In situ self-assembled cell reservoir hydrogel for maneuvering multistage radioimmunotherapy

Domain: chemistry
Published: 2026-01-21
Editorial outcome: major_revision

Abstract
Radiotherapy (RT) is a clinical mainstay of cancer treatment that triggers tumor-specific immune responses. However, the effectiveness is usually hampered due to the hypoxic tumor microenvironment (TME) and the ambivalent impact of RT on the immune landscape of tumors. Herein, we develop an injectable hydrogel encapsulating interleukin-12 (IL-12)/anti-CTLA-4 (aCTLA-4) co-engineered red blood cells (RBC), which is in situ self-assembled within the TME to increase oxygen supply and instigate sequential aCTLA-4/IL-12 release, thus achieving Ba/O2 self-compensated radiosensitization and activating multistage immune responses. Once in the acidic TME, the in situ injected BaO2 undergoes hydrolysis to generate H2O2 and Ba2+, followed by the rapid reaction of Ba2+ with sodium alginate to afford a biocompatible hydrogel. Meanwhile, catalase presented on RBC converts H2O2 into O2, thereby alleviating hypoxia-induced radioresistance and inducing O2-mediated pore formation on RBC membrane for rapid release of aCTLA-4 to relieve tumor immunosuppression. Subsequently, IL-12 anchored on RBC is dilatorily released and interacts with T/NK cells within the TME to induce IFN-γ-dependent antitumor immunity. Taken together, the in situ self-assembled cell reservoir hydrogel offers a futuristic avenue to realize multistage radioimmunotherapy for effective tumor regression by programmable immunoregulation with significant clinical value.

Introduction
Radiotherapy (RT) is a prominent clinical treatment modality for cancer by focusing a local beam of radiation at the tumor site1,2. Extensive investigations have recently revealed its dual natures in provoking DNA double-strand breaks (DSBs) in tumor cells as well as eliciting the release of damage-associated molecular patterns (DAMPs)3, thereby promoting the recruitment and maturation of dendritic cells (DCs) by instigating systemic antitumor immunity4. Of note, the effectiveness of RT is often restricted by tumor hypoxia5, which competitively reduces the DNA radicals (DNA·) to produce DNA-OO·6,7, thus resulting in less damage fixation and decreased DSBs. Moreover, due to the differential RT sensitivity among immune cells, RT may cause a direct decrease in the ratio of cytotoxic T cells to regulatory T cells (Tregs)8, accompanied by the upregulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)9. This inevitably restrains the DC migration and inhibits the transportation of tumor antigens to tumor-draining lymph nodes (TDLNs)10, thus greatly attenuating the therapeutic efficacy of RT. Consequently, it is a pressing priority to explore unparalleled combinational therapeutic approaches to increase intratumoral oxygen supply and alleviate tumor immunosuppression for ameliorating therapeutic outcomes of RT.

Cancer immunotherapy has been extensively investigated as a vital part of recent advent and revolutionized the profound outlook through immune checkpoint blockade (ICB) strategies11. Anti-CTLA-4 (aCTLA-4) serves as an immune modulator to inhibit CTLA-4 function12,13, thus dampening the activity of Tregs while simultaneously promoting DC maturation and even facilitating the migration of both cDC1 and cDC2 to TDLNs for CD8+ T and CD4+ T cells activation14,15. In recent years, significant research interests have been attracted to the concurrent administration of aCTLA-4 with RT to harness the potential synergism between the two treatment modalities16. Despite promising preclinical results and substantial mechanism evidence of the synergism, a majority of studies at the clinical level fail to verify the positive effect of the combined treatments17, possibly due to the simple immune checkpoint blockade insufficient to relieve cytotoxic T cell depletion and NK cell exhaustion within the TME18. Interleukin-12 (IL-12), a crucial immune cytokine, emerges as a pivotal immunostimulatory agent capable of stimulating both the innate and adaptive immune systems19. As one of the direct downstream molecules of IL-12, IFN-γ, secreted by natural killer (NK) and T cells20, promotes the recruitment of immune cells into tumors as well as instigates the DC cell-intrinsic IL-12/IFN-γ positive feedback loop21, thus bolstering antitumor responses via immune engine refueling. Hence, the integrated utilization of IL-12 and aCTLA-4 exhibits immense potential in augmenting antitumor efficacy by synergistic combination with RT22. Nevertheless, a major concern arises from the direct injection of the mixture of IL-12 and aCTLA-4 (IL/aC), which may result in uncontrolled activation of immune cells and an IL-12-induced cytokine storm23. Therefore, we postulate that orchestrating an in situ IL-12/aCTLA-4 multisite-fettering system could be a promising strategy to facilitate the application of dual immunostimulatory agents in combination with RT, thereby maximizing antitumor responses while minimizing adverse effects.

Hydrogels, defined as water-swollen and insoluble three-dimensional (3D) crosslinked networks similar to the tumor extracellular matrix24, can offer physical support for engineered cells and slow release of loaded cargos25, thus holding great potential in enabling long-term therapeutic effects and efficient tumor intervention. In comparison to synthetic hydrogels, natural hydrogels exhibit superior biodegradability26 and unexceptionable biocompatibility27, which are advantageous for applications requiring high biological compatibility and environmental sustainability. Sodium alginate (Alg), a natural polymer that remains stable within the biological environment, bears a strong resemblance to the extracellular matrix28, thus demonstrating low immunogenicity and excellent biocompatibility29. Furthermore, Alg can be degraded into nontoxic polysaccharides that do not participate in normal metabolic processes30,31. Due to the multiple merits of this natural polymer discussed above, Alg-based hydrogel is a potentially attractive alternative as a drug-encapsulating depot with negligible biosafety concerns32,33. However, traditional Alg-based hydrogels generally chelate with metal ions intrinsic in the body and lack precise control over the architecture formation34, which may result in drug leakage and potential toxicity. Hence, the development of a TME-responsive in situ-formed Alg-based hydrogel emerges as an urgent demand for spatiotemporally controlled drug release and unleashed antitumor efficacy.

In this study, we devise an in situ self-assembled cell reservoir crosslinked network based on the introduction of barium dioxide (BaO2) into the synthesis of injectable hydrogels with locoregional delivery of IL-12/aCTLA-4 co-engineered red blood cells (IL-12/aCTLA-4@RBC-BaO2-Alg hydrogel, IL/aC@RBAH) to initiate multistage immune effects for amplified synergistic radioimmunotherapy (RIT) (Fig. 1). Upon intratumoral injection of the hydrogel precursors, BaO2 undergoes rapid hydrolysis within the acidic TME to release H2O2 and Ba2+, which quickly reacts with Alg to form hydrogels to entrap multiple cargos within the TME. Simultaneously, the generated H2O2 is decomposed under the catalysis of catalase (CAT) presented on RBC to produce oxygen for endogenous O2 furnishment. This multifaceted process not only alleviates tumor hypoxia-induced radioresistance, but also triggers pore formation on RBC membrane (RBCM) for rapid release of aCTLA-4. Subsequently, IL-12 anchored on RBC initiates activation of cytotoxic T/NK cells in the TME, with mitigated potential immunotoxicity by the immobilization on RBC surface and steady in situ retention of the hydrogel. The sequential aCTLA-4/IL-12 releasing hydrogel unleashes a therapeutic window in step wise modulating immune networks, signified by significantly enhanced DC homing, boosted antitumor T cell infiltration, and NK cell recruitment for garnering an immunostimulatory TME. Notably, the synergy between Ba/O2 dual-compensated RT and sequential aCTLA-4/IL-12 release potentiates a benefit in both innate and adaptive immune activation to reshape the immune landscape of the TME and bolster the attack on both poorly immunogenic 4T1 triple-negative breast cancer (TNBC) and Panc02 pancreatic cancer. Collectively, the meticulous design of engineered RBC-encapsulating hydrogels offers a facile and feasible approach to synergize radiotherapy with immunotherapy to garner multistage radioimmunotherapy for reinvigored antitumor immune responses and effective tumor regression.

Results
Design and characterization of IL/aC@RBC and BaO2
Numerous studies have demonstrated that native RBC (nRBC) harbors a range of active enzymes, notably CAT35,36, which catalyzes the production of oxygen from H2O2 for radiosensitization. Conventional methodologies for RBCM extraction led to substantial loss or inactivation of CAT37. In contrast, RBC processed through alternate hypotonic and hypertonic dialysis (designated as RBC) would preserve the structural integrity and enzyme activity of nRBC. Furthermore, in order to mask the immunotoxicity of IL-12 and facilitate sequential aCTLA-4/IL-12 release, RBC was engineered with IL-12 on the surface and aCTLA-4 within the cell for multisite drug encapsulation (Fig. 2a). Firstly, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed that the composition of nRBC proteins was mostly reserved within IL/aC@RBC (Fig. 2b). In addition, to evaluate the influence of IL-12 feeding time points on the loading capacities of IL-12/aCTLA-4@RBC (IL/aC@RBC), IL-12 and aCTLA-4 were respectively labeled with AMCA and FITC to obtain IL-12-AMCA and aCTLA-4-FITC, as affirmed by the SDS-PAGE protein analysis and ultraviolet–visible (UV–Vis) absorption changes (Supplementary Fig. 1). Concurrently, sulfo-SMCC was connected with IL-12-AMCA to afford Mal-IL-12-AMCA, whose maleimide group was further clicked with DSPE-PEG2000-SH to generate DSPE-PEG2000-IL-12-AMCA for efficient IL-12 anchorage on aCTLA-4-encapsulated RBC (aC@RBC). Subsequently, IL/aC@RBC-1, IL/aC@RBC-2, and IL/aC@RBC-3 were prepared by varying the feeding time points of DSPE-PEG2000-IL-12-AMCA, which were respectively fed along with aCTLA-4-FITC, immediately subsequent to aCTLA-4-FITC, and followed by hypertonic sealing of aC@RBC. Compared with the fluorescence intensities of IL-12-AMCA and aCTLA-4-FITC in IL/aC@RBC-1 and IL/aC@RBC-2, that of IL-12-AMCA in IL/aC@RBC-3 was significantly elevated, concomitant with negligible discrepancy in the intensities of aCTLA-4-FITC (Supplementary Fig. 2). The result suggested superior IL-12/aCTLA-4 co-loading capabilities of IL/aC@RBC-3. Therefore, IL/aC@RBC-3 (denoted as IL/aC@RBC) was applied for subsequent experiments.

To visualize the loading sites of IL-12 and aCTLA-4, confocal laser scanning microscopy (CLSM) was used to analyze the colocalization of IL-12-AMCA and aCTLA-4-FITC with DiI-labeled RBCM (RBCM-DiI). The results revealed that IL-12 was effectively anchored on RBC surface, whereas aCTLA-4 was proficiently encapsulated into the cytosol of RBC (Fig. 2c, d). Moreover, the UV–Vis spectra presented the characteristic absorption peaks of both IL-12-AMCA and aCTLA-4-FITC in IL/aC@RBC, evidencing excellent dual-loading of IL-12 and aCTLA-4 in RBC, with corresponding encapsulation efficiency at 22.54% and 11.52%, respectively (Supplementary Fig. 3). Further investigations into the stability of IL/aC@RBC illustrated that IL-12-AMCA remained bound to RBC surface with aCTLA-4-FITC being retained inside IL/aC@RBC for an extended period of up to 7 days (Supplementary Fig. 4), confirming steady IL-12/aCTLA-4 co-loading capability of IL/aC@RBC. Next, to visualize the integrity of RBC in IL/aC@RBC, scanning electron microscopy (SEM) was employed to observe the morphology change. Although some IL/aC@RBC underwent a morphological transformation from the typical biconcave disc shape of nRBC to a spherical form with co-encapsulation of IL-12 and aCTLA-4, the images showed excellent integrity of RBC following the facile alternate hypotonic and hypertonic dialysis method (Fig. 2e). For further CAT enzyme activity evaluation, H2O2 catalytic activities were respectively monitored in nRBC, RBC, IL/aC@RBC and RBCM. The result indicated a negligible loss of CAT enzyme activity in RBC, yet a substantial decrease in RBCM compared to nRBC (Fig. 2f), indicating magnificent CAT preservation by using the alternate hypotonic and hypertonic dialysis method. Besides, a slight reduction in the enzyme activity value was observed in IL/aC@RBC, which was merely attributed to the elevation of total protein level with negligible loss in the active unit of CAT itself. Moreover, the catalytic O2 production from nRBC and IL/aC@RBC were quantitatively investigated to affirm CAT reservation on IL/aC@RBC. The results manifested that the O2 production from IL/aC@RBC and nRBC exhibited negligible difference, with the amount at 0.085 mg and 0.092 mg, respectively (Fig. 2g), demonstrating that the IL-12/aCTLA-4 encapsulation would not disrupt the CAT activity of RBC for O2 generation. Furthermore, the bioactivity of IL-12 in IL-12-anchored RBC (IL@RBC) was evaluated by the IFN-γ production during splenocyte activation. The half-maximal effective concentration (EC50) of IL@RBC was determined to be 2.302 pM, which was similar to that of free IL-12 at 1.698 pM (Fig. 2h), demonstrating effective preservation of IL-12 bioactivity in IL@RBC.

Given the lack of precise control over the drug payload and skeleton construction, most Alg-based hydrogels, chelating with metal ions intrinsic in the body38, show poor specificity between tumors and the surrounding tissues. Due to the high X-ray absorption coefficient of Ba element with a small K-edge value (37 keV) for nice X-ray attenuation character at low operating voltage39,40, BaO2 was chosen as a precursor to construct an acidity-responsive self-assembled hydrogel within the TME. During in situ injection, BaO2 was introduced for selective hydrolysis into Ba2+ and H2O2 in the acidic environment, subsequently facilitating the rapid in situ hydrogel formation through the cross-linking interaction between Ba2+ and Alg. Herein, a modified hydrolyzation-precipitation approach was employed to synthesize BaO2. Briefly, with the assistance of polyvinylpyrrolidone (PVP) as a colloidal stabilizer, BaCl2 was reacted with choline hydroxide to construct Ba(OH)2 as an intermediate product, which was further mixed with H2O2 in an alkaline methanol solution to afford BaO2 (Supplementary Fig. 5). Intriguingly, the morphology of the as-synthesized BaO2 was influenced by the feeding mass ratios of both PVP:H2O2 and H2O2:BaCl2. With the gradual decrease in the feeding mass ratio of PVP:H2O2 from 20:1 to 12:1, the morphology of the BaO2 products gradually changed from a smooth spherical shape to a rough bamboo-like shape with severe aggregation (Supplementary Fig. 6). While the ratio of PVP:H2O2 was further decreased to 2:1, bamboo-like BaO2 products with uniform dispersion were obtained. Owing to the low yield of spherical BaO2 nanoparticles formed at a high ratio of PVP:H2O2 (20:1), the feeding mass ratio of PVP:H2O2 (2:1) was ultimately chosen to synthesize smooth bamboo-like BaO2 for subsequent studies. Moreover, the influence of H2O2 on the formation of BaO2 was further investigated. At a feeding mass ratio of H2O2:BaCl2 = 1.2:1, the as-prepared BaO2 exhibited irregular morphologies with large aggregation. With an increased ratio of H2O2:BaCl2 to 1.8:1, the BaO2 products transitioned to a uniform, bamboo-like structure. Conversely, an elevation of the ratio of H2O2:BaCl2 to 3.6:1 resulted in the formation of agglomerated nanoparticles with a shortened bamboo-like structure. Consequently, a feeding mass ratio of H2O2:BaCl2 at 1.8:1 was determined optimal choice for the synthesis of BaO2. Overall, BaO2 was meticulously synthesized at the feeding mass ratio of BaCl2:H2O2:PVP = 1:1.8:3.6 for further experiments with the morphology, dispersion, and yield into consideration.

Next, high-resolution transmission electron microscopy (TEM) was employed to determine the size of bamboo-like BaO2 (Fig. 2i), which revealed that the average length was approximately 140 nm (Supplementary Fig. 7). Besides, both the energy-dispersive X-ray spectroscopy (EDS) element mapping and spectrum presented the physical composition of BaO2, including O and Ba, each of which was uniformly distributed within the skeleton of BaO2 (Supplementary Fig. 8). Moreover, the X-ray diffraction (XRD) pattern revealed that the as-prepared BaO2 exhibited characteristic diffraction peaks aligned with those of tetragonal BaO2 crystal, confirming the similar crystallinity structure of as-synthesized BaO2 to the standard one (Supplementary Fig. 9). As expected, the valence states presented the coexistence of Ba2+ and O− within BaO2, as evidenced by the Ba 3d peaks at 794.8/779.4 eV and O 1s peak at 530.4 eV in the X-ray photoelectron spectroscopy (XPS) spectra (Fig. 2j and Supplementary Fig. 10). To further investigate the acidity responsiveness of BaO2, a comprehensive study was conducted on its degradation behavior under various acidic conditions. It is revealed that the Ba2+ release and H2O2 generation from BaO2 were notably slow in the neutral buffer (Fig. 2k, l). By contrast, a significant increase in the release rates was observed in the acidic buffer at pH 6.5 (Fig. 2k, l), probably because the acidic environment provided an abundance of protons to facilitate the rapid decomposition of BaO2. Furthermore, the IL/aC@RBC treated with BaO2 presented notable pores with distortions in appearance in a mildly acidic environment (pH 6.5) (Supplementary Fig. 11), which was attributed to CAT-mediated O2 production from BaO2-derived H2O2. Excitingly, the methyl thiazolyl tetrazolium (MTT) assay revealed negligible cytotoxicity of BaO2 to both 4T1 and Panc02 cells at a high concentration of even 150 μg/mL (Supplementary Fig. 12), suggesting relatively high biosafety of BaO2. Collectively, all these findings demonstrated that biocompatible BaO2 was successfully prepared and could respond to the acidic TME to generate sufficient Ba2+ and H2O2 for subsequent in situ hydrogel formation and local O2 supply.

Construction and characterization of biodegradable IL/aC@RBAH
Previous studies have reported that Alg could chelate with metal ions to form crosslinked hydrogels for efficient encapsulation of biomacromolecules41,42 and cells43,44. Herein, to construct a self-assembled IL/aC@RBC reservoir hydrogel in response to the acidic TME, we introduced the scrupulously orchestrated BaO2 as the hydrogel precursor. Inspired by the above acidity-responsiveness of BaO2, we speculated that BaO2 could undergo hydrolysis in the acidic environment, resulting in the generation of Ba2+ and H2O2. Then, Ba2+ reacted rapidly with Alg to form a biocompatible 3D crosslinked network to in situ encapsulate IL/aC@RBC for constructing IL/aC@RBAH. Meanwhile, the generated H2O2 was catalyzed by primordial CAT on RBC to increase O2 supply, which also induced pore formation on RBCM for rapid aCTLA-4 release. Subsequently, with the slow degradation of the constructed Ba-Alg hydrogel, IL-12 anchored on RBC was steadily released through the enlarged crosslinked channels of the hydrogel (Fig. 3a). To testify the acidity-responsive self-assembly characteristics, the formation of IL/aC@RBAH was monitored at different pH values. As expected, IL/aC@RBAH was successfully gelated at pH 6.5, indicating that the generated Ba2+ from decomposed BaO2 could effectively chelate with Alg under acidic conditions (Fig. 3b). To gain further insight into the rheological and mechanical properties of IL/aC@RBAH, we conducted dynamic oscillation tests at first. The gelatin time of IL/aC@RBAH at pH 6.5 and 7.4 was measured using a rheometer. As shown in Fig. 3c, the hydrogel formed in merely 2.39 min at pH 6.5, while no gel formation was observed at pH 7.4, which demonstrated that the IL/aC@RBAH hydrogel could be rapidly formed in response to the acidic condition. Moreover, IL/aC@RBAH fed with various concentrations of BaO2 underwent strain amplitude sweeps and dynamic oscillatory frequency sweeps. It is noteworthy that a significant enhancement in the mechanical strength was observed when the feeding concentration of BaO2 increased from 2 to 6 mg/mL (Fig. 3d, e), which indicated increased toughness and mechanical strength of the Ba-Alg crosslinked network. With a further increase in the feeding concentrations of BaO2 from 6 to 8 mg/mL, the elevation of mechanical strength was less pronounced. Consequently, IL/aC@RBAH with feeding BaO2 at 6 mg/mL was selected for further experiments with both biosafety and rheological properties of Ba-Alg hydrogel into consideration. In order to observe the in situ encapsulation of multiple cargos within the Ba-Alg hydrogel, IL-12, aCTLA-4, and RBCM were labeled with AMCA, FITC, and DiI, respectively. Both the fluorescences of IL-12-AMCA (blue) and aCTLA-4-FITC (green) were successfully overlapped with RBCM-DiI (red), and all of them presented a uniform distribution within the hydrogel, as visualized via CLSM observation (Fig. 3f). The findings manifested successful IL-12/aCTLA-4 engineering of RBC, which was uniformly distributed in the Ba-Alg hydrogel to yield IL/aC@RBAH. Meanwhile, the cryo-scanning electron microscopy (cryo-SEM) image of IL/aC@RBAH showed that IL/aC@RBC with smoothly spherical morphology lay in the crosslinked networks structure of the hydrogel (Fig. 3g). All these results verified IL/aC@RBAH to be an excellent IL/aC@RBC reservoir hydrogel.

