J and BALB/c mice were randomly assigned to five groups, receiving saline or PBS, OMV, αPD-L1, OPCM, and the combination of αPD-L1 and OPCM on 1 st , 4 th , 8 th , 12 th . All mice were weighed and tumor volume was assessed once every other day. The length (L) and width (W) of tumors were measured by Vernier calipers to calculate the tumor volume (V) through the formula: V = (L × W 2 )/2. Photos were taken on the 1st, 5th, 10th, 15th, and 20th day. The observation for tumor treatment lasted for 20 days. Subsequently, the mice were sacrificed and the tumor tissues were extracted and weighed to calculate the tumor inhibition rate. After that, the tumor tissues extracted from C57BL/6J mice were fixed with 4% paraformaldehyde, followed by hematoxylin and eosin (H&E) staining assays and the immunofluorescent staining of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA). The average FL signals of TUNEL and PCNA were calculated by ImageJ. Simultaneously, survival curves of C57BL/6J mice were monitored every other day for up to 60 days after the first treatment. The mice were considered dead when the tumor volume reached 1500 mm 3 . 4.5.3. Evaluation of ferroptosis and cGAS-STING activation After 13 days, tumor tissues of each group were extracted and sent for IF staining to assess the expression of GPX4, DHE, HIF-1α, p-STING, p-TBK1, p-IRF3, CRT, and HMGB1, IFN-γ. To detect the expression of DHE, the tumor tissues of each group were frozen in -80℃ refrigerator and then sent for frozen slicing and staining of DHE working solution. Finally, the tumor sections were observed under a FL microscopy (NIKON DS-U3, Japan). 4.5.4. Evaluation of the immune cell in the TME To study the immunostimulatory effect of the OMV-based nanodrugs, tumors, tumor-draining lymph nodes (TDLNs) and spleens were harvested from C57BL/6J mice 12 days after first treatment in different groups for immune cell analysis. Immediately after tumor tissue extraction, the TDLNs and spleens were obtained. These tissues were cut into small pieces, and digested with collagenase A (1 mg/mL), DNAase I (0.5 mg/mL) and HAase (1 mg/mL) for 30 min at 37°C. Afterward, the tissues and TDLNs were filtered to obtain single cells by 40 μm cell strainers, while spleens were filtered to obtain single cells by 70 μm cell strainers. Afterwards, the collected cells were centrifuged at 1500 rpm for 5 min and then suspended in red blood cell lysis buffer for 2 min to remove residual red blood cells. Subsequently, 1% FBS was added into the mixed cell suspension to stop digestion, followed by centrifugation at 1500 rpm for 5 min to obtain the purified cells from tumor tissues, TDLNs, and spleens. Next, the cells were stained with the corresponding fluorescence-labeled antibodies as follows: FITC anti-mouse CD11c, PE anti-mouse CD80, APC anti-mouse CD86, PE anti-mouse CD3, APC anti-mouse CD8a, FITC anti-mouse F4/80, PerCP/Cyanine5.5 anti-mouse/human CD11b, APC anti-mouse CD206, APC anti-mouse NK1.1. Finally, samples were detected by FCM. Simultaneously, tumor tissues in each group were collected for ELISA to detect the intratumoral secretion level of ATP, HMGB1, and type 1 IFNs, while serums in each group were collected for ELISA to detect the secretion level of IL-6, TNF-α, IL-10, and IL-12. 4.5.6. The establishment of tumor re-challenged models and lung metastasis models The C57BL/6J subcutaneous models were established by the aforementioned method, followed by the same therapeutic interventions. On Day 21, primary tumors in the right flanks of mice were completely excised by surgery. After that, all mice were retained for one week. On day 30, 5×10 6 Hepa1-6 cells were rechallenged into the mice’s left flanks of mice contralateral to the primary tumor site. The rechallenged tumor growth curves and weight changes were monitored once every other day from day 37 to day 51. The L and W of tumors were measured by Vernier calipers to calculate the tumor volume with the same aforementioned calculation formula. Photos were taken on the 37 d, 40 d, 43 d, 46 d, 49 d and 52 d. The observation for tumor treatment lasted for 15 days. On Day 52, all mice were sacrificed and the tumors, TDLNs, and spleens were collected. All tumor samples were weighed and photographed. TDLNs and spleens underwent the aforementioned procedure to obtain single cell suspension. Next, the cells from the tumor tissues of different treatment groups were stained with PE anti-mouse CD3 and APC anti-mouse CD8a, FITC anti-mouse CD44 and PerCP/Cyanine 5.5 anti-mouse CD62L according to the manufacturer’s instructions. Finally, FCM was conducted to analyze the ratio of effector memory T (T em ) cells in the spleens. To establish the distant lung metastasis model, the subcutaneous models were established by the aforementioned method and treated by the same treatments. On day 21, to mimick the process of lung metastasis, the mice in each group were injected Hepa1-6 cells (1×10 6 ) by tail vein. On the 28 d, all mice were sacrificed and the lung were extracted, followed by staining with Bouin’s solution and HE staining to observe the lung metastasis condition. 4.5.7. FLI, MRI, and pharmacokinetics of OPCM In vivo FLI was demonstrated using a NIRF imaging system (NightOWL II LB983, Germany) (Exc/Em = 748/780 nm). OPCM@DiR (30 mg kg −1 ) were injected into the H22 tumor cell-bearing BALB/c mice intravenously. Then, the mice underwent FLI after certain time intervals (0, 2, 4, 8, 24, and 48 h) post injection. Afterward, the mice were sacrificed, followed by extraction of the tumors and main organs for biodistribution analysis. To detect the in vitro MR imaging performance of OPCM, OPCM with different concentrations (Mn: 0.13, 0.26, 0.77, 1.55, 2.58 mM) were scanned using a 3.0 T MRI scanner (MAGNETOM Prisma, Siemens Healthineers Inc., Munich, Germany). The samples were prepared in 2 mL centrifuge tube, followed by the acquisition of the T 1 weighted images (T 1 WI) using the MRI scanner. The T 1 WI parameters were listed as follows: fast field echo (FFE), TR = 650 ms, TE = 11 ms, and slice thickness = 1.4 mm. The T 1 relaxation rate (r 1 ) was calculated by the linear fitting of the inverse T 1 relaxation times as a function of Mn concentration. The T 1 relaxation rates of the acquired images were obtained through the T1 mapping data from Syngo.via software. For the MRI experiment in vivo, the MRI performance of the OPCM was demonstrated in H22 tumor cell-bearing BALB/c mice. A 3.0 T mouse MRI coil from Chenguang Medical Technology Company was used. The T 1 WI of the tumor area was captured at different time points (0, 2, 4, 8, 12 and 24 h) after the intravenous injection of the BSA-mFe@Len NPs (30 mg/kg). The T 1 WI parameters were set as follows: TR = 650 ms; TE = 11 ms; slice thickness = 1.5 mm; FOV = 80 mm. To determine the in vivo pharmacokinetics of the OPCM, the mice were first injected with the OPCM. Then, the mice were sacrificed at each predetermined times (0, 1, 2, 4, 8, 12, 24, 48, 72 h), followed extraction of tumor tissues and major organs (heart, liver, spleen, lung, kidney). All tissues were collected, weighed, dissolved with aqua regia (HCl: HNO 3 = 3:1), evaporated, and re-dissolved in 1% HNO 3 . The Cu and Mn concentration was estimated by ICP-MS. 4.5.8. Bio-safety of the OPCM The bio-compatibility of the OPCM in vivo was evaluated in C57 BL6/J (male, 6-8 weeks). The mice were randomly divided into six groups (control group and 1, 3, 7, 14, 21 d group after injection of OPCM, n=3). On Day 21, all mice were sacrificed. Blood samples were collected for routine blood and biochemical examinations. The major organs (heart, liver, spleen, lung, kidney) were collected for H&E analysis. 4.6. Statistical analysis All quantitative data are shown as the mean ± SD. Statistical analysis was performed using GraphPad 10.1 (La Jolla, CA, USA). A Student’s unpaired or paired t-test was used to analyze the significant differences between the two groups. We used the one unpaired multiple t-test and analysis of variance (ANOVA) for the analysis of the differences between multiple groups. Statistical tests were double-sided, and values with P < 0.05 were considered statistically significant. Declarations Author contributions Ying Luo is the first author, and Zhongsheng Xu are the parallel first author. They contributed equally to this study, including conceptualization, methodology, investigation, validation, data analysis. Specifically, Ying Luo is responsible for processing raw data and writing the manuscript, while Zhongsheng Xu is responsible for reviewing and revising the draft. Qianying Du, Lian Xu, and Yi Wang contributed partially to the methodology part. Jie Xu, Junrui Wang, Sijin Chen, Wenli Zhang, and Bo Liu contributed partially to the study administration part. Jia Liu contributed partially to the Funding part. Dajing Guo * , and Yun Liu * are the corresponding author and contributed mainly to the Funding part and paper revision. Funding This work was supported by the National Natural Science Foundation of China (82271970), Chongqing Medical Scientific Research Project (Joint Project of Chongqing Health Commission and Science and Technology Bureau) (Grant No. 2022ZDXM026), Senior Medical Talents Program of Chongqing for Young and Middle-aged, Chongqing Returned Overseas Students’ Entrepreneurship and Innovation Support Program (cx2021006), CQMU Program for Youth Innovation in Future Medicine (W0171), and the Kuanren Doctoral supervisor Cultivation Program of the second affiliated hospital of Chongqing Medical University. This work was also supported by the General Program of Chongqing Natural Science Foundation (cstc2021jcyj-msxmX0040) and the National Science Foundation for Young Scholars (82102063). Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References a)J. M. Llovet, R. K. Kelley, A. Villanueva, A. G. Singal, E. Pikarsky, S. Roayaie, R. Lencioni, K. Koike, J. Zucman-Rossi, R. S. Finn, Nat Rev Dis Primers 2021 , 7, 6; b)A. Vogel, T. Meyer, G. Sapisochin, R. Salem, A. Saborowski, Lancet 2022 , 400, 1345. a)F. Xu, T. Jin, Y. Zhu, C. Dai, J Exp Clin Cancer Res 2018 , 37, 110; b)Y. Zheng, S. Wang, J. Cai, A. Ke, J. Fan, Biochim Biophys Acta Rev Cancer 2021 , 1876, 188638. R. Donne, A. Lujambio, Hepatology 2023 , 77, 1773. a)M. Toyofuku, N. Nomura, L. Eberl, Nat Rev Microbiol 2019 , 17, 13; b)M. Toyofuku, S. Schild, M. Kaparakis-Liaskos, L. Eberl, Nat Rev Microbiol 2023 , 21, 415. a)Q. Chen, H. Bai, W. Wu, G. Huang, Y. Li, M. Wu, G. Tang, Y. Ping, Nano Lett 2020 , 20, 11; b)M. Z. Zou, Z. H. Li, X. F. Bai, C. J. Liu, X. Z. Zhang, Nano Lett 2021 , 21, 8609; c)F. Meng, L. Li, Z. Zhang, Z. Lin, J. Zhang, X. Song, T. Xue, C. Xing, X. Liang, X. Zhang, J Extracell Vesicles 2022 , 11, e12289; d)L. Chen, X. Ma, W. Liu, Q. Hu, H. Yang, Nano Lett 2023 , 23, 8725; e)G. Liu, N. Ma, K. Cheng, Q. Feng, X. Ma, Y. Yue, Y. Li, T. Zhang, X. Gao, J. Liang, L. Zhang, X. Wang, Z. Ren, Y. X. Fu, X. Zhao, G. Nie, Nat Nanotechnol 2023 . a)B. A. Flood, E. F. Higgs, S. Li, J. J. Luke, T. F. Gajewski, Immunol Rev 2019 , 290, 24; b)K. M. Garland, T. L. Sheehy, J. T. Wilson, Chem Rev 2022 , 122, 5977; c)Z. J. Long, J. D. Wang, J. Q. Xu, X. X. Lei, Q. Liu, Mol Ther 2022 , 30, 1006; d)E. N. Chin, A. Sulpizio, L. L. Lairson, Trends Cell Biol 2023 , 33, 189. a)N. Cheng, R. Watkins-Schulz, R. D. Junkins, C. N. David, B. M. Johnson, S. A. Montgomery, K. J. Peine, D. B. Darr, H. Yuan, K. P. McKinnon, Q. Liu, L. Miao, L. Huang, E. M. Bachelder, K. M. Ainslie, J. P. Ting, JCI Insight 2018 , 3; b)J. Guo, L. Huang, Trends Pharmacol Sci 2022 , 43, 957. a)D. Cen, Q. Ge, C. Xie, Q. Zheng, J. Guo, Y. Zhang, Y. Wang, X. Li, Z. Gu, X. Cai, Adv Mater 2021 , 33, e2104037; b)Z. Li, Z. Chu, J. Yang, H. Qian, J. Xu, B. Chen, T. Tian, H. Chen, Y. Xu, F. Wang, ACS Nano 2022 , 16, 15471. a)C. Wang, Y. Guan, M. Lv, R. Zhang, Z. Guo, X. Wei, X. Du, J. Yang, T. Li, Y. Wan, X. Su, X. Huang, Z. Jiang, Immunity 2018 , 48, 675; b)R. M. Hooy, G. Massaccesi, K. E. Rousseau, M. A. Chattergoon, J. Sohn, Nucleic Acids Res 2020 , 48, 4435; c)X. Sun, Y. Zhang, J. Li, K. S. Park, K. Han, X. Zhou, Y. Xu, J. Nam, J. Xu, X. Shi, L. Wei, Y. L. Lei, J. J. Moon, Nat Nanotechnol 2021 , 16, 1260; d)J. Yan, G. Wang, L. Xie, H. Tian, J. Li, B. Li, W. Sang, W. Li, Z. Zhang, Y. Dai, Adv Mater 2022 , 34, e2105783; e)Y. Zhu, Z. Yang, Z. Pan, Y. Hao, C. Wang, Z. Dong, Q. Li, Y. Han, L. Tian, L. Feng, Z. Liu, Sci Adv 2022 , 8, eabo5285. a)W. Wu, Y. Pu, X. Lu, H. Lin, J. Shi, Adv Healthc Mater 2021 , 10, e2001819; b)Y. Yang, K. Hu, P. Zhang, P. Zhou, X. Duan, H. Sun, S. Wang, Small 2021 , 17, e2100927; c)M. Tang, Z. Zhang, T. Sun, B. Li, Z. Wu, Adv Healthc Mater 2022 , 11, e2201733. a)Q. Xiang, C. Yang, Y. Luo, F. Liu, J. Zheng, W. Liu, H. Ran, Y. Sun, J. Ren, Z. Wang, Small 2022 , 18, e2107809; b)Z. Zhao, S. Dong, Y. Liu, J. Wang, L. Ba, C. Zhang, X. Cao, C. Wu, P. Yang, ACS Nano 2022 , 16, 20400; c)X. Jia, J. Wang, E. Wang, Adv Mater 2023 , e2309261; d)D. Li, E. Ha, Z. Zhou, J. Zhang, Y. Zhu, F. Ai, L. Yan, S. He, L. Li, J. Hu, Adv Mater 2023 , e2308747. a)S. Guo, W. Xiong, J. Zhu, J. Feng, R. Zhou, Q. Fan, Q. Zhang, Z. Li, J. Yang, H. Zhou, P. Yi, Y. Feng, S. Yang, X. Qiu, Y. Xu, Z. Shen, Biomaterials 2023 , 302, 122300; b)J. Liu, J. Zhan, Y. Zhang, L. Huang, J. Yang, J. Feng, L. Ding, Z. Shen, X. Chen, Adv Mater 2023 , e2309562. J. Wang, W. Song, X. Wang, Z. Xie, W. Zhang, W. Jiang, S. Liu, J. Hou, Y. Zhong, J. Xu, H. Ran, D. Guo, Biomaterials 2021 , 277, 121100. L. Kennedy, J. K. Sandhu, M. E. Harper, M. Cuperlovic-Culf, Biomolecules 2020 , 10. B. Niu, K. Liao, Y. Zhou, T. Wen, G. Quan, X. Pan, C. Wu, Biomaterials 2021 , 277, 121110. Y. Yang, S. Karakhanova, W. Hartwig, J. G. D'Haese, P. P. Philippov, J. Werner, A. V. Bazhin, J Cell Physiol 2016 , 231, 2570. R. Tang, J. Xu, B. Zhang, J. Liu, C. Liang, J. Hua, Q. Meng, X. Yu, S. Shi, J Hematol Oncol 2020 , 13, 110. Y. Luo, J. Wang, L. Xu, Q. Du, N. Fang, H. Wu, F. Liu, L. Hu, J. Xu, J. Hou, Y. Zhong, Y. Liu, Z. Wang, H. Ran, D. Guo, Acta Biomater 2022 , 154, 478. a)O. Y. Kim, H. T. Park, N. T. H. Dinh, S. J. Choi, J. Lee, J. H. Kim, S. W. Lee, Y. S. Gho, Nat Commun 2017 , 8, 626; b)W. Nie, A. Jiang, X. Ou, J. Zhou, Z. Li, C. Liang, L. L. Huang, G. Wu, H. Y. Xie, Biomaterials 2024 , 304, 122396. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files 0325SupportingInformation1.docx Scheme1.jpg Scheme 1. Schematic illustration of the synthesis of OPCM and OPCM-induced pleiotropic immune mobilization strategy in synergy with αPD-L1. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4183359","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288572458,"identity":"c1d63ba1-c5bf-44bb-a2b7-bebe9adb09d8","order_by":0,"name":"Ying Luo","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Luo","suffix":""},{"id":288572459,"identity":"d73517d1-ef9b-479b-838d-047c14b180e8","order_by":1,"name":"Zhongsheng Xu","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing 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University","correspondingAuthor":false,"prefix":"","firstName":"Sijin","middleName":"","lastName":"Chen","suffix":""},{"id":288572466,"identity":"d4c17fb4-63e3-41eb-ac61-e2ebc75395ae","order_by":8,"name":"Wenli Zhang","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenli","middleName":"","lastName":"Zhang","suffix":""},{"id":288572467,"identity":"c112b268-6450-4d08-9469-6f3fd43daf96","order_by":9,"name":"Bo Liu","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Liu","suffix":""},{"id":288572468,"identity":"f86164c4-9e95-4a4f-82df-96d2c6cc5145","order_by":10,"name":"Jia Liu","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Liu","suffix":""},{"id":288572469,"identity":"22a98711-c3b5-4df0-a898-d12466d1c9db","order_by":11,"name":"Dajing Guo","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dajing","middleName":"","lastName":"Guo","suffix":""},{"id":288572470,"identity":"ae04d9d1-b234-4298-97bd-4d7b556e32dc","order_by":12,"name":"Yun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYHACxgMJDDZQNhuReoBa0kjVwsBwmAQtBjdyDxx42HZenu/aGQOGD2WHGfhnNxDSkpdwILHttuHM2zkGjDPOHWaQuHOAkJYcA5CWBAOgFmbetsMMBhIJRGk5B9HylwQtByBaGInRInnmjcGBhHPJQL+kFRzsOZfOI3GDgBa+4zmGD3+U2cnz3U7e+OBHmbUc/wwCWhQOwFgHwIiBB796IJBvQNIyCkbBKBgFowArAABCdkry0Emm7wAAAABJRU5ErkJggg==","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yun","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-03-28 15:44:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4183359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4183359/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54428600,"identity":"62f6edcb-c471-4018-8043-47f4730daca1","added_by":"auto","created_at":"2024-04-10 10:05:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":182323,"visible":true,"origin":"","legend":"\u003cp\u003e(a) TEM of OMV, OP, and OPCM. Scale bar: 100 nm. (b) Zeta potential of OMV, PM, OP, OCM, and OPCM. Data are shown as the mean values ± SD (n = 5). (c) SDS-PAGE assay of OMV, PM, OP, and OPCM. (d) Elemental mapping of OPCM. Scale bar: 100 nm. (e, f, g) XPS survey spectra and XPS spectra of Cu2P and Mn2P. (h, i, j) CV curves of OP@Cu, OP@Mn, and OPCM of various electrodes in the non-Faradaic capacitive range at the scan rate of 10~100mV s\u003csup\u003e-1\u003c/sup\u003e. (k, i, m) Cell viability of M2 TAMs, JAWSII, Hepa1-6 cells after co-incubation with OMV, OCM, or OPCM. Data are shown as the mean values ± SD (n = 5). (n, o) FCM analysis of Hepa1-6 cells apoptosis after various treatments and corresponding quantitative analysis. Data are shown as the mean values ± SD (n = 3). All the statistical significance was analyzed by ANOVA. ∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, ∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, ∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.001, ∗∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/7406711a5fa4297c461bbeab.jpg"},{"id":54428603,"identity":"7916e7fe-bfbe-4e63-909c-c5a8cf6b716f","added_by":"auto","created_at":"2024-04-10 10:05:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164861,"visible":true,"origin":"","legend":"\u003cp\u003e(a) CLSM of ROS analysis induced by PBS, OMV, OCM, OPCM, and OPCM+Fer respectively. Scale bar: 50 μm. (b) LPO stained with green fluorescent BODIPY-C11 in Hepa1-6 cells after incubation with PBS, OMV, OCM, OPCM, OPCM + Fer, scale bar: 50 μm. (c) WB analysis of GPX4 expression in Hepa1-6 cells after treatment with PBS, OMV, OCM, or OPCM, respectively. (d) CLSM images of γ-H2AX and CRT expression in Hepa1-6 cells after treatment with PBS, OMV, OP, OCM, or OPCM. Scale bar: 50 μm. (e) The schematic diagram of ferroptosis-induced cGAS-STING signaling pathway activation. (f, g) WB analysis of key protein expression in the cGAS-STING signaling pathway and the corresponding quantitative analysis. Data are shown as the mean values ± SD (n = 3). (h) The ratio of phosphorylated proteins versus non-phosphorylated proteins in the cGAS-STING signaling pathway in different treatment groups. Data are shown as the mean values ± SD (n = 3). All the statistical significance was analyzed by ANOVA. ∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, ∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, ∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.001, ∗∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/a37297ce316f7b9a6e20037e.jpg"},{"id":54428607,"identity":"d2374b0a-92fd-45ed-93c5-0ca278ca90bc","added_by":"auto","created_at":"2024-04-10 10:05:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139249,"visible":true,"origin":"","legend":"\u003cp\u003e(a, b) The schematic diagram of the co-culture system of Hepa1-6 cells with DCs, and TAMs, respectively. (c, d) Representative FCM results of DC maturation after different treatments and the corresponding quantitative analysis. Data are shown as the mean values ± SD (n = 3). (e, f, g, h, i) ELISA test of the secretion level of ATP, HMGB1, Type 1 IFN, IL-6, and TNF-α in the coculture system of Hepa1-6 cells and DCs. Data are shown as the mean values ± SD (n = 3). (j, k, l, m) Representative FCM results of the expression of CD80 and CD206 in TAMs after different treatments and the corresponding quantitative analysis. Data are shown as the mean values ± SD (n = 3). (n, o, p, q, r) ELISA test of the secretion level of ATP, HMGB1, Type 1 IFN, IL-10, and IL-12, respectively. Data are shown as the mean values ± SD (n = 3). All the statistical significance was analyzed by ANOVA. ∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, ∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, ∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.001, ∗∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/24ff5d4c602af12dfb34482a.jpg"},{"id":54428601,"identity":"9015321a-497e-44f2-bf0c-38e9ee770801","added_by":"auto","created_at":"2024-04-10 10:05:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":159206,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic diagram of the experimental procedure for exploring antitumor effects of different treatments on Hepa1-6 cell inoculated C57BL/6J mice and H22 cell inoculated BALB/c mice. (b) Average body weight change of mice during the observation duration. Data are shown as the mean values ± SD (n = 5). (c) The growth curve of relative tumor volume in different treatment groups. Data are shown as the mean values ± SD (n = 5). (d) The digital photograph of extracted tumors at sacrifice on the 21\u003csup\u003est\u003c/sup\u003e day. Data are shown as the mean values ± SD (n = 5). (e) The individual tumor growth curve in each treatment group. Data are shown as the mean values ± SD (n = 5). (f) The representative PCNA, TUNEL, and HE staining of tumor slices after different treatments. Scale bar: 50 μm. (g, h) The quantitative analysis of PCNA FL intensity and TUNEL FL intensity after various treatments. (i) Representative MR gray images and pseudo-color images of the tumor-bearing mice at different time points after the \u003cem\u003ei.v.\u003c/em\u003e injection of OPCM. Data are shown as the mean values ± SD (n = 3). All the statistical significance was analyzed by ANOVA. ∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, ∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, ∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.001, ∗∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/cb7e3824ff644b798b0ca8b6.jpg"},{"id":54428602,"identity":"6ab73bc2-fb4c-41bc-b791-e364af8a4567","added_by":"auto","created_at":"2024-04-10 10:05:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":122583,"visible":true,"origin":"","legend":"\u003cp\u003e(a) IF staining of GPX4, DHE, HIF-1α in tumor sections after various treatments. Scale bar: 50 μm. (b) IF staining of p-STING, p-TBK1, and p-IRF3 in tumor sections after various treatments. Scale bar: 50 μm. (c) IF staining of DAMPs (CRT, and HMGB1) in tumor sections after various treatments. Scale bar: 50 μm. (d, e, f) Quantitative FL analysis of GPX4, DHE, and HIF-1α. Data are shown as the mean values ± SD (n = 3). (g, h, i) Quantitative FL analysis of p-STING, p-TBK1, and p-IRF3. Data are shown as the mean values ± SD (n = 3). (j, k) Quantitative FL analysis of CRT and HMGB1. Data are shown as the mean values ± SD (n = 3). All the statistical significance was analyzed by ANOVA. ∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, ∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, ∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.001, ∗∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/e705bdea37c7f9239cd88f43.jpg"},{"id":54428605,"identity":"f028f94f-e2a3-49d4-b63d-b6940bea3b95","added_by":"auto","created_at":"2024-04-10 10:05:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":191681,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Representative FCM results of matured DCs (CD11c\u003csup\u003e+\u003c/sup\u003eCD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e) within tumors, TDLNs, and spleens after different treatments. (b) Representative FCM results of CTLs (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e) within tumors, TDLNs, and spleens after different treatments. (c, d) Quantitative analysis of DC maturation and CTLs proportion within tumors, TDLNs, and spleens after different treatments. Data are shown as the mean values ± SD (n = 3). (e) Representative FCM results of M2 TAMs and M1 TAMs within tumors after different treatments. (f, g) Quantitative analysis of M1 TAMs and M2 TAMs within tumors after different treatments. Data are shown as the mean values ± SD (n = 3). (h, i) Representative FCM results of NK cells within tumors after different treatments and the corresponding quantitative analysis. Data are shown as the mean values ± SD (n = 3). (j, k) The secretion level of IL-6, TNF-α, IL-10, IL-12, ATP, HMGB1, and Type 1 IFN within mouse serums or tumors after various treatments. Data are shown as the mean values ± SD (n = 3). All the statistical significance was analyzed by ANOVA. ∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, ∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, ∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.001, ∗∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/1c018b84054a399e3e1c1e83.jpg"},{"id":54428599,"identity":"890db3c8-d4fa-4b03-b2c5-0771aff47a1d","added_by":"auto","created_at":"2024-04-10 10:05:15","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":146635,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The schematic diagram of the experimental procedure for re-challenged mouse models. (b) The body weight change of re-challenged mice in different groups. Data are shown as the mean values ± SD (n = 3). (c) The contra-lateral tumor growth curve of re-challenged mice in different groups. Data are shown as the mean values ± SD (n = 3). (d) The tumor weight of extracted contra-lateral tumors in different groups. Data are shown as the mean values ± SD (n = 3). (e) The digital photograph of the extracted contra-lateral tumors in different groups. (f, g) Representative FCM results of T\u003csub\u003eem\u003c/sub\u003e cells (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e-\u003c/sup\u003e) within TDLNs and the corresponding quantitative analysis. Data are shown as the mean values ± SD (n = 3). (h, i) Representative FCM results of T\u003csub\u003eem\u003c/sub\u003e cells (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e-\u003c/sup\u003e) within spleens and the corresponding quantitative analysis. Data are shown as the mean values ± SD (n = 3). (j) The schematic diagram of the experimental procedure for lung metastasis models. (k) Representative photographs of lung tissue in mice. (i) H\u0026amp;E staining of lung tissue in mice. All the statistical significance was analyzed by ANOVA. ∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05, ∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01, ∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.001, ∗∗∗∗ \u003cem\u003ep \u0026lt; \u003c/em\u003e0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/bbb37b059fe8b73a53d726a1.jpg"},{"id":55264642,"identity":"32d1b3cf-0fa3-4401-9d86-058da8b5fda0","added_by":"auto","created_at":"2024-04-25 01:45:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1882195,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/35ffc31e-65a9-46a8-8256-770dfdcf6670.pdf"},{"id":54428606,"identity":"0ea364e0-1faf-4075-97aa-39917604ffae","added_by":"auto","created_at":"2024-04-10 10:05:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5882867,"visible":true,"origin":"","legend":"","description":"","filename":"0325SupportingInformation1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/601bdea18dc8841640c94ab6.docx"},{"id":54428598,"identity":"dce4935a-ade6-4e69-853e-428b1e8490fa","added_by":"auto","created_at":"2024-04-10 10:05:15","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":175724,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic illustration of the synthesis of OPCM and OPCM-induced pleiotropic immune mobilization strategy in synergy with αPD-L1.\u003c/p\u003e","description":"","filename":"Scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4183359/v1/309141a4c70198fb49fa2979.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biomineralized bacterial outer membrane vesicles exert pleiotropic immunoferroptotic effects on immune-deserted liver cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn clinical settings, late-stage liver cancer patients display low response rates to a variety of treatments, including chemotherapy, radiotherapy, and anti-angiogenetic therapy.\u003csup\u003e[1]\u003c/sup\u003e Immunotherapy, such as immune checkpoint therapy, is an evolving therapeutic paradigm for cancer treatment, however, its clinical response rate for liver cancer is only estimated to be around 15%, which entails further improvement.\u003csup\u003e[2]\u003c/sup\u003e The low response rate to mainstream immunotherapy is mainly attributed to the severe immunosuppression of liver cancer featured by poor immunogenicity, T-cell malfunction, infiltration of immunosuppressive cells owing to the insufficient release of tumor-associated antigens (TAAs), high expression of programmed cell death 1 ligand 1 (PD-L1), and a large number of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and tumor-associated macrophages (TAMs).\u003csup\u003e[3]\u003c/sup\u003e As a result, mono-immunotherapy, like immune checkpoint inhibitors (anti-PD-1/PD-L1 antibody) which mainly mobilize the body's CD8\u003csup\u003e+\u003c/sup\u003e T cells to exert a tumoricidal effect, naturally showed sub-optimal effects on liver cancer treatment. Thus, it is imperative to seek a pleiotropic immune mobilization strategy that can induce highly immunogenic tumor cell death and mobilize both the innate and adaptive immune systems to suppress liver cancer growth, metastasis, and recurrence.\u003c/p\u003e\n\u003cp\u003eBacteria-derived outer membrane vesicle (OMV), secreted by Gram-negative bacteria, a neotype immune adjuvant or immune vaccine, holds great promise for boosting tumor immunotherapy.\u003csup\u003e[4]\u003c/sup\u003e OMV can initiate strong innate immune responses because OMV contains numerous pathogen-associated molecular patterns (PAMPs), such as peptidoglycan, lipopolysaccharide (LPS) and flagellin, and a growing body of evidence confirmed that OMV administration effectively mobilized multiple immune cells to resist tumor growth \u003cem\u003evia\u0026nbsp;\u003c/em\u003evarious approaches, like promoting dendritic cell (DC) maturation and TAM re-polarization, inducing immunogenic pyroptosis, and even evoking trained anti-tumor immunity.\u003csup\u003e[5]\u003c/sup\u003e Nevertheless, the mere administration of OMV only moderately suppresses tumor growth in numerous studies and entails combination treatment to strengthen antitumor immunity since the OMV can only serve as an immune adjuvant to increase the immunogenicity of tumor cells and activate the innate immune system to inhibit tumor growth. Even worse, intravenous administration of pristine OMV can cause systemic inflammatory reactions, attacking major functional organs. Therefore, optimization of pristine OMVs and exploiting OMV as a vector to load synergistic therapeutic agents are needed to trigger a comprehensive immune attack on liver cancer cells by mobilizing multiple immune cells from both the innate and adaptive immune systems.\u003c/p\u003e\n\u003cp\u003eIn recent years, several lines of evidence confirmed that cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway plays a crucial role in awakening both the innate and adaptive immune systems by triggering the production of type 1 interferons (IFNs) and other inflammatory cytokines, leading to natural killer (NK) cell activation, TAMs repolarization, DC maturation, and cytotoxic T cell (CTL) infiltration in the tumor microenvironment (TME).\u003csup\u003e[6]\u003c/sup\u003e Numerous STING agonists have been proposed to activate this popular signaling pathway for antitumor purposes.\u003csup\u003e[7]\u003c/sup\u003e Other than that, therapeutic agents that can produce intracellular reactive oxygen species (ROS) have been employed to assist in cGAS-STING activation in synergy with STING agonists since ROS can attack cellular DNA and result in cytosol DNA exposure, which can be recognized by monomeric cGAS to produce cGAMP, thereby priming the cGAS-STING signaling pathway.