Artificial Macrophage with Hierarchical Nanostructure for Biomimetic Reconstruction of Antitumor Immunity

Highlights An artificial macrophage with hierarchical nanostructure (BaSO4@ZIF-8/TRF NMΦ) is constructed as an alternative to immunoactive macrophages. The Zn2+ chemical messenger as an “artificial cytokine” is released from the artificial macrophage to induce tumor anoikis and enhance immunogenicity. The artificial macrophage can efficiently capture tumor antigens for antigen presentation and T cell activation to fabricate long-term antitumor immunity, successfully mimicking the basic functions of natural immunoactive macrophage. Supplementary Information The online version contains supplementary material available at 10.1007/s40820-023-01193-4.

• The Zn 2+ chemical messenger as an "artificial cytokine" is released from the artificial macrophage to induce tumor anoikis and enhance immunogenicity.
• The artificial macrophage can efficiently capture tumor antigens for antigen presentation and T cell activation to fabricate long-term antitumor immunity, successfully mimicking the basic functions of natural immunoactive macrophage.
ABSTRACT Artificial cells are constructed from synthetic materials to imitate the biological functions of natural cells.By virtue of nanoengineering techniques, artificial cells with designed biomimetic functions provide alternatives to natural cells, showing vast potential for biomedical applications.Especially in cancer treatment, the deficiency of immunoactive macrophages results in tumor progression and immune resistance.To overcome the limitation, a BaSO 4 @ZIF-8/transferrin (TRF) nanomacrophage (NMΦ) is herein constructed as an alternative to immunoactive macrophages.Alike to natural immunoactive macrophages, NMΦ is stably retained in tumors through the specific affinity of TRF to tumor cells.Zn 2+ as an "artificial cytokine" is then released from the ZIF-8 layer of NMΦ under tumor microenvironment.Similar as proinflammatory cytokines, Zn 2+ can trigger cell anoikis to expose tumor antigens, which are selectively captured by the BaSO 4 cavities.Therefore, the hierarchical nanostructure of NMΦs allows them to mediate immunogenic death of tumor cells and subsequent antigen capture for T cell activation to fabricate long-term antitumor immunity.