Next, the degradation of IL/aC@RBAH was detected at pH 6.5, mimicking the mildly acidic TME. The result revealed a two-phase degradation pattern of IL/aC@RBAH, comprising an initial rapid phase and a subsequent slower rate, with the degradation process reaching equilibrium over time (Fig. 3h). Within the first day of immersion, the residual liquid and BaO2, which were originally presented on the hydrogel surface during preparation, exhibited rapid depletion, indicative of the swift degradation kinetics of the hydrogel. Subsequently, the degradation rate stabilized, with approximately 70% degradation observed after 14 days (Fig. 3h). The cumulative release behaviors of IL-12 and aCTLA-4 from IL/aC@RBAH were also investigated to prove the sequential release performance. It was obvious that aCTLA-4 was rapidly released (Fig. 3i), which demonstrated a primary liberation of aCTLA-4 through the O2-induced pores on RBCM. Afterward, IL-12 anchored on RBC was dilatorily released through the enlarged crosslinked channels of the degrading IL/aC@RBAH. Notably, the release rate of aCTLA-4 was significantly accelerated at pH 6.5 compared to that at pH 7.4, which was possibly attributed to the acidity-induced residual BaO2 degradation to trigger H2O2 production for continuous O2-mediated pore formation on RBC. Contrarily, the pores on RBC were no longer increased at pH 7.4, thus delaying the release kinetics of aCTLA-4 in the neutral environment. Moreover, the cumulative release of Ba2+ was further investigated. After 48 h of incubation at pH 6.5, approximately 50% of Ba2+ was liberated from the network structure of IL/aC@RBAH, slightly higher than that in the neutral environment (Fig. 3j), possibly due to the continuous degradation of superfluous BaO2 in the hydrogel in the mildly acidic condition. To further evaluate its biocompatibility, we conducted a calcein-AM/propidium iodide (PI) co-staining assay, showing negligible red fluorescence compared with green signals (Fig. 3k), which revealed little toxicity of IL/aC@RBAH, fitting well with the result of the MTT assay conducted on 4T1 and Panc02 cells (Supplementary Fig. 13). Overall, all the results above underscored that the acidity-responsive self-assembled IL/aC@RBAH hydrogel exhibited excellent biocompatibility and controlled sequential drug release.

In vitro Ba/O2 dual-compensated radiosensitization of IL/aC@RB
IL/aC@RBC was dexterously combined with BaO2 for IL/aC@RB to initiate O2 production for Ba/O2 self-compensated radiosensitization. Mechanically, BaO2 could respond to the acidic environment to provoke H2O2 production, which was then catalyzed by CAT inherent on RBC for O2 supply. Meanwhile, Ba element with a high X-ray absorption coefficient could ally with self-supplied O2, thus inducing Ba/O2 dual-compensated radiosensitization for augmented antitumor efficacy. To verify the underlying mechanism of dual-compensated radiosensitization, a colony formation assay was conducted to evaluate the sensitivity of 4T1 cells to RT with different formulations in vitro. The result showed that RT+IL/aC@RBC exhibited an imperceptible change in the cytotoxicity of 4T1 cells compared to the sole RT group (Supplementary Fig. 14), demonstrating that single IL/aC@RBC intervention scarcely promoted the efficacy of RT. Nevertheless, the treatment of RT+BaO2 considerably decreased the cloning numbers of 4T1 cells compared to the groups of RT and RT+IL/aC@RBC, suggesting the ameliorated RT by the existence of Ba element in BaO2. Interestingly, the cell viability was further constrained with the slightest clones in the RT+IL/aC@RB group, inferring excellent radiosensitization induced by the joint of Ba element and O2 from the alliance of RBC with BaO2. Furthermore, the intracellular ROS level was evaluated by employing 2’,7’-dichlorofluorescein diacetate (DCFH-DA) as a ROS probe45 and investigated via both CLSM and flow cytometry. As expected, RT+BaO2-treated cells presented moderately elevated ROS generation in contrast to the RT and RT+IL/aC@RBC groups, which indicated mild radiosensitization by Ba element from BaO2 (Supplementary Figs. 15 and 16). Remarkably, a significantly stronger fluorescence signal of ROS could be detected in the RT+IL/aC@RB group compared to that in the RT, RT+BaO2, and RT+IL/aC@RBC groups (Supplementary Figs. 15 and 16), attributing to effective Ba/O2-supplied radiosensitization through Ba element introduction and in situ O2 generation. Collectively, these findings indicated that IL/aC@RB could be deemed to be an efficient Ba/O2 depot for radiosensitization via the tactical incorporation of RBC with BaO2.

Immunogenic cell death (ICD) induced by RT causes the release of a serious of DAMPs, including calreticulin (CRT), high mobility group box 1 protein (HMGB1), and adenosine triphosphate (ATP), thus collectively activating the immunogenicity of tumor cells for promoted antigen presentation46. Therefore, the exposure of CRT, and the release of both HMGB1 and ATP after different treatments were analyzed to further substantiate amplified ICD effects of Ba/O2 dual-compensated radiosensitization. As expected, the immunofluorescence staining showed that RT+BaO2 could increase the CRT exposure on 4T1 cells, accompanied by HMGB1 release from cell nuclei in 4T1 cells in contrast to sole RT and RT+IL/aC@RBC (Supplementary Figs. 17 and 18). Noteworthily, the irradiated cells in the RT+IL/aC@RB group displayed the strongest CRT signals on the membrane surface and the lowest HMGB1 retention in the nucleus of 4T1 cells (Supplementary Figs. 17 and 18), affirming boosted in vitro ICD induction efficacy of Ba/O2-driven radiosensitization. In addition, concomitant with the decreased intracellular ATP content, the extracellular level of ATP in the RT+IL/aC@RB group was nearly 2.08-fold, 2.02-fold, and 1.24-fold of that in RT, RT+IL/aC@RBC, and RT+BaO2 groups, respectively (Supplementary Fig. 19). These results jointly clarified prominent ICD efficacy by RT+IL/aC@RB-mediated radiosensitization.

In situ retention and O2 self-compensation of IL/aC@RBAH
The in vivo degradation of the IL/aC@RBAH was evaluated by monitoring its residues within the skin of mice over a period of 14 days. Consistent with the in vitro degradation behaviors (Fig. 3h), the hydrogel displayed a progressive deterioration with slight debris observed on the 14th day post-implantation (Fig. 4a). To further assess the local retention ability of IL/aC@RBAH, we introduced a near-infrared (NIR) fluorescent probe Cypate and intratumorally injected the mixture of Cypate-labeled IL-12 and aCTLA-4 (IL-Cp&aC), IL-Cp/aC@RBC, or IL-Cp/aC@RBAH into the tumors of orthotopic 4T1 tumor-bearing mice for real-time monitoring in situ retention via whole-animal in vivo fluorescence imaging system (IVIS). By comparison with the rapid fluorescence elimination of IL-Cp&aC within 1 day and IL-Cp/aC@RBC within 5 days, strong Cypate fluorescence of IL-Cp/aC@RBAH was still detected on day 14 post injection (Fig. 4b, c), similar to the local retention behaviors of the formulations implanted under the skin (Supplementary Fig. 20). The difference claimed the prominent in situ retention capability of IL/aC@RBAH compared to free IL-12 and IL/aC@RBC, possibly due to the in situ IL-12/aCTLA-4 multisite-fettering system. Moreover, the IL-12 retention in the orthotopic 4T1 tumors was also monitored. Predictably, the IL-12 levels in the IL-aC (IL-12 prior to aCTLA-4) and aC-IL (IL-12 subsequent to aCTLA-4) groups both remarkably declined in one day (Supplementary Fig. 21), indicating rapid diffusion loss of free IL-12 from tumors. Compared to the time-dependent moderate reduction in the IL&aC@RBAH group (mixture of IL-12 and aCTLA-4 in RBC) due to the pore formation on RBC, the majority of IL-12 content still remained at the tumor site on 3 days post-injection in the IL/aC@RBAH group, possibly attributed to the excellent hydrogel stability and effective IL-12 anchorage on the RBC surface. Besides, the biodistribution of BaO2 in orthotopic 4T1 tumor-bearing mice was detected. Once IL/aC@RBAH was injected into the tumors, the level of Ba in the tumors was rapidly elevated at 0 h, followed by a moderate reduction at 24 h post-injection (Supplementary Fig. 22). During the subsequent 2-day monitoring period, the Ba content in the tumors maintained over 20% ID/g with a slight declining trend, manifesting the favorable in situ retention capability of IL/aC@RBAH with minimal leakage of Ba. Contrarily, the levels of Ba in the livers and kidneys were both imperceptible post the local administration during 72 h of monitoring, indicative of excellent in situ retention capacity of IL/aC@RBAH. To further visualize the RBC distribution in orthotopic 4T1 tumor-bearing mice post local injection, the RBC in IL/aC@RBAH was labeled with DiR (a cell membrane probe) to obtain DiR-RBC for real-time monitoring. During the 72 h of the tracking process, the total fluorescence of DiR-RBC at the tumor site remained persistently above 8×109 p/s with imperceptible changes (Supplementary Fig. 23). Moreover, the tumors and major organs, including heart, liver, spleen, lung, and kidney, were collected at 72 h for ex vivo imaging. As anticipated, in sharp contrast to the negligible fluorescence detected in the major organs, substantial DiR signals were tracked in the tumors. Collectively, all those results confirmed the brilliant in situ retention capability of IL/aC@RBAH with favorable stability.

The excellent long-term retention performance of IL/aC@RBAH might favor to restrict the potential toxicity and enhance the biocompatibility of IL-12. In light of the prolonged in situ retention characteristic inherent to IL/aC@RBAH in vivo, the concentrations of aCTLA-4 and IL-12 in the serum were testified. The concentrations of these immunological agents exhibited a significant decline in comparison to those in the direct intratumoral administration aCTLA-4 of and IL-12 (Fig. 4d, e), signifying the rapid in situ information of IL/aC@RBAH to afford low leakage and in situ retention of both aCTLA-4 and IL-12. Considering the reported toxicity of free IL-12 to liver damage and kidney injury47, liver damage indexes, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as kidney function indexes compromising blood urea nitrogen (BUN) and creatinine (CREA), were respectively measured. Compared to the liver and kidney indexes in the IL&aC group, in which the mixture of IL-12 and aCTLA-4 was injected intratumorally, those indexes in the RBC/BaO2/Alginate hydrogel (RBAH) and IL/aC@RBAH groups were lower and maintained within the normal range (Fig. 4f–i), probably ascribed to the biosafety of RBAH, firm immobilization of IL-12 on RBCM, and steady retention of IL/aC@RBAH within the TME. Collectively, these findings provided compelling evidence supporting the favorable biosafety of IL/aC@RBAH.

The outcome of RT is severely hampered by the hypoxic TME48,49. It is anticipated that the designed IL/aC@RBAH possessed the ability of O2 self-production for tumor hypoxia amelioration. To unveil the underlying mechanism of O2 self-supply by IL/aC@RBAH, we assessed the vascular saturated oxygen (sO2) levels in tumors after local injection of IL/aC@RBC and IL/aC@RB. The result showed that IL/aC@RBC without BaO2 hardly afforded a statistically significant change in oxygen levels (Fig. 4j), possibly originating from restricted H2O2 in TME. Excitingly, with the incorporation of BaO2, IL/aC@RB could engender a notable elevation in the intratumoral sO2 levels (Fig. 4j), which affirmed that the integration of BaO2 into IL/aC@RB facilitated the generation of H2O2 and subsequent breakdown of H2O2 by CAT for O2 provision. Moreover, tumors injected with IL/aC@RB and IL/aC@RBAH were monitored over a 48-h period. The in situ O2 production from H2O2 by IL/aC@RB was slightly increased during the first 24 h (Fig. 4k). However, the sO2 content was decreased within 48 h, possibly owing to the rapid O2 generation and consumption. In contrast, the sO2 levels in the tumors subjected to IL/aC@RBAH showed an increase in sO2 levels within 48 h, which was attributed to the slow diffusion of H2O2 and the long-term retention of IL/aC@RBC during the hydrogel formulation. All the results above suggested that the IL/aC@RBAH could be an effective mean to alleviate tumor hypoxia over an extended period. The expression of hypoxia-inducible factor-1α (HIF-1α) is recognized as an indicator of hypoxic conditions within biological systems50–52. In both the IL/aC@RB and IL/aC@RBAH groups, a discernible decrease in the HIF-1α expression was observed, and the tumors exposed to IL/aC@RBAH showed negligible signals of HIF-1α in comparison to those treated with BaO2 or IL/aC@RBC alone (Fig. 4l and Supplementary Fig. 24). The results collectively evidenced the efficacy of IL/aC@RBAH in persistently increasing O2 supply within the TME, and palliating intratumoral hypoxia dilemma. Moreover, the expression levels of γ-H2Aχ in tumor tissues were measured to examine the DNA damage induced by different treatments. Compared to the fluorescence intensity of γ-H2Aχ in the RT and RT+IL/aC@RA groups, that in the RT+IL/aC@BaO2/Alg (RT+IL/aC@BAH) group was significantly elevated (Fig. 4m and Supplementary Fig. 25), potentially attributed to augmented X-ray absorption of Ba element for radiosensitization. Notably, the green signal of γ-H2Aχ was the utmost in the RT+IL/aC@RBAH group, thus demonstrating excellent Ba/O2 dual-compensated radiosensitization for instigating DNA damage by the ingenious marriage of RBC and BaO2.

In vivo antitumor efficacy and multistage immune responses in 4T1 tumor-bearing mice
Motivated by the aforementioned findings, we proceeded to assess the antitumor efficacy of IL/aC@RBAH in vivo. Orthotopic 4T1 tumor-bearing mice were randomly allocated into six groups: saline (G1), RT (G2), RT+RBAH (G3), RT+aC@RBAH (G4), RT+IL@RBAH (G5), and RT+IL/aC@RBAH (G6). Prior to exposure to X-ray irradiation (8 Gy) in pre-designed groups, these mice were respectively intratumorally administered with saline, RBAH, aC@RBAH, IL@RBAH, and IL/aC@RBAH. The tumor volumes were recorded every other day and corresponding bioluminescence intensities were monitored every week. It was found that the tumors in the RT+RBAH group were moderately inhibited compared to those in the RT group (Fig. 5a–d), probably due to Ba/O2-supplied radiosensitization by RBAH. With aCTLA-4- or IL-12-engineered RBC encapsulation within RBAH in the RT+aC@RBAH or RT+IL@RBAH group, the mice showed longer survival with more notable tumor suppression in contrast to those in the RT+RBAH group (Fig. 5a–e), possibly attributed to the potential antitumor immune efficacy by the rapid release of aCTLA-4 or sustained liberation of IL-12 alone. As expected, the administration of RT+IL/aC@RBAH resulted in superior tumor regression and prolonged lifespan of at least 2 months in 60% of the mice compared with the RT+aC@RBAH and RT+IL@RBAH groups (Fig. 5a–e). Moreover, the necrosis and proliferation of tumor cells were respectively assessed by H&E staining and Ki67 immunofluorescence. Similarly, tumors derived from the RT+IL/aC@RBAH group manifested the most extensive necrosis and weakest green fluorescence of Ki67 (Fig. 5f–i and supplementary Fig. 26), indicative of the most pronounced therapeutic effect of RT+IL/aC@RBAH-mediated RIT for effective tumor regression. These findings verified the exceptional antitumor effect of RT+IL/aC@RBAH in the synergy of Ba/O2-supplied radiosensitization and aCTLA-4/IL-12-mediated multistage immunotherapy.

The biotoxicities of different treatments were further evaluated by monitoring the body weights of mice, which presented no discernible discrepancy among these groups (Supplementary Fig. 27). The typical hematoxylin and eosin (H&E) staining of major organs including heart, liver, spleen, lung and kidney from mice after different treatments were also observed, which showed excellent biosafety of all these treatments with negligible histomorphological changes (Supplementary Fig. 28). Besides, long-term toxicities were measured to affirm the cytokine storm mitigation of IL-12 by IL/aC@RBAH. Healthy BALB/c mice were randomly allocated into 4 groups and subcutaneously injected with saline, IL&aC (the mixture of IL-12 and aCTLA-4), RB (the mixture of RBC and BaO2), and IL/aC@RBAH once a week. During 2 months of continuous treatments, the mice treated with IL/aC@RBAH exhibited negligible body weight changes similar to those in the saline group (Supplementary Fig. 29a), indicating minimal systemic toxicity or metabolic perturbations induced by IL/aC@RBAH. Moreover, the blood and major organs were collected after 2 months of monitoring. The blood biochemical parameters of BUN (Supplementary Fig. 29b), CREA (Supplementary Fig. 29c), and ALT (Supplementary Fig. 29d) were maintained within the normal range in all the groups. Moreover, distinct from the abnormal AST level in the RB and IL&aC groups, that in the IL/aC@RBAH group was significantly lowered within safe bounds (Supplementary Fig. 29e). All the above results evidenced the normal hepatic and renal function in the IL/aC@RBAH-treated mice. Additionally, H&E analysis presented negligible histomorphological abnormalities of major organs in the IL/aC@RBAH group, in sharp contrast to moderate inflammation of the liver tissues in the IL&aC and RB groups (Supplementary Fig. 29f), probably triggered by the burst release of aCTLA-4/IL-12 and Ba2+. All the above studies confirmed the favorable long-term biosafety of IL/aC@RBAH with negligible risks of IL-12-related cytokine storm.

Besides, we conducted a comprehensive investigation into the impact of RT+IL/aC@RBAH on the in situ immunomodulatory TME. An established orthotopic 4T1 tumor model was employed to harvest tumors to scrutinize the immune cell composition within the TME. Remarkably, the treatment of RT+IL/aC@RBAH exhibited a notable elevation in the proportion of CD8+ T cells in company with the increased level of CD8+ IFN-γ+ T cells within the tumors in comparison to the treatments of RT+RBAH, RT+aC@RBAH, and RT+IL@RBAH (Fig. 5j, k and Supplementary Fig. 30). The results illuminated exceptional RT-associated antitumor immune responses triggered by the synergistic therapeutic strategy of aCTLA-4 and IL-12. In addition, the proportion of Th1 cells (CD4+ IFN-γ+ T cells) was increased, while that of Tregs was decreased in the RT+IL/aC@RBAH group (Fig. 5l, m and Supplementary Fig. 30), indicating effective comovements of aCTLA-4 and IL-12 by IL/aC@RBAH in combination with RT. Meanwhile, both the ratios of CD8+ T/Treg and Th1/Treg were significantly increased in the RT+IL/aC@RBAH group, which were approximately twofold higher than those in the RT+aC@RBAH and RT+IL@RBAH groups, and nearly threefold increase compared to those in the RT+RBAH group (Fig. 5n, o and Supplementary Fig. 30). Moreover, RT+IL/aC@RBAH was also verified to significantly enhance the proportion of NK cells and stimulate NK cells to produce IFN-γ (Fig. 5p, q and Supplementary Fig. 31). Furthermore, the cellular populations within TDLNs were investigated. Typically, RT+aC@RBAH triggered moderately upregulated infiltration of cDC1 and cDC2 in TDLNs, a better result distinct from the treatments of RT, RT+RBAH, and RT+IL@RBAH (Fig. 5r and Supplementary Fig. 32), demonstrating the enhanced DC homing efficacy of aCTLA-4. Notably, after exposure to RT+IL/aC@RBAH, both the levels of cDC1 and cDC2 in TDLNs were significantly elevated compared to those in the RT+IL@RBAH and RT+aC@RBAH groups. These findings collectively underscored the potent activation of antitumor immune responses and thus the robust therapeutic efficacy achieved by RT+IL/aC@RBAH-mediated RIT in 4T1 breast tumors.