\u003csup\u003e[8]\u003c/sup\u003e To exert the full potential of the cGAS-STING signaling pathway for antitumor purposes, multiple strategies have been applied to induce cytosolic DNA exposure and sensitize protein activities simultaneously. Among all STING agonists, Mn\u003csup\u003e2+\u003c/sup\u003e has received vast attention since it can not only catalyze Fenton-like reactions to produce intracellular ROS, leading to DNA damage and cytosol DNA exposure, but also independently activates cGAS, enhances dsDNA-binding capability of cGAS, accelerates the overall catalytic activity of dsDNA-bound cGAS, and induces the phosphorylation of both TBK1 and p65 in a STING-independent manner, leading to synergistic cGAS-STING activation and pleiotropic immune mobilization.\u003csup\u003e[9]\u003c/sup\u003e Nonetheless, the catalytic efficiency of Mn\u003csup\u003e2+\u003c/sup\u003e is relatively low as compared to the other transitional metals, such as Cu, Fe, and Co, thereby requiring further improvement.\u003csup\u003e[10]\u003c/sup\u003e Hence, we hypothesize that the addition of another transitional metal element with high catalytic efficiency, such as Cu, may help boost ROS production and cGAS-STING activation. In addition to the immunostimulatory effect induced by metal ions with univalent state, metal ions with mixed valence state that possess multiple enzyme-like activities were reported to exert greater tumoricidal effects.\u003csup\u003e[11]\u003c/sup\u003e For example, Mn\u003csup\u003e2+\u003c/sup\u003e and Cu\u003csup\u003e+\u003c/sup\u003e in a low valence state possess peroxidase (POD)-like activities while Mn\u003csup\u003e3+\u003c/sup\u003e, Mn\u003csup\u003e4+\u003c/sup\u003e, and Cu\u003csup\u003e2+\u003c/sup\u003e in a high valence state possess catalase (CAT)/glutathione peroxidase (GPX)-like activities. Thus, we hypothesized that Mn and Cu with mixed valence states possess ternary enzyme-like activities (POD, GPX, CAT), resulting in ROS production, GPX4 degradation, and O\u003csub\u003e2\u003c/sub\u003e generation, ultimately resulting in non-ferrous ferroptosis and hypoxia alleviation. Ferroptotic tumor cells can release abundant intracellular cytokines, damaged associated antigen molecules (DAMPs), and TAAs to promote DC maturation, while hypoxia alleviation can reduce intratumoral hypoxia-inducible factor 1α (HIF-1α) expression, leading to TAM polarization. Robust IFN-γ secreted by CTL and NK cells as a result of the activated STING pathway can inhibit the xc-system of tumor cells and further suppress the bio-activity of GPX4, thereby enhancing lipid peroxidation (LPO) accumulation and exacerbating ferroptosis of tumor cells.\u003csup\u003e[12]\u003c/sup\u003e Herein, a closed-loop therapeutic paradigm was formulated between ferroptosis and cGAS-STING activation. However, the introduction of abundant free metal ions into human bodies poses a serious health threat, which will lead to metal toxicity and organ dysfunction. Precise transportation of metal ions into the targeted tumor sites and preventing premature leakage of metal ions are crucial for developing a bio-safe and feasible technique for metallo-immunotherapy.\u003c/p\u003e\n\u003cp\u003eInspired by the fact that OMV is negatively charged and embedded with vast biomolecules and proteins just as cytomembrane, we hypothesized that OMV may serve as a favorable nanocarrier to deposit positively charged metal ions. Herein, in this study, we first harvested OMVs from Escherichia coli (\u003cem\u003eE. coli\u003c/em\u003e), followed by Cu and Mn deposition. Afterward, to shield the pristine OMVs from the body's immune defense, platelet membrane (PM) was adsorbed on the surface of the OMVs by physical extrusion. Mechanistically, upon the stealth effect of PM, OPCM selectively accumulated at the tumor site, followed by phagocytosis by tumor cells. Once getting into the tumor cells, Cu and Mn ions were subsequently released from the OMVs into the cytoplasm. On the one hand, glutathione (GSH) depletion, ROS production, and O\u003csub\u003e2\u003c/sub\u003e generation can be achieved due to the mixed valence state of Cu and Mn, leading to down-regulation of HIF-1α, inactivation of GPX4, accumulation of lipid peroxidation, and cytosol DNA exposure, leading to the alleviation of tumor hypoxia, initiation of tumor ferroptosis, and activation of the cGAS-STING signaling pathway. On the other hand, Mn\u003csup\u003e2+\u003c/sup\u003e boosted the activation of the cGAS-STING signaling pathway by sensitizing the activities of various molecules in this molecular pathway. As a result, abundant immunogenic factors (CRT, ATP, HMGB1), TAAs, and type 1 IFNs can be produced and released due to broken cell membranes induced by LPO accumulation, and further awaken both the innate and adaptive immune systems by activating NK cells, re-polarizing M2 TAMs, promoting DC maturation, and recruiting cytotoxic T cells (CTLs) into the TME. Abundant IFN-γ secretion as a result of the massive immune stimulation further exacerbates tumor ferroptosis by down-regulating the GPX4 pathway, forming a closed-loop therapy. Specifically, Mn\u003csup\u003e2+\u003c/sup\u003e possesses magnetic resonance imaging (MRI) performance and can realize theranostic purposes in a TME-responsive manner. This paradigm was proved to significantly amplify the therapeutic effect of anti-PD-L1 antibody (αPD-L1) to treat hepatocellular carcinoma (HCC) in two mouse models. As a proof of concept, this is the first study that applied OMVs as metal ion transporters by one-step biomineralization and identified the critical role of both Cu and Mn in inducing ferroptosis and ferroptosis-enhanced cGAS-STING activation for tumor metallo-immunotherapy.\u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cp\u003e2.1. Characterization of various OMV-based nanodrugs\u003c/p\u003e\n\u003cp\u003eFirstly, pristine OMV from\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e was extracted as previously reported, followed by one-step biomineralization of Cu and Mn ions, and coating of PM.\u003csup\u003e[5c]\u003c/sup\u003e Next, a range of input concentrations of CuCl\u003csub\u003e2\u003c/sub\u003e and MnCl\u003csub\u003e2\u003c/sub\u003e, ranging from 50 mM to 800 mM, were applied to assess the optimal loading capacity (LC) of OMVs for both Cu and Mn ions. The results of inductively coupled plasma-mass spectrometry (ICP-MS)\u0026nbsp;indicated a dose-dependent increase of LC for both Cu and Mn. When the input concentration of Cu and Mn reached 800 mM, the LC reached ~6% for both metals \u003cstrong\u003e(Figure S1)\u003c/strong\u003e. The molar ratio of Cu and Mn in the OPCM sample was determined to be approximately 1.2:1 by ICP-MS. Based on this result, 800 mM input of Cu and Mn was chosen to synthesize OPCM for the following experiments. As shown in Figure 1a, the morphology of pristine OMVs was spherical under\u0026nbsp;transmission electron microscopy (TEM). After the coating of PM, a double membrane structure was identified under TEM. Beyond that, sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) assay confirmed that protein bands from the original PM and OMV were reserved in the OPCM, indicating the co-existence of both PM and OMV (Figure 1c). In addition, TEM mapping was done to verify the elemental composition of the ultimate OPCM. As indicated in Figure 1d, the C, O, Cu, and Mn elements were shown around the OPCM. Dynamic light scattering (DLS) revealed that the hydrodynamic size of OMVs, OP, OCM, and OPCM was 228, 396.9, 412.3, and 506.6 nm, respectively\u0026nbsp;(Figure S2). The results of DLS demonstrated that the coating of PM and deposition of Cu and Mn ions enlarged the diameter of OMVs, which might be ascribed to the aggregation of OPCM induced by metal ion deposition. The zeta potentials of OMV, PM, OP, OCM, and OPCM were all negative, among which OP displayed the most negative value, and OCM displayed the least negative value, indicating the successful loading of Cu and Mn ions in the OPCM (Figure 1b). The elemental composition of the OPCM was detected further by X-ray photoelectron spectroscopy (XPS), which revealed the co-existence of Cu and Mn elements in the OPCM (Figure 1e). Interestingly, after analysis of Cu2P and Mn2P, we identified two strong binding energy peaks (932.18 and 934.28 eV) corresponding to Cu\u003csup\u003e+\u003c/sup\u003e and Cu\u003csup\u003e2+\u003c/sup\u003e, and three strong binding energy peaks (640.88, 646.18, and 642.28 eV) that were assigned to Mn\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e3+\u003c/sup\u003e, and Mn\u003csup\u003e4+\u003c/sup\u003e (Figure 1f, g). The presence of Cu and Mn ions with mixed valence states was believed to be correlated with a series of redox reactions that took place in the process of metal ion biomineralization due to the presence of polysaccharide, protein molecules, and biomacromolecules anchored on the surface of pristine OMV. The\u0026nbsp;X-ray powder diffraction (XRD)\u0026nbsp;pattern showed the amorphous structure of OPCM (Figure S3). All the above results demonstrated that OPCM was successfully synthesized \u003cem\u003evia\u0026nbsp;\u003c/em\u003eone-step biomineralization.\u003c/p\u003e\n\u003cp\u003eIn addition, the electrochemically active area (ESCA) of biomineralized OMVs was measured through the double-layer capacitance method \u003cstrong\u003e(Figure 1h, i, j, and Figure S4)\u003c/strong\u003e. Compared with the mono-metallic biomineralized OMV, the dua-metallic biomineralized OMV obtained the highest value of ESCA (0.0481 mF/cm\u003csup\u003e2\u003c/sup\u003e), indicating that the dual-metallic biomineralized OMV enabled the most abundant active sites, which was mainly attributed to the introduced Cu ions with high catalytic activity. Metal ions in a low valence state with POD-like activities decompose H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into hydroxyl radical (•OH) under an acidic environment, while metal ions in a high valence state with CAT/GPX-like activities, promote O\u003csub\u003e2\u003c/sub\u003e production and GSH degradation into oxidized glutathione disulfide (GSSG), thereby alleviating intratumoral hypoxia and preventing ROS elimination.\u003csup\u003e[13]\u003c/sup\u003e Owning to the co-existence of Cu\u003csup\u003e+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e3+\u003c/sup\u003e, and Mn\u003csup\u003e4+\u003c/sup\u003e, high content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and GSH in tumor cells and TME were supposed to be catalyzed into O\u003csub\u003e2\u003c/sub\u003e, ·OH, and GSSG. Based on the results of the electrochemical test, the factual catalytic activity of OPCM was validated \u003cem\u003evia\u0026nbsp;\u003c/em\u003e3,3,5,5-tetramethylbenzidine (TMB), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and a dissolved oxygen monitor. TMB was applied to evaluate the POD-like activity of OPCM. The intermediate ·OH could oxidize TMB from colorless to blue with obvious characteristic light absorption at about 575 nm (Figure S5a). Absorption at this wavelength was enhanced in a concentration-dependent manner with the addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which indicated that OPCM had a favorable chemo-dynamic effect \u003cem\u003evia\u0026nbsp;\u003c/em\u003ethe Fenton-like reaction. A dissolved oxygen monitor was employed to confirm the CAT-like activity of OPCM. Figure\u0026nbsp;S5b showed a time-dependent change of O\u003csub\u003e2\u003c/sub\u003e generation in three groups with different concentrations of OPCM + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, among which 400 μg/mL OPCM displayed the maximum O\u003csub\u003e2\u003c/sub\u003e increase, suggesting the ability of OPCM to catalyze O\u003csub\u003e2\u003c/sub\u003e generation for alleviating intratumoral hypoxia status. To evaluate the GPX-like activity of OPCM, DTNB was chosen as an indicator, since DTNB can measure GSH content due to the weakening of its characteristic UV–Vis absorption peak at 412 nm when reacting with GSH. As revealed by Figure S5c, a time-dependent decrease pattern of GSH content was identified, which indicated that the as-synthesized OPCM possessed GPX-like activity and may potentiate ROS production and GPX4-dependent ferroptosis in tumor cells. Taken together, the obtained OPCM could not only decompose H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e but also could deplete GSH, leading to simultaneous O\u003csub\u003e2\u003c/sub\u003e generation, and ROS production, implying great promise for anti-tumor application. Subsequently, the TME-responsive release behavior of OPCM was measured by ICP-MS.\u0026nbsp;Figure S6\u0026nbsp;showed distinct acid-responsive release behavior of Cu and Mn, which may be attributed to the acid-responsive degradation of PM and OMV. The results proved that Cu and Mn ions could be released in tumor cells accurately and efficiently.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEncouraged by the ternary enzyme-like activities, the biosafety and tumor-killing effect of OMV-based nanodrugs were further evaluated at the cellular level. M2 TAMs and JAWSII cells were chosen to test the biocompatibility of OMV-based nanodrugs on non-tumor cells. As revealed in \u003cstrong\u003eFigure 1k\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;1l\u003c/strong\u003e, CCK-8 results showed that the pristine OMV, OCM, and OPCM had no obvious cytotoxicity on M2 TAMs and JAWSII cells even when the concentration reached 300 μg/mL. The cytotoxicity of OCM on RAW264.7 and JAWSII cells was determined to be lower than that of OPCM, and higher than that of OMV, which might be ascribed to PM-mediated intracellular uptake of metal-deposited OPCM. To test the antitumor effect of OPCM on Hepa1-6 cells, different concentrations of OMV, OCM, and OPCM were incubated with Hepa1-6 cells for 24 h. Figure 1m\u0026nbsp;revealed that the pristine OMV at 300 μg/mL lowered the viability of Hepa1-6 cells to about 70%, OCM at 300 μg/mL lowered the viability to about 60%, OPCM at the same concentration considerably lowered the viability to around 45%, suggesting the effective tumor-killing effect of bimetallic biomineralized OPCM. To summarize, compared with the cytotoxicity on RAW264.7 and JAWSII cells, OMV, OCM, and OPCM exerted stronger cytotoxicity on Hepa1-6 cells, which might be attributed to excessive GSH and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ein tumor cells. Furthermore, the existence of PM on the outer shell of OPCM facilitated intracellular uptake of OPCM due to the ligand-receptor interaction, leading to more metal ion intake compared with non-tumor cells. Based on the results of CCK-8, 300 μg/mL of OMV, OP, OCM, and OPCM were used to evaluate other cellular performance.\u003c/p\u003e\n\u003cp\u003eEffective intracellular uptake of OPCM in tumor cells is a prerequisite for achieving desirable antitumor efficacy. Therefore, the intracellular uptake of DiI-labeled OPCM was assessed by\u0026nbsp;confocal laser scanning microscopy (CLSM).\u0026nbsp;\u003cstrong\u003eFigure S7a\u003c/strong\u003e displayed a time-dependent increase of red fluorescence (FL) signals around the DAPI-stained nuclei, suggesting that the DiI-labeled OPCM was effectively engulfed by Hepa1-6 cells. This phenomenon was also verified by flow cytometry (FCM) as demonstrated in Figure S7b and S7c, suggesting that tumor cells could effectively engulf OPCM.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the inhibitory effect of various OMV-based nanomaterials on tumor cell growth, Calcein-AM (green)/propidium iodide (PI) (red) kit and Annexin V-FITC/PI assay were conducted and observed by both FL microscopy and FCM. According to the results of live/dead cell staining under FL microscopy, the control, OMV, and OP groups did not exhibit obvious cell damage, indicating that OMV and OP without metal ions were biocompatible \u003cstrong\u003e(Figure S8)\u003c/strong\u003e. Notably, the OCM group displayed a decreased number of live cells and a significantly increased number of dead cells, suggesting that the biomineralization of Cu and Mn on the OMVs increased cytotoxicity on cancer cells. Compared with the above four groups, the last OPCM group exhibited the least Calcein-AM-stained live cells and the most prevalent PI-stained dead cells, indicating that OPCM possessed the strongest anticancer efficacy. The results of FCM were in line with the result of live/dead cell staining.\u0026nbsp;(Figure 1n, and o).