Introduction
Biomimetics utilizes synthetic materials to mimic the biological functions or processes in nature, which has attracted interdisciplinary research interest [1][2][3].As a typical example of biomimetics, artificial cells are constructed from synthetic materials to imitate all or part of the biological functions of natural cells [4].The synthetic and tunable structures of artificial cells allow them to adequately exert various physicochemical interactions with cells [5].Moreover, the functions of artificial cells can be flexibly designed and adjusted according to practical requirements [6].Besides, artificial cells are facile to be produced on a large scale with low cost [7].All these merits make artificial cells a promising alternative to natural cells in the treatment of diseases [8].Especially in cancer immunity, the deficiency of natural immunoactive macrophages gives rise to tumor progression and immune resistance [9].Moreover, the residence of immunosuppressive macrophages impairs the infiltration of T cells and drives T cell exhaustion in tumors via the secretion of anti-inflammatory cytokines [10,11].As a result, neither innate nor adaptive antitumor immunity can be activated to defend against cancer [12,13].To supplement immunoactive macrophages, researchers have paid tremendous endeavors to develop chimeric antigen receptor (CAR) cells via cell engineering, such as CAR-macrophages [14].CAR cells are engineered to express tumor-specific receptors on immune cells by gene editing technology, but its application is restricted by elaborate construction, high cost and tumor heterogeneity [15][16][17].Thus, developing artificial macrophages as an alternative to immunoactive macrophages would be more preferable for cancer therapy.
To date, artificial macrophages have been rarely fabricated to mimic natural macrophages, probably due to the following challenges [18].Firstly, the biological functions of natural macrophages highly rely on the pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukins (ILs), which can mediate tumor apoptosis and antitumor immunity [19,20].The ability of releasing cytokines to initiate immunity is a prerequisite for the design of artificial macrophages.Second, antigen capture is a key pivotal step for natural macrophages in mediating antigen presentation and T cell activation [21].In most cases, antigen capture is accomplished by natural macrophages via nonspecific phagocytosis [22].However, few artificial macrophages have been successfully constructed to fulfill the above requirements.
With the advances in nanotechnology, nanomaterials have been developed to mimic versatile cellular functions [23,24].Especially, nanomaterials with the component of chemical messengers, such as Ca 2+ , Mg 2+ , Zn 2+ and H 2 S, are highly effective in regulating physiological processes and cellular communication [25].The trace variation of chemical messengers may play regulatory roles in either antitumor therapy or immune activation, similar to those of pro-inflammatory cytokines [26].For example, intracellular Ca 2+ or Zn 2+ overload can effectively impede tumor growth, and activate antitumor specific immune response to arrest tumor metastasis [27,28].Given the pivotal roles in immune regulation, chemical messengers are expected to be "artificial cytokines" of the synthetic macrophages.Besides, nanomaterials have been utilized as nanocarriers for the transfer of bioactive cargoes, such as drugs, enzymes, genes, and even antigens [29].Nanomaterials with high loading capacity lay a foundation for the antigen capture of nanoscaled artificial macrophages.
Herein, a BaSO 4 @ZIF-8/TRF nanomacrophage (NMΦ) with hierarchical nanostructure is constructed as an alternative to immunoactive macrophages (Fig. 1).Upon residence in tumor via transferrin (TRF), the BaSO 4 @ZIF-8/TRF NMΦ can release Zn 2+ as an "artificial cytokine" to reverse tumor immunosuppression and induce tumor anoikis.The released tumor antigens can be selectively captured by the cavities of BaSO 4 nanoparticles, which is similar to the antigen capturing process of immunoactive macrophages.Thus, the BaSO 4 @ZIF-8/TRF NMΦ successfully mimics the basic functions of immunoactive macrophage, including tumor residence, cytokine release, antigen capture and immune activation.As a result, the NMΦ efficiently mediates macrophage polarization and T cell activation to fabricate systemic antitumor immunity.Besides, the immune memory established by the NMΦ can work against tumor recurrence.
The absorption spectra were measured on a UV-Vis spectrometer (UV-2450, Shimadzu, Japan).The pore size was characterized with an automatic BET specific surface area and porosity analyzer (ASAP2020 HD88 type, Micromeritics, USA).

Cell Culture and Animal Model
All the cells were cultured in complete DMEM medium with 10% fetal bovine serum and 1% streptomycin/penicillin at 37 °C in a 5% CO 2 atmosphere.Animal experiments were carried out under protocols (No.XMSB-2022-0103) approved by the Department of Laboratory Animals (Central South University).

In vivo Immune Responses
Female BALB/c mice (6-week-old) were subcutaneously injected with 4T1 tumor cells to fabricate bilateral tumors model.When the primary tumor volume was over 50 mm 3 , mice received different treatments (n = 5 in each group): (1) PBS, (2) BaSO 4 , (3) ZIF-8/TRF, (4) BaSO 4 @ZIF-8, (5) BaSO 4 @ZIF-8/TRF.All the nanoparticles were intratumorally injected into the primary tumors at the dosage of 5 mg kg −1 .On day 3 post injection, mouse blood was collected and the serum cytokines (TNF-α, IL-6, IL-10, and INF-γ) were detected with corresponding ELISA assay kits.On day 5 post injection, Zinquin was injected into the primary tumors at the dosage of 2 mg kg −1 for Zn 2+ detection.Moreover, the spleens and primary tumors were isolated to prepare single-cell suspensions.All the cells were fixed with 4% paraformaldehyde and blocked in 5% bovine serum albumin (BSA).To detect dendritic cell maturation, the splenic cell suspensions were stained with FITC-conjugated CD86, PE-conjugated CD80, and APC-conjugated CD11c at 4 °C for 12 h.To detect T cell activation, the splenic cell suspensions were stained with PE-conjugated CD4, APC-conjugated CD8a, and FITC-conjugated CD3 at 4 °C for 12 h.To detect the intratumoral macrophages, cell suspensions of 1 3 primary tumors were stained with FITC-conjugated CD86, APC-conjugated CD206 and PE-conjugated F4/80 at 4 °C for 12 h.All the cell suspensions were washed with PBS three times and analyzed by flow cytometry.In addition, the distant tumors were sliced and subjected to CD3/CD4/CD8 immunohistological staining and FoxP3/CD3 immunofluorescent staining.