Inspired by the above excellent antitumor immune responses within the TME triggered by RT with multiplex immunotherapy by aCTLA-4 and IL-12, we further exploited the underlying mechanism of IL/aC@RBAH-mediated RIT. Previous studies have reported that RT-induced ICD may promote antitumor immune responses by tumor antigen presentation. Nevertheless, owing to the concomitant upregulation of CTLA-4 and differential RT sensitivities among immune cells in the TME53,54, we speculated that the ratio of CD8+ T/Treg would be decreased in tumors due to the stronger radioresistance of Treg than CD8+ T cells (Fig. 6a). After exposure to RT, the proportion of CTLA-4 in CD3+ T cells was remarkably increased (Fig. 6b). As speculated, the ratio of CD8+ T/Treg within tumors was also significantly downregulated following single RT, accompanied by a discernible decrease in the overall number of CD8+ T cells and a slight decline in the quantity of Treg compared to those in the saline group (Fig. 6c, d). The increased CTLA-4 expression and decreased CD8+ T/Treg ratio would potentiate DC tolerance and prevent antigen presentation for constrained antitumor immune effects55. Due to the immunosuppressive TME in the initial phase after single RT, simple RIT is limited without blocking immune checkpoints first. Therefore, we introduced a stepwise immune network regulation strategy for sequentially promoting antigen presentation and activating immune cells to generate multistage immune effects. Initially, the rapid release of aCTLA-4 from IL/aC@RBAH might relieve RT-induced immunosuppressive TME and promote the migration of DCs to TDLNs to present tumor antigens for priming T cells. Subsequently, the dilatory release of IL-12 from IL/aC@RBAH would further promote the antitumor effector cell infiltration and elevate IFN-γ production to initiate fortified antitumor immunity.

To affirm the above speculated multistage immune mechanism of RT+IL/aC@RBAH, we comprehensively tested the kinetics of aCTLA-4 and IL-12 release, as well as the respective effects of their administration sequence on immune cell populations and function. IL-12 and aCTLA-4 were jointly entrapped in RBC to obtain IL&aC@RBAH as control. The release behaviors of IL-12 and aCTLA-4 from IL&aC@RBAH and IL/aC@RBAH in the mildly acidic environment (pH 6.5) were investigated. As expected, both IL-12 and aCTLA-4 in IL&aC@RBAH were released rapidly in a similar trend within 48 h (Supplementary Fig. 33), possibly due to the simple diffusion of the mixture of IL-12 and aCTLA-4 from the formed pores on RBC. Contrarily, IL/aC@RBAH presented a sequential release behavior with rapid release of approximately 40% aCTLA-4 from the oxygen-induced pores on the RBC membrane within 48 h and slow release of less than 20% IL-12 from the enlarged channels in the crosslinked network of the degrading hydrogel within 48 h. Collectively, all those results affirmed the sequential timing and kinetics of aCTLA-4 and IL-12 release from IL/aC@RBAH. To further unveil the impact of sequential aCTLA-4/IL-12 release on the multistage immune effects, we investigated the immune cell populations in orthotopic 4T1 tumor-bearing mice exposed to different treatments. Briefly, the orthotopic 4T1-tumor bearing mice were randomly divided into 5 groups and then respectively treated with saline, RT, RT+IL-aC (IL-12 prior to aCTLA-4), RT+IL&aC (the mixture of IL-12 and aCTLA-4), and RT+aC-IL (IL-12 subsequent to aCTLA-4). Compared to the slight upregulation of CD8+ T cells induced by RT-mediated ICD, the intratumoral level of those cells was moderately upregulated in the RT+IL-aC and RT+IL&aC groups (Fig. 6e), demonstrating the modest immune activation in the primary and relay stages. This limitation was likely attributed to the rapid diffusion and loss of IL-12 from tumors, which was insufficient to sustain downstream activation during the relay stage. Of note, the RT+aC-IL treatment elicited the utmost increase in CD8+ T cells, along with the lowest Treg ratio and the highest NK cell level among all the groups (Fig. 6f, g), indicating the strongest antitumor immune initiation by the sequential aCTLA-4/IL-12 supply. Moreover, the IFN-γ production in CD8+ T cells, CD4+ T cells, and NK cells in the RT+aC-IL group were all at the highest level among all the groups (Fig. 6h–j), further affirming the most potent immune responses through aCTLA-4-mediated primary immunosuppression alleviation and relay IL-12-induced immunoactivation. It is noteworthy that the RT+aC-IL treatment likewise elicited the uppermost level of mature DCs, which was almost 1.44- and 1.27-fold higher than that in the RT+IL-aC and RT+IL&aC groups (Fig. 6k). The result further elucidated the most powerful antigen-presenting initiation of the sequential aCTLA-4/IL-12 supply in the RT+aC-IL treatment for antitumor immunity through the DC cell-intrinsic IL-12/IFN-γ positive feedback loop. Given the intratumoral immune responses elicited by RT+aC-IL, the impacts of sequential cytokine supply on antigen presentation process were further investigated through the immune cell populations in the tumor-draining lymph nodes (TDLNs) of orthotopic 4T1-tumor bearing mice subjected to different treatments. The amounts of both cDC1 and cDC2 in TDLNs were maximally elevated in the RT+aC-IL group, quite different from those in the other groups (Fig. 6l, m). Meanwhile, both the levels of CD8+ T cell infiltration and activation were at the peak in the RT+aC-IL group (Fig. 6n and Supplementary Fig. 34), further demonstrating the supreme antitumor initiation by the sequential aCTLA-4/IL-12 supply. Overall, all the results firmly affirmed the virtues of systemic multistage immune responses induced by the sequential aCTLA-4/IL-12 release from RT+IL/aC@RBAH.

Building on the multistage immune response initiated by RT+IL/aC@RBAH, local injection of IL-12 and aCTLA-4 plus RT (RT+IL&aC) were further set as a control for comprehensive therapeutic efficacy evaluation. The orthotopic 4T1 tumor-bearing mice were randomly allocated into 6 groups and respectively treated with saline, RT, RT+RBAH, IL&aC, RT+IL&aC, and RT+IL/aC@RBAH. Compared to the slight tumor inhibition in the RT group, the tumor volumes in the RT+RBAH and IL&aC groups were moderately suppressed (Supplementary Fig. 35a–d), elucidating the inadequate antitumor efficacy of the sole Ba/O2- supplied radiosensitization by RBAH or the single antitumor immune responses induced by IL&aC. Comparatively, the RT+IL&aC treatment showed much more prominent tumor inhibition than the RT+RBAH and IL&aC groups, possibly ascribed to the combined therapeutic strategy of RT and immunotherapy. Notably, the mice in the RT+IL/aC@RBAH group presented the utmost tumor regression with a prolonged lifetime of at least 2 months in about 80% of the mice (Supplementary Fig. 35e), thus substantiating the supreme antitumor efficacy of RT+IL/aC@RBAH-induced multistage RIT. In addition, the immune cell populations in the RT+IL&aC and RT+IL/aC@RBAH groups were characterized to elucidate the immunological mechanisms underlying the superior antitumor efficacy of multistage RIT. In contrast to the modest increases in intratumoral CD8+ T cells and NK cells induced by RT+IL&aC, a marked accumulation of both effector populations was instigated by RT+IL/aC@RBAH (Supplementary Fig. 36a, b). Concordantly, Tregs were reduced to the lowest levels, approximately 0.46-fold relative to those in the RT+IL&aC group (Supplementary Fig. 36c), thereby sharpening the intratumoral effector-to-suppressor balance. These findings were aligned with hydrogel-enabled sequential release of aCTLA-4 followed by IL-12, producing early relief of checkpoint and Treg constraints and later reinforcement of immune cell-mediated antitumor activities. Furthermore, the migration of both cDC1 and cDC2 to TDLNs was highest in the RT+IL/aC@RBAH group, exceeding that in the RT+IL&aC group (Supplementary Fig. 36d, e), consistent with enhanced antigen transport and cross-priming, thus providing a mechanistic basis for the superior initiation of antitumor immunity conferred by the sequential release of aCTLA-4 and IL-12 from the hydrogel.

Tumor relapse is the major cause of cancer-related mortality56. Given 60% of tumor regression by RT+IL/aC@RBAH in the above in vivo antitumor results (Fig. 5a–e), we pondered whether the therapeutic strategy of RT+IL/aC@RBAH could prevent tumor relapse. Herein, orthotopic 4T1 tumor-bearing mice with complete tumor regression after receiving the RT+IL/aC@RBAH treatment were rechallenged with 4T1 tumor cells in the right mammary fat pad to simulate the relapse. Following the re-implantation, the tumor regrowth process was monitored until day 52 following primary tumor inoculation (Fig. 6o). Strikingly, RT+IL/aC@RBAH-treated mice exhibited superior tumor suppression with almost no tumor occurrence (Fig. 6p). However, discernible tumor growth in uncontrollable trends was monitored in the 4T1-implanted healthy mice in the control group. The result verified that the treatment of RT+IL/aC@RBAH instigated long-term immunity against tumor occurrence. Emerging studies have unveiled effector memory T (Tem) cells as the direct antigen-primed cells that migrated into peripheral tissues to initiate inflammatory responses or cytotoxic effects57. However, central memory T (Tcm) cells, produced by naive T cells after antigen activation, could not directly initiate cytotoxic antitumor effects until secondary stimulation triggered effective differentiation into Tem57. To further explore the mechanism of durable immune responses elicited by RT+IL/aC@RBAH, we distinguished the levels of memory T cells, comprising Tem and Tcm in CD4+ T cells and CD8+ T cells within spleens. Excitingly, the numbers of CD8+ Tem and CD4+ Tem cells were entirely increased by the treatment of RT+IL/aC@RBAH, along with the lower ratio of Tcm/Tem in both CD8+ T cells and CD4+ T cells compared to those in the control group (Fig. 6q and supplementary Fig. 37). Taken together, the RT+IL/aC@RBAH-mediated RIT strategy based on Ba/O2 self-compensated radiosensitization and sequential aCTLA-4/IL-12 release could effectively elicit the establishment of tumor-specific immune memory against tumor relapse. Although short-term antigenic or inflammatory exposure prompts immune cells to undergo memory differentiation, persistent stimulation drives them toward an exhausted phenotype. Immune cell exhaustion is characterized by diminished effector function and increased inhibitory receptor expression, which is associated with detrimental drug resistance58,59. Studies have shown that key cytokines driving immune cell exhaustion in vivo include IL-10, IFN-I, TGF-β, and IL-23, while IL-12 often acts to reverse or alleviate the exhaustion in the majority of settings60–63. Despite its generally restorative role in the immune recovery, sustained intratumoral IL-12 exposure at a high level from the hydrogel might paradoxically promote immune cell exhaustion, accompanied by heightened metabolic demand and increased susceptibility to activation-induced dysfunction. Nevertheless, the critical driving factor of exhaustion remains persistent antigen exposure64. By rapidly suppressing tumor growth and thereby markedly reducing the antigen exposure of intratumoral immune cells, RT+IL/aC@RBAH is expected to prevent immune cell exhaustion, thereby outweighing any potential IL-12-associated pro-exhaustion effect.

Immune checkpoint blockade (ICB) targeting PD-1/PD-L1, a pivotal approach to inhibiting immune evasion, has achieved durable clinical benefits and favorable antitumor efficacy when combined with RT65. Hence, the RT+IL/aC@RBAH treatment was compared with RT combined with conventional PD-1/PD-L1-targeting ICB therapy to evaluate its deeper antitumor potential. Distinct from the rapid tumor progression in the saline group, the tumor volumes in the RT+aPD-1 and RT + aPD-L1 groups were moderately suppressed (Supplementary Fig. 38a–c), indicating the deficient antitumor efficacy of RT combined with ICB therapy. Intriguingly, the RT+IL/aC@RBAH treatment showed much more prominent tumor inhibition than the RT+aPD-1 and RT+aPD-L1 groups, thus illustrating the superior antitumor efficacy of RT+IL/aC@RBAH-mediated multistage RIT over RT combined with ICB therapy. To further elucidate the underlying immune mechanisms, the immune effects of RT+IL/aC@RBAH were compared with those of RT combined with either aPD-1 or aPD-L1. Relative to the moderate changes in immune cell levels observed with RT+aPD-1 and RT+aPD-L1, RT+IL/aC@RBAH markedly increased CD8⁺ T cells and NK cells infiltration while reducing intratumoral Tregs, thereby enhancing the effector-to-suppressor balance in the TME (Supplementary Fig. 38d–f). Taken together, these results indicated that RT synergized with the sequential release of IL-12 and aCTLA-4 was superior over its combination with anti-PD-1/PD-L1 in priming and amplifying antitumor immunity and in reshaping the TME toward an effector-dominant state that favored immune-mediated antitumor efficacy.

In vivo antitumor mechanism of RT+IL/aC@RBAH in 4T1 tumor-bearing mice
To elucidate the underlying mechanism governing the modulation of antitumor immune responses by the treatment of RT+IL/aC@RBAH, we conducted a comprehensive analysis of the transcriptomic landscape post-treatment. Differential expression gene (DEGs) analysis, in conjunction with enrichment analysis, including Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, was employed to delineate the transcriptomic alterations within tumors from orthotopic 4T1 tumor-bearing mice following diverse therapeutic interventions. Compared with the control group, there were 1071 differentially expressed genes (DEGs) with 849 upregulated ones and 222 downregulated ones in the RT+IL/aC@RBAH group (Fig. 7a). In comparison to the components in the RT group, a discernible gene expression disparity was also signified in the tumors after exposure to RT+IL/aC@RBAH (Fig. 7b). Analysis of the Volcano plot revealed significant expression differences in 770 genes, comprising 557 upregulated genes and 213 downregulated genes after treatment with RT+IL/aC@RBAH. The group cluster heatmap also delineated that there were gene expression distinctions among the control, RT, and RT+IL/aC@RBAH groups (Fig. 7c). To further explore these transcriptional changes, the enrichment analysis was conducted to compare the control or RT group with the RT+IL/aC@RBAH group. The DEGs in the RT+IL/aC@RBAH group, as shown by the KEGG enrichment analysis, were enriched in antitumor immune response-associated pathways, including cytokine-cytokine receptor interaction, NOD-like receptor, TNF, MAPK, B cell receptor, NF-κB, natural killer cell-mediated cytotoxicity, and Toll-like receptor signaling pathway (Supplementary Fig. 39a). In particular, the KEGG pathway enrichment also revealed pronounced upregulation of immune response-related and inflammatory signaling pathways in the RT+IL/aC@RBAH group compared to the RT group, notably encompassing cytokine-cytokine receptor interaction, TNF, IL-17, NOD-like receptor, and chemokine signaling pathways (Fig. 7d, e). In addition, GO enrichment analysis manifested that the upregulated genes in the RT+IL/aC@RBAH group compared to control group were mainly involved in response to IFN-γ, B cell activation, lymphocyte-mediated immunity, T cell proliferation, cell activation involved in immune response, and lymphocyte proliferation (Supplementary Fig. 39b). Consistently, the pronounced enrichment of related genes in immune-related signaling pathways under the RT+IL/aC@RBCH treatment in the contrast to the RT group (Fig. 7f). Notably, the genes, such as Il21, Cxcl2, Il1a, Ccl3, Il1b, Il1f9, Il6, Tnf, and Il12a, were significantly upregulated in the RT+IL/aC@RBAH group, which were in accordance with the activation of leukocyte migration, leukocyte cell-cell adhesion, cell chemotaxis, cytokine-mediated signaling pathway, and T cell activation. Furthermore, in comparison with control group, the multistage RIT initiated by RT+IL/aC@RBAH could activate antitumor immune effect-related signaling pathways, such as C-type lectin receptor signaling pathway, natural killer cell-mediated cytotoxicity, TNF signaling pathway, and NOD-like receptor signaling pathway (Supplementary Fig. 40), as evidenced by the Gene Set Enrichment Analysis (GESA). Notably, as further presented by the GSEA, quite different from the RT group, RT+IL/aC@RBAH could collectively upregulate immune response-related pathways, including Toll-like receptor signaling pathway (Fig. 7g), NOD-like receptor signaling pathway (Fig. 7h), IL-17 signaling pathway (Fig. 7i), and chemokine signaling pathway (Fig. 7j). The upregulation of these immune-related pathways delineated above was attributed to the Ba/O2-supplied radiosensitization and multistage immune activation by sequential aCTLA-4/IL-12 release. In summary, the presented transcriptomic profiles provided reliable evidence that the synergistic RIT strategy based on sequential aCTLA-4/IL-12 release and Ba/O2-aided radiosensitization facilitated the potentiation of multistage immune activation and thus induction of tumor eradication.

In vivo antitumor efficacy and multistage immune responses in Panc02 tumor-bearing mice
To validate the broad applicability of the treatment of RT+IL/aC@RBAH, a highly aggressive subcutaneous Panc02 tumor model was established to assess the potential of RT+IL/aC@RBAH-mediated multistage RIT. When the tumor volume reached approximately 100 mm3, C57BL/6 mice bearing Panc02-luciferase (Panc02-luc) tumors were randomly divided into six groups and the treatments were commenced as delineated above. Different from the cases of saline, RT, and RT+RBAH, the treatments of RT+aC@RBAH and RT+IL@RBAH could respectively lead to considerably deferred tumor growth (Fig. 8a–d). Remarkably, RT+IL/aC@RBAH resulted in a superior therapeutic outcome with substantial tumor suppression compared to other treatments (Fig. 8a–d), possibly induced by locally multistage immune activation via sequential release of aCTLA-4 and IL-12. Owing to the excellent tumor inhibition efficacy, the treatment of RT+IL/aC@RBAH generally resulted in a discernibly prolonged lifespan of mice (Fig. 8e). Body weights of all the treated mice were recorded every 4 days and no obvious weight changes were observed during the surveillance period (Supplementary Fig. 41). Moreover, a histopathological examination of tumor specimens was conducted to determine the in vivo antitumor efficacy of RT+IL/aC@RBAH by H&E staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) immunofluorescence. Both of the staining results revealed predominant tumor tissue apoptosis and necrosis with the most extensive damage of tumor cells in the RT+IL/aC@RBAH group (Fig. 8f–i and supplementary Fig. 42). To reveal the underlying mechanism of the therapeutic efficacy, the immunophenotyping of tumors from mice subjected to different treatments was examined. As expected, after the synergistic RIT by RT+IL/aC@RBAH, the numbers of NK and CD8+ T cells were increased, accompanied by decreased Tregs compared with those in the other groups (Fig. 8j–l and supplementary Fig. 43). The results collectively demonstrated efficient tumor immunomodulation by Ba/O2-mediated radiosensitization and multistage immune responses. Moreover, compared to the other groups, the treatment of RT+IL/aC@RBAH significantly increased the IFN-γ levels in CD4+ T, CD8+ T, and NK cells (Fig. 8m–o and supplementary Fig. 43). Overall, all these findings comprehensively affirmed excellent antitumor immune responses and robust treatment efficacy by the synergistic therapeutic strategy of RT+IL/aC@RBAH-induced multistage RIT in pancreatic tumors.