\u003c/p\u003e\n\u003cp\u003eTaken together, the as-synthesized OPCM was successfully synthesized, which possessed a typical membrane structure with vast bacterial biomacromolecules and platelet proteins, ternary enzyme-like activities (POD-like, CAT-like, and GPX-like activities), pH responsiveness, and\u0026nbsp;considerable anti-tumor effect, which might be due to the metal ion-induced tumoricidal activity and the PM-mediated intracellular uptake.\u003c/p\u003e\n\u003cp\u003e2.2. OPCM-initiated immunogenic ferroptosis and cGAS-STING activation\u003c/p\u003e\n\u003cp\u003eGiven the favorable ternary enzyme-like activities, distinct cellular uptake behavior, and effective tumoricidal effect of OPCM validated in the above studies, the antitumor therapeutic mechanism of OPCM was further evaluated at the cellular level. First, ·OH generation was detected using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe since non-fluorescent DCFH-DA can be converted into fluorescent DCF by ROS oxidization. Specifically, a small-molecule lipophilic antioxidant, Fer, was added to the OPCM group to antagonize ferroptosis induction. The result of CLSM revealed that OMV group displayed negligible DCFH-FITC FL signals, while the OCM and OPCM groups exhibited greater DCFH-FITC FL signals, with the OPCM showing the strongest FL signals which were reduced by the addition of Fer, suggesting that the biomineralization of both Cu and Mn potentiated ROS production in Hepa1-6 cells and the initiation of ferroptosis\u003cstrong\u003e\u0026nbsp;(Figure 2a)\u003c/strong\u003e. Accordingly, a similar result was observed via FCM (Figure S9). It is reported that the overproduction of ROS results in LPO and mitochondrial membrane damage, leading to ferroptosis. Therefore, OPCM-induced LPO was assessed by a BODIPY-C11 fluorescent probe (581/591 nm) which is a lipid-soluble fluorescent indicator of LPO. During the LPO, the BODIPY-C11 dye changes from the aggregate state with red FL to the monomer state emitting green FL. The pristine OMVs had little effect on the LPO of Hepa1-6 cells, while cells cultured with the OCM and OPCM emitted more intense green FL signals, suggesting that the biomineralization of Cu and Mn ions on the OMVs induced LPO and played a crucial role in initiating tumor ferroptosis (Figure 2b). Similar to the result of ROS detection, the intensity of LPO was reduced by the addition of Fer. These results collectively demonstrated that OPCM successfully induced cancer cell ferroptosis as shown by ROS production and LPO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperiments \u003cem\u003ein vitro\u003c/em\u003e showed that the OPCM was capable of consuming GSH which is an endogenous antioxidant.\u003csup\u003e[14]\u003c/sup\u003e Depletion of GSH in tumor cells inactivates GPX4, resulting in an imbalance of redox reactions and then the accumulation of LPO, and subsequent ferroptosis.\u003csup\u003e[15]\u003c/sup\u003e Therefore, we further determined whether the OPCM activated the GPX4-related ferroptotic pathway. As shown in\u003cstrong\u003e\u0026nbsp;Figure S10\u003c/strong\u003e, the OCM and OPCM groups exhibited distinct reduced levels of intracellular GSH content compared to the control and OMV groups, demonstrating the GPX-like activity of OPCM. To test whether GSH consumption induced by OPCM leads to GPX4 degradation, a western blot (WB) was conducted. The result showed that the expression of GPX4 protein was declined in the OCM and OPCM groups compared to the control and OMV group, suggesting that OPCM contributed to ferroptosis \u003cem\u003evia\u0026nbsp;\u003c/em\u003eGPX4 degradation in addition to ROS production (Figure 2c and Figure S11). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was reported that overexpression of intracellular ROS broke the integrity of the mitochondrial membrane, leading to the decrease of mitochondrial membrane potential (MMP) and the release of mitochondrial DNA (mtDNA).\u003csup\u003e[16]\u003c/sup\u003e To further assess OPCM-induced mitochondrial damage, the mitochondrial function was evaluated by JC-1 dye that can monitor MMP change. When the positive charge of cell membrane potential declines, the JC-1 dye changes from the aggregate state (red color fluorescence) to the scattered state (green color fluorescence). According to the results of CLSM, compared with the control and OMV group, cells cultured with OCM and OPCM displayed a distinct increase in green FL signals and a decrease in red FL signals, suggesting that the JC-1 dye changed from the aggregate state to the scattered state \u003cstrong\u003e(Figure S12)\u003c/strong\u003e. The result of FCM was in accordance with the CLSM results as shown in\u0026nbsp;Figure S13, illustrating that OPCM treatment broke the mitochondrial membrane integrity and declined the MMP of Hepa1-6 cells, which might result in the release of mtDNA into the cytosol. To confirm DNA damage, γ-H2AX was used as a DNA damage marker. According to the immunofluorescence (IF) results of γ-H2AX, OCM and OPCM exhibited greatly enhanced green γ-H2AX FL signals compared to the control, OMV, and OP groups, hinting that the bimetallic biomineralized OCM and OPCM effectively induced DNA damage which may lead to cytosol dsDNA exposure (Figure 2d, Figure S14).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, cytosol dsDNA exposure is not only a result of intracellular ROS attack but also an activator of the cGAS-STING signaling pathway which plays a prominent role in the cancer immunity cycle by producing pro-inflammatory cytokines, such as type 1 IFN. Hypothetically, the cGAS-STING signaling pathway could also be synergistically activated by Mn\u003csup\u003e2+\u003c/sup\u003e anchored on the OPCM as reported by previous studies. Hence, the expression levels of STING, p-STING, TBK1, p-TBK1, IRF3, and p-IRF3 were measured and corresponding statistical analyses were conducted. The schematic diagram of cGAS-STING activation is displayed in \u003cstrong\u003eFigure 2e\u003c/strong\u003e. As demonstrated in Figure 2f and 2g, the OMV treatment had little impact on the expression level of all the above six proteins just as the control group. The bimetallic biomineralized OCM and OPCM showed insignificant influence on the expression of non-phosphorylated proteins (STING, TBK-1, IRF3), but augmented the expression of phosphorylated proteins (p-STING, p-TBK1, and p-IRF3). Through statistical analysis, the expression ratio of phosphorylated proteins versus non-phosphorylated proteins was significantly increased in the OCM and OPCM groups compared with the control and OMV groups, suggesting that the bimetallic biomineralized OCM and OPCM successfully activated the cGAS-STING signaling pathway (Figure 2h).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo summarize, GSH depletion and ROS generation could be achieved due to the mixed valence states of Cu and Mn in OCM or OPCM, thus enabling tumor cells to undergo non-ferrous ferroptosis \u003cem\u003evia\u0026nbsp;\u003c/em\u003einactivation of GPX4 and accumulation of LPO. In this process, the mitochondrial membrane was broken and the intranuclear DNA and mitochondrial DNA were damaged, leading to the cytosol mtDNA and dsDNA exposure, which might be conducive to activating the cGAS-STING signaling pathway. Notably, ferroptotic cancer cells are known to be immunogenic as the affected cells could release TAAs, and damage-associated molecular patterns (DAMPs), ultimately leading to antitumor immune activation. Therefore, the immunostimulatory effect of OPCM will be assessed and discussed in the following sections.\u003c/p\u003e\n\u003cp\u003e2.3. Exploration of ICD and immune cell regulation \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFerroptosis was reported to be immunogenic as numerous studies proved that abundant DAMPs (ATP, CRT, and HMGB1) are released from cell membrane rupture due to LPO accumulation, which promotes immune stimulation, such as DC maturation and TAM re-polarization.\u003csup\u003e[17]\u003c/sup\u003e In addition, the end-product of the cGAS-STING signaling pathway, type 1 IFN, was reported to mobilize multiple immune cells to exert tumoricidal effects. Furthermore, OMVs were also confirmed to be a strong immune adjuvant for immune stimulation. Therefore, the immune stimulatory effects of OPCM were examined on DCs and TAMs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3a and 3b\u003c/strong\u003e elucidated the co-culture system of Hepa1-6 cells and DCs or TAMs respectively. The Hepa1-6 cells were placed on the upper chamber, while DCs or TAMs were seeded on the bottom of the six-well plate.\u0026nbsp;As demonstrated in the FCM results, compared to the control group, the proportion of matured DCs was increased by 3.1-, 2.6-, 5.4-, and 6.6-fold in the OMV, OP, OCM, and OPCM groups, respectively, which showed that all the OMV-based nanodrugs were capable of inducing ICD and therefore promoting DC maturation and activating cellular antitumor immunity (Figure 3c, 3d). Notably, the population of CD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells in the OPCM group was the largest and 6.6-fold higher than that of the control group, suggesting that metal ions with mixed valence states played a dominant role in DC maturation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cytokine secretion level was also measured to confirm the ICD effect and DC maturation. Firstly, the ICD effect of OPCM on CRT expression was examined by IF staining.\u0026nbsp;As revealed by \u003cstrong\u003eFigure S15\u003c/strong\u003e, the OCM and OPCM groups displayed more green FL signals in comparison with the control group, OMV group, and OP group, suggesting the exposure of CRT induced by OCM and OPCM. Subsequently,\u0026nbsp;we decided to further measure the secretion level of another two DAMPs, ATP and HMGB1, \u003cem\u003evia\u0026nbsp;\u003c/em\u003eenzyme-linked immunosorbent assay (ELISA). Type 1 IFN, and typical pro-inflammatory cytokines secreted by matured DCs, IL-6 and TNF-α were also tested \u003cem\u003evia\u0026nbsp;\u003c/em\u003eELISA. The secretion levels of ATP and HMGB1 were distinctly higher in the OCM and OPCM groups than other groups, confirming the ICD effect of metal-deposited OMVs (Figure 3e, 3f). The secretion level of type 1 IFN was consistent with the results of ATP and HMGB1 (Figure 3g). There was a little variation of type 1 IFN among the control, OMV, and OP group. The OCM and OPCM groups remarkably enhanced the secretion level of type 1 IFN, suggesting the activation of the cGAS-STING signaling pathway. IL-6 and TNF-α were also higher in the OCM and OPCM groups than other groups, illustrating the effective DC maturation induced by bimetallic biomineralized OMVs (Figure 3h, 3i). Notably, the secretion levels of IL-6 and TNF-α were slightly higher in the OMV and OP groups than those in the control group, which might be ascribed to the intrinsic immunostimulatory effect of OMV on immature DCs.\u003c/p\u003e\n\u003cp\u003eNext, the immunostimulatory effect of OMV-based nanodrugs on M2 TAMs was assessed. The OMV, OP, OCM, and OPCM groups all displayed higher CD80\u003csup\u003e+\u003c/sup\u003e cells than the control group, among which the OPCM displayed the highest proportion of CD80\u003csup\u003e+\u003c/sup\u003e cells among which the OPCM displayed the highest proportion of CD80+ cells (74.2%) which indicated the enhancement of M1 TAMs \u003cstrong\u003e(Figure 3j, 3k)\u003c/strong\u003e. Conversely, the number of CD206\u003csup\u003e+\u003c/sup\u003e cells was significantly lowered in all four intervention groups, among which the OPCM group displayed the minimum number of CD206\u003csup\u003e+\u003c/sup\u003e cells, suggesting that all the OMV-based nanodrugs were capable of decreasing the number of M2 TAMs (Figure 3l, 3m). The secretion levels of ATP, HMGB1, and type 1 IFN in the coculture system of Hepa1-6 and TAMs followed the same pattern just as that of Hepa1-6 and DCs. The bimetallic biomineralized OCM and OPCM significantly enhanced the secretion of ATP, HMGB1, and type 1 IFN (Figure 3n, 3o, 3p). In addition, we measured classical anti-inflammatory cytokine, IL-10, secreted by M2 TAMs, and classical pro-inflammatory cytokine, IL-12, secreted by M1 TAMs, to further verify the TAM re-polarization effect induced by OCM and OPCM. As expected, the secretion of IL-10 was significantly reduced in the OCM and OPCM groups, while the secretion of IL-12 was remarkably enhanced in these two groups, suggesting the successful re-polarization of M2 TAMs into M1 TAMs (Figure 3q, 3r).\u003c/p\u003e\n\u003cp\u003eIn short, the as-synthesized OMV, OP, OCM, and OPCM could all promote DC maturation and TAM re-polarization with the OPCM exerting the greatest immune activation effect. The potent immunostimulatory effect of OPCM might be ascribed to the collective effort induced by\u0026nbsp;PAMPs\u0026nbsp;anchored on OMVs and Cu and Mn-induced cGAS-STING activation and immunogenic ferroptosis. Inspired by the results of the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eexperiments, we hypothesized that the OPCM might serve as a potent immune adjuvant to exert antitumor immunity in combination with typical immunotherapy in the following \u003cem\u003ein vivo\u003c/em\u003e studies.\u003c/p\u003e\n\u003cp\u003e2.4. Exploration of anti-tumor effect \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBased on the satisfactory inhibitory effect of OMV-based nanodrugs on tumor cells \u003cem\u003ein vitro\u003c/em\u003e, the in-depth anti-tumor effect \u003cem\u003ein vivo\u003c/em\u003e was further investigated in two mouse models, including\u0026nbsp;C57BL/6J and BALB/c mice. To be specific, Hepa1-6 cells were inoculated into the right flank of C57BL/6J mice, while H22 cells were inoculated into the right flank of\u0026nbsp;BALB/c\u0026nbsp;mice.\u003csup\u003e[18]\u003c/sup\u003e The treatment protocol is depicted in\u003cstrong\u003e\u0026nbsp;Figure 4a\u003c/strong\u003e. As a crucial immunosuppressive factor in the TME, high expression of PD-L1 could trigger malfunction of CTL and compromise T cell-related tumoricidal effect. Encouraged by the efficient tumor-killing effect as demonstrated in the above \u003cem\u003ein vitro\u003c/em\u003e studies, a commonly used immunotherapeutic agent, αPD-L1, was applied to synergize with OPCM to boost the pleiotropic immune stimulation effect of OPCM and concurrently induce tumoricidal effects on liver cancer models. The mice were divided into five groups, including the control, OMV, αPD-L1, OPCM, and the combination of αPD-L1 and OPCM. Firstly, we analyzed the antitumor effect of the four intervention groups on C57BL/6J mice. The body weight of mice in all groups showed no abnormalities, suggesting that all the treatments exhibited no obvious systemic toxicity (Figure 4b). As revealed by the growth of tumor relative volume, the combination group\u0026nbsp;(αPD-L1 + OPCM) presented a higher tumor inhibiting capacity compared with the other groups (Figure 4c). The results of the individual tumor growth curve, the representative photos of mice during treatment, the photograph and average weight of extracted tumors in each group, and the corresponding tumor inhibition rates were consistent with the outcome of relative tumor volume as shown in Figure 4d, 4e, S16, and S17. Afterward,\u0026nbsp;terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL),\u0026nbsp;proliferating cell nuclear antigen (PCNA), and\u0026nbsp;hematoxylin and eosin (H\u0026amp;E)\u0026nbsp;staining assays were conducted to assess the extent of tumor apoptosis, tumor proliferation, and tumor necrosis. As demonstrated in\u0026nbsp;Figure 4f, 4g, 4h, prevalent red PCNA FL signals were shown in the control group. The PCNA FL signals were slightly diminished in the OMV and αPD-L1 groups, while the FL signals were largely reduced in the OPCM and the combination groups, suggesting a lower cell proliferation in the two groups. Conversely, the results of TUNEL staining showed the opposite outcomes, with the combination group showing the greatest green TUNEL FL signals and the control group showing the least FL signals, suggesting that the combination group presented the highest cell death ratio. The H\u0026amp;E staining results were consistent with the TUNEL and PCNA staining assays, in which excessive apoptosis and necrosis were found in the OPCM and combination groups, implying the effective antitumor effect induced by the combination of OPCM and αPD-L1. The survival time was significantly prolonged in all intervention groups compared with the control group, suggesting the long-term antitumor protection of the OMV-based nanodrugs and/or αPD-L1 (Figure S18). Notably, there was only one mouse dead in the combination group, suggesting the effective therapeutic capacity and the long-term protective effect of the combination therapeutic paradigm. In summary, the above data suggested that the therapeutic effect of pristine OMVs was moderate, while the Cu and Mn deposited on the OMVs augmented the therapeutic efficacy of pristine OMVs for liver cancer, which was further strengthened by the combination with αPD-L1.