Antitumor Therapy in vivo
4T1-Luc tumor cells were subcutaneously injected into mice to fabricate bilateral tumor model.When the primary tumor volume was over 50 mm 3 , mice were divided into eight groups (n = 6 in each group): (1) PBS; (2) BaSO 4 ; (3) anti-PD-1; (4) ZIF-8/TRF; (5) BaSO 4 @ZIF-8; (6) BaSO 4 @ ZIF-8/TRF; (7) ZIF-8/TRF + anti-PD-1; (8) BaSO 4 @ZIF-8/ TRF + anti-PD-1.All the nanoparticles were intratumorally injected into the primary tumors at the dosage of 5 mg kg −1 .Anti-PD-1 was intravenously injected into mice at the dosage of 0.75 mg kg −1 on day 1, 4, and 7. To monitor tumor growth, tumor bioluminescence images were captured after intravenous injection of D-luciferin (100 mg kg −1 ) every 5 days by an IVIS Lumina imaging system (PerkinElmer, USA).The tumor volumes of mice were recorded by a vernier caliper every other day and calculated according to the following formula: tumor volume = (width 2 × length)/2; meanwhile the body weights of mice were measured.On day 15 post injection, blood cells were harvested for blood biochemistry analysis on an auto hematology analyzer (HF-3800, HLIFE, China) and a blood chemistry analyzer (Pointcare V2, MNCHIP, China).All tumors were subjected to H&E and TUNEL staining for histopathological analysis.Main organs (liver, heart, spleen, lung, kidney) were also excised and stained with H&E.To calculate survival rate, mice received the above treatments and were fed for 60 days.When tumor volume was over 2000 mm 3 , mice were euthanized according to ethical guidelines.The survival rates of mice were calculated on day 60 post-injection.
For tumor anoikis analysis, the harvested primary tumors in different groups were divided into two halves.One half of the tumors were fixed and sliced for immunofluorescence staining of E-cadherin and integrin β1.The other half was prepared into single-cell suspension for western blotting analysis following the aboved-mentioned procedures.

Immune Memory Effect
4T1 breast tumor-bearing mice received the above-mentioned treatments for 15 days (n = 10 in each group).Residual tumors were removed via surgical excision under anesthesia.On day 28 post-treatment, mice were intravenously injected with 4T1-Luc cells (1 × 10 6 ).On day 32, the spleens of one half of the mice were harvested to obtain single-cell suspensions.Following tissue homogenization and erythrocyte lysis, the cells were stained with FITC-conjugated CD44, PerCP/Cy5.5-conjugatedCD62L and PE-conjugated CD8a.On day 42 post-treatment, D-luciferin (100 mg kg −1 ) was intravenously injected into the mice, and the bio-luminescence images were captured by the IVIS Lumina imaging system.Finally, the lungs of mice were collected and the tumor metastases were histologically examined via H&E staining.

Biodistributions in vivo
4T1 tumor cells were subcutaneously injected into mice to fabricate unilateral tumor model.4T1 breast tumor-bearing mice received intratumoral injection of Cy5.5-BaSO 4 @ ZIF-8 or Cy5.5-BaSO 4 @ZIF-8/TRF at a dose of 5 mg kg −1 .The nanoparticles in vivo were tracked through fluorescence imaging on the IVIS Lumina imaging system.At 24 h post injection, mice were euthanized for organ collection.The biodistributions of nanoparticles in the main organs were detected on the IVIS Lumina imaging system.For pharmacokinetics study of BaSO 4 @ZIF-8 nanoparticles, Cy5.5-BaSO 4 @ZIF-8 and Cy5.5-BaSO 4 @ZIF-8/TRF were intravenously injected into mice at a dose of 5 mg kg −1 .The nanoparticles in vivo were tracked through fluorescence imaging.At 72 h post injection, the main organs of mice were harvested for the Ba content analysis.

Statistical Analysis
All the data were expressed as the mean ± standard deviation (SD).The experiments were replicated three times, unless otherwise stated.The normality of data was examined by Shapiro-Wilk tests in SPSS Statistics 26.One-way ANOVA of variance was utilized to evaluate significance of difference.* p < 0.05, and ** p < 0.01.