Furthermore, antitumor immune cell infiltration within tumors was also visualized by immunofluorescence. Evidently, the treatment of RT+aC@RBAH and RT+IL@RBAH could temperately relieve RT-induced tumor immunosuppression by increasing CD4+ T and CD8+ T cell infiltration (Fig. 9a–c). Intriguingly, both above antitumor T cells were uppermost elevated in the tumors subjected to the therapeutic modality of RT+IL/aC@RBAH (Fig. 9a–c), emphasizing amplified antitumor immunity through the remedy of Ba/O2-enhanced RT with sequential aCTLA-4/IL-12 release. Similarly, the impact of RT+IL/aC@RBAH on the immune cellular compositions of TDLNs and spleens in Panc02 tumor-bearing mice was decoded to determine whether systemic antitumor responses were induced. Resembled with the case of 4T1 breast tumor-bearing mice, cDC1 and cDC2 cells in TDLNs were respectively sorted to investigate the capacities of RT+IL/aC@RBAH to induce antigen presentation. As predicted, the RT+IL/aC@RBAH group exhibited a significant increase in the numbers of cDC1 and cDC2 cells in TDLNs compared with the other groups (Supplementary Figs. 44 and 45), demonstrating that systemic antitumor efficacy was remarkably enhanced by the tactics of aCTLA-4-alleviated immunosuppression and IL-12-induced immunoactivation. Moreover, similar to the results of the previous antitumor immunity in subcutaneous Panc02 tumors, the RT+IL/aC@RBAH treatment exhibited significantly elevated IFN-γ secretion by CD4+ T, CD8+ T, and NK cells in spleens (Fig. 9d–i and supplementary Fig. 46). The levels of IFN-γ secretion in CD8+ T cells were approximately 1.5-fold higher than those in the RT+IL@RBAH and RT+aC@RBAH groups, and increased even more than 2.6 times compared to the saline group (Fig. 9f, g and supplementary Fig. 46). All the findings collectively corroborated that RT+IL/aC@RBAH elicited remarkable systemic immune responses and robust antitumor efficacy for prompt regression of multiple tumors.

Methods
Chemicals and reagents
Polyvinylpyrrolidone, choline hydroxide solution, glutathione, adenosine triphosphate, creatinine, 7-Amino-4-methyl-3-coumarinylacetic acid, fluorescein isothiocyanate, and sodium pyruvate were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Sodium alginate and barium chloride were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) was purchased from Sigma-Aldrich. DSPE-PEG2000-SH was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. Mouse IFN-γ (Interferon Gamma) ELISA kit (Cat# E-EL-M0048) was purchased from Elabscience. Catalase assay kit (Cat# S0051), calreticulin rabbit monoclonal antibody (Cat# AF1666, Clone N/A, 1:100), HMGB1 rabbit polyclonal antibody (Cat# AF0180, Clone N/A, 1:100), ATP assay kit (Cat# S0026), and Reactive oxygen species assay kit (Cat# S0033S) were purchased from Beyotime Biotechnology. Horseradish peroxidase (HRP) (Cat# P8020), BCA protein assay kit (Cat# PC0020), live/dead cell double stain kit (Cat# CA1630), and red blood cell lysis buffer (Cat #R1010) were purchased from Beijing Solarbio Science & Technology Co., Ltd. Recombinant Mouse IL-12 (Cat# CM39) was purchased from Novoprotein. InVivoMAb anti-mouse CTLA-4 (CD152) antibody (Cat# BE0131, BioX Cell) was purchased from BioXcell. The aCTLA-4 ELISA detection kit (Cat# HS1392-Mu) was purchased from Shanghai Hengyuan Biotechnology Company. Purified Rat anti-CD16/CD32 Antibody (Cat# 553141, Clone 2.4G2, 1:1000), fixable viability stain 510 (Cat# 564406), and BV421 anti-CD8α Antibody (Cat# 563898, Clone 53-6.7, 1:100) were purchased from BD Bioscience. FITC anti-CD45 Antibody (Cat# 103108, Clone 30-F11, 1:200), APC-cy7 anti-CD4 Antibody (Cat# 100526, Clone RM4-5, 1:20), APC anti-CD11c Antibody (Cat# 117310, Clone N418, 1:80), PE-cy7 anti-CD25 Antibody (Cat# 102016, Clone PC61, 1:20) PE anti-IFN-γ Antibody (Cat# 505807, Clone XMG1.2, 1:80), and APC anti-CD49b Antibody (Cat# 108909, Clone DX5, 1:80) were purchased from BioLegend. PE-EF610 anti-CD44 Antibody (Cat# 61-0441-82, Clone IM7, 1:20), SB600 anti-CD62L Antibody (Cat# 63-0621-80, Clone MEL-14, 1:150), FITC anti-CD11b Antibody (Cat# 11-0112-81, Clone M1/70, 1:100), PE anti-MHC Ⅱ Antibody (Cat# 12-5321-82, Clone M5/114.15.2, 1:1000), and EF450 anti-CD103 Antibody (Cat# 48-1031-82, Clone 2E7, 1:20) were purchased from Ebioscience.

Material characterizations
The morphologies of samples were observed by transmission electron microscopy (TEM, JEM-2100F, Japan) and scanning electron microscopy (SEM, JSM-7900F, JEOL, Japan). The element mapping (Ba, O) of BaO2 nanoparticles was characterized by energy-dispersive spectroscopy (EDS, FEI Talos F200X G2). X-ray diffraction (XRD) patterns of synthesized BaO2 nanoparticles and standard pattern of BaO2 were tested by X-ray diffractometer (X’ Pert Pro MPD, PANalytical B.V., Netherlands). The presence and valence state of the elements (Ba and O) in BaO2 nanoparticles were analyzed by X-ray photospectrometer (XPS, EscaLab 250Xi, Thermo, USA).

Cells and animals
4T1 breast cancer cell line (purchased from the American Type Culture Collection, ATCC, Cat# CRL-2539) and Panc02 pancreatic ductal adenocarcinoma cell line (purchased from Procell, Cat# CL-0736) were respectively cultivated in RPMI 1640 medium (Gibico) supplemented with 10% FBS (Bioind) and DMEM (Gibco) supplemented with 10% FBS (Bioind) at 37 °C in a humidified atmosphere with 5% CO2.
Female BALB/c and male C57BL/6 mice were purchased from Yangzhou University (Yangzhou, China) and were 6–8 weeks old at the beginning of each experiment. All the mice were fed in the pathogen-free facility under a 12 h light/dark cycle at 20–26 °C and 40–70% humidity, with free access to food and water.

Ethics statement
All animal studies were performed under the guidelines for the care and use of laboratory animals in research approved by China Pharmaceutical University (SYXK(Su) 2023-0018) and in accordance with all relevant ethical regulations. According to the institutional ethical regulations, subcutaneous tumors must not exceed a maximal volume of 1500 mm³. We confirm that no experimental animals exceeded this limit during the study.

Preparation of IL-12-AMCA-DSPE and IL-12-Cp-DSPE
To visualize the IL-12 anchorage on RBC in vitro and in situ retention of IL/aC@RBAH in vivo, IL-12 was respectively labeled with AMCA and Cypate. Briefly, AMCA (2.00 mg, 8.58 μmol) or Cypate (5.32 mg, 8.58 μmol) was dissolved in DMSO (100 μL). Then, NHS (1.19 mg, 10.30 μmol) and EDCI (1.97 mg, 10.30 μmol) were added and reacted at room temperature for 2 h to afford activated AMCA or Cypate. Subsequently, 5 mg of IL-12 was added into carbonate buffer solution at pH 9.2 (250 μL) and mixed with 25 μL of activated AMCA/Cypate. After stirring in dark overnight at 4 °C, the as-prepared IL-12-AMCA and IL-12-Cp were respectively ultrafiltered (3.5 kD molecular weight cut off (MWCO)), Millipore Sigma) to clear away the free AMCA/Cypate. Subsequently, IL-12-AMCA or IL-12-Cp was mixed with sulfo-SMCC (33 μg, 0.075 μmol) in PBS for 2 h, and further ultrafiltered (3.5 kD MWCO, Millipore Sigma) to afford Mal-IL-12-AMCA or Mal-IL-12-Cp.
5 mg of DSPE-PEG2000-SH was dissolved in DMSO (50 μL) and Mal-IL-12-AMCA or Mal-IL-12-Cp was dissolved in PBS (500 μL). 1.8 μL of DSPE-PEG2000-SH was subsequently added into Mal-IL-12-AMCA or Mal-IL-12-Cp solution and stirred at room temperature for 2 h. Finally, the resultant DSPE-PEG2000-IL-12-AMCA or DSPE-PEG2000-IL-12-Cp product was concentrated via ultrafiltration (3.5 kD MWCO, Millipore Sigma) and stored at 4 °C for use.

Preparation of aCTLA-4-FITC
100 μg of aCTLA-4 was dissolved in Na2CO3-NaHCO3 buffer at pH 9.0 (0.1 M, 100 μL). After 10 min of stirring at 4 °C, 1.03 μg of FITC was added and further stirred at 4 °C for 18 h. Finally, NH4Cl (5 M, 1 μL) was added to cease the reaction. Free FITC was removed by ultrafiltration (3.0 kD molecular weight cutoff (MWCO), Millipore Sigma) with the endpoint as determined by the visual clarity of the filtrate.

Preparation of IL/aC@RBC
aC@RBC was prepared via an optimized “alternate hypotonic and hypertonic dialysis” approach as reported71. Briefly, the whole blood was collected from healthy BALB/c mice and mixed with PBS buffer with 5% EDTA. The obtained RBC was isolated by centrifugation (800×g, 4 °C, 5 min) three times and resuspended in PBS buffer at 70% hematocrit. Then 500 μL of RBC suspension and 85 μL of FITC labeled aCTLA-4 (aCTLA-4-FITC, 5 mg/mL) were mixed and dialyzed (3.5 kD MWCO) against 40 mL of hypotonic buffer (deionized water containing 10 mM NaHCO3, 10 mM Na2HPO4, 20 mM glucose, 2 mM ATP, and 3 mM reduced glutathione (GSH)) at 4 °C for 90 min. Subsequently, the buffer was replaced with 45 mL of PBS buffer for 5 min of balance and then dialyzed with 50 mL of hypertonic buffer containing 10% PIGPA-NaCl buffer (deionized water containing 100 mM sodium pyruvate, 100 mM inosine, 100 mM glucose, 33 mM NaH2PO4, 12% (w/v) NaCl, and 2 mM ATP, pH 7.4) at 37 °C for 30 min. The resealed aC@RBC was collected by centrifugation (1000×g, 4 °C, 10 min) and washed 3 times with PBS buffer to remove unentrapped aCTLA-4.
For efficient IL-12 anchorage and aCTLA-4 encapsulation in RBC for IL/aC@RBC, the loading capacities were investigated by varying the feeding time points of aCTLA-4.
IL/aC@RBC-1 was synthesized as follows: 500 μL of RBC suspension (4×109/mL), 85 μL of aCTLA-4-FITC (5 mg/mL), and 100 μL of DSPE-PEG2000-IL-12-AMCA (5 mg/mL) were mixed, and then dialyzed (3.5 kD MWCO) against 40 mL of hypotonic buffer. Subsequently, the buffer was replaced with 35 mL of PBS buffer for balance and then dialyzed with 50 mL of hypertonic buffer for further 30 min. The resealed IL/aC@RBC was collected by centrifugation (1000×g, 4 °C, 10 min) and washed 3 times with PBS buffer to remove unentrapped aCTLA-4-FITC and DSPE-PEG2000-IL-12.
IL/aC@RBC-2 was synthesized as follows: 500 μL of RBC suspension (4×109/mL) was mixed with 85 μL of aCTLA-4-FITC (5 mg/mL) and then dialyzed (3.5 kD MWCO) against 40 mL of hypotonic buffer. Subsequently, the buffer was substituted with 35 mL of PBS buffer for balance. The resultant aC@RBC was added with 100 μL of DSPE-PEG2000-IL-12-AMCA (5 mg/mL) and dialyzed by 50 mL of hypertonic buffer. The resealed IL/aC@RBC was collected by centrifugation (1000×g, 4 °C, 10 min) and washed 3 times with PBS buffer to remove unentrapped aCTLA-4-FITC and DSPE-PEG2000-IL-12.
IL/aC@RBC-3 was synthesized as follows: 500 μL of RBC suspension (4×109/mL) and 85 μL of aCTLA-4-FITC (5 mg/mL) were mixed and then dialyzed (3.5 kD MWCO) against 40 mL of hypotonic buffer. Subsequently, the buffer was substituted with 35 mL of PBS buffer for balance and then dialyzed by 40 mL of hypertonic buffer. The resealed aC@RBC was collected by centrifugation (1000×g, 4 °C, 10 min) and washed 3 times with PBS buffer to remove unentrapped aCTLA-4-FITC. Then the resultant aC@RBC was added with 100 μL of IL-12-AMCA-DSPE (5 mg/mL) and incubated in PBS for IL-12 intercalation at 37 °C. The engineered IL/aC@RBC was collected by centrifugation (1000×g, 4 °C, 10 min) and washed 3 times with PBS buffer to remove unentrapped DSPE-PEG2000-IL-12.

In vitro bioactivity assessment of Catalase (CAT) and IL-12
For CAT bioactivity evaluation, native RBC (nRBC) and RBC membrane (RBCM) were extracted as previous studies. Briefly, the whole blood was harvested from healthy BALB/c mice, collected in a tube, and then centrifuged (800×g, 4 °C, 5 min) to discard plasma, leukocytes, and platelets in the upper layers. The precipitates were resuspended and washed three times by pre-cooled PBS buffer for nRBC. The resultant nRBC was diluted 40 times by pre-cooled lysate (0.01 M Tris-HCl, pH 8.0) containing 0.02 mM PMSF and stood at 4 °C for 2 h. Subsequently, RBCM was obtained by centrifugation (10,000×g, 4 °C, 30 min), washed three times, and stored at −80 °C for further use. RBC and IL/aC@RBC were prepared via the optimized “alternate hypotonic and hypertonic dialysis” approach as introduced in “preparation of IL/aC@RBC”. Finally, the catalase bioactivities of nRBC, RBC, RBCM, and IL/aC@RBC were respectively assessed by Catalase Assay Kit (Beyotime, Shanghai, China), and normalized by the whole protein in the samples. In addition, the O2 generation from IL/aC@RBC and native RBC (nRBC) were quantitatively investigated to affirm CAT reservation on IL/aC@RBC. Briefly, 109 cells of IL/aC@RBC and nRBC were respectively dispersed in 5 mL of PBS buffer containing 500 μM H2O2. Then, the changes in O2 concentration were determined in 2 min by a dissolved oxygen analyzer.
For IL-12 bioactivity assessment, splenocytes were extracted for the evaluation of IFN-γ production stimulated by free IL-12 and IL@RBC. Briefly, spleens were collected from healthy BALB/c mice. The splenocytes were extracted and processed to remove RBC by lysis buffer (Beyotime, Shanghai, China). The obtained cells were seeded in a 96-well plate and subjected to free IL-12 or IL@RBC at pre-designated concentrations at 37 °C for 48 h. The culture supernatants were respectively collected for IFN-γ evaluation by ELISA (Mouse IFN-γ ELISA, Elabscience), and normalized by the whole protein in the samples.

Preparation of BaO2
Polyvinyl pyrrolidone (PVP, 0.35 g, 6.03 μmol) was ultrasonically dissolved in 15 mL of methanol, followed by the addition of BaCl2 (0.1 g, 0.48 mmol) and further stirring for complete dissolution. Then 20 μL of choline hydroxide (44% wt) was added to the solution. After 30 min of reaction, the mixture was dripped with 6 mL of 3% H2O2 at a constant rate of 0.05 mL/min and then stirred at room temperature for 1 h. The resultant BaO2 products were collected by centrifugation (10,000×g, 4 °C, 15 min). After discarding the supernatant, the precipitates were resuspended by 15 mL of methanol and washed three times to afford white precipitates for further use.

Determination of Ba2+ and H2O2 generation from BaO2
In vitro release of Ba2+ from BaO2 was measured using the dialysis bag diffusion method. Typically, 1 mg of BaO2 nanoparticles in 1 mL of PBS (pH 7.4) were respectively dialyzed (1 kD MWCO) against 19 mL of PBS at pH 7.4 and pH 6.5. The dialysis was performed at 37 °C with a shaking speed of 100 rpm. At pre-designed time points of 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h, 1 mL of dialysate was collected and replaced with 1 mL of fresh medium. The obtained samples were diluted with 3% HNO3 and the Ba2+ concentration was quantified by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS, iCAP RQplus, Thermo Scientific, USA).
To evaluate the acidity-induced generation of H2O2, 100 μL of BaO2 nanoparticles (0.1 mg/mL) was incubated with horseradish peroxidase (HRP) in 1.5 mL of PBS at pH 7.4 and pH 6.5, respectively. At pre-designed time points of 0, 4, 10, 20, and 30 min, 200 μL of tetramethylbenzidine (TMB) was added for 2–3 min of incubation. The absorption at 652 nm was measured by UV–vis spectrophotometer (3J1-0015, HITACHI, Japan) and H2O2 production was calculated according to the established standard curve of commercial H2O2 (0.05-1.0 mM).

Rheological test
Rheological characterizations of IL/aC@RBAH were observed using a rheometer (Discovery HR-2-TA, TA Instrument, American) with a parallel-plate geometry of 12 mm diameter and 0.6 mm gap at 25 °C. The changes in storage modulus and loss modulus of IL/aC@RBAH were monitored at a constant strain of 2.5% and a fixed frequency of 1 Hz for 20 min. Strain amplitude sweeps were conducted from (1% to 1000%) at a fixed frequency of 1 Hz. Dynamic oscillatory frequency sweeps were performed from 1 to 32 rad/s at a constant strain of 2.5% to estimate the elastic and viscous properties of IL/aC@RBAH.

In vitro degradation determination of IL/aC@RBAH hydrogel
The degradation profile of IL/aC@RBAH was investigated relying on the mass loss during the degradation process. Typically, BaO2 (0.1 mg), 3% Alg (100 μL), and IL/aC@RBC (2×108 cells in 100 μL) were mixed and incubated in PBS at pH 6.5 for IL/aC@RBAH formation. Afterward, the hydrogel was washed by deionized water three times and then dried under vacuum at −40 °C for 4–6 h. The initial mass of lyophilized IL/aC@RBAH was indicated as W0. Subsequently, the lyophilized IL/aC@RBAH was swollen in PBS at pH 6.5 and incubated at 37 °C. At designated time points on the 0.25, 0.5, 1.0, 1.5, 2, 4, 6, 8, 10, and 14 days, the hydrogel was collected, dried, and weighed (Wd). The degradation percentage (D%) of IL/aC@RBAH was calculated according to the formula as follows:
D(%) = (W0 - Wd) / W0 × 100%

Release of IL-12, aCTLA-4 and Ba2+ from IL/aC@RBAH
IL-12 and aCTLA-4 in IL/aC@RBAH were respectively labeled with AMCA and FITC to obtain IL-12-AMCA and aCTLA-4-FITC. The release behaviors of IL-12 and aCTLA-4 were examined according to the previous report72. A 40 µm cell strainer containing IL/aC@RBAH was embedded in a six-well plate with 5 mL of PBS at pH 6.5 and pH 7.4. After 0, 1, 2, 4, 6, 8, 16, 24, and 48 h of incubation, the concentrations of IL-12-AMCA and aCTLA-4-FITC in PBS were respectively detected by a microplate reader (Molecular Devices, USA) at the absorption wavelength of 350 nm and 488 nm.
In vitro release of Ba2+ from IL/aC@RBAH was investigated via the dialysis method. Briefly, IL/aC@RBAH formed in PBS (pH 6.5) was washed with deionized water three times, and then 1 mL of the sample was dialyzed (3 kD MWCO) against 19 mL of PBS (pH 6.5) at 37 °C in a shaking culture incubator. After 0, 1, 2, 4, 6, 8, 16, 24, and 48 h of incubation, 1 mL of dialysate was taken and replenished with an equal volume of fresh buffer. The released Ba2+ amount was determined by ICP-MS (iCAP RQplus, Thermo Scientific, USA).