\u003c/p\u003e\n\u003cp\u003eSince the TME of liver cancer is complex and heterogeneous, another hepatocellular carcinoma (HCC) cell line model, H22 cells, was chosen to establish\u0026nbsp;BALB/c\u0026nbsp;tumor-bearing mouse models to confirm the therapeutic potential of the OMV-based nanodrugs for HCC treatment. Similar to the therapeutic outcomes of the C57BL/6J tumor-bearing mouse model, all intervention groups displayed obvious tumor inhibition, with the combination group showing the greatest tumor suppression effect\u003cstrong\u003e\u0026nbsp;(Figure S19)\u003c/strong\u003e. The body weight was stable in different groups, showing no unusual changes during the treatment processes. The representative digital images of H22 tumor-bearing mice, the digital photograph of dissected tumors in different groups 20 d post-treatment, the extracted tumor weight, the individual tumor growth curve, and the tumor inhibition rates elucidated the same outcomes.\u003c/p\u003e\n\u003cp\u003eApart from eliciting the high therapeutic efficacy, OPCM is also able to achieve TME-responsive T\u003csub\u003e1\u003c/sub\u003e weighted MR imaging due to the presence of Mn\u003csup\u003e2+\u003c/sup\u003e. We first demonstrated the MRI performance of OPCM \u003cem\u003ein vitro\u003c/em\u003e. As revealed by \u003cstrong\u003eFigure S20a\u003c/strong\u003e, Mn concentration-dependent 1/T\u003csub\u003e1\u003c/sub\u003e increase was found in all PBS systems containing 500 μg/mL OPCM. The T\u003csub\u003e1\u003c/sub\u003e relaxation rate was 0.7854 mM\u003csup\u003e-1\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e in pH 7.4 PBS system, 2.210 mM\u003csup\u003e-1\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e in pH 6.0 PBS system, 2.434 mM\u003csup\u003e-1\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e in pH 5.0 PBS system, and 2.636 mM\u003csup\u003e-1\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e in pH 5.0 + 10 mM GSH PBS system, among which the PBS system with pH 5.0 + 10 mM GSH displayed the strongest T1 signal and the largest T\u003csub\u003e1\u003c/sub\u003e relaxation rate (Figure S20b). The above data indicated that OPCM released more Mn\u003csup\u003e2+\u003c/sup\u003e in acidic environment with high GSH content, suggesting that the OPCM might release abundant Mn\u003csup\u003e2+\u003c/sup\u003e in the TME. Then, we further detected its imaging capability\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e. As shown in Figure 4i and Figure S21, the OPCM displayed favorable T\u003csub\u003e1\u003c/sub\u003e-weighted MRI performance. After \u003cem\u003ei.v.\u003c/em\u003e injection of OPCM, the T\u003csub\u003e1\u003c/sub\u003e signal at the tumor site displayed a time-dependent increase until the 4\u003csup\u003eth\u003c/sup\u003e hour and then started to decrease until the 24\u003csup\u003eth\u003c/sup\u003e hour.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo summarize, OPCM exhibited a strong antitumor effect on liver cancer models, and the effect was further amplified by αPD-L1, which might be attributed to the synergistic effect of immunogenic ferroptosis, cGAS-STING activation, and salvation of T-cell. The mechanism and the alternation of the tumor immune microenvironment will be discussed in detail in the following parts.\u003c/p\u003e\n\u003cp\u003e2.5. Exploration of immunogenic ferroptosis and cGAS-STING activation \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo further clarify the underlying mechanism of OPCM-based treatments on the TME of liver cancer, the tumor tissues in each group were collected and some important proteins and markers were investigated \u003cem\u003ein vivo\u003c/em\u003e. In the\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e study, the OPCM was demonstrated to possess POD-like, CAT-like, and GPX-like activities. Moreover, in cellular studies, OPCM was proven to induce ferroptosis and activate the cGAS-STING signaling pathway, leading to the release of DAMPs and type 1 IFN. Hence, we conducted IF staining to verify the expression of GPX4, DHE, HIF-1α, p-STING, p-TBK1, p-IRF3, CRT, and HMGB1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs expected, the red GPX4 signals were largely reduced in the OPCM and combination group compared with the control group, while the intervention of OMV and αPD-L1 had no significant influence on the expression of GPX4, suggesting that Cu and Mn ions in the OPCM played the key role in degrading GPX4 (Figure 7a, 7d). Contrary to the result of GPX4, the dihydroethidium (DHE) frozen staining revealed that the OPCM and the combination groups greatly enhanced the ROS generation, while OMV had little impact on the ROS level, indicating the POD-like activity of OPCM \u003cstrong\u003e(Figure 5a, 5e)\u003c/strong\u003e. Since the OPCM was capable of catalyzing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate O\u003csub\u003e2\u003c/sub\u003e, a cellular hypoxia indicator, HIF-1α, was chosen to detect the status of tumor hypoxia. According to the IF results\u0026nbsp;(Figure 5a, 5f), the tumors in the control group displayed high expression of HIF-1α. The intervention of OMV and αPD-L1 showed negligible effects on reducing tumor hypoxia, while the bimetallic biomineralized OPCM and the combination treatments significantly reduced tumor HIF-1α signals, illustrating the intratumoral O\u003csub\u003e2\u003c/sub\u003e generation capability of OPCM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious cellular experiments demonstrated that DNA damage induced by OPCM could activate the cGAS-STING signaling pathway, therefore we further evaluated the influence of OPCM on the cGAS-STING pathway \u003cem\u003ein vivo\u003c/em\u003e. As expected, the treatment with OMV or αPD-L1 exhibited negligible effect on the expression of p-STING, p-TBK1, and p-IRF3, while the bimetallic biomineralized OPCM group and the combination group markedly enhanced the expression of the three phosphorylated proteins, corroborating that Cu and Mn deposited on the OPCM activated the cGAS-STING signaling pathway \u003cstrong\u003e(Figure 5b, 5g, 5h, 5i)\u003c/strong\u003e. As reported in previous studies, CTLs and NK cells are capable of secreting IFN-γ which exacerbates ferroptosis by inhibiting system xc\u003csup\u003e-\u003c/sup\u003e and downregulating the expression of intracellular GPX4.\u003csup\u003e[12]\u003c/sup\u003e In our study, the administration of αPD-L1 was believed to activate CTLs, while OPCM was demonstrated to activate the cGAS-STING signaling pathway which further promoted DC maturation, CTL, and NK activation, resulting in abundant release of IFN-γ. As revealed by the IF staining of IFN-γ in the tumor tissues (Figure S22), the administration of OMV slightly enhanced the expression of IFN-γ which may be attributed to the immunostimulatory effect of OMV, while the αPD-L1, OPCM, αPD-L1 + OPCM groups displayed significantly enhanced expression of IFN-γ, and the combination group showed the greatest expression, suggesting that the co-administration of αPD-L1 and OPCM activated the immunoferroptosis pathway to the greatest level. As observed in the above IF staining of GPX4, the administration of αPD-L1 down-regulated GPX4 expression. Moreover, the co-administration of both OPCM and αPD-L1 reduced the expression of GPX4 to the least as compared with other groups, confirming the immunoferroptosis pathway activated by the synergistic immunoferroptotic effect of αPD-L1 and OPCM.\u003c/p\u003e\n\u003cp\u003eTo confirm the immunogenic effect of OPCM-induced ferroptosis, the expression levels of CRT and HMGB1 were measured by IF\u003cstrong\u003e\u0026nbsp;(Figure 5c, 5j, 5k)\u003c/strong\u003e. As shown by the IF results, the OMV and αPD-L1 slightly enhanced CRT expression and reduced the expression of intracellular HMGB1 in comparison with the control group, while the OPCM and the combination group drastically increased the expression of CRT and downregulated intracellular HMGB1, elucidating that OPCM induced immunogenic ferroptosis.\u003c/p\u003e\n\u003cp\u003eTo summarize, the OPCM exerted obvious POD-like, GPX-like, and CAT-like activities \u003cem\u003ein vivo\u003c/em\u003e, resulting in GPX4/ROS-dependent immunogenic ferroptosis, tumor hypoxia alleviation, and cGAS-STING activation. Moreover, OPCM and αPD-L1-induced immune activation further amplified the intensity of ferroptosis by IFN-γ, which forms a closed-loop therapy cycle. The alteration of tumor cell signaling pathways and transformation of tumor metabolism may explain the mechanism of the potent antitumor effects of OPCM.\u003c/p\u003e\n\u003cp\u003e2.6. Immune cell evaluation \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo delineate the immune landscape of HCC after various treatments, the infiltration level of various antitumor immune cells, including DCs, CTLs, M1 TAMs, M2 TAMs, and NK cells were analyzed by FCM. The matured DCs (CD11c\u003csup\u003e+\u003c/sup\u003eCD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e) are crucial competent antigen professional cells (APCs) and regulate the adaptive antitumor immunity, thereby cross-priming CTLs. To investigate DC maturation, tumor tissues, tumor-draining lymph nodes (TDLNs), and spleens were harvested post-treatment for FCM analysis\u0026nbsp;\u003cstrong\u003e(Figure 6a, 6c)\u003c/strong\u003e. Compared with the control group with ~12.8% intratumoral CD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells, the proportion of matured DCs showed a remarkable increase in the OPCM and combination groups, particularly in the latter group with 3.9-fold increase. Similarly, the combination group displayed the highest frequency of CD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells in the TDLNs (~41.3%) and spleen (~33.9%). Notably, the population of matured DCs in the OMV groups from the tumor (~28.7%), TDLNs (~24.9%), and spleen (~27.6%), was also obviously higher than that in the control group, which might be ascribed to the intrinsic immunostimulatory of OMVs on DCs as reported by previous research.\u003csup\u003e[19]\u003c/sup\u003e The FCM results of DC analysis in different organs clarified that OPCM was capable of promoting DC maturation which was necessary to initiate cellular immunity, and the enhancement was more pronounced in co-administration of OPCM and αPD-L1, which might be ascribed to the concerted immunostimulatory efforts of the PAMPs anchored on the OMV, abundant DAMPs outflow induced by ferroptosis, and cGAS-STING activation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMatured DCs are professional APCs that can process tumor antigens and present them to naive T cells, thereby activating T-cell function and differentiating them into CTLs. Since the OMV-based nanodrugs were proven to promote DC maturation successfully, therefore we decided to analyze the proportion of tumoricidal CTLs within tumors, TDLNs, and spleens. As revealed by the FCM outcomes \u003cstrong\u003e(Figure 6b, 6d)\u003c/strong\u003e, compared to the control group with 6.86% CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in the tumors, 14.1% CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in the TDLNs, and 6.84% CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleens, the treatment of OMV and αPD-L1 slightly increased the proportion of CTLs, while the combination group displayed the remarkable increase of CTLs with 3.3-fold increase in the tumor, 3.2-fold increase in the TDLNs, and 2.8-fold increase in the spleen, suggesting that the as-synthesized OPCM in combination with αPD-L1 could effectively mobilize a large number of intratumoral and systematic T cells, and prompt them differentiating into CTLs to exert tumoricidal effects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOwing to the O\u003csub\u003e2\u003c/sub\u003e generation capability of Mn and Cu ions with high valence state and immunostimulatory capacity (DAMPs and inflammatory cytokines induced by OPCM, and PAMPs carried by OMVs) of OPCM, protumoral M2 TAMs can be polarized into antitumoral M1 phenotype to awaken innate antitumor immunity. To test this hypothesis, the phenotype and proportion of M1 TAMs and M2 TAMs were verified\u0026nbsp;\u003cstrong\u003e(Figure 6e, 6f, and 6g)\u003c/strong\u003e.\u0026nbsp;As demonstrated by FCM results, the frequency of M2 TAMs, F4/80\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e cells, was largely decreased in the OPCM and combination groups, while the proportion of M1 TAMs, F4/80\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eCD80\u003csup\u003e+\u003c/sup\u003e cells, was markedly promoted in these two groups as compared with the control group, confirming the TAM re-polarization effect of OPCM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to DCs, CTLs, and TAMs, the proportion of another innate tumoricidal immune cell, NK cell was also detected since numerous studies proved that the activation of the cGAS-STING signaling pathway could increase the population of intratumoral NK cells \u003cem\u003evia\u0026nbsp;\u003c/em\u003ethe production of type 1 IFN. As expected, the OPCM and combination groups significantly increased the frequency of intratumoral NK cells by 4.3-fold and 5.7-fold as compared with the control group, indicating that the as-synthesized OPCM were able to mobilize NK cells to exert antitumor immunity \u003cstrong\u003e(Figure 6h and 6i)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eApart from FCM analysis of the immune cell population, some crucial cytokines and immunogenic factors were detected to corroborate the alteration of the tumor immune microenvironment by ELISA. On the one hand, we collected mouse serums post-therapeutic treatment to detect the secretion level of IL-6, TNF-α, and IL-12. \u003cstrong\u003eFigure 6j\u0026nbsp;\u003c/strong\u003eshowed that the serum content of pro-inflammatory cytokines, IL-6, TNF-α, and IL-12 were enhanced in all intervention groups as compared to the control group with the combination group showing the greatest enhancement, while the serum content of anti-inflammatory cytokines, IL-10 and IL-12, were downregulated in the intervention groups as compared to the control group, with the combination group displaying the lowest level of IL-10 and IL-12, indicating the effective DC maturation and TAM re-polarization effects induced by OPCM. On the other hand, we extracted tumor tissues post-treatment to detect the intratumoral content of ATP, HMGB1, and type 1 IFNs (Figure 6j, 6k). As expected, the expression levels of these three factors were markedly increased in the treatment groups with the combination group displaying the highest promotion, suggesting the induction of ICD and activation of cGAS-STING signaling pathway.\u003c/p\u003e\n\u003cp\u003eTaken together, all the above results demonstrated that the OPCM could serve as a potent immunoadjuvant to stimulate the immunosuppressive TME by mobilizing and activating multiple immune cells from the innate and adaptive immune systems to inhibit tumor growth in combination with αPD-L1.