Artificial Cytokine Role of Zn 2+
The immune response of macrophages highly relies on cytokine secretion to regulate tumor cell death and immune cell activation [33].To verify the possibility of Zn 2+ as an artificial proinflammatory cytokine, we investigated Zn 2+ release from the NMΦs in response to tumor pathological conditions, and its regulation on tumor cell death and immune activation.The PXRD data shows that the diffraction peaks of ZIF-8 disappear when BaSO 4 @ZIF-8 nanoparticles are exposed to tumor acidic condition (pH 6.5) (Fig. 2e).Due to the acid-responsive decomposition of ZIF-8, 83.7% of Zn 2+ ions can be released from BaSO 4 @ ZIF-8/TRF at tumor acidic condition.By contrast, the Zn 2+ release was barely detected under normal physiological condition (pH 7.4) (Fig. 2g).To investigate Zn 2+ release in vitro, BaSO 4 @ZIF-8/TRF NMΦs were co-incubated with tumor cells and normal cells, respectively.Similarly, the cumulative release rates of Zn 2+ from BaSO 4 @ZIF-8/ TRF NMΦs under tumor acidic and normal physiological conditions were 67% and 16%, respectively.Comparatively, Ba 2+ release was negligible either at tumor acidic conditions or at normal physiological conditions, indicating the high stability of BaSO 4 (Fig. S8).The intracellular Zn 2+ flux was further investigated in tumor cells (4T1), and normal cells (3T3 and L02) in vitro.The cellular Zn 2+ was stained with Zinquin as a fluorescent probe and quantified with flow cytometry (Figs.3a & S9).When 4T1 tumor cells were treated with BaSO 4 @ZIF-8/TRF for 12 h, the cellular Zinquin fluorescence was approximately increased by 10 times, which is much higher than those of 3T3 and L02 cells.To confirm Zn 2+ accumulation in tumor cells, chloroquine (CQ) as a typical Zn 2+ inhibitor was supplemented to scavenge free Zn 2+ .Obviously, the intracellular Zinquin fluorescence of tumor cells returned to normal level.Thus, BaSO 4 @ZIF-8/TRF NMΦ can release Zn 2+ ions under tumor acidic conditions, similar to the cytokines secreting profiles of macrophages.
Proinflammatory cytokines are able to mediate tumor cell death via multiple apoptotic pathways [34][35][36].To determine the tumor-killing activity of Zn 2+ as an artificial proinflammatory cytokine, cell viability assay was carried out on 4T1, 3T3 and L02 cells.After the treatment of BaSO 4 @ZIF-8/ TRF NMΦs, the cell viabilities of 3T3 and L02 cells maintained above 80% (Figs.3b & S10); while more than 70% of 4T1 tumor cells were killed after the same treatments.The tumor-killing activity of BaSO 4 @ZIF-8/TRF NMΦs was also validated by the live-dead staining (Fig. S11).Obviously, the majority of tumor cells were dead after the treatment of BaSO 4 @ZIF-8/TRF.Notably, the toxicity was also observed in the groups of ZIF-8/TRF and BaSO 4 @ZIF-8 but not in the group of BaSO 4 nanoparticles, indicating the Zn 2+ -mediated tumor cytotoxicity.To confirm this assumption, CQ was used as an inhibitor to scavenge free Zn 2+ .As expected, tumor cell viability was recovered to 91% in the co-presence of BaSO 4 @ZIF-8/TRF NMΦs and CQ, verifying the cytotoxic role of Zn 2+ .Therefore, Zn 2+ can mimic the function of proinflammatory cytokines to mediate tumor cell death.