In vitro biosafety evaluation
For the cytotoxicity analysis of BaO2, 4×103 4T1 cells and Panc02 cells were plated in each well of 96-well plates and incubated at 37 °C for 24 h. Different concentrations of BaO2 (0, 20, 40, 60, 80, 100, and 150 μg/mL) were added for 24 h of incubation. Afterward, MTT solution (0.5 mg/mL) was added and cocultured at 37 °C for 4 h. Then medium containing MTT was removed and replaced with 150 μL of DMSO in each well. After 30 min of constant temperature shaking at 37 °C, the optical density (OD) at 490 nm was detected. The experiment was repeated independently 5 times and the cell viability was calculated according to the formula: cell viability (%) = ODsample / ODcontrol × 100%.
For the biocompatibility evaluation of IL/aC@RBAH, hydrogel leaching solution was collected and diluted into different concentrations (0, 6.25%, 12.5%, 25%, 50%, and 100%). 4T1 cells and Panc02 cells were seeded in 96-well plates and incubated at 37 °C. After 24 h of culture, hydrogel leaching solutions at various concentrations were added and cocultured for 24 h. Subsequently, MTT solution (0.5 mg/mL) was added and incubated at 37 °C for 4 h. Then medium containing MTT was removed and replaced with 150 μL of DMSO in each well. After 30 min of constant temperature shaking at 37 °C, the OD at 490 nm was detected. The experiment was repeated independently 5 times for statistical analysis.
IL/aC@RBAH cytotoxicity was further investigated by live/dead cell staining assay. Briefly, sterile IL/aC@RBAH was placed in 48-well plates and 1×104 4T1 cells were seeded in the wells containing hydrogels, followed by 24 h of incubation at 37 °C. Subsequently, the cells were stained with calcein-AM (1 μg/mL) and PI (1 μg/mL) for confocal laser scanning microscopy (CLSM, Olympus Corporation, Japan) observation.

Colony formation assay
The cell viability exposed to different treatments was investigated via colony formation assay. Typically, one hundred cells were seeded in culture dishes (60 mm in diameter) and incubated at 37 °C for 24 h. Subsequently, cells were respectively subjected to saline, RT, RT+BaO2, RT+IL/aC@RBC, and RT+IL/aC@RB. The X-ray irradiation intensity was 1 Gy. Then cells were incubated for 14 days and replaced with fresh culture medium every 2–3 days. After colony formation in each dish, the cells were washed with PBS several times and fixed by 4% polyformaldehyde for 30 min at room temperature. Finally, colonies were dyed with 0.1% crystal violet for 30 min and washed by PBS for visual observation. The colonies were counted and calculated for survival fraction analysis.

Detection of intracellular ROS production
The ROS production inside cells was investigated by Reactive Oxygen Species Assay Kit (DCFH-DA, ROS fluorescence probe). 4T1 cells were cultured in confocal dishes and 12-well plates for 24 h at 37 °C and then added with DCFH-DA solution (10 μM). Subsequently, cells were treated with saline, RT, RT+BaO2, RT+IL/aC@RBC, and RT+IL/aC@RB, respectively cells and then incubated with DCFH-DA solution (10 μM) for 20 min at 37 °C. Afterward, the cells in 12-well plates were trypsinized and collected for flow cytometry. Meanwhile, the cells in confocal dishes were washed and stained with DAPI for observation by CLSM (Olympus Corporation, Japan).

In vivo degradation tests and in situ retention tests
In vivo degradation tests: The mixture of Ba2+, IL/aC@RBC, and Alg was injected into the right thorax of female BALB/c mice (6–8 weeks old, n=3 mice per group). On days 7 and 14 after in situ administration, a small incision of about 1 cm was created on the injected site, and the residual hydrogel was photographed.
In situ retention tests: For real-time monitoring of in situ retention of IL/aC@RBAH in female BALB/c mice (6–8 weeks old, n=3 mice per group), the IL-12 was pre-labeled by Cypate for IL-Cp&aC, IL-Cp/aC@RBC, and IL-Cp/aC@RBAH. At 1, 2, 3, 5, 7, 9, 11, and 14 days after subcutaneous injection and intratumoral injection, the fluorescence intensities were monitored by the IVIS Spectrum In Vivo Imaging System (IVIS Lumina XR, USA).

In vivo safety analysis
For cytokine analysis of serum, the orthotopic 4T1 tumor-bearing mice were intratumorally injected with saline, IL-12, aCTLA-4, and IL/aC@RBAH (n=3 mice per group), respectively. After 4 h, the blood samples were collected and centrifuged (800×g, 4 °C, 10 min) to remove cells. The concentrations of IL-12 and aCTLA-4 in the serum samples were determined by IL-12 and aCTLA-4 ELISA kit, and measured by microplate reader (Molecular Devices, USA).
For blood biochemical analysis, the orthotopic 4T1 tumor-bearing mice were intratumorally injected with saline, IL/aC, RBAH, and IL/aC@RBAH (n=3 mice per group), respectively. After 3 days, the blood samples were acquired for quiescence at 4 °C overnight and further centrifugation (800×g, 4 °C, 10 min) to remove cells. The serum samples were collected and sent to Servicebio Biotechnology Co., Ltd for analysis of liver function (Aspartate aminotransferase, ALT; Alanine aminotransferase, AST) and renal function (Creatinine, CREA; Blood urea nitrogen, BUN). The major organs (heart, liver, spleen, lung, and kidney) from the mice subjected to different treatments were collected for H&E staining analysis.
For long-term biosafety analysis, healthy BALB/c mice were randomly allocated into 4 groups (n=3 mice per group) and subcutaneously injected with saline, IL&aC (the mixture of IL-12 and aCTLA-4), RB (the mixture of RBC and BaO2), and IL/aC@RBAH once a week. During 2 months of continuous treatments, the body weights of the mice were recorded every 7 days. After 2 months of monitoring, the blood and major organs were collected. Liver damage indexes involving alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as kidney function indexes compromising blood urea nitrogen (BUN) and creatinine (CREA), were respectively detected. In addition, the major organs, including heart, liver, spleen, lung, and kidney, were collected for H&E analysis.

Evaluation of intratumoral oxygen contents
The female BALB/c mice (6–8 weeks old, n=3 mice per group) were orthotopically inoculated with 4T1 cells (1×106 cells per mouse) to establish a 4T1 mouse breast tumor model. When the tumor volume reached approximately 100 mm3, the mice were subjected to treatments of IL/aC@RBC or IL/aC@RB. For quantification of intratumoral vascular oxygen saturation levels, the ratios of deoxygenated hemoglobin (λ=750 nm) versus oxygenated hemoglobin (λ=850 nm) were recorded, and the 2D photoacoustic (PA) images were monitored by PA imaging system (Vevo® LAZR, Fujifilm VisualSonics, Canada). In addition, for oxygen production sustainability evaluation, the orthotopic 4T1 tumor-bearing mice were respectively treated with IL/aC@RB and IL/aC@RBAH. At 0, 24, and 48 h post-treatment, the tumors were photographed by PA imaging system (Vevo® LAZR, Fujifilm VisualSonics, Canada) and the intratumoral vascular oxygen saturation levels were recorded.
For further evaluation of the intratumoral hypoxia relief, the orthotopic 4T1 tumor-bearing female BALB/c mice (6–8 weeks old, n=3 mice per group) were respectively treated with saline, BaO2, IL/aC@RBC, IL/aC@RB, and IL/aC@RBAH. After 24 h, animals were sacrificed, and the tumors were collected. Subsequently, these tissues were successively frozen, sectioned, air-dried, fixed in acetone, and blocked. Immunofluorescence staining of HIF-1α and CD31 was conducted and then observed by CLSM (Olympus Corporation, Japan).

In vivo antitumor study on 4T1 tumor models and Panc02 tumor models
For orthotopic 4T1 tumor therapy, 1×106 4T1 expressing luciferase (4T1-luc) cells were injected into the left mammary fat pad of female BALB/c mice (6–8 weeks old, n=5 mice per group). After the tumor volume reached 100 mm3, the mice were randomly divided into 6 groups (5 mice per group) and respectively treated with saline (G1), RT (G2), RT+RBAH (G3), RT+aC@RBAH (G4), RT+IL@RBAH (G5), and RT+IL/aC@RBAH (G6) (an equivalent aCTLA-4 amount of 500 μg/kg, IL-12 amount of 250 μg/kg, BaO2 amount of 150 μg/kg, and Alg amount of 3%). The X-ray irradiation treatment (8 Gy) in the pre-designed groups was imposed twice at the tumor sites after 0 and 2 days of in situ administration. On the 7, 14, and 21 days after the tumor inoculation, the tumor growth was monitored by the IVIS Spectrum In Vivo Imaging System (IVIS Lumina XR, USA). The tumor sizes and body weight were recorded every 2 days. The survival of mice was monitored over a 2-month period. The tumor volume (V) was calculated following the formula V=0.5×W2×L, where L represents the long diameter of the tumor and W represents the width of the tumor. The tumors from the mice were collected for histological analysis, including H&E staining and Ki67 staining.
For evaluating the antitumor efficacy of RT+IL/aC@RBAH-induced multistage RIT, an orthotopic 4T1 tumor model was established. After the tumor volume reached 100 mm3, the female BALB/c mice were randomly divided into 6 groups (6–8 weeks old, n=5 mice per group) and respectively treated with saline, RT, RT+RBAH, IL&aC, RT+IL&aC, and RT+IL/aC@RBAH (an equivalent aCTLA-4 amount of 500 μg/kg, IL-12 amount of 250 μg/kg, BaO2 amount of 150 μg/kg, and Alg amount of 3%). The X-ray irradiation treatment (8 Gy) in the pre-designed groups was imposed twice at the tumor sites after 0 and 2 days of in situ administration. On the 7, 14, and 21 days after the tumor inoculation, the tumor growth was monitored by the IVIS Spectrum In Vivo Imaging System. The tumor sizes and survival time were also recorded. The tumor volume (V) was calculated following the formula V=0.5×W2×L.
For orthotopic 4T1 tumor relapse, 1×106 4T1 cells were injected into the left mammary fat pad of female BALB/c mice (6–8 weeks old, n=5 mice per group). After 10 days, the mice were treated with RT+IL/aC@RBAH. Following 2 more days, the mice were re-treated with RT. On day 26, the mice presenting CR after RT+IL/aC@RBAH treatment were rechallenged with 1×106 4T1 cells into the right mammary fat pad. Meanwhile, healthy mice were orthotopically injected with 1×106 4T1 cells into the right flank of mice as the control. The tumor growth was tracked every 2 days and calculated following the formula V=0.5×W2×L.
For comparing the antitumor efficacy between RT+aPD-1/aPD-L1 and RT+IL/aC@RBAH, an orthotopic 4T1 tumor model was established in female BALB/c mice. After the tumor volume reached 100 mm3, the mice were randomly divided into 4 groups (6–8 weeks old, n=5 mice per group) and respectively treated with saline, RT+aPD-1 (10 mg/kg), RT+aPD-L1 (10 mg/kg), and RT+IL/aC@RBAH. The X-ray irradiation treatment (8 Gy) in the pre-designed groups was imposed twice at the tumor sites after 0 and 2 days of in situ administration. On the 7, 14, and 21 days after the tumor inoculation, the tumor growth was monitored by the IVIS Spectrum In Vivo Imaging System. The tumor growth was tracked every 2 days and calculated following the formula V=0.5×W2×L.
For subcutaneous Panc02 tumor treatment, 1×106 Panc02 cells were injected into the right back of male C57BL/6 mice (6–8 weeks old, n=5 mice per group). When subcutaneous tumor volume reached approximately 100 mm3, the mice were subjected to different treatments as designed in the orthotopic 4T1 tumor therapy. On days 7, 14, and 21 after the tumor inoculation, the tumor growth was monitored in real-time by the IVIS Spectrum In Vivo Imaging System (IVIS Lumina XR, USA). The tumor sizes and survival time were also tracked every 2 days. The tumors were taken for histological analysis, including H&E staining and TUNEL staining.

In vivo antitumor immune response analysis
For localized and systemic immune response analysis, orthotopic 4T1 tumor models were established in female BALB/c mice, and subcutaneous Panc02 tumor models were established in male C57BL/6 mice. When the tumors reached approximately 100 mm3, the mice were randomly divided into 6 groups (n=3 mice per group) and treated with saline, RT, RT+RBAH, RT+aC@RBAH, RT+IL@RBAH, and RT+IL/aC@RBAH, respectively. Tumors and TDLNs were both collected 4 days after different treatments. The tissues were rinsed by PBS, minced and digested by Hank’s buffer containing DNase (0.1 mg/mL) and collagenase (1 mg/mL) for 45 min at 37 °C. Then the cells were filtered through 70 μm nylon cell strainers and centrifuged (500×g, 4 °C, 5 min) to obtain single-cell suspensions. Afterward, 1 mL of erythrocyte lysate was added to each sample and lysed for 3 min at room temperature. Then, the cells were washed, blocked by anti-CD16/32 antibody for 5 min and stained with fixable viability stain 510 for 15 min at room temperature. Subsequently, fluorescein-labeled antibodies were added and incubated at 4 °C for 30 min, followed by washing with PBS. For the intracellular staining, the cell suspension was fixed and permeabilized by the fixation/permeabilization buffer (Thermo Fisher Scientific). Then the processed cells were stained with intracellular fluorescein-labeled antibodies and detected by flow cytometry.
For multistage immune effect analysis, the immune cell populations in orthotopic 4T1 tumor-bearing mice exposed to different treatments were investigated. Briefly, the orthotopic 4T1-tumor bearing female BALB/c mice were randomly divided into 5 groups (n=3 mice per group) and then respectively treated with saline, RT, RT+IL-aC (IL-12 prior to aCTLA-4), RT+IL&aC (the mixture of IL-12 and aCTLA-4), and RT+aC-IL (IL-12 subsequent to aCTLA-4). Tumors and TDLNs were collected at 4 days after different treatments. The samples were processed as described above. Afterward, the processed cells were stained with intracellular fluorescein-labeled antibodies and detected by flow cytometry.
To further elucidate the antitumor efficacy between RT+IL&aC and RT+IL/aC@RBAH, the immune cell populations in orthotopic 4T1 tumor-bearing female BALB/c mice exposed to different treatments were investigated. Briefly, the orthotopic 4T1-tumor bearing mice were randomly divided into 3 groups (n=3 mice per group) and then respectively treated with saline, RT+IL&aC, and RT+IL/aC@RBAH. The tumors were collected at 4 days after different treatments. The samples were processed as described above. Subsequently, the processed cells were stained with intracellular fluorescein-labeled antibodies and detected by flow cytometry.
For long-term immune memory evaluation, the orthotopic 4T1 rechallenge model was established as designed above. Briefly, 1×106 4T1 cells were injected into the left mammary fat pad of female BALB/c mice (n=3 mice per group). After 10 days, the mice were treated with RT+IL/aC@RBAH. The X-ray irradiation treatment (8 Gy) was imposed twice at the tumor sites after 0 and 2 days of the treatment. On day 26, the mice presenting CR after RT+IL/aC@RBAH treatment were rechallenged with 1×106 4T1 cells into the right mammary fat pad. Meanwhile, healthy mice were orthotopically injected with 1×106 4T1 cells into the right flank of mice as the control. On day 52, the spleens were collected and processed as above for analysis by flow cytometry.
To compare the antitumor efficacy between RT+aPD-1/aPD-L1 and RT+IL/aC@RBAH, the immune cell populations in orthotopic 4T1 tumor-bearing female BALB/c mice exposed to different treatments were investigated. Briefly, the orthotopic 4T1-tumor bearing mice were randomly divided into 4 groups (n=3 mice per group) and then respectively treated with saline, RT+aPD-1 (10 mg/kg), RT+aPD-L1 (10 mg/kg), and RT+IL/aC@RBAH. The tumors and TDLNs were collected at 4 days after different treatments. The samples were processed as described above. Afterward, the processed cells were stained with intracellular fluorescein-labeled antibodies and detected by flow cytometry.

Transcriptome analysis
An orthotopic 4T1 tumor model was established for transcriptome analysis. Briefly, 1×106 4T1 cells were injected into the left mammary fat pad of female BALB/c mice. After the tumor size reached 100 mm3, the orthotopic 4T1 tumor-bearing mice were randomly divided into 3 groups (n=3 mice per group) and exposed to different treatments of saline, RT, and RT+IL/aC@RBAH, respectively. The tumor tissues were harvested on day 3 after treatments. Total RNA was extracted for transcriptome sequencing and analysis containing correlation analysis, differential analysis, GO analysis, KEGG analysis, and GSEA analysis on a Novogene platform (https://magic.novogene.com/).