\u003c/p\u003e\n\u003cp\u003e2.7. Long-term antitumor immune memory effect \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe activation of the tumor immune microenvironment is not only conducive to killing primary tumors but also helps the body build up long-term immune memory against tumor recurrence and metastasis. Encouraged by the effective tumor suppression effect and the immunostimulatory effect of the combinational therapeutic paradigm (αPD-L1 and OPCM) achieved on the primary tumor models, tumor-re-challenged mouse and lung metastasis models were established to verify the long-term antitumor immune memory. The schematic diagrams of the establishment of both tumor models are shown in \u003cstrong\u003eFigure 7a and 7j\u003c/strong\u003e. The body weight of re-challenged mouse models was recorded for 2 weeks since the 37\u003csup\u003eth\u003c/sup\u003e day. The tumor weight of all re-challenged mouse models remained stable during the observation process (Figure 7b). Regarding the re-challenged tumor growth, the average tumor volume in the four intervention groups was smaller than that in the control group with 250 mm\u003csup\u003e3\u003c/sup\u003e (Figure 7c). Notably, the combination group showed the slowest tumor growth with an average tumor volume of around 70 mm\u003csup\u003e3\u003c/sup\u003e, indicating that the combination treatment possessed long-term resistance to tumor recurrence. The results of extracted tumor weight, the photograph of extracted tumors, and the representative digital photos of re-challenged mice were consistent with the outcomes of tumor volume growth, indicating that the OMV-based nanodrugs and/or αPD-L1 established long-term antitumor immune memory and thus provided long-term protection against tumor recurrence (Figure 7d, 7e, S23).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, the immune mechanism was further studied. TDLNs and spleens were extracted and digested to obtain a single-cell suspension, followed by FCM detection, to quantify the population of effector memory T cells (T\u003csub\u003eem\u003c/sub\u003e). As revealed by the FCM results of the TDLNs, compared with only 19.1% of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e-\u003c/sup\u003e cells, the other four intervention groups displayed enhanced proportion of T\u003csub\u003eem\u003c/sub\u003e, particularly in the OPCM and combination groups with 1.9-fold, and 2.2-fold increase, respectively\u0026nbsp;\u003cstrong\u003e(Figure 7f, 7g)\u003c/strong\u003e. Similarly, the FCM results of the spleens displayed the same outcome, with the combination group showing the highest proportion of T\u003csub\u003eem\u003c/sub\u003e (Figure 7h, 7i). These results indicated that the OMV-based nanodrugs combined with αPD-L1 could induce long-lasting antitumor immune memory and systemic immunity, which was beneficial for suppressing tumor recurrence via recognition of “old antigens”.\u003c/p\u003e\n\u003cp\u003eTo further verify that\u0026nbsp;the combination paradigm of OPCM and αPD-L1 can suppress tumor metastasis, we established lung metastasis models by injecting Hepa1-6 tumor cells into the mice after various treatments through the tail vein. One week after tumor cells injection, the lung tissues of all mice were obtained, photographed, and sent for H\u0026amp;E staining to observe lung metastasis nodules. As revealed by \u003cstrong\u003eFigure 7k\u003c/strong\u003e and \u003cstrong\u003e7l\u003c/strong\u003e, as compared with the four intervention groups, the H\u0026amp;E staining of lung sections in the control group showed a higher degree of canceration in which numerous metastasis nodules were found in-homogeneously, indicating the effective suppression of metastasis induced by OMV-based nanodrugs and/or αPD-L1 due to the activation of antitumor immune response.\u003c/p\u003e\n\u003cp\u003eIn short, we demonstrated that the OMV-based nanodrugs could not only suppress the primary tumor growth by ferroptosis induction and pleiotropic immune cell mobilization but also induced long-term antitumor immune protection against tumor recurrence and metastasis \u003cem\u003evia\u0026nbsp;\u003c/em\u003eevoking T\u003csub\u003eem\u003c/sub\u003e locally and systemically.\u003c/p\u003e\n\u003cp\u003e2.8. Evaluation of biodistribution and biosafety\u003c/p\u003e\n\u003cp\u003eEffective accumulation of OPCM in tumor sites is a prerequisite for achieving a favorable anti-tumor effect. Hence, the biodistribution of OPCM was analyzed using an \u003cem\u003ein vivo\u003c/em\u003e FL imaging system. After\u003cem\u003e\u0026nbsp;i.v.\u003c/em\u003e DiR-labeled OPCM administration, a clear and gradually increasing FL signal was observed at the tumor site from 2 h to 24 h, and the FL signal reached a maximum at 24-h post-injection and reduced at 48-h post-injection as shown in\u0026nbsp;\u003cstrong\u003eFigures S24a and S24b\u003c/strong\u003e, verifying the efficient tumor accumulation of OPCM owing to the enhanced permeability and retention effect. Moreover, the mice were sacrificed at 48-h post-injection, followed by extraction of tumor tissues and major organs, including the spleen, heart, liver, lung, and kidney.\u003cem\u003e\u0026nbsp;Ex vivo\u003c/em\u003e FL imaging was done to capture the FL images of these tissues. The results showed that the liver displayed the highest FL signals, followed by the lung, tumor, spleen, kidney, and heart, illustrating the \u003cem\u003ein vivo\u003c/em\u003e biodistribution of DiR-labeled OPCM (Figure S24c, S24d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further detect the \u003cem\u003ein vivo\u003c/em\u003e accumulation and distribution of Cu and Mn ions in tumor tissues and major organs, ICP-MS was conducted to measure the Cu and Mn content of each tissue at different time intervals \u003cstrong\u003e(Figure S24f, S24g, S24h, S24i, S24j, S24k)\u003c/strong\u003e. The results showed that Cu and Mn ions preferentially accumulate in the lung, heart, and liver, which might be due to the rapid blood supply and the capture by the reticuloendothelial system, followed by metabolization and elimination of the kidney over time. Notably, the maximum contents of Mn and Cu were found in the tumor at the 4\u003csup\u003eth\u003c/sup\u003e hour, which was in accordance with the result of the MRI with the 4\u003csup\u003eth\u003c/sup\u003e hour showing the maximum imaging intensity.\u003c/p\u003e\n\u003cp\u003eTo evaluate biological safety, 6-8-week-old C57BL/6J mice were treated by\u003cem\u003e\u0026nbsp;i.v.\u0026nbsp;\u003c/em\u003einjection of OPCM at the 0\u003csup\u003eth\u003c/sup\u003e d, 1\u003csup\u003eth\u003c/sup\u003e d, 3\u003csup\u003eth\u003c/sup\u003e d, 7\u003csup\u003eth\u003c/sup\u003e d, 14\u003csup\u003eth\u003c/sup\u003e d, 21\u003csup\u003eth\u003c/sup\u003e d. At the 21st d, all mice were sacrificed and the whole blood samples, blood serums, and major organs were obtained to detect hematology parameters, liver function, and renal function markers, as well as histology observation.\u0026nbsp;\u003cstrong\u003eFigure S24k and S24l\u003c/strong\u003e displayed that all hematology parameters and biochemical indexes were within the normal range, suggesting negligible toxicity to the liver and kidney with good safety. As demonstrated by Figure S24m the H\u0026amp;E staining of major organs showed no morphological abnormalities, implying no acute and chronic pathological toxicities and adverse events. The above results illustrated that OPCM was bio-compatible and posed no obvious threat to major organs, making it a feasible alternative for future clinical translation.\u003c/p\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn conclusion, we successfully developed Cu and Mn-deposited OPCM \u003cem\u003evia\u0026nbsp;\u003c/em\u003eone-step biomineralization, which possessed favorable biocompatibility, TME responsiveness, and POD/CAT/GPX-like activities, leading to the initiation of tumor ferroptosis and cGAS-STING activation for antitumor immunity. This study provided a state-of-art paradigm for treating immunosuppressive liver cancer, since the OPCM successively initiated ferroptosis and activated the cGAS-STING signaling pathway, leading to pleiotropic immune cell mobilization, including DCs, CTLs, M1 TAMs, M2 TAMs, NK cells, and T\u003csub\u003eem\u003c/sub\u003e. Moreover, OPCM and \u0026alpha;PD-L1-induced immune activation further amplified the intensity of ferroptosis by IFN-\u0026gamma; secretion, which formed a closed-loop therapy cycle. As a result, the systematic immunostimulation led to both primary anti-tumor immunity and long-term anti-tumor immune memory, suppressing tumor recurrence and metastasis, thus providing an innovative combination therapeutic strategy for advanced liver cancer treatment.\u003c/p\u003e"},{"header":"4. Experimental section","content":"\u003cp\u003e\u003cstrong\u003e4.1. Chemical and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMnCl\u003csub\u003e2\u003c/sub\u003e·4H\u003csub\u003e2\u003c/sub\u003eO and CuCl\u003csub\u003e2\u003c/sub\u003e·2H\u003csub\u003e2\u003c/sub\u003eO were obtained from Sigma Aldrich (USA). BeyoPure™ LB Broth (premixed powder), protease inhibitor cocktail, and Ethylene Diamine Tetraacetic Acid (EDTA) were bought from Beyotime Biotechnology (Jiangsu, China). HEPES-NaOH was bought from Bio-sharp company. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 30%), 5,5’-dithiobis-(2-nitrobenzoic acid) (C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, DTNB), and 3,3',5,5'-Tetramethylbenzidine (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003e, TMB) were acquired from Aladdin (Shanghai, China). The Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Boster (Wuhan, China). Streptomycin-penicillin, trypsin, DAPI staining agent, Cell Counting Kit-8 (CCK8), DiI, DNA Damage Assay Kit by γ-H2AX Immunofluorescence, and JC-1 dye were obtained from Beyotime Biotechnology (Jiangsu, China). 1,1-dioctadecyl-3,3,3,3-tetramethylindo tricarbocyanine iodide (DiR) was bought from AAT Bioquest (CA, USA). Annexin V-FITC/PI Apoptosis Kit was bought from Elascience Biotechnology (Wuhan, China). Calcein-AM/PI Double Staing Kit and ROS assay kit were bought from Dojindo Laboratories (Kumamoto, Japan). BODIPY-C11 was obtained from ThermoFisher (USA). Anti-glutathione peroxidase 4 antibody was bought from Abcam (USA). Mouse-Reactive STING Pathway Antibody Sampler Kit was acquired from CST (USA). Calreticulin recombinant antibody was obtained from Proteintech (Wuhan, China). The enzyme-linked immunosorbent assays (ELISAs), including ATP, HMGB1, IL-6, TNF-α, Type 1 IFN, IL-10, and IL-12 were acquired from MEIMIAN (Jiangsu, China) or Bioswamp (Wuhan, China). αPD-L1 antibody was bought from Bio X cell (USA). FITC CD11c antibody, PE anti-mouse CD80 antibody, and APC anti-mouse CD86 antibody, and APC anti-mouse CD206, PE anti-mouse CD3, APC anti-mouse CD8a, FITC anti-mouse F4/80, PerCP/Cyanine5.5 anti-mouse/human CD11b, APC anti-mouse NK1.1, and APC anti-mouse CD8a, FITC anti-mouse CD44 and PerCP/Cyanine 5.5 anti-mouse CD62L were purchased from Biolegend (San Diego, CA, USA). All the chemicals were used as purchased without further purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Synthesis of OMV-based nanodrugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.1. Acquirement of OMVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOMVs was obtained according to a previously reported method. Briefly, Escherichia coli was inoculated into 250 mL LB medium and the medium was placed in a rotary shaker (180 rpm) with 37°C overnight. When OD600 of the LB medium reached 1.2, the medium was centrifuged at 5000 rpm for 10 min to collect bacteria. Afterwards, HM lysis buffer was prepared and the formula was as follows: 0.25 M sucrose, 1 mM Ethylene Diamine Tetraacetic Acid (EDTA), 20 mM HEPES-NaOH (pH 7.4), and 1х protease inhibitor cocktail. The collected bacteria were washed three times by PBS and then suspended in the HM lysis buffer. Then, the mixed solution was transferred to the ultrasonic cell disruptor in an ice bath for ultrasonic decomposition (power: 30%, time: 10 min). Subsequently, the whole solution was centrifuged at 3000 g for 5 min to eliminate the remaining bacteria and large bacterial fragments. After centrifugation, the supernatant was aspirated carefully and further underwent centrifugation at 15000 g for 30 min to collect the sediment. The sediment was resuspended in a cold HM buffer, passing through 0.8 μm and 0.45 μm filters at least 5 times in turn to get the OMVs. To quantify OMVs, the obtained OMVs solutions were dried at 60℃ and then weighed in a precision balance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2. Preparation of platelet membrane (PM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst of all, whole blood was obtained from mice and then centrifuged at 200 g for 10 min at room temperature. Next, the upper platelet (PLT)-rich plasma was collected and further centrifuged at 800 g for 15 min at room temperature to collect the sediment. Cold PBS containing 1 mM EDTA and protease inhibitor was employed to suspend the PLT sediment. The mixed PLT solution was transferred thrice from room temperature to -80℃ refrigerator to obtain PLT fragments. Then, the PLT lysis was centrifuged at 4000 g for 30 min at RT to collect the sediment. Finally, the sediment was washed 3 times with PBS to obtain the pristine PM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3. Preparation of OP, OP@Cu, OP@Mn, OCM, OPCM, and DiI or DiR-labeled OPCM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain PM-coated OMVs (OP), PM and OMV were dispersed in saline with a mass ratio of 1:1. Then, the mixed solutions were put into an ultrasonic bath under 20℃ for 5 minutes and physically extruded 11 times by an Avanti mini extruder through a porous polycarbonate membrane (400 nm). The mixture was centrifuged (4°C, 12 000 rpm for 30 min) to obtain the OP. To obtain Cu and Mn bio-mineralized OMVs (OCM), different input concentrations of Cu and Mn (50 mM, 100 mM, 200 mM, 400 mM, 800 mM) were separately mixed with 10 mg/mL OMVs to assess the optimal input amount of CuCl\u003csub\u003e2\u003c/sub\u003e and MnCl\u003csub\u003e2\u003c/sub\u003e by ICP-MS. After confirming the best input of CuCl\u003csub\u003e2\u003c/sub\u003e (800 mM) and MnCl\u003csub\u003e2\u003c/sub\u003e (800 mM), PM was mixed with the synthesized OCM to obtain OPCM. OPCM was dispersed and stored in saline for future use. OP@Cu and OP@Mn were prepared by mixing OMV, CuCl\u003csub\u003e2\u003c/sub\u003e or MnCl\u003csub\u003e2\u003c/sub\u003e, and PM similar to the preparation of OCM. DiI or DiR-labeled OPCM was prepared by mixing OPCM and DiI or DiR by physical stirring (250 rpm) for 1 h at 37℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. Characterization of various OMV-based nanodrugs, enzyme-like activities, and pharmacokinetics of OPCM in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.1. Characterization of various OMV-based nanodrugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of various OMV-based nanodrugs was characterized by transmission electron microscopy (TEM). The elemental mapping of C, O, Cu, and Mn were also examined post TEM. The hydrodynamic diameters and zeta potentials of the OMV-based nanodrugs were analyzed by a surface zeta potential and particle size analyzer (Zetasizer Nano ZS90, Britain). Inductively coupled plasma-mass spectrometry (ICP-MS, Agilent 7700(MS), USA) was applied to quantify the content of Cu and Mn in OCM and OPCM. X-ray photoelectron spectroscopy (XPS) measurements of the valence states of Cu and Mn were obtained using the K-Alpha XPS System (Thermo Scientific K-Alpha, USA). The X-ray powder diffraction (XRD) pattern was obtained using an X-ray diffractometer (Rigaku Ultima IV, Japan).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.2. Enzyme-like activities of OPCM in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the POD-like activity of OPCM, different concentrations of OPCM (50, 200, 300, 500, and 600 μg/mL) was incubated with 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution for 30 min at a 96-well plate. Then, 4 µL of TMB (80 × 10\u003csup\u003e-3\u003c/sup\u003e M) was added into the above solutions, followed by measurement of the wavelength absorption from 400 nm to 600 nm in a microplate reader (MultiSkan GO, Thermo scientific, USA). To track O\u003csub\u003e2\u003c/sub\u003e production, 0, 200, and 400 μg/mL OPCM were suspended in ultra-pure water and then sealed by paraffin. After 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was injected into the airtight system, a dissolved oxygen meter (JPBJ-608, Shanghai Oustor Industrial Co.) was used to measure the dissolved oxygen content for 15 min. To detect the consumption of GSH induced by OPCM, the 5, 5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) solution was used. In brief, OPCM (500 µg/mL) were dissolved in PBS and reacted with GSH (5 mM). Afterward, the sediment was removed by centrifugation (13,000 rpm, 10 min) for various incubation durations (30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h) to collect the supernatant. Finally, DTNB solution (3 mg/mL, 10 µL) was added into the supernatant in a 96-well plate, the absorbance at 412 nm was measured by a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.3. Pharmacokinetics of OPCM in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo detect the pH-responsive behavior of OPCM, OPCM was suspended in PBS solution with pH 5.0, pH 6.0, and pH 7.4. Then, the above PBS solutions containing OPCM were placed in a shaker (150 rpm, 37℃). 200 uL solutions were taken out of the three solutions at each time point (15min, 30min, 1 h, 4 h, 8 h, 24 h, and 48 h). Afterward, the 200 µL solutions were centrifuged at 15000 rpm for 5min to obtain the supernatant for ICP-MS to detect the level of Cu and Mn.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. Cellular experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.1. Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 cells, Hepa1-6 cells and H22 cells were purchased from Boster (Wuhan, China). JAWSII cell was obtained from MeisenCTCC (Zhejiang, China). RAW264.7 cells, Hepa1-6 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL) under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37°C. H22 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e. JAWSII cells were cultured in RPMI-1640 medium supplemented with 20% FBS, 1% penicillin– streptomycin, and 5 ng/mL GM-CSF at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e. It is worth noting that RAW264.7 cells and JAWSII are half adherent and half suspended, H22 cells are fully suspended, while Hepa1-6 cells are fully adherent. The culture medium of Hepa1-6 cells, H22 cells, and RAW264.7 cells were sub-cultured every two days, while the JAWSII cells were sub-cultured every three days. M2 TAMs were obtained by culturing RAW264.7 cells with interleukin 4 (IL- 4) (40 ng/mL) overnight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.2. Cytotoxicity of various OMV-based nanodrugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJAWII and M2 TAMs were selected to verify the bio-compatibility of the OMV-based nanodrugs. Hepa1-6 cells were chosen to examine the anti-tumor effect of the OMV-based nanodrugs. All cell lines were seeded in 96-well plates at a density of 1 × 10\u003csup\u003e4\u003c/sup\u003e cells per well. M2 TAMs were obtained with the aforementioned method. After cell attachment, JAWSII, M2 TAMs, and Hepa1-6 cells were treated with gradient concentrations of OMV, OCM, and OPCM (50, 100, 150, 200, 250, 300 μg/mL) for 24 h. After repeated washing, the cell viability was measured by the CCK-8 assay following the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.3. The intracellular uptake of DiI-labeled OPCM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phagocytosis of DiI-labeled OPCM was explored in Hepa1-6 cells under confocal laser scanning microscopy (CLSM) (Nikon, Tokyo, Japan) and flow cytometry (FCM) (FACS Vantage SE, Becton Dickinson, San Jose, CA, USA). For CLSM characterization, Hepa1-6 cells (1 × 10\u003csup\u003e5\u003c/sup\u003e cells per well) were separately cultured in con-focal dishes under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃, followed by cell attachment overnight. M2 TAMs were obtained by the aforementioned method. Next, the culture medium was discarded and replaced with fresh FBS-free DMEM containing DiI-labeled OPCM, followed by co-incubation for 30 min, and 1, 2, 3, and 4 h. Then, the confocal dishes were removed from the cell incubater and rinsed with PBS thrice. Afterwards, the cells were fixed with 1 mL 4% formalin for 15 min. Subsequently, the cells were washed with PBS three times and counter-stained with 100 μL DAPI staining solution for another 15 min. Finally, the cells were washed with PBS and then visualized by CLSM to take fluorescence (FL) images to observe the cellular uptake of DiI-labeled OPCM in Hepa1-6 cells. FCM characterization was also applied for quantitative analysis. Hepa1-6 cells (2 × 10\u003csup\u003e5\u003c/sup\u003e cells per well) were cultured in 6-well plates under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃ respectively. Then, the medium was discarded and replaced with FBS-free DMEM containing DiI-labeled OPCM, followed by co-incubation for 30 min, and 1, 2, 3, and 4 h. Then, cells were collected by trypsin and suspended in 300 μL PBS solution to undergo FCM analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.4. Antitumor effect induced by various OMV-based nanodrugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further detect the antitumor effect of OMV-based nanodrugs on Hepa1-6 cells, live/dead cell staining and FCM were conducted to confirm the viability of Hepa1-6 cells after various treatments. Hepa1-6 cells (1 × 10\u003csup\u003e5\u003c/sup\u003e cells per well) were cultured in six-well plate under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃, followed by cell attachment overnight. Afterwards, 300 μg/mL of OMV, OP, OCM, and OPCM suspending in fresh FBS-free DMEM were added into the six-well plate respectively. After co-incubation for 24 h, the cells were sent for live/dead cell staining based on the manufacturer’s instructions, followed by observation under an FL microscope (Nikon Ti-S, Tokyo, Japan). Simultaneously, the cells were also collected by trypsin, followed by staining of Annexin V-FITC and PI in PBS solution based on the manufacturer’s instructions. Finally, FCM was performed to analyze the cell apoptotic rates of Hepa1-6 cells in various treatment groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.5. The detection of cellular ROS generation, lipid peroxidation (LPO), and mitochondrial membrane potential (MMP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepa1-6 cells (1 × 10\u003csup\u003e5\u003c/sup\u003e cells per well) were separately cultured in con-focal dishes under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃, followed by cell attachment overnight. Then, the Hepa1-6 cells were divided into 5 groups and incubated with PBS, OMV, OCM, OPCM, or OPCM + Fer for 24 h respectively. Afterwards, all cells were stained with DCFH-DA for detection of ROS. The cells were also stained with JC-1 dye and bodipy-C11 dye to observe variations in MMP and LPO respectively. All dishes were visualized by CLSM. In addition, cells were collected for FCM analysis of MMP. MMP was calculated as the ratio of the JC-1 aggregate/monomer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.6. Intracellular evaluation of GSH content and GPX4 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepa1-6 cells (1 × 10\u003csup\u003e5\u003c/sup\u003e cells per well) were seeded in six-well plate under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃, followed by cell attachment overnight. Afterwards, PBS, 300 μg/mL of OMV, OCM, and OPCM suspending in fresh FBS-free DMEM were added into the six-well plate respectively. After co-incubation for 24 h, cells were harvested to detect the concentration of GSH and GSSG via GSSG/GSH Quantification Kit on the one hand. On the other hand, proteins were extracted from cells to detect the expression level of GPX4 by western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.7. Intracellular detection of immunogenic cell death and cGAS-STING activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepa1-6 cells (1 × 10\u003csup\u003e5\u003c/sup\u003e cells per well) were plated in con-focal dishes under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃, followed by cell attachment overnight. Afterwards, PBS, 300 μg/mL of OMV, OP, OCM, and OPCM suspending in fresh FBS-free DMEM were added into the con-focal dishes respectively. After co-incubation for 24 h, immunofluorescence staining of intracellular γ-H2AX and CRT was conducted to detect DNA damage and CRT exposure. Simultaneously, all cells were seeded in in six-well plate and then treated with PBS, 300 μg/mL of OMV, OCM, and OPCM suspending in fresh FBS-free DMEM for 24 h. Afterwards, proteins were extracted from cells to detect the expression level of STING, p-STING, TBK1, p-TBK1, IRF3, and p-IRF3 by western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.8. Immune stimulation experiments induced by various OMV-based nanodrugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA coculture transwell system (6-well plate, 0.4 μm-sized microporous membrane) was used to evaluate the immunostimulatory effect of OMV-based nanodrugs on immature DCs and M2 TAMs. JAWSII cells were seeded on the bottom of the coculture transwell system, while the Hepa1-6 cells were placed on the upper chamber. After cell attachment overnight, the old culture medium was replaced by FBS free DMEM medium containing 300 μg/mL OMV, OP, OCM, and OPCM. 24 h after co-incubation, the JAWSII cells were obtained and stained with FITC CD11c antibody, PE anti-mouse CD80 antibody, and APC anti-mouse CD86 antibody following the manufacturer’s instruction. Finally, the JAWSII cells underwent FCM analysis to determine the proportion of matured DCs. In addition, the supernatant of the coculture transwell system was collected and sent for ELISA to detect the secretion level of ATP, HMGB1, Type 1 IFN, IL-6, and TNF-α.\u003c/p\u003e\n\u003cp\u003eSimilar to the experiment of JAWSII cells, RAW264.7 cells were seeded on the bottom of the coculture transwell system, while the Hepa1-6 cells were placed on the upper chamber. Notably, the RAW264.7 cells were first stimulated to differentiate into M2 TAMs by 40 ng/mL IL4. After cell attachment overnight, the old culture medium was replaced by FBS free DMEM medium containing 300 μg/mL OMV, OP, OCM, and OPCM. 24 h after co-incubation, the JAWSII cells were obtained and stained with PE anti-mouse CD80 antibody, and APC anti-mouse CD206 antibody following the manufacturer’s instruction. Finally, the RAW264.7 cells underwent FCM analysis to determine the proportion of M1 TAMs and M2 TAMs. In addition, the supernatant of the coculture transwell system was collected and sent for ELISA to detect the secretion level of ATP, HMGB1, Type 1 IFN, IL-10, and IL-12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5. Animal experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5.1. Establishment of syngeneic HCC mouse models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Ethics Committee of Chongqing Medical University and Institutional Animal Care. The permit number for the animal experiments is Research Ethics Review No. 223 (2023). C57BL/6J and BALB/c mice (6-8 weeks, male) were bought from the experimental animal center of Chongqing Medical University. Subcutaneous syngeneic C57BL/6J HCC mouse models were developed by injecting 5 × 10\u003csup\u003e6\u003c/sup\u003e Hepa1-6 cells suspended in 100 μL PBS into the right flanks of 6-8-week-old male C57BL/6J mice (5 mice per group). H22 tumor-bearing BALB/c mice were established by injecting 2×10\u003csup\u003e6\u003c/sup\u003e H22 cells suspended in 100 μL PBS into the right flanks of 6-8-week-old male BALB/c mice (3 mice per group).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5.2. Evaluation of antitumor effect in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe schematic diagram of therapeutic and observation process for subcutaneous syngeneic HCC mouse models was shown in Figure 6a. Subcutaneous tumors of C57BL/6J and BALB/c mice reached a measurable size 7 d post tumor cell inoculation. Then, the tumor-bearing C57BL/6J and BALB/c mice were randomly assigned to five groups, receiving saline or PBS, OMV, αPD-L1, OPCM, and the combination of αPD-L1 and OPCM on 1\u003csup\u003est\u003c/sup\u003e, 4\u003csup\u003eth\u003c/sup\u003e, 8\u003csup\u003eth\u003c/sup\u003e, 12\u003csup\u003eth\u003c/sup\u003e. All mice were weighed and tumor volume was assessed once every other day. The length (L) and width (W) of tumors were measured by Vernier calipers to calculate the tumor volume (V) through the formula: V = (L × W\u003csup\u003e2\u003c/sup\u003e)/2. Photos were taken on the 1st, 5th, 10th, 15th, and 20th day. The observation for tumor treatment lasted for 20 days. Subsequently, the mice were sacrificed and the tumor tissues were extracted and weighed to calculate the tumor inhibition rate. After that, the tumor tissues extracted from C57BL/6J mice were fixed with 4% paraformaldehyde, followed by hematoxylin and eosin (H\u0026amp;E) staining assays and the immunofluorescent staining of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA). The average FL signals of TUNEL and PCNA were calculated by ImageJ. Simultaneously, survival curves of C57BL/6J mice were monitored every other day for up to 60 days after the first treatment. The mice were considered dead when the tumor volume reached 1500 mm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e4.5.3. \u003cstrong\u003eEvaluation of ferroptosis and cGAS-STING activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 13 days, tumor tissues of each group were extracted and sent for IF staining to assess the expression of GPX4, DHE, HIF-1α, p-STING, p-TBK1, p-IRF3, CRT, and HMGB1, IFN-γ. To detect the expression of DHE, the tumor tissues of each group were frozen in -80℃ refrigerator and then sent for frozen slicing and staining of DHE working solution. Finally, the tumor sections were observed under a FL microscopy (NIKON DS-U3, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5.4. Evaluation of the immune cell in the TME\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the immunostimulatory effect of the OMV-based nanodrugs, tumors, tumor-draining lymph nodes (TDLNs) and spleens were harvested from C57BL/6J mice 12 days after first treatment in different groups for immune cell analysis. Immediately after tumor tissue extraction, the TDLNs and spleens were obtained. These tissues were cut into small pieces, and digested with collagenase A (1 mg/mL), DNAase I (0.5 mg/mL) and HAase (1 mg/mL) for 30 min at 37°C. Afterward, the tissues and TDLNs were filtered to obtain single cells by 40 μm cell strainers, while spleens were filtered to obtain single cells by 70 μm cell strainers. Afterwards, the collected cells were centrifuged at 1500 rpm for 5 min and then suspended in red blood cell lysis buffer for 2 min to remove residual red blood cells. Subsequently, 1% FBS was added into the mixed cell suspension to stop digestion, followed by centrifugation at 1500 rpm for 5 min to obtain the purified cells from tumor tissues, TDLNs, and spleens. Next, the cells were stained with the corresponding fluorescence-labeled antibodies as follows: FITC anti-mouse CD11c, PE anti-mouse CD80, APC anti-mouse CD86, PE anti-mouse CD3, APC anti-mouse CD8a, FITC anti-mouse F4/80, PerCP/Cyanine5.5 anti-mouse/human CD11b, APC anti-mouse CD206, APC anti-mouse NK1.1. Finally, samples were detected by FCM. Simultaneously, tumor tissues in each group were collected for ELISA to detect the intratumoral secretion level of ATP, HMGB1, and type 1 IFNs, while serums in each group were collected for ELISA to detect the secretion level of IL-6, TNF-α, IL-10, and IL-12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5.6. The establishment of tumor re-challenged models and lung metastasis models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe C57BL/6J subcutaneous models were established by the aforementioned method, followed by the same therapeutic interventions. On Day 21, primary tumors in the right flanks of mice were completely excised by surgery. After that, all mice were retained for one week. On day 30, 5×10\u003csup\u003e6\u003c/sup\u003e Hepa1-6 cells were rechallenged into the mice’s left flanks of mice contralateral to the primary tumor site. The rechallenged tumor growth curves and weight changes were monitored once every other day from day 37 to day 51. The L and W of tumors were measured by Vernier calipers to calculate the tumor volume with the same aforementioned calculation formula. Photos were taken on the 37 d, 40 d, 43 d, 46 d, 49 d and 52 d. The observation for tumor treatment lasted for 15 days. On Day 52, all mice were sacrificed and the tumors, TDLNs, and spleens were collected. All tumor samples were weighed and photographed. TDLNs and spleens underwent the aforementioned procedure to obtain single cell suspension. Next, the cells from the tumor tissues of different treatment groups were stained with PE anti-mouse CD3 and APC anti-mouse CD8a, FITC anti-mouse CD44 and PerCP/Cyanine 5.5 anti-mouse CD62L according to the manufacturer’s instructions. Finally, FCM was conducted to analyze the ratio of effector memory T (T\u003csub\u003eem\u003c/sub\u003e) cells in the spleens. To establish the distant lung metastasis model, the subcutaneous models were established by the aforementioned method and treated by the same treatments. On day 21, to mimick the process of lung metastasis, the mice in each group were injected Hepa1-6 cells (1×10\u003csup\u003e6\u003c/sup\u003e) by tail vein. On the 28 d, all mice were sacrificed and the lung were extracted, followed by staining with Bouin’s solution and HE staining to observe the lung metastasis condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5.7. FLI, MRI, and pharmacokinetics of OPCM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vivo FLI was demonstrated using a NIRF imaging system (NightOWL II LB983, Germany) (Exc/Em = 748/780 nm). OPCM@DiR (30 mg kg\u003csup\u003e−1\u003c/sup\u003e) were injected into the H22 tumor cell-bearing BALB/c mice intravenously. Then, the mice underwent FLI after certain time intervals (0, 2, 4, 8, 24, and 48 h) post injection. Afterward, the mice were sacrificed, followed by extraction of the tumors and main organs for biodistribution analysis.