BaSO 4 @ZIF-8/TRF NMΦ Mediated Tumor Anoikis
To understand the antitumor mechanism of BaSO 4 @ZIF-8/ TRF NMΦ, cytoskeleton of tumor cells was examined with fluorescent phalloidin as a specific probe.As shown in Fig. 3c, cytoskeleton collapse and cell detachment of the tumor cells were clearly found after the treatment of BaSO 4 @ZIF-8/TRF NMΦs, which are the characteristics of anoikis [37].To further confirm the anoikis of tumor cells, cellular adhesion-related proteins were examined through Western blotting.Evidently, as typical adhesionrelated membrane proteins, E-cadherin and integrin β1 are individually down-regulated by 55% and 67% in the presence of BaSO 4 @ZIF-8/TRF NMΦs, respectively (Figs. 3d & S12).In general, cell adhesion was closely associated with cJUN NH2-terminal kinase (JNK) pathway [38].In the presence of BaSO 4 @ZIF-8/TRF NMΦs, the reduced expression of adhesion-related proteins results in the 3.4fold upregulation of JNK.As the downstream of JNK pathway, phosphorylated BMF and BIM are elevated by BaSO 4 @ZIF-8/TRF NMΦs, thereby activating BAK/BAX apoptotic pathway [39].As a result, caspase-3 as an intracellular apoptosis effector is up-regulated 2.6-fold, while the expression of antiapoptotic Bcl-2 is suppressed by nearly 50% (Fig. S12).Based on the above evidence, tumor anoikis induced by BaSO 4 @ZIF-8/TRF NMΦs can be mediated by the JNK and BAK/BAX signaling pathways.It is noteworthy that the activation of JNK and BAK/BAX pathways was also observed in the groups of ZIF-8/TRF and BaSO 4 @ZIF-8, but not in the group of BaSO 4 , suggesting the pivotal role of Zn 2+ in the processes.According to recent studies, cell adhesion is influenced by intracellular Ca 2+ oscillations, which are highly activated in the presence of Zn 2+ overload [40,41].Thus, tumor anoikis was deduced to be mediated by Zn 2+ released from the decomposition of BaSO 4 @ ZIF-8/TRF NMΦs.To validate the assumption, CQ as a Zn 2+ scavenger was supplemented together with BaSO 4 @ ZIF-8/TRF NMΦs.As expected, the expression of either E-cadherin or integrin β1 was recovered back to the normal level (Fig. S12); meanwhile, the activation of JNK and BAK/ BAX pathways was suppressed.Therefore, the anoikis of tumor cells in the presence of BaSO 4 @ZIF-8/TRF NMΦs was mediated by Zn 2+ release via activating JNK and BAK/ BAX pathways (Fig. 3e).
The fabrication of adaptive immunity requires the immunogenic death of tumor cells to release damage associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), calreticulin (CRT) and high-mobility group protein B1 (HMGB1) [44].Natural macrophages can secrete proinflammatory cytokines such as tumor necrosis factor (TNF) to initiate immunogenic cell death of tumors [45].Therefore, we examined the potential of Zn 2+ as an artificial proinflammatory cytokine in triggering the release of DAMPs from tumor cells.Upon co-incubation with BaSO 4 @ZIF-8/TRF, the ATP release from 4T1 tumor cells gradually increased fourfold, as compared with the control group (Fig. 4a).According to the immunofluorescence staining of tumor cells, CRT was recruited from cytoplasm and aggregated on the plasma membrane; meanwhile, HMGB1 was translocated from the nucleus to the cytoplasm (Figs.4b & S17).Thus, with Zn 2+ as an artificial proinflammatory cytokine, BaSO 4 @ZIF-8/TRF NMΦs are able to trigger the immunogenic cell death of tumors to expose tumor antigens, which is similar to natural macrophages.