Statistics and reproducibility
All numeric data are presented as mean ± SD deviation from at least three parallel experiments. No data were excluded from the analyses. The significance between two groups was determined by the two-sided unpaired students’ t-test. Two-way analysis of variance (ANOVA) with Turkey’s post hoc test was employed for multiple comparisons. Survival analysis was performed by log-rank (Mantel–Cox) test. All statistical analyses were performed using GraphPad Prism (v9.5.0). P values of less than 0.05 were considered significant. All flow cytometry data were analyzed on NovoExpress (v1.5.0, Agilent Technologies) and FlowJo software (v10, BD Biosciences). Living imaging software (v4.4, Perkin Elmer) was used to analyse bioluminescent and fluorescent images. Microscopic images were analyzed by Olympus Micro (FV30S-SW) and ImageJ (Version 1.52k). Sex was not considered as a biological variable within each tumor model because 4T1 studies used only female mice and Panc02 studies used only male mice. Therefore, sex-disaggregated analysis within models is not applicable.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Re: “Hierarchical brake-releasing and accelerator-pushing butterfly effect of in situ cell reservoir hydrogel for maneuvering multistage radioimmunotherapy.”\nThe pain purpose of this manuscript is to describe a novel hydrogel to potentiate and enhance the effects of radiation therapy and immunotherapy.\nFrom a stylistic perspective - there is, in my opinion, too may “buzzwords” and too much “jargon” in the paper. Words like “strategic” and “hierarchical” are overused and slightly distracting.\nThe term “butterfly effect” is not well-defined.\nA breast cancer orthotopic model was used to demonstrate the efficacy of the hydrogel. The same is shown in pancreatic cell models.\nOverall, this is a well-studied and thought-provoking model to study the effects of hydrogel on", "reviewer_2": "I have reviewed the manuscript entitled “Hierarchical brake-releasing and accelerator-pushing butterfly effect of in situ cell reservoir hydrogel for maneuvering multistage radioimmunotherapy”. I have the following comments:\nStrengths:\n  The paper presents an approach for multistage radioimmunotherapy (RIT) using an in situ cell reservoir hydrogel.\n  The hydrogel is composed of red blood cells (RBCs) engineered to carry interleukin-12 (IL-12) and anti-CTLA-4 (aCTLA-4), and barium dioxide (BaO2) for in situ gelation and oxygen production.\n  The hydrogel addresses the limitations of radiotherapy (RT) and immunotherapy by:\nImproving tumor oxygenation through Ba/O2 dual-compensation.\nSequentially releasing aCTLA-4 to block immune checkpoints and IL-12 to activate immune cells, creating a multistage immune response.\n  The approach demonstrates significant antitumor efficacy and improved survival in both 4T1 breast cancer and Panc02 pancreatic cancer models.\n  Transcriptomic analysis reveals activation of immune response-related pathways, supporting the multistage immune activation mechanism.\nWeaknesses and Suggestions for Improvement:\n  The nano material design of hydrogel and RBCs is less innovative, which has been reported a lot. It is unclear why the research focus on CTLA-4 and IL-12, why not PD-1/PD-L1 and other cytokines?\n  Mechanism of Action: While the authors propose a mechanism for the multistage immune response, further studies are needed to validate this mechanism. For example, experiments could be conducted to investigate the timing and kinetics of aCTLA-4 and IL-12 release, as well as their respective effects on immune cell populations and function.\n  Comparison with Other Therapies: The paper would benefit from a direct comparison with other RT and immunotherapy combinations, including those using different aCTLA-4 and IL-12 formulations and delivery methods. This would provide a clearer understanding of the advantages and limitations of the proposed approach.\n  Toxicity and Safety: While the paper mentions that the treatments were well-tolerated and showed no significant toxicity, a more comprehensive assessment of safety is needed. This could include studies on the biodistribution of BaO2 and RBCs, as well as long-term toxicity studies.\n  Clinical Translation: The paper should discuss the potential challenges and considerations for clinical translation of the approach, such as the scalability of RBC engineering and the production of BaO2.\nAdditional Suggestions:\n  The paper could benefit from a more detailed discussion of the potential applications of the approach for different types of cancer.\n  The authors could consider exploring the use of other cytokines or immunomodulatory agents in combination with IL-12 and aCTLA-4.\n  The paper could be improved by including a more detailed discussion of the economic and logistical considerations for the proposed approach.\nOverall, this paper presents a promising approach for multistage RIT with the potential to improve outcomes for cancer patients. Further studies are needed to fully understand the mechanism of action, optimize the treatment regimen, and assess the safety and feasibility for clinical translation.", "reviewer_3": "This is a complex study addressing the intrinsic drawbacks of radiotherapy in treating cancer. The authors provided comprehensive in vitro and in vivo data to demonstrate the success of their hydrogel system. Many control groups are included. Overall, this is a well designed study with a good level of innovation.\nThe title of the paper sounds more like a newspaper article title, instead of a research paper. Lots of concepts are introduced here: e.g.\nHierarchical, \"brake-releasing\", \"accelerator-pushing\", \"butterfly effect\", \"double edged sword\". None of them are accurate scientific terms. I suggest the authors to revise their title to accurately capture the study results. The schematic in Figure 1 is also too complicated.\nI think a good control would be the co-administration of IL12 and CTL4 antibody. Also, many IL12 clinical trials failed due to uncontrolled toxicity in cytokine storm. Can the authors discuss how their system would mitigate this side effect?", "reviewer_4": "Dr. Fan et al. developed an injectable gel encapsulating interleukin-12 and anti-CTLA-4 co-engineered red blood cells. This gel was self-assembled in situ within tumor microenvironments to increase levels of O2, anti-CTLA-4, and IL-12, thereby activating multistage immune responses. While they present an interesting strategy, their approach is quite complex and it is unclear whether this is translationally relevant, especially since they give local injection. I have the following comments.\n1. The authors claimed that \"In response to the acidic TME, the in situ injected BaO2 undergoes hydrolysis to generate H2O2 and Ba2+, followed by the rapid reaction of Ba2+ with sodium alginate (Alg) to afford a biocompatible hydrogel.\" However, both in vivo degradation and in situ retention studies were performed on healthy mice (Fig. 4a-c), showing good retention in vivo for up to 14 days. How did the gel form under normal physiological conditions (pH 7.4)? This seemed inconsistent with Fig. 3b and the authors' hypothesis. Moreover, without acid stimulation, how did the system degrade? If BaO2 could decompose under pH 7.4, how to ensure the long-term stability of this gel system?\n2. Please plot the gelatin time of the system vs. pH.\n3. The authors only mentioned that 500 μL of RBCs was used for the gel system. Then how much O2 could be converted by catalase from these cell mimics? Could the authors provide more detailed preparation and measurement protocols, including the specific amounts of RBCs used and the quantification of O2? A quantitative measurement of O2 generation, rather than enzyme activity, is the key evidence to support the hypothesis.\n4. The authors claimed to have used aCTLA-4 but did not indicate its source. They mentioned that PD-L1 (B7-H1) (Cat# BE0101) was purchased from BioXcell, although this study did not utilize it. Please clarify the source of aCTLA-4 and the relevance of PD-L1 in the context of this study. Besides, were the FITC-aCTLA4 get? Please clarify aCLTA-4 ELISA detection kit in serum as the kit was quite rare.\n5. Detailed protocols for labeling both IL-12 and aCTLA-4 should be provided. Please also include spectral data (such as NMR, IR, Mass, GPC, etc.) to confirm labeling and modification. A release study in PBS (pH 7.0) is also needed. Additionally, since aCTLA-4 has a molecular weight of 150 kDa, how could it be detected in dialysates from 3 kD MWCO dialysis devices?\n6. Did the H2O2 produced by BaO2 hydrolysis and the ROS induced by RT directly damage tumor tissue? If so, what was the killing efficiency, and how does this compare to immune-mediated killing?\n7. The authors claimed O2-mediated pore formation in RBC membranes. Please provide direct evidence to support this claim, rather than relying solely on release data, to improve the rigor of the draft. Additionally, will H2O2 and ROS trigger pore formation on the RBC membrane, and how did the authors design the system to avoid this?\n8. The authors claimed that there was a sequence of immune actions by the gel system, that ultimately achieved a butterfly effect, a feature of the paper. Please provide more evidence to illustrate this effect, including data on the sequential initiation of the immune response in vivo. Presently, this hypothesis is not consistent with in vivo data. The authors claimed that ‘rapid release of aCTLA-4 to relieve tumor immunosuppression. Subsequently, IL-12 anchored on RBC, acting as a “relay baton”, is dilatorily released and interacts with T/NK cells within the TME to induce IFN-γ-dependent antitumor immunity’. However, in Fig5i-p, comparing G4 and G5, aCTLA-4 alone did not significantly increase T cells, and IL-12 alone also did not significantly increase IFN-γ.\n9. Local injection of IL-2 and aCTLA-4 plus RT should be included as a control.\n10. Will local RT treatment and/or ROS damage the gel structure? If so, how could cargo burst release be avoided?\n11. In Fig. 2b, compared to nRBC, what proteins (MWCO 25 kDa) were lost in RBC? It is necessary to add a set of IL/aC@RBC for comparison.\n12. Please provide high-resolution TEM images of the BaO2 products (Fig. S5). It is difficult to conclude that a shortened bamboo-like structure exists for BaO2 here, as well as the particle size distribution.\n13. The authors should indicate the gel position in the PA images. Additionally, will the intertumoral-injected gels with cells affect PA imaging? RBC is also rich in O2 and sonification will help O2 release from cell mimics.\n14. In Figs. 4l-m, 5f, and 9a, the dense tumor cell nuclei (DAPI staining) showed no signs of reduced tumor cell proliferation following RT+IL/aC@RBAH treatment. Additionally, the HE staining appeared to be collected from the tumor margin.\n15. What were the encapsulation efficiency and loading capacity of IL-12 and aCTLA4?\n16. For transcriptome analysis, a PBS group is needed as a control."}, "author_response": "Detailed responses to the reviewers’ comments\nReviewer #1 (radiosensitization):\n“Hierarchical brake-releasing and accelerator-pushing butterfly effect of in situ cell\nreservoir hydrogel for maneuvering multistage radioimmunotherapy.” The pain purpose\nof this manuscript is to describe a novel hydrogel to potentiate and enhance the effects\nof radiation therapy and immunotherapy.\n- From a stylistic perspective - there are, in my opinion, too many “buzzwords” and too\nmuch “jargon” in the paper. Words like “strategic” and “hierarchical” are overused and\nslightly distracting. The term “butterfly effect” is not well-defined.\nResponse: Thanks for the reviewer’s kind suggestion. Considering the scientificity and\npreciseness of this article, buzzwords like “strategic” and “hierarchical” have been\nremoved to straightforward describe our design and results. In our study, the “butterfly\neffect” is defined to be multistage immune effects induced by the proposed therapeutic\nmodality of RT + IL/aC@RBAH. To avert unnecessary confusion, we have substituted\nthe term “butterfly effect” with “multistage effect” and supplemented relevant\ndescription in the revised manuscript.\n- A breast cancer orthotopic model was used to demonstrate the efficacy of the hydrogel.\nThe same is shown in pancreatic cell models. Overall, this is a well-studied and thought-\nprovoking model to study the effects of hydrogel on.\nResponse: Many thanks for the reviewer’s endorsement of our work.\nReviewer #2 (radiosensitization):\nI have reviewed the manuscript entitled “Hierarchical brake-releasing and accelerator-\npushing butterfly effect of in situ cell reservoir hydrogel for maneuvering multistage\nradioimmunotherapy”. I have the following comments:\nStrengths:\nThe paper presents an approach for multistage radioimmunotherapy (RIT) using an in\nsitu cell reservoir hydrogel. The hydrogel is composed of red blood cells (RBCs)\nengineered to carry interleukin-12 (IL-12), anti-CTLA-4 (aCTLA-4), and barium\ndioxide (BaO ) for in situ gelation and oxygen production. The hydrogel addresses the\n2\nlimitations of radiotherapy (RT) and immunotherapy by: Improving tumor oxygenation\nthrough Ba/O dual-compensation. Sequentially releasing aCTLA-4 to block immune\n2\ncheckpoints and IL-12 to activate immune cells, creating a multistage immune response.\nThe approach demonstrates significant antitumor efficacy and improved survival in\nboth 4T1 breast cancer and Panc02 pancreatic cancer models. Transcriptomic analysis\nreveals activation of immune response-related pathways, supporting the multistage\nimmune activation mechanism.\nResponse: Thanks for the reviewer’s commendation on our study.\nWeaknesses and Suggestions for Improvement:\n- The nano material design of hydrogel and RBCs is less innovative, which has been\nreported a lot. It is unclear why the research focuses on CTLA-4 and IL-12, why not\nPD-1/PD-L1 and other cytokines?\nResponse: Thanks for the reviewer’s valuable questions. Despite the fact that hydrogels\nand RBCs have been reported a lot in previous studies, both of them in our research\nhave been innovatively renovated and engineered for extended applications. In this\nstudy, we innovatively devise an in situ self-assembled hydrogel based on the strategic\nintroduction of sodium alginate (Alg) and barium dioxide (BaO ). Once injected into\n2\nthe tumor, the acidity-responsive BaO can degrade into Ba2+, which rapidly reacts with\n2\nbiocompatible Alg to form an in situ self-assembled cell reservoir crosslinked network\nin the acidic tumor microenvironment rather than neutral normal tissues, thus realizing\neffective multidrug encapsulation that is not equipped by traditional hydrogels slowly\nchelating with intrinsic metal ions in the body[1]. Besides, the concurrently generated\nH O can be catalyzed into O by the catalase (CAT) intrinsic on RBCs, thereby\n2 2 2\ncollectively achieving Ba/O self-compensated radiosensitization.\nvitro production, have garnered considerable attraction as an intelligent drug delivery\nsystem[2]. Different from traditional strategies for encapsulating a single drug within\nRBCs [3], we innovatively develop a multisite-fettering approach to entrap aCTLA-4 in\nthe interior and anchor IL-12 on the membrane. Notably, the optimized “alternate\nhypotonic and hypertonic dialysis” method is elaborately introduced to encapsulate\naCTLA-4 with both the structural integrity and enzyme activity of RBCs into account.\nBeyond being a dual-cargo carrier, those engineered RBCs with preserved CAT\nbioactivity can catalyze the production of O from H O for radiosensitization. Overall,\n2 2 2\nthe material design of hydrogel and RBCs is much more innovative than simple in situ\ncompartmental encapsulation of multiple cargos and can realize sequential aCTLA-\n4/IL-12 release for multistage antitumor immune responses without biosafety concerns.\nDue to the differential RT sensitivity among immune cells, RT causes a direct\ndecrease in the ratio of cytotoxic T cells to regulatory T cells (Tregs), accompanied by\nthe upregulation of CTLA-4 in the initial stage[4-6]. The elevated CTLA-4 expression\ninevitably restrains dendritic cell (DC) migration and antigen presentation, thus greatly\nattenuating the therapeutic efficacy of RT. Besides, IL-12, as a critical\nimmunostimulatory agent in both innate and adaptive immune responses, can promote\nthe proliferation and enhance the function of NK/T cells to shape an immune-activating\ntumor microenvironment[7]. To modulate the multistage immune landscape of RT, we\nscrupulously combine aCTLA-4 with IL-12 in a temporal manner, thus compensating\nprimary immunosuppression and instigating relay immunoactivation during the\nmultistage of RT, which is also the main reason why we focus on aCTLA-4 and IL-12\ninstead of PD-1/PD-L1 or other cytokines.\nReferences:\n[1] Zhao, Z. et al. Capturing magnesium ions via microfluidic hydrogel microspheres\nfor promoting cancellous bone regeneration. ACS Nano 15, 13041-13054 (2021).\n[2] Liu, H. et al. Red blood cells-derived components as biomimetic functional\nmaterials: Matching versatile delivery strategies based on structure and function. Bioact\nMater 47, 481-501 (2025).\n[3] Li, Y. et al. Clinical progress and advanced research of red blood cells based drug\ndelivery system. Biomaterials 279,121202 (2021).\n[4] Wirsdörfer, F. et al. Combining Radiotherapy and Immunotherapy in Lung Cancer:\nCan We Expect limitations due to altered normal tissue toxicity? Int J Mol Sci 20, 24\n(2018).\n[5] Qu, Y. et al. 2-Gy whole-body irradiation significantly alters the balance of\nCD4+CD25- T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice. Cell\nMol Immunol 7, 419-427 (2010).\n[6] Persa, E. et al. The effect of ionizing radiation on regulatory T cells in health and\ndisease. Cancer Lett 368, 252-261 (2015).\n[7] Zou, Z. et al. Anti-PD-1 cis-delivery of low-affinity IL-12 activates intratumoral\nCD8+ T cells for systemic antitumor responses. Nat Commun 15, 4701 (2024).\n- Mechanism of Action: While the authors propose a mechanism for the multistage\nimmune response, further studies are needed to validate this mechanism. For example,\nexperiments could be conducted to investigate the timing and kinetics of aCTLA-4 and\nIL-12 release, as well as their respective effects on immune cell populations and\nfunction.\nResponse: Thanks for the reviewer’s constructive advice. To investigate the timing and\nkinetics of aCTLA-4 and IL-12 release, IL-12 and aCTLA-4 were jointly entrapped in\nRBCs to obtain IL&aC@RBAH as control. The release behaviors of IL-12 and aCTLA-\n4 from IL&aC@RBAH and IL/aC@RBAH in the mildly acidic environment (pH 6.5)\nwere investigated. As expected, both IL-12 and aCTLA-4 in IL&aC@RBAH were\nreleased rapidly in a similar trend within 48 h (Fig. R1), possibly due to the simple\ndiffusion of the mixture of IL-12 and aCTLA-4 from the formed pores on RBCs.\nContrarily, IL/aC@RBAH presented a sequential release behavior with rapid release of\napproximately 40% aCTLA-4 from the oxygen-induced pores on the RBC membrane\nwithin 48 h and slow release of less than 20% IL-12 from the enlarged channels in the\ncrosslinked network of the degrading hydrogel within 48 h. Collectively, all those\nresults affirmed the sequential timing and kinetics of aCTLA-4 and IL-12 release from\nIL/aC@RBAH, thus building the foundation for the multistage initiation of immune\nresponses in vivo.\nFig. R1 Release profiles of IL-12 and aCTLA-4 from IL&aC@RBAH and\nIL/aC@RBAH at pH 6.5 within 48 h. n = 3, mean ± SD.\nNotably, in our initial submission, we investigated the respective effects of IL-12\nand aCTLA-4 on systemic immune responses, as presented in Figs. 5j-r. Herein, to\nfurther unveil the impact of sequential aCTLA-4/IL-12 release on the multistage\nimmune effects, we investigated the immune cell populations in orthotopic 4T1 tumor-\nbearing mice exposed to different treatments. Briefly, the orthotopic 4T1-tumor bearing\nmice were randomly divided into 5 groups and then respectively treated with saline, RT,\nRT + IL-aC (IL-12 prior to aCTLA-4), RT + IL&aC (the mixture of IL-12 and aCTLA-\n4), and RT + aC-IL (IL-12 subsequent to aCTLA-4). Compared to the slight\nupregulation of CD8+ T cells induced by RT-mediated immunogenic cell death (ICD),\nthe intratumoral level of those cells was moderately upregulated in the RT + IL-aC and\nRT + IL&aC groups (Figs. R2a, b), demonstrating the modest immune activation in the\nprimary and relay stages. This limitation was likely attributed to the rapid diffusion and\nloss of IL-12 from tumor, which was insufficient to sustain downstream activation\nduring the relay stage. Of note, the RT + aC-IL treatment elicited the utmost increase in\nCD8+ T cells, along with the lowest Treg ratio and the highest NK cell level among all\nthe groups (Figs. R2c, d), indicating the strongest antitumor immune initiation by the\nsequential aCTLA-4/IL-12 supply parallel to IL/aC@RBAH. Moreover, the IFN-γ\nproduction in CD8+ T cells, CD4+ T cells, and NK cells in the RT + aC-IL group were\nall at the highest level among all the groups (Figs. R2e-g), further affirming the most\npotent immune responses through aCTLA-4-mediated primary immunosuppression\nalleviation and relay IL-12-induced immunoactivation. It is noteworthy that the RT +\naC-IL treatment likewise elicited the uppermost level of mature DCs, which was almost\n1.44- and 1.27-fold higher than that in the RT + IL-aC and RT + IL&aC groups (Figs.