\u003c/p\u003e\n\u003cp\u003eTo detect the in vitro MR imaging performance of OPCM, OPCM with different concentrations (Mn: 0.13, 0.26, 0.77, 1.55, 2.58 mM) were scanned using a 3.0 T MRI scanner (MAGNETOM Prisma, Siemens Healthineers Inc., Munich, Germany). The samples were prepared in 2 mL centrifuge tube, followed by the acquisition of the T\u003csub\u003e1\u003c/sub\u003e weighted images (T\u003csub\u003e1\u003c/sub\u003eWI) using the MRI scanner. The T\u003csub\u003e1\u003c/sub\u003eWI parameters were listed as follows: fast field echo (FFE), TR = 650 ms, TE = 11 ms, and slice thickness = 1.4 mm. The T\u003csub\u003e1\u003c/sub\u003e relaxation rate (r\u003csub\u003e1\u003c/sub\u003e) was calculated by the linear fitting of the inverse T\u003csub\u003e1\u003c/sub\u003e relaxation times as a function of Mn concentration. The T\u003csub\u003e1\u003c/sub\u003e relaxation rates of the acquired images were obtained through the T1 mapping data from Syngo.via software. For the MRI experiment in vivo, the MRI performance of the OPCM was demonstrated in H22 tumor cell-bearing BALB/c mice. A 3.0 T mouse MRI coil from Chenguang Medical Technology Company was used. The T\u003csub\u003e1\u003c/sub\u003eWI of the tumor area was captured at different time points (0, 2, 4, 8, 12 and 24 h) after the intravenous injection of the BSA-mFe@Len NPs (30 mg/kg). The T\u003csub\u003e1\u003c/sub\u003eWI parameters were set as follows: TR = 650 ms; TE = 11 ms; slice thickness = 1.5 mm; FOV = 80 mm.\u003c/p\u003e\n\u003cp\u003eTo determine the in vivo pharmacokinetics of the OPCM, the mice were first injected with the OPCM. Then, the mice were sacrificed at each predetermined times (0, 1, 2, 4, 8, 12, 24, 48, 72 h), followed extraction of tumor tissues and major organs (heart, liver, spleen, lung, kidney). All tissues were collected, weighed, dissolved with aqua regia (HCl: HNO\u003csub\u003e3\u003c/sub\u003e = 3:1), evaporated, and re-dissolved in 1% HNO\u003csub\u003e3\u003c/sub\u003e. The Cu and Mn concentration was estimated by ICP-MS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5.8. Bio-safety of the OPCM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bio-compatibility of the OPCM in vivo was evaluated in C57 BL6/J (male, 6-8 weeks). The mice were randomly divided into six groups (control group and 1, 3, 7, 14, 21 d group after injection of OPCM, n=3). On Day 21, all mice were sacrificed. Blood samples were collected for routine blood and biochemical examinations. The major organs (heart, liver, spleen, lung, kidney) were collected for H\u0026amp;E analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll quantitative data are shown as the mean ± SD. Statistical analysis was performed using GraphPad 10.1 (La Jolla, CA, USA). A Student’s unpaired or paired t-test was used to analyze the significant differences between the two groups. We used the one unpaired multiple t-test and analysis of variance (ANOVA) for the analysis of the differences between multiple groups. Statistical tests were double-sided, and values with P \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYing Luo is the first author, and Zhongsheng Xu are the parallel first author. They contributed equally to this study, including conceptualization, methodology, investigation, validation, data analysis. Specifically, Ying Luo is responsible for processing raw data and writing the manuscript, while Zhongsheng Xu is responsible for reviewing and revising the draft. Qianying Du, Lian Xu, and Yi Wang contributed partially to the methodology part. Jie Xu, Junrui Wang, Sijin Chen, Wenli Zhang, and Bo Liu contributed partially to the study administration part. Jia Liu contributed partially to the Funding part. Dajing Guo\u003csup\u003e*\u003c/sup\u003e, and Yun Liu\u003csup\u003e*\u0026nbsp;\u003c/sup\u003eare the corresponding author and contributed mainly to the Funding part and paper revision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82271970), Chongqing Medical Scientific Research Project (Joint Project of Chongqing Health Commission and Science and Technology Bureau) (Grant No. 2022ZDXM026), Senior Medical Talents Program of Chongqing for Young and Middle-aged, Chongqing Returned Overseas Students\u0026rsquo; Entrepreneurship and Innovation Support Program (cx2021006), CQMU Program for Youth Innovation in Future Medicine (W0171), and the Kuanren Doctoral supervisor Cultivation Program of the second affiliated hospital of Chongqing Medical University. This work was also supported by the General Program of Chongqing Natural Science Foundation (cstc2021jcyj-msxmX0040) and the National Science Foundation for Young Scholars (82102063).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ea)J. M. Llovet, R. K. Kelley, A. Villanueva, A. G. Singal, E. Pikarsky, S. Roayaie, R. Lencioni, K. Koike, J. Zucman-Rossi, R. S. Finn, \u003cem\u003eNat Rev Dis Primers\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 7, 6; b)A. Vogel, T. Meyer, G. Sapisochin, R. Salem, A. Saborowski, \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 400, 1345.\u003c/li\u003e\n\u003cli\u003ea)F. Xu, T. Jin, Y. Zhu, C. Dai, \u003cem\u003eJ Exp Clin Cancer Res\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, 37, 110; b)Y. Zheng, S. Wang, J. Cai, A. Ke, J. Fan, \u003cem\u003eBiochim Biophys Acta Rev Cancer\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 1876, 188638.\u003c/li\u003e\n\u003cli\u003eR. Donne, A. Lujambio, \u003cem\u003eHepatology\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 77, 1773.\u003c/li\u003e\n\u003cli\u003ea)M. Toyofuku, N. Nomura, L. Eberl, \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 17, 13; b)M. Toyofuku, S. Schild, M. Kaparakis-Liaskos, L. Eberl, \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 21, 415.\u003c/li\u003e\n\u003cli\u003ea)Q. Chen, H. Bai, W. Wu, G. Huang, Y. Li, M. Wu, G. Tang, Y. Ping, \u003cem\u003eNano Lett\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 20, 11; b)M. Z. Zou, Z. H. Li, X. F. Bai, C. J. Liu, X. Z. Zhang, \u003cem\u003eNano Lett\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 21, 8609; c)F. Meng, L. Li, Z. Zhang, Z. Lin, J. Zhang, X. Song, T. Xue, C. Xing, X. Liang, X. Zhang, \u003cem\u003eJ Extracell Vesicles\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 11, e12289; d)L. Chen, X. Ma, W. Liu, Q. Hu, H. Yang, \u003cem\u003eNano Lett\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 23, 8725; e)G. Liu, N. Ma, K. Cheng, Q. Feng, X. Ma, Y. Yue, Y. Li, T. Zhang, X. Gao, J. Liang, L. Zhang, X. Wang, Z. Ren, Y. X. Fu, X. Zhao, G. Nie, \u003cem\u003eNat Nanotechnol\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003ea)B. A. Flood, E. F. Higgs, S. Li, J. J. Luke, T. F. Gajewski, \u003cem\u003eImmunol Rev\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 290, 24; b)K. M. Garland, T. L. Sheehy, J. T. Wilson, \u003cem\u003eChem Rev\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 122, 5977; c)Z. J. Long, J. D. Wang, J. Q. Xu, X. X. Lei, Q. Liu, \u003cem\u003eMol Ther\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 30, 1006; d)E. N. Chin, A. Sulpizio, L. L. Lairson, \u003cem\u003eTrends Cell Biol\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 33, 189.\u003c/li\u003e\n\u003cli\u003ea)N. Cheng, R. Watkins-Schulz, R. D. Junkins, C. N. David, B. M. Johnson, S. A. Montgomery, K. J. Peine, D. B. Darr, H. Yuan, K. P. McKinnon, Q. Liu, L. Miao, L. Huang, E. M. Bachelder, K. M. Ainslie, J. P. Ting, \u003cem\u003eJCI Insight\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, 3; b)J. Guo, L. Huang, \u003cem\u003eTrends Pharmacol Sci\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 43, 957.\u003c/li\u003e\n\u003cli\u003ea)D. Cen, Q. Ge, C. Xie, Q. Zheng, J. Guo, Y. Zhang, Y. Wang, X. Li, Z. Gu, X. Cai, \u003cem\u003eAdv Mater\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 33, e2104037; b)Z. Li, Z. Chu, J. Yang, H. Qian, J. Xu, B. Chen, T. Tian, H. Chen, Y. Xu, F. Wang, \u003cem\u003eACS Nano\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 16, 15471.\u003c/li\u003e\n\u003cli\u003ea)C. Wang, Y. Guan, M. Lv, R. Zhang, Z. Guo, X. Wei, X. Du, J. Yang, T. Li, Y. Wan, X. Su, X. Huang, Z. Jiang, \u003cem\u003eImmunity\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, 48, 675; b)R. M. Hooy, G. Massaccesi, K. E. Rousseau, M. A. Chattergoon, J. Sohn, \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 48, 4435; c)X. Sun, Y. Zhang, J. Li, K. S. Park, K. Han, X. Zhou, Y. Xu, J. Nam, J. Xu, X. Shi, L. Wei, Y. L. Lei, J. J. Moon, \u003cem\u003eNat Nanotechnol\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 16, 1260; d)J. Yan, G. Wang, L. Xie, H. Tian, J. Li, B. Li, W. Sang, W. Li, Z. Zhang, Y. Dai, \u003cem\u003eAdv Mater\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 34, e2105783; e)Y. Zhu, Z. Yang, Z. Pan, Y. Hao, C. Wang, Z. Dong, Q. Li, Y. Han, L. Tian, L. Feng, Z. Liu, \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 8, eabo5285.\u003c/li\u003e\n\u003cli\u003ea)W. Wu, Y. Pu, X. Lu, H. Lin, J. Shi, \u003cem\u003eAdv Healthc Mater\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 10, e2001819; b)Y. Yang, K. Hu, P. Zhang, P. Zhou, X. Duan, H. Sun, S. Wang, \u003cem\u003eSmall\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 17, e2100927; c)M. Tang, Z. Zhang, T. Sun, B. Li, Z. Wu, \u003cem\u003eAdv Healthc Mater\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 11, e2201733.\u003c/li\u003e\n\u003cli\u003ea)Q. Xiang, C. Yang, Y. Luo, F. Liu, J. Zheng, W. Liu, H. Ran, Y. Sun, J. Ren, Z. Wang, \u003cem\u003eSmall\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 18, e2107809; b)Z. Zhao, S. Dong, Y. Liu, J. Wang, L. Ba, C. Zhang, X. Cao, C. Wu, P. Yang, \u003cem\u003eACS Nano\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 16, 20400; c)X. Jia, J. Wang, E. Wang, \u003cem\u003eAdv Mater\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, e2309261; d)D. Li, E. Ha, Z. Zhou, J. Zhang, Y. Zhu, F. Ai, L. Yan, S. He, L. Li, J. Hu, \u003cem\u003eAdv Mater\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, e2308747.\u003c/li\u003e\n\u003cli\u003ea)S. Guo, W. Xiong, J. Zhu, J. Feng, R. Zhou, Q. Fan, Q. Zhang, Z. Li, J. Yang, H. Zhou, P. Yi, Y. Feng, S. Yang, X. Qiu, Y. Xu, Z. Shen, \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 302, 122300; b)J. Liu, J. Zhan, Y. Zhang, L. Huang, J. Yang, J. Feng, L. Ding, Z. Shen, X. Chen, \u003cem\u003eAdv Mater\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, e2309562.\u003c/li\u003e\n\u003cli\u003eJ. Wang, W. Song, X. Wang, Z. Xie, W. Zhang, W. Jiang, S. Liu, J. Hou, Y. Zhong, J. Xu, H. Ran, D. Guo, \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 277, 121100.\u003c/li\u003e\n\u003cli\u003eL. Kennedy, J. K. Sandhu, M. E. Harper, M. Cuperlovic-Culf, \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 10.\u003c/li\u003e\n\u003cli\u003eB. Niu, K. Liao, Y. Zhou, T. Wen, G. Quan, X. Pan, C. Wu, \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 277, 121110.\u003c/li\u003e\n\u003cli\u003eY. Yang, S. Karakhanova, W. Hartwig, J. G. D\u0026apos;Haese, P. P. Philippov, J. Werner, A. V. Bazhin, \u003cem\u003eJ Cell Physiol\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 231, 2570.\u003c/li\u003e\n\u003cli\u003eR. Tang, J. Xu, B. Zhang, J. Liu, C. Liang, J. Hua, Q. Meng, X. Yu, S. Shi, \u003cem\u003eJ Hematol Oncol\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, 13, 110.\u003c/li\u003e\n\u003cli\u003eY. Luo, J. Wang, L. Xu, Q. Du, N. Fang, H. Wu, F. Liu, L. Hu, J. Xu, J. Hou, Y. Zhong, Y. Liu, Z. Wang, H. Ran, D. Guo, \u003cem\u003eActa Biomater\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 154, 478.\u003c/li\u003e\n\u003cli\u003ea)O. Y. Kim, H. T. Park, N. T. H. Dinh, S. J. Choi, J. Lee, J. H. Kim, S. W. Lee, Y. S. Gho, \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, 8, 626; b)W. Nie, A. Jiang, X. Ou, J. Zhou, Z. Li, C. Liang, L. L. Huang, G. Wu, H. Y. Xie, \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, 304, 122396.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"outer membrane vesicle (OMV), metal ions, ferroptosis, cCAS-STING, immunotherapy, liver cancer","lastPublishedDoi":"10.21203/rs.3.rs-4183359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4183359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe immune landscape of late-stage liver cancer is featured by severe immunosuppression that is characterized by poor immunogenicity, T-cell exhaustion, and infiltration of a large number of immunosuppressive cells, leading to compromised therapeutic efficacy of mainstream immunotherapies. Herein, we developed a pleiotropic immunoferroptotic mobilization strategy to treat intractable advanced liver cancer. In this study, immunogenic bacteria-derived outer membrane vesicles (OMVs) were exploited as a vector to deposit Cu and Mn with mixed valence states \u003cem\u003evia \u003c/em\u003eone-step biomineralization, followed by platelet membrane camouflage to enhance the circulation time and reduce systemic side effects of the metal biomineralized OMVs, which are denoted as OPCM. Mechanistically, the metal-deposited OPCM possesses POD, CAT, and GPX-like activities, thereby stimulating immunogenic ferroptosis, cGAS-STING activation, and tumor hypoxia alleviation, ultimately leading to pleiotropic immunoferroptotic mobilization to combat tumor growth, recurrence, and metastasis. Notably, the combination with typical αPD-L1 augmented the tumor suppression effect, since the administration of αPD-L1 not only rescued exhausted T cells, but also amplified the intensity of ferroptosis due to IFN-γ secretion by activated T cells. Overall, the metal biomineralized OPCMs in combination with αPD-L1 formed a closed-loop therapy that cycles from immunotherapy and ferroptosis therapy, providing new insights for treating immunosuppressive liver cancer.\u003c/p\u003e","manuscriptTitle":"Biomineralized bacterial outer membrane vesicles exert pleiotropic immunoferroptotic effects on immune-deserted liver cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 10:05:09","doi":"10.21203/rs.3.rs-4183359/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"738a730e-1065-4c3c-afe8-31535537a3ef","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-24T02:43:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-10 10:05:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4183359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4183359","identity":"rs-4183359","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}