Antigen Capture of BaSO 4 @ZIF-8/TRF NMΦs
In addition to cytokine secretion, antigen capture is another feature of macrophages [46].After the decomposition of ZIF-8 shell in an acidic tumor condition, tremendous voids with average diameter of c.a. 20 nm were exposed on the BaSO 4 nanoparticles (Figs.4c & S18).Previous studies have confirmed that Ba possesses high affinity to cellular proteins, especially Ca-enriched membrane protein [47].In many cases, Ba and Ca share the same binding sites of membrane proteins, thereby allowing Ba to substitute Ca for membrane protein binding [48].Thus, BaSO 4 nanoparticles are assumed to be capable of binding membrane proteins for capturing tumor associated antigens.The selective affinity of BaSO 4 @ZIF-8/ TRF NMΦs to tumor cells was investigated by SEM.Due to the binding ability of Ba to membrane proteins, bare BaSO 4 nanoparticles were recruited to the surface of 4T1 tumor cell or L02 normal cell, which is lack of selectivity (Fig. 4d).For BaSO 4 @ZIF-8 or BaSO 4 @ZIF-8/TRF NMΦs, the ZIF-8 shell blocked the direct interaction of Ba to membrane proteins, thus only a little of them was found on the surface of L02 cells.However, the ZIF-8 shell was selectively decomposed under tumor acidic microenvironment to recover the interaction of BaSO 4 with membrane proteins.As a result, BaSO 4 @ZIF-8 or BaSO 4 @ZIF-8/TRF NMΦs are specifically bound to the surface of 4T1 tumor cells.Because of the specific affinity of TRF to tumor cells, the accumulation of BaSO 4 @ZIF-8/ TRF NMΦs on the 4T1 tumor cell membrane was over twofold higher than that of BaSO 4 @ZIF-8 nanoparticles.To further assess the capturing selectivity, the binding capacity of BaSO 4 nanoparticles to membrane proteins was quantified in the presence of serum proteins (e.g.bovine serum albumin, BSA).When exposed to physiological environments (pH 7.4), the binding ability of BaSO 4 @ZIF-8/TRF NMΦs to TRF is 63.8 mg g −1 , but its binding ability to cell protein or serum protein is less than 12.0 mg g −1 (Fig. 4e).By contrast, under tumor acidic conditions, nearly 80% of TRFs were desorbed from BaSO 4 @ZIF-8/TRF NMΦs.Meanwhile, the binding capacity of BaSO 4 @ZIF-8/TRF NMΦs towards membrane proteins was increased to 44.9 mg g −1 , much higher than that of serum proteins, suggesting that BaSO 4 @ZIF-8/TRF NMΦs exhibit selective enrichment of tumor cell proteins, which is similar to the selective capture of tumor antigens by natural macrophages.The selective binding of BaSO 4 to membrane proteins (e.g.CRT and E-cadherin) was further investigated through molecular dynamics (MD) simulation.Either membrane proteins or serum proteins can interact with BaSO 4 by van der Waals interactions and Coulomb force.However, the binding energy of BaSO 4 with CRT and E-cadherin was about 1.6-and 2.4-fold lower than that of BaSO 4 with serum protein, respectively (Figs. 4f & S19).Thus, BaSO 4 nanoparticles possess specific binding ability to membrane proteins for capturing tumor antigens.
In the process of anoikis, the detached cancer cells are likely to migrate and recolonized, resulting in anoikis resistance and tumor metastasis [49].To prevent cancer cell recolonization, various cell adhesion inhibitors have been developed, such as oligopeptides or small molecular complexes [50].In this work, BaSO 4 nanoparticles are demonstrated to be with high affinity to E-cadherin (Fig. 4g).Therefore, BaSO 4 nanoparticles can act as a cell adhesion inhibitor to prevent cancer cell recolonization after ZIF-8/TRF-mediated anoikis.Our experimental results confirm that BaSO 4 nanoparticles enhance tumor anoikis mediated by ZIF-8/TRF, although its cytotoxicity is weak (Fig. S10).