\nR2h, i). The result further elucidated the most powerful antigen-presenting initiation of\nthe sequential aCTLA-4/IL-12 supply in the RT + aC-IL treatment for antitumor\nimmunity through the DC cell-intrinsic IL-12/IFN-γ positive feedback loop.\nFig. R2 a, b Representative flow cytometry plots (a) and quantification (b) of cytotoxic\nT lymphocytes (CD8+ in CD45+CD3+ cells) in orthotopic 4T1 tumors after different\ntreatments. c, d Quantitative analyses of Tregs (CD4+CD25+ in CD45+CD3+ cells) (c)\nand NK cells (CD3-CD49b+ in CD45+ cells) (d) in orthotopic 4T1 tumors after different\ntreatments. e-g Quantification of IFN-γ production by CD8+ T cells (IFN-γ+ in\nCD45+CD3+CD8+ cells) (e), CD4+ T cells (IFN-γ+ in CD45+CD3+CD4+ cells) (f), and\nNK cells (IFN-γ+ in CD45+CD3-CD49b+ cells) (g). h, i Representative flow cytometry\nplots (h) and quantification (i) of mature DCs (CD80+CD86+ in CD11c+MHC Ⅱ+ cells)\nin orthotopic 4T1 tumors after different treatments. Data in b-g, i were presented as\nmean ± SD. n = 3 biologically independent samples in b-g, i. P values were calculated\nvia student’s t-test.\nGiven the intratumoral immune responses elicited by RT + aC-IL, the impacts of\nsequential cytokine supply on antigen presentation process were further investigated\nthrough the immune cell populations in the tumor-draining lymph nodes (TDLNs) of\northotopic 4T1-tumor bearing mice subjected to different treatments. Both the levels of\nCD8+ T cell infiltration and activation were at the peak in the RT + aC-IL group (Figs.\nR3a-d). Meanwhile, the amounts of both cDC1 and cDC2 to TDLNs were maximally\nelevated in the RT + aC-IL group, quite different from those in the other groups (Figs.\nR3e, f), further demonstrating the supreme antitumor initiation by the sequential\naCTLA-4/IL-12 supply. Overall, all the results firmly affirmed the virtues of systemic\nmultistage immune responses induced by the sequential aCTLA-4/IL-12 release from\nRT + IL/aC@RBAH. We have included Fig. R1 as Supplementary Fig. 33, Figs. R2b-\ng and Fig. R2i as Figs. 6e-k, Fig. R3c as Supplementary Fig. 34 and Figs. R3d-f as\nFigs. 6l-n. Relevant description has been added in the revised manuscript.\nFig. R3 a Representative flow cytometry plots of cytotoxic T lymphocytes (CD8+ in\nCD45+CD3+ cells) in TDLNs of orthotopic 4T1 tumor-bearing mice post different\ntreatments. b Representative flow cytometry plots of activated CD8+ T lymphocytes\n(CD69+ in CD3+CD8+ cells) in TDLNs of orthotopic 4T1 tumor-bearing mice post\ndifferent treatments. c-f Quantitative analyses of cytotoxic T lymphocytes (CD8+ in\nCD45+CD3+ cells) (c), activated CD8+ T lymphocytes (CD69+ in CD3+CD8+ cells) (d),\ncDC1 (CD103+ in CD11c+MHC Ⅱ+ cells) (e) and cDC2 (CD11b+ in CD11c+MHC Ⅱ+\ncells) (f) in TDLNs of orthotopic 4T1 tumor-bearing mice post different treatments.\nData in c-f were presented as mean ± SD. n = 3 biologically independent samples in c-\nf. P values were calculated via student’s t-test.\n- Comparison with Other Therapies: The paper would benefit from a direct comparison\nwith other RT and immunotherapy combinations, including those using different\naCTLA-4 and IL-12 formulations and delivery methods. This would provide a clearer\nunderstanding of the advantages and limitations of the proposed approach.\nResponse: Thanks for the reviewer’s helpful suggestions. In this study, we develop an\nin situ self-assembled cell reservoir hydrogel to initiate multistage radioimmunotherapy.\nDistinct from the simple combination of RT with aCTLA-4 or IL-12 in the previous\nstudies[1, 2], the proposed approach of RT + IL/aC@RBAH enhances radiosensitivity\nand realizes chronologically augmented antitumor immune responses. Specifically, the\ndesigned IL/aC@RBAH can provide a Ba2+ depot and initiate O production for Ba/O\n2 2\ndual-compensated radiosensitization. Distinguished from the traditional delivery of a\nsimple cytokine mixture or the sequential local administration of aCTLA-4 and IL-12,\nthe in situ IL-12/aCTLA-4 multisite-fettering system (IL/aC@RBAH) could facilitate\nthe sequential release of aCTLA-4 and IL-12 via a single administration (Fig. R1),\nthus eliciting multistage antitumor immunity for effective tumor regression (Figs. R2,\nR3).\nOn the other hand, recombinant IL-12 has failed in clinical trials, owing to the\nuncontrolled cytokine release syndrome and liver damage[3]. The systemic delivery of\nIL-12 by novel nanoparticles has been recently reported a lot, but the potential toxicity\nstill exists with elevated IL-12 concentration in the serum[4]. Moreover, aCTLA-4 alone\nexhibits grade 3-4 immune-related serious side effects (irAEs) in about 54% of patients,\nwhich greatly limits its clinical use[5, 6]. In this study, leveraging the rapid in situ\nformation and dilatory degradation of the IL/aC@RBAH hydrogel, aCTLA-4 and IL-\n12 can be steadily and slowly released in the tumors, thus avoiding the potential\ncytokine storm and the toxicity of free aCTLA-4 and IL-12 to liver damage and kidney\ninjury (Figs. 4d-i).\nOverall, the therapeutic modality of RT + IL/aC@RBAH compensates for the\nshortcomings of other RT and immunotherapy combinations by minimizing off-target\neffects and reversing RT-induced periodical tumor immunosuppression. In the future,\nthe self-assembled reservoir hydrogel is promising to be a generic platform to offer\nphysical support for diverse engineered cells with compartmentalized encapsulation of\nvarious cytokines, thus breaking through its limitation in the radiosensitive tumors or\nthe ones highly expressing CTLA-4. Following the reviewer’s kind suggestions, we\nhave added the relevant description to expand its future perspectives on multiple tumor\ntypes in the revised manuscript.\nReferences:\n[1] Twyman-Saint Victor C, et al. Radiation and dual checkpoint blockade activate non-\nredundant immune mechanisms in cancer. Nature, 520, 373-377 (2015).\n[2] Mills, BN. et al. Stereotactic body radiation and interleukin-12 combination therapy\neradicates pancreatic tumors by repolarizing the immune microenvironment. Cell Rep\n29, 406-421.e5 (2019).\n[3] Eisenbeis C F, et al. Phase I study of the sequential combination of interleukin-12\nand interferon alfa-2b in advanced cancer: evidence for modulation of interferon\nsignaling pathways by interleukin-12. J Clin Oncol 23, 8835-8844 (2005).\n[4] Wang, P. et al. Re-designing Interleukin-12 to enhance its safety and potential as an\nanti-tumor immunotherapeutic agent. Nat Commun 8, 1395 (2017).\n[5] Postow, MA. et al. Immune-related adverse events associated with immune\ncheckpoint blockade. N Engl J Med 378, 158-168 (2018).\n[6] Poto, R. et al. Holistic approach to immune checkpoint inhibitor-related adverse\nevents. Front Immunol 13, 804597 (2022).\n- Toxicity and Safety: While the paper mentions that the treatments were well-tolerated\nand showed no significant toxicity, a more comprehensive assessment of safety is\nneeded. This could include studies on the biodistribution of BaO and RBCs, as well as\n2\nlong-term toxicity studies.\nResponse: Thanks for the reviewer’s valuable advice. Following the reviewer’s kind\nsuggestions, comprehensive assessments of biosafety, including long-term toxicities,\nas well as the biodistribution of BaO and RBCs, have been conducted. Briefly, healthy\nsaline, IL&aC (the mixture of IL-12 and aCTLA-4), RB (the mixture of RBC and BaO ),\n2\nand IL/aC@RBAH once a week. During 2 months of continuous treatments, the body\nweights of the mice were recorded every 7 days. In comparison to the slightly declined\nbody weight in the IL&aC and RB groups, the mice treated with IL/aC@RBAH\nexhibited negligible body weight changes similar to those in the saline group (Fig. R4a),\nindicating minimal systemic toxicity or metabolic perturbations induced by\nIL/aC@RBAH. After 2 months of monitoring, the blood and major organs were\ncollected. Liver damage indexes involving alanine aminotransferase (ALT) and\naspartate aminotransferase (AST), as well as kidney function indexes compromising\nblood urea nitrogen (BUN), creatinine (CREA), were respectively detected. The blood\nbiochemical parameters of BUN (Fig. R4b), CREA (Fig. R4c) and ALT (Fig. R4d)\nwere maintained within the normal range in all the groups. Moreover, compared to the\nabnormal AST level in the RB and IL&aC groups, that in the IL/aC@RBAH group was\nsignificantly lowered within safe bounds (Fig. R4e), evidencing the normal hepatic and\nrenal function in the IL/aC@RBAH-treated mice. In addition, the H&E-stained images\npresented negligible histomorphological abnormalities of major organs in the\nIL/aC@RBAH group, distinct from moderate inflammation of the liver tissues in the\nIL&aC and RB groups (Fig. R4f), possibly caused by the burst release of aCTLA-4/IL-\n12 and Ba2+. Collectively, all the above studies confirmed the favorable long-term\nbiosafety of IL/aC@RBAH.\nFig. R4 a Body weight changes of healthy BALB/c mice after different treatments once\na week for 2 months. b, c Kidney function indexes including BUN (b) and CREA (c)\nin healthy BALB/c mice exposed to different treatments once a week for 2 months. d,\ne Liver function indexes comprising ALT (d) and AST (e) in healthy BALB/c mice\nexposed to different treatments once a week for 2 months. The normal range of liver\nfunction indexes was marked by dash lines. f H&E staining analysis of major organs\n(heart, liver, spleen, lung, and kidney) excised from healthy BALB/c mice exposed to\ndifferent treatments once a week for 2 months. Scale bar, 100 μm. Data in a-e were\npresented as mean ± SD. n = 3 biologically independent samples in a-e.\nTo monitor the biodistribution of BaO in orthotopic 4T1 tumor-bearing mice, the\n2\nelement Ba in major organs and tumors was detected by inductively coupled plasma-\nmass spectrometry (ICP-MS). Once IL/aC@RBAH was injected into the tumors, the\nlevel of Ba in the tumors was rapidly elevated at 0 h, followed by a moderate reduction\nat 24 h post-injection (Fig. R5). During the subsequent 2-day monitoring period, the\nBa content in the tumors maintained over 20% ID/g with a slight declining trend,\nmanifesting the favorable in situ retention capability of IL/aC@RBAH with minimal\nleakage of Ba. Contrarily, the levels of Ba in the livers and kidneys were both\nimperceptible post the local administration during 72 h of monitoring, indicative of\nexcellent in situ retention capacity of IL/aC@RBAH.\nFig. R5 Ba contents in major organs and tumors, indicative of in vivo distribution of\nBaO after intratumoral administration of IL/aC@RBAH in orthotopic 4T1 tumor-\n2\nbearing mice. n = 3, mean ± SD.\nTo visualize the RBC distribution in orthotopic 4T1 tumor-bearing mice post local\ninjection, the RBC in IL/aC@RBAH was labeled with DiR (a cell membrane probe) to\nobtain DiR-RBC for real-time monitoring through the in vivo imaging system (IVIS).\nDuring the 72 h of the tracking process, the total fluorescence of DiR-RBC at the tumor\nsite remained persistently above 8 × 109 p/s with imperceptible changes (Fig. R6).\nMoreover, the tumors and major organs, including heart, liver, spleen, lung and kidney,\nwere collected at 72 h for ex vivo imaging. As anticipated, in sharp contrast to the\nnegligible fluorescence detected in the major organs, substantial DiR signals were\ntracked in the tumors, further evidencing the excellent intratumoral retention of\nIL/aC@RBAH.\nFig. R6 a, b Whole-animal in vivo fluorescence imaging (a) and corresponding\nfluorescence quantification (b) of orthotopic 4T1 tumor-bearing mice after intratumoral\nadministration of IL/aC@RBAH. The RBC in IL/aC@RBAH is labeled with DiR for\nspatial tracking and visualization. c, d Ex vivo fluorescence imaging (c) and\ncorresponding fluorescence quantification (d) of major organs and tumors from\northotopic 4T1 tumor-bearing mice at 72 h post-injection of IL/aC@RBAH. Data in b,\nd were presented as mean ± SD. n = 3 biologically independent samples in b, d.\nOverall, the above results confirmed the favorable biosafety of IL/aC@RBAH,\nbenefiting from the in situ retention of the hydrogel for low risks of cargo leakage.\nFollowing the reviewer’s kind suggestions, we have respectively included Fig. R4 as\nSupplementary Fig. 29 and Figs. R5, R6 as Supplementary Figs. 22, 23 in the revised\nsupplementary information and added relevant discussions to improve the biosafety\nassessments in the revised manuscript.\n- Clinical Translation: The paper should discuss the potential challenges and\nconsiderations for clinical translation of the approach, such as the scalability of RBC\nengineering and the production of BaO .\nIL/aC@RBAH approach offers a powerful and versatile paradigm for the development\nof generic in situ cancer therapy. In terms of clinical translation, the chemically\nengineered RBCs exhibit enhanced safety and controllability, with significantly lower\nproduction costs compared to genetically engineered cells. Notably, the quality and\nyield of RBCs are both stable and reproducible at laboratory and bench scales. All the\nvirtues of RBC engineering endow itself with potential scalability. In addition, the\nintroduced BaO nanoparticles present highly consistent bamboo-like morphology and\n2\nexcellent batch-to-batch uniformity during our study. The degraded nontoxic\npolysaccharides from Alg-based hydrogel do not participate in normal metabolic\nprocesses, which are conducive to the development requiring high biological\ncompatibility and environmental sustainability. Despite the above advantages of our\napproach, the challenges still persist in the immature large-scale RBC production and\nlow BaO production yields. The follow-up efforts should be focused on pilot-scale\n2\noptimization and rigorous control of scale-up parameters to facilitate clinical translation.\nIn the revised manuscript, we have included pertinent discussions relevant to the\npotential opportunities and challenges of clinical translation of the approach.\nAdditional Suggestions:\n- The paper could benefit from a more detailed discussion of the potential applications\nof the approach for different types of cancer.\nResponse: Thanks for the reviewer’s valuable suggestions. Leveraging the merit of\nsubcutaneous injectability, the cell reservoir hydrogel in the study, featuring excellent\nbiosafety, compartmentalized encapsulation and sequential drug release, warrants\npotential development in the therapeutic application of solid tumors, including skin\ncancer, breast cancer, prostate cancer, and oral carcinoma. Moreover, given the\npredominant clinical use of RT as a local treatment, the in situ cell reservoir hydrogel\ncombined with RT in the study holds great potential in ameliorating multistage\nantitumor immune responses for effective tumor eradication. With respect to superficial\ntumors like skin or oral cancers, percutaneous hydrogel injection can facilitate in situ\nrapid formation, followed by localized RT to suppress tumor progression. In cases of\ndeeper-seated malignancies like pancreatic cancer or prostate cancers, the hydrogel can\nbe precisely administered via minimally invasive surgery approaches to directly inhibit\ntumorigenesis or be deemed to be an adjuvant therapy to prevent postoperative\nrecurrence. In the future, this designed hydrogel will open new therapeutic avenues in\nthe application of deeper tumors like hepatocellular carcinoma, gastric cancer, and\ncolon cancer through the integration of precise localized delivery and advanced\ninterventional techniques. Relevant discussions have been incorporated into the revised\nmanuscript.\n- The authors could consider exploring the use of other cytokines or immunomodulatory\nagents in combination with IL-12 and aCTLA-4.\nResponse: Thanks for the reviewer’s helpful comments. The cancer-immunity cycle is\na multi-step biological process comprising several key stages, including antigen release\nfrom dying tumor cells, antigen presentation, priming and activation of immune cells,\nimmune cell trafficking and infiltration, and the final tumor recognition and\nelimination[1]. Given the multi-step nature of the cancer-immunity cycle, multiplexed\ncytokines targeting the immune pathways in different stages need to be developed to\ntemporally coordinate combinational immunotherapy for enhanced antitumor\nresponses. In our study, the meticulous design of sequential aCTLA-4 and IL-12 release\nis introduced to significantly enhance the antitumor immune efficacy and guarantee the\nbiosafety of the treatment regime. Briefly, aCTLA-4 primarily promotes the antigen\npresentation phase by specifically blocking immune checkpoint molecules CTLA-4 on\nT cells. Subsequently, IL-12 enhances the immune effects in the relay stage by\npotentiating the function of NK cells, CD8+ T cells, and CD4+ T cells to promote IFN-\nγ secretion for boosted tumoricidal activities.\nTo further broaden its therapeutic applications, future studies may be focused on\ncombining the current regimen with agents targeting other phases of the cancer-\nimmunity cycle. On the one side, ICD inducers, such as radiotherapy or anthracycline-\nbased chemotherapeutic agents, could be incorporated to amplify tumor antigen\nrelease[2]. On the other side, immune cell chemoattractants, including CXCL9 and\nCXCL10, might be co-administered to facilitate the infiltration of peripherally primed\nT cells into tumor tissues[3]. Additionally, immune adjuvants like Toll-like receptor\nagonists could be utilized to prompt intratumoral antigen-presentation, thereby further\npotentiating antitumor immune responses[4]. Those multi-pronged combinatorial\napproaches would systematically optimize each critical step of the cancer-immunity\ncycle for superior antitumor immune outcomes. The pertinent discussion has been\nsupplemented in the revised manuscript.\nReferences:\n[1] Mellman, I. et al. The cancer-immunity cycle: Indication, genotype, and\nimmunotype. Immunity, 56, 2188–2205 (2023).\n[2] Hayashi, K. et al. Tipping the immunostimulatory and inhibitory DAMP balance to\novercome immunogenic cell death resistance in pancreatic cancer. Nat Commun 11,\n6299 (2020).\n[3] Lim, R. et al. CXCL9/10‑engineered dendritic cells promote T cell activation and\nenhance immune checkpoint blockade for lung cancer. Cell Rep Med 5, 101479 (2024).\n[4] Zhang, Q. et al. Intratumoral sustained release of a TLR7/8 agonist via biomaterial\ndepot enhances dendritic cell-mediated antigen presentation and synergizes with\ncheckpoint blockade to drive potent antitumor immunity. Nat Mater 22, 428–439\n(2023).\n- The paper could be improved by including a more detailed discussion of the economic\nand logistical considerations for the proposed approach.\nResponse: Thanks for the reviewer’s insightful comments. The therapeutic system in\nour study is composed of RBC modified with dual cargos, barium peroxide, and sodium\nalginate. Economically, the engineered RBCs exhibit distinct advantages[1], including\nwide availability, high prevalence, and low-cost chemical modification. Additionally,\nmodified RBCs maintain stable morphological properties for up to 42 days at 2-6 °C[2],\nfacilitating convenient storage and transportation. Moreover, the barium peroxide\n(BaO ) is synthesized via a hydrolytic precipitation method, which also offers the\n2\nadvantages of low cost and economical production[3]. However, BaO exhibits limited\n2\nstability in deionized water (Fig. 2k), necessitating storage in alcoholic solvents or\nsuitable preservation systems for transportation and long-term storage, with immediate\npreparation preceding therapeutic administration. Moreover, sodium alginate exhibits\nexcellent potential as a biomaterial due to its high structural stability, mature production\nprocesses, and wide availability from brown algae (e.g., laminaria japonica, lessonia,\netc.)[4]. This low-cost material offers convenient transportability and superior economic\nbenefits. In this study, alginate-based hydrogels, featuring low production costs and\nFDA-approved excellent biocompatibility[5], illuminate a promising feasibility for\nclinical translation.\nIn this study, aCTLA-4 was successfully loaded into RBCs to generate aC@RBC\nvia hypotonic dialysis, which is a well-established method benefiting from existing\nautomatic loading instruments, thereby reducing production costs and enhancing\nclinical translation potential. Subsequently, IL-12 was anchored onto the surface of\nRBCs through membrane insertion to yield IL/aC@RBC, which maintained structural\nintegrity after 7-day storage at 4 °C (Supplementary Fig. 4), indicating suitability for\nextended transportation and storage. However, the membrane insertion approach faces\nthree critical limitations, involving high-cost lipid reagents, suboptimal antibody\nconjugation efficiency, and low insertion efficiency of lipid-conjugated antibodies, thus\ncontributing to high production costs and operational complexity. Therefore, further\nmethodology optimization and automatic production system development of the\nmembrane insertion approach are in demand for clinical translation.\nCollectively, the elaborately designed self-assembled hydrogel is a facile platform\nwith comprehensive economic and logistical considerations. Following the reviewer’s\nkind suggestions, we have included relevant discussion in the revised manuscript to\npropel its clinical translation.\nReferences:\n[1] Nguyen, PHD. et al. Advances in Drug Delivery Systems Based on Red Blood Cells\nand Their Membrane-Derived Nanoparticles. ACS Nano 17, 5187-5210 (2023).\n[2] Koch, CG. et al. Real Age: Red Blood Cell Aging During Storage. Ann Thorac Surg\n107, 973-980 (2019).\n[3] Zhang, M. et al. Radiation-assisted metal ion interference tumor therapy by barium\nperoxide-based nanoparticles. Mater Horiz 6, 1034-1040 (2019).\n[4] Tagliapietra, B. L. et al. Brown algae and their multiple applications as functional\ningredient in food production. Food Res Int 167, 112655 (2023).\n[5] Basu, T. et al. Cross-linker-free sodium alginate and gelatin hydrogels: a multiscale\nbiomaterial design framework. J Mater Chem B 10, 3614-3623 (2022).\nOverall, this paper presents a promising approach for multistage RIT with the potential\nto improve outcomes for cancer patients. Further studies are needed to fully understand\nthe mechanism of action, optimize the treatment regimen, and assess the safety and\nfeasibility for clinical translation.\nResponse: Thanks for the reviewer’s commendation on our research. According to the\nreviewer’s valuable suggestions, we have supplemented a series of studies on the\nsequential release kinetics of aCTLA-4/IL-12, as well as the multistage initiation of\nimmune responses in vivo to fully understand the mechanism of action and the merits\nof the treatment regimen. Moreover, long-term toxicity studies and the biodistribution\nof BaO and RBCs have been conducted to evidence the safety and feasibility of clinical\n2\ntranslation. Furthermore, the innovation, comparison with other therapies, potential\napplications in different types of cancer, and further combinational application, as well\nas the economic and logistical advantages of the treatment regime have been discussed\nin detail to enhance the scientificity and clinical value of the manuscript.\nReviewer #3 (biomaterials):\nThis is a complex study addressing the intrinsic drawbacks of radiotherapy in treating\ncancer. The authors provided comprehensive in vitro and in vivo data to demonstrate\nthe success of their hydrogel system. Many control groups are included. Overall, this is\na well-designed study with a good level of innovation.\nResponse: Thanks for the reviewer’s endorsement of our study.\n- The title of the paper sounds more like a newspaper article title, instead of a research\npaper. Lots of concepts are introduced here: e.g. “Hierarchical”, “brake-releasing”,\n“accelerator-pushing”, “butterfly effect”, “double edged sword”. None of them are\naccurate scientific terms. I suggest the authors to revise their title to accurately capture\nthe study results. The schematic in Figure 1 is also too complicated.\nResponse: Thanks for the reviewer’s constructive comments on the title and schematic.\nFollowing the reviewer’s kind suggestions, we have revised the title as “In situ self-\nassembled cell reservoir hydrogel for maneuvering multistage radioimmunotherapy” to\naccurately present the study results. Moreover, the schematic was simplified as Fig. R7\nto improve the readability and preciseness of the article. The simplified schematic was\nincluded as Fig. 1 in the revised manuscript.\nFig. R7 Schematic of the in situ self-assembled cell reservoir hydrogel (IL/aC@RBAH)\nto maneuver multistage radioimmunotherapy (RIT). a Upon intratumoral injection of\nthe hydrogel precursors, BaO reacts with hydrogen ions within the TME to produce\n2 2 2 2\non RBC to supply oxygen for aerobic RT to augment sequential aCTLA-4/IL-12 release.\nb In the primary stage of RIT, aCTLA-4 is introduced to alleviate the tumor\nimmunosuppression, thus primarily inducing DC homing and T cell activation.\nAfterward, IL-12 can initiate the immunoactivation in the relay stage to instigate the\nproduction of IFN-γ from T/NK cells for boosted antitumor immune responses.\n- I think a good control would be the co-administration of IL-12 and CTLA-4 antibody.\nAlso, many IL-12 clinical trials failed due to uncontrolled toxicity in cytokine storm.\nCan the authors discuss how their system would mitigate this side effect?\nResponse: Thanks for the reviewer’s valuable advice. Following the reviewer’s kind\nsuggestions, co-administration of IL-12 and CTLA-4 antibody (IL&aC) has been set as\na control for a comprehensive evaluation of the therapeutic efficacy of RT +\nIL/aC@RBAH. The orthotopic 4T1 tumor-bearing mice were randomly allocated into\n6 groups and treated with saline, RT, RT + RBAH, IL&aC, RT + IL&aC, and RT +\nIL/aC@RBAH. Distinct from the slight tumor inhibition in the RT group, the tumor\nvolumes in the RT + RBAH and IL&aC groups were moderately suppressed (Figs.\nR8a-d), indicating the deficient antitumor efficacy of the sole Ba/O -supplied\n2\nradiosensitization by RBAH or the single antitumor immune responses induced by\nIL&aC. Contrarily, the RT + IL&aC treatment showed much more prominent tumor\ninhibition than the RT + RBAH and IL&aC groups, possibly ascribed to the combined\ntherapeutic strategy of RT and immunotherapy. Intriguingly, the mice in the RT +\nIL/aC@RBAH group presented the uppermost tumor regression with a prolonged\nlifetime of at least 2 months in about 80% of the mice (Fig. 8e), thus illustrating the\nsupreme antitumor efficacy of RT + IL/aC@RBAH-induced multistage RIT. According\nto the reviewer’s kind advice, we have supplemented relevant description in the revised\nmanuscript and included Fig. R8 as Supplementary Fig. 35 in the revised\nsupplementary information.\nFig. R8 a, b Whole-animal in vivo bioluminescence imaging (a) and corresponding\nbioluminescence quantification (b) of orthotopic 4T1-luciferase (4T1-luc) tumor-\nbearing mice after treated with saline, RT, RT + RBAH, IL&aC, RT + IL&aC, and RT\n+ IL/aC@RBAH, respectively. c, d Average tumor growth curves (c) and individual\ntumor volume dynamics (d) of orthotopic 4T1-luc tumor-bearing mice subjected to\ndifferent treatments. e Survival curves of orthotopic 4T1-luc tumor-bearing mice\nsubjected to different treatments. Data in b, c were presented as mean ± SD. n = 5\nbiologically independent samples in b-e. Statistical significance was determined using\ntwo-way ANOVA (c).\nDue to the merits of in situ formation and dilatory degradation of IL/aC@RBAH,\nIL-12 could be lodged at the tumor site with low leakage into serum (Fig. 4e), thus\naverting the side effects of IL-12. To evaluate the in situ retention capability of\nIL/aC@RBAH, the orthotopic 4T1 tumor-bearing mice were randomly allocated into 4\ngroups and intratumorally injected with IL-aC (IL-12 prior to aCTLA-4), aC-IL (IL-12\nsubsequent to aCTLA-4), IL&aC@RBAH (mixture of IL-12 and aCTLA-4 in RBC),\nand IL/aC@RBAH, respectively. The changes in the IL-12 levels in tumors were\nmonitored for 3 days post-injection. Predictably, the IL-12 levels in the IL-aC and aC-\nIL groups both remarkably declined in one day (Fig. R9), indicating rapid diffusion\nloss of free IL-12 from tumors. Compared to the time-dependent moderate reduction in\nthe IL&aC@RBAH group due to the pore formation on RBC, the majority of IL-12 content\nremained at the tumor site on 3 days post-injection in the IL/aC@RBAH group,\npossibly attributed to the effective anchorage of IL-12 on the RBC surface.\nFig. R9 IL-12 contents in orthotopic 4T1 tumors post different treatments for various\ntime periods. n = 3, mean ± SD. P values were calculated via student’s t-test.\nIn addition, long-term toxicities have been measured to affirm the cytokine storm\nmitigation of IL-12 by IL/aC@RBAH. Healthy BALB/c mice were randomly allocated\ninto 4 groups and subcutaneously injected with saline, IL&aC (the mixture of IL-12\nand aCTLA-4), RB (the mixture of RBC and BaO ), and IL/aC@RBAH once a week.\nexhibited negligible body weight changes similar to those in the saline group (Fig. R4a),\nindicating minimal systemic toxicity or metabolic perturbations induced by\nIL/aC@RBAH. Moreover, the blood and major organs were collected after 2 months\nof monitoring. The blood biochemical parameters of BUN (Fig. R4b), CREA (Fig. R4c)\nand ALT (Fig. R4d) were maintained within the normal range in all the groups.\nMoreover, distinct from the abnormal AST level in the RB and IL&aC groups, that in\nthe IL/aC@RBAH group was significantly lowered within safe bounds (Fig. R4e).\nAll the above results evidenced the normal hepatic and renal function in the IL/aC@RBAH-\ntreated mice. Additionally, H&E analysis presented negligible histomorphological\nabnormalities of major organs in the IL/aC@RBAH group, in sharp contrast to\nmoderate inflammation of the liver tissues in the IL&aC and RB groups (Fig. R4f),\nprobably triggered by the burst release of aCTLA-4/IL-12 and Ba2+. All the above\nstudies confirmed the favorable long-term biosafety of IL/aC@RBAH with negligible\nrisks of IL-12-related cytokine storm. Following the reviewer’s kind suggestions, we\nhave included Fig. R9 as Supplementary Fig. 21 and Fig. R4 as Supplementary Fig.\n29 in the revised supplementary information and added relevant discussions to improve\nthe biosafety assessments in the revised manuscript.\nFig. R4 a Body weight changes of healthy BALB/c mice after different treatments once\na week for 2 months. b, c Kidney function indexes including BUN (b) and CREA (c)\nin healthy BALB/c mice exposed to different treatments once a week for 2 months. d,\ne Liver function indexes comprising ALT (d) and AST (e) in healthy BALB/c mice\nexposed to different treatments once a week for 2 months. The normal range of liver\nfunction indexes was marked by dash lines. f H&E staining analysis of major organs\n(heart, liver, spleen, lung, and kidney) excised from healthy BALB/c mice exposed to\ndifferent treatments once a week for 2 months. Scale bar, 100 μm. Data in a-e were\npresented as mean ± SD. n = 3 biologically independent samples in a-e.\nReviewer #4 (cancer nanomedicine, also covers immune):\nDr. Fan et al. developed an injectable gel encapsulating interleukin-12 and anti-CTLA-\n4 co-engineered red blood cells. This gel was self-assembled in situ within tumor\nmicroenvironments to increase levels of O , anti-CTLA-4, and IL-12, thereby\n2\nactivating multistage immune responses. While they present an interesting strategy,\ntheir approach is quite complex and it is unclear whether this is translationally relevant,\nespecially since they give local injection.\nResponse: Thanks for the reviewer’s constructive comments. Despite the complexity\nof the therapeutic strategy in multistage immunity, the local injection of IL/aC@RBAH\nis convenient and feasible in various solid tumors, including the orthotopic 4T1 tumor\nand subcutaneous Panc02 tumor models in our study. Moreover, due to the local\ninjection of IL/aC@RBAH, the therapeutic approach exhibited excellent biosafety with\nnegligible systemic toxicity (Figs. 4d-i), which would be beneficial to its potential\nclinical translation. Skin cancer, as one of the most prevalent malignancies worldwide,\ndemonstrates particularly high mortality due to its aggressive and metastatic nature[1].\nBeyond the substantial economic burden, those subcutaneous cancers, including basal\ncell carcinoma, squamous-cell carcinoma, and melanoma, impose cumbersome\npsychological distress that significantly impairs patients’ quality of life[2]. Current\nclinical practice prioritizes surgical excision as the primary treatment modality.\nHowever, incomplete tumor resection with residual malignant tissues generally triggers\nsustained proliferation, substantially elevating the risk of local recurrence[3]. Owing to\nthe merit of intratumoral injectability of the self-assembled hydrogel, the therapeutic\nmodality of RT + IL/aC@RBAH holds great promise for different types of skin cancers\nin the future, thus exemplifying a potential shift in clinical translation.\nEconomically, the engineered RBCs exhibit distinct advantages[4], including wide\navailability, high prevalence, and low-cost chemical modification. Modified RBCs\nmaintain stable morphological properties for up to 42 days at 2-6 °C[5], facilitating\nconvenient storage and transportation. Moreover, the chemically engineered RBCs\nexhibit enhanced safety and controllability, with significantly lower production costs\ncompared to genetically engineered cells. Notably, the quality and yield of RBCs are\nboth stable and reproducible at laboratory and bench scales. All the virtues of RBC\nengineering endow itself with potential scalability. Additionally, the introduced barium\nperoxide (BaO ) is synthesized via a hydrolytic precipitation method, which also offers\n2\nthe advantages of low cost and economical production[6]. The as-prepared BaO\n2\nnanoparticles present a highly consistent bamboo-like morphology and excellent batch-\nto-batch uniformity during our study. Moreover, sodium alginate exhibits excellent\npotential as a biomaterial due to its high structural stability, mature production\nprocesses, and wide availability from brown algae (e.g., laminaria japonica, lessonia,\netc.)[7]. This low-cost material offers convenient transportability and superior economic\nbenefits. Moreover, the degraded nontoxic polysaccharides from Alg-based hydrogel\ndo not participate in normal metabolic processes, which are conducive to the\ndevelopment requiring high biological compatibility and environmental sustainability.\nTherefore, alginate-based hydrogels, featuring low production costs and FDA-approved\nexcellent biocompatibility[8], illuminate a promising feasibility for clinical translation.\nIn terms of bioengineering, aCTLA-4 was successfully loaded into RBCs to\ngenerate aC@RBC via hypotonic dialysis, which is a well-established method\nbenefiting from existing automatic loading instruments, thereby reducing production\ncosts and enhancing clinical translation potential. Subsequently, IL-12 was anchored\nonto the surface of RBCs through membrane insertion to yield IL/aC@RBC, which\nmaintained structural integrity after 7-day storage at 4 °C (Supplementary Fig. 4),\nindicating suitability for extended transportation and storage.\nCollectively, the elaborately designed self-assembled hydrogel is a simple and\nviable platform with comprehensive economic and logistical considerations. Following\nthe reviewer’s kind suggestions, we have included relevant discussion in the revised\nmanuscript to propel its clinical translation.\nReferences:\n[1] Hasan, N. et al. Skin cancer: understanding the journey of transformation from\nconventional to advanced treatment approaches. Mol Cancer 22, 168 (2023).\n[2] Emery, J. et al. Management of common clinical problems experienced by survivors\nof cancer. Lancet 399, 1537-1550 (2022).\n[3] Xie, H. et al. Validation of the proposed international association for the study of\nlung cancer residual tumor classification to upgrade extracapsular extension of tumor\nin nodes from R0 to incomplete resection. J Thorac Oncol 19,130-140 (2024).\n[4] Nguyen, PHD. et al. Advances in Drug Delivery Systems Based on Red Blood Cells\nand Their Membrane-Derived Nanoparticles. ACS Nano 17, 5187-5210 (2023).\n[5] Koch, CG. et al. Real Age: Red Blood Cell Aging During Storage. Ann Thorac Surg\n107, 973-980 (2019).\n[6] Zhang, M. et al. Radiation-assisted metal ion interference tumor therapy by barium\nperoxide-based nanoparticles. Mater. Horiz 6, 1034-1040 (2019).\n[7] Tagliapietra, B. L. et al. Brown algae and their multiple applications as functional\ningredient in food production. Food Res Int 167, 112655 (2023).\n[8] Basu, T. et al. Cross-linker-free sodium alginate and gelatin hydrogels: a multiscale\nbiomaterial design framework. J Mater Chem B 10, 3614-3623 (2022).\nI have the following comments.\n- The authors claimed that “In response to the acidic TME, the in situ injected BaO\n2\nundergoes hydrolysis to generate H O and Ba2+, followed by the rapid reaction of Ba2+\n2 2\nwith sodium alginate (Alg) to afford a biocompatible hydrogel.” However, both in vivo\ndegradation and in situ retention studies were performed on healthy mice (Figs. 4a-c),\nshowing good retention in vivo for up to 14 days. How did the gel form under normal\nphysiological conditions (pH 7.4)? This seemed inconsistent with Fig. 3b and the\nauthors’ hypothesis. Moreover, without acid stimulation, how did the system degrade?\nIf BaO could decompose under pH 7.4, how to ensure the long-term stability of this\n2\ngel system?\nResponse: Thanks for the reviewer’s professional comments. Considering the sight\ninterference of tumor inoculation on the observation of hydrogel volume changes,\nthe\nIL/aC@RBAH hydrogel had to be implanted under the skins of healthy mice for\nperiodic surgical monitoring of in vivo degradation. In regard to the hydrogel formation\nin the skin (Fig. 4a and Supplementary Fig. 20, which are Figs. 4a-c in the original\nmanuscript), IL/aC@RBAH was in vitro immersed in the mildly acidic saline (pH 6.5)\nand then subcutaneously injected into healthy BALB/c mice for in vivo degradation\nevaluation. Moreover, the feasibility of the experiment was proven by the gelation\nprocess of IL/aC@RBAH in saline at pH 6.5. Notably, the hydrogel could be formed\nfor merely 2.39 min in the buffer at pH 6.5 (Fig. R10), which could be of benefit to the\nrapid formation of the IL/aC@RBAH hydrogel under the skins in the acidic condition.\nFig. R10 Changes of G’ and G’’ of IL/aC@RBAH at pH 7.4 and pH 6.5.\nAs to the degradation behavior of the hydrogel in the physiological environment\n(pH 7.4) (Fig. 4a), the natural polymer sodium alginate (Alg), cross-linked with Ba2+\nin IL/aC@RBAH, could slowly react with metal ions (like Ca2+) in the body[1], thus\ninducing dilatory degradation into nontoxic polysaccharides. The biocompatible\ndegradation process, irrelevant to acidity, persisted for nearly two weeks, thereby\nevidencing the relative stability of the hydrogel and mitigating the potential risks of\nlong-term accumulation. Collectively, the investigation of in vivo degradation and in\nsitu retention studies were consistent with our hypothesis of the acidity-responsive\nformation and dilatory biodegradability of the IL/aC@RBAH hydrogel. To avert\nunnecessary confusion, we have supplemented relevant description in the revised\nmanuscript.\nAdditionally, to further consummate the hypothesis of acidity-responsive\nformation and intratumoral long-term retention of the hydrogel, the mixture of Cypate-\nlabeled IL-12 and aCTLA-4 (IL-Cp&aC), IL-Cp/aC@RBC, and IL-Cp/aC@RBAH\nwere respectively injected into the tumors of orthotopic 4T1 tumor-bearing mice for\nreal-time monitoring via whole-animal in vivo fluorescence imaging. Similar to the in\nsitu retention behaviors of the formulations injected in the skin (Supplementary Fig.\n20), IL-Cp&aC was rapidly eliminated from the tumors within 1 day, along with fast\nclearance of IL-Cp/aC@RBC within 5 days (Fig. R11). Consistently, visible Cypate\nfluorescence of IL-Cp/aC@RBAH could still be tracked at the tumor site on day 14\npost-injection. Collectively, all those results confirmed the brilliant in situ retention\ncapability of IL/aC@RBAH with favorable stability. In the revised manuscript, we have\nincluded Fig. R10 as Fig. 3c, Fig. R11 as Figs. 4b, c and added relevant description.\nFig. R11 (a) Whole-animal in vivo fluorescence imaging and (b) corresponding\nfluorescence quantification of orthotopic 4T1 tumor-bearing mice after intratumoral\ninjection of IL-Cp&aC, IL-Cp/aC@RBC, and IL-Cp/aC@RBAH.\nReferences:\n[1] Zhao, Z. et al. Capturing magnesium ions via microfluidic hydrogel microspheres\nfor promoting cancellous bone regeneration. ACS Nano 15, 13041-13054 (2021).\n- Please plot the gelatin time of the system vs. pH.\nResponse: Thanks for the reviewer’s valuable advice. The gelatin time of\nIL/aC@RBAH at pH 6.5 and 7.4 was measured using a rheometer (Discovery HR-2,\nTA Instruments, USA) equipped with a 12-mm-diameter parallel-plate geometry at a\n0.6 mm gap with constant temperature at 25 °C. As shown in Fig. R10, the hydrogel\nformed in merely 2.39 min in the buffer at pH 6.5, while no gel formation was observed\nat pH 7.4. This result demonstrated that the hydrogel of IL/aC@RBAH could be rapidly\nformed in response to the acidic condition. In the revised manuscript, we have included\nFig. R10 as Fig. 3c and added relevant description.\nFig. R10 Changes of G’ and G’’ of IL/aC@RBAH at pH 7.4 and pH 6.5.\n- The authors only mentioned that 500 μL of RBCs was used for the gel system. Then\nhow much O could be converted by catalase from these cell mimics? Could the authors\n2\nprovide more detailed preparation and measurement protocols, including the specific\namounts of RBCs used and the quantification of O ? A quantitative measurement of O\n2 2\ngeneration, rather than enzyme activity, is the key evidence to support the hypothesis.\nResponse: Thanks for the reviewer’s constructive question. 500 μL of RBCs that the\nreviewer mentioned were merely utilized for the synthesis of IL/aC@RBC. In brief,\n500 μL of RBC suspension (4 × 109/mL) and 85 μL of aCTLA-4 (5 mg/mL) were mixed\nand then dialyzed (3.5 kD MWCO) against 40 mL of hypotonic buffer. Subsequently,\nthe buffer was substituted with 35 mL of PBS buffer for balance and then dialyzed by\n40 mL of hypertonic buffer. The resealed aC@RBC was collected by centrifugation\n(1000 g, 4 °C, 10 min) and washed 3 times with PBS buffer to remove unentrapped\naCTLA-4. Then the resultant aC@RBC was added with 100 μL of DSPE-PEG2000-\nIL-12 (5 mg/mL) and incubated in PBS for IL-12 intercalation at 37°C. The engineered\nIL/aC@RBC was collected by centrifugation (1000 g, 4 °C, 10 min) and washed 3 times\nwith PBS buffer to remove unentrapped DSPE-PEG2000-IL-12. For the synthesis of\nthe final gel system of IL/aC@RBAH, IL/aC@RBCs (109/mL) were mixed with BaO\n2\n(6 mg/mL) and Alg (3% w/v) in the mildly acidic environment (pH 6.5). The detailed\npreparation approaches have been supplemented in the revised manuscript.\nAccording to the reviewer’s valuable suggestions, the O generation from 109 cells\n2\nof IL/aC@RBC and native RBC (nRBC) were quantitatively investigated to affirