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 CuCl2 and MnCl2, 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 Cu2+, and three strong binding energy peaks (640.88, 646.18, and 642.28 eV) that were assigned to Mn2+, Mn3+, and Mn4+ (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/cm2), 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 H2O2 into hydroxyl radical (•OH) under an acidic environment, while metal ions in a high valence state with CAT/GPX-like activities, promote O2 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+, Cu2+, Mn2+, Mn3+, and Mn4+, high content of H2O2 and GSH in tumor cells and TME were supposed to be catalyzed into O2, ·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 H2O2, 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 O2 generation in three groups with different concentrations of OPCM + H2O2, among which 400 μg/mL OPCM displayed the maximum O2 increase, suggesting the ability of OPCM to catalyze O2 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 H2O2 but also could deplete GSH, leading to simultaneous O2 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 H2O2 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 Mn2+ 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 T1 weighted MR imaging due to the presence of Mn2+. We first demonstrated the MRI performance of OPCM in vitro. As revealed by Figure S20a, Mn concentration-dependent 1/T1 increase was found in all PBS systems containing 500 μg/mL OPCM. The T1 relaxation rate was 0.7854 mM-1s-1 in pH 7.4 PBS system, 2.210 mM-1s-1 in pH 6.0 PBS system, 2.434 mM-1s-1 in pH 5.0 PBS system, and 2.636 mM-1s-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 T1 relaxation rate (Figure S20b). The above data indicated that OPCM released more Mn2+ in acidic environment with high GSH content, suggesting that the OPCM might release abundant Mn2+ in the TME. Then, we further detected its imaging capability in vivo. As shown in Figure 4i and Figure S21, the OPCM displayed favorable T1-weighted MRI performance. After i.v. injection of OPCM, the T1 signal at the tumor site displayed a time-dependent increase until the 4th hour and then started to decrease until the 24th 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 H2O2 to generate O2, 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 O2 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 O2 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 37th 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 mm3 (Figure 7c). Notably, the combination group showed the slowest tumor growth with an average tumor volume of around 70 mm3, 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 (Tem). 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 Tem, 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 Tem (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 Tem 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 4th hour, which was in accordance with the result of the MRI with the 4th 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 0th d, 1th d, 3th d, 7th d, 14th d, 21th 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.