Reconstruction of Antitumor Immunity in vivo
The outstanding performance of BaSO 4 @ZIF-8/TRF NMΦs encourages us to evaluate their therapeutic efficiency in bilateral 4T1 tumor-bearing mice.To suppress immune tolerance of tumors, PD-1 antibody as a typical immune checkpoint blockade agent was administrated during the treatments.Upon injection of BaSO 4 @ZIF-8/TRF NMΦs, the expressions of adhesion-related proteins (i.e., integrin β1 and E-cadherin) were sharply downregulated (Fig. 6a  and b).Notably, the adhesion-related proteins were not significantly varied after treatment with αPD-1 or BaSO 4 alone.To confirm tumor anoikis in vivo, the expressions of anoikis-related proteins were determined via Western blotting assay (Figs.6c & S31).After the treatment with ZIF-8/TRF, BaSO 4 @ZIF-8 and BaSO 4 @ZIF-8/TRF, the JNK signaling pathway was activated to promote BMF phosphorylation and BIM expression.Subsequently, BAX/BAK pathway was involved to mediate the anoikis, accompanied with the downregulation of Bcl-2 and overexpression of caspase-3.Therefore, BaSO 4 @ZIF-8/TRF NMΦ can elicit tumor anoikis by virtue of Zn 2+ release, which is similar to the cytotoxic process of macrophages via cytokine secretion.
Tumor growth was continuously monitored by bioluminescence imaging (Fig. 6d).Obviously, both the primary or distant tumors grew rapidly regardless of the treatment with anti-PD-1 alone or BaSO 4 nanoparticles, indicating their faint antitumor activity (Fig. 6e and f).By contrast, for mice treated with BaSO 4 @ZIF-8/TRF NMΦs, the bioluminescence intensity and tumor volumes of primary tumors were remarkably reduced by 70% on day 15 (Fig. S32).The high antitumor efficacy of BaSO 4 @ZIF-8/TRF NMΦs could be ascribed to tumor anoikis.When anti-PD-1 was co-administrated with BaSO 4 @ZIF-8/TRF NMΦs, the tumor suppression rate further reached 95% due to the activation of antitumor immunity.Moreover, cytotoxic T cells and helper T cells were able to migrate into distant tumors for immunological attack, thereby inhibiting the growth of distant tumors by 90% (Fig. S33).After the treatment of BaSO 4 @ZIF-8/TRF NMΦs with anti-PD-1, obvious nucleus condensation and DNA fragmentations of dead cells were found from histological analysis of tumor slices (Figs.S34 & S35).Because of the immune modulating role of BaSO 4 @ZIF-8/TRF NMΦs, the mice survival rate was substantially increased to 83% within 60 days (Fig. 6g).During the treatments, all the mice behaved normally without significant loss of body weight (Fig. S36).
To evaluate the immune memory effect, tumor-bearing mice were rechallenged with intravenous injection of 4T1-Luc tumor cells after various treatments (Fig. 7a).For blank and BaSO 4 groups, bright bioluminescence was detected in the chest area on day 42, and tremendous metastatic nodules were distributed over 75% of the lung surface.In mice treated with BaSO 4 @ZIF-8/TRF and anti-PD-1, the bioluminescence signal of tumor metastasis was almost undetectable (Fig. 7b), and lung metastasis was completely suppressed (Fig. 7c-e).Flow cytometry analysis reveals fivefold increase of the population of effector memory T cells (T EM ) in the group of BaSO 4 @ZIF-8/ TRF + anti-PD-1.Therefore, BaSO 4 @ZIF-8/TRF NMΦs can successfully elicit the long-term immune memory effect to defend against tumor recurrence (Fig. 7f and g).According to the H&E staining results, the main organs kept normal histological structures (Fig. S37).Moreover, the blood biochemistry indexes were within the normal ranges (Fig. 7h).BaSO 4 @ZIF-8/TRF NMΦs can be metabolized by the body within 72 h even after intravenous injection into the mice (Fig. S38).All these aspects suggest the high biosafety of BaSO 4 @ZIF-8/TRF NMΦs for practical applications.

Conclusions
In summary, with BaSO 4 @ZIF-8/TRF as a paradigm, we present an artificial macrophage to mimic macrophagetumor interactive pattern for antitumor immune activation.BaSO 4 @ZIF-8/TRF NMΦs are able to selectively accumulate surrounding tumor cells and gradually release Zn 2+ into tumor microenvironment.Zn 2+ as a chemical messenger depressed the expression of adhesion proteins on cell membranes to induce tumor immunogenic death via anoikis.As the disintegration of ZIF-8 shell, a large number of mesopores were left on the BaSO 4 nanoparticles, providing cavities for capturing tumor antigens.As a result, BaSO 4 nanoparticles bearing tumor antigens promote the macrophage polarization and subsequent T cell recruitment for immune activation.Together with immune checkpoint inhibitors, BaSO 4 @ZIF-8/TRF NMΦ can elicit systemic antitumor immunity against distant metastasis.Moreover, the immune memory effect is successfully achieved to prevent tumor relapse.Overall, the BaSO 4 @ZIF-8/TRF NMΦ successfully simulates the basic biological functions of natural macrophages, including tumor retention, cytokine release, antigen capture and immune activation.The artificial macrophages with hierarchical nanostructure provide a promising strategy to overcome tumor immunosuppression, and raise the prospect of artificial cells in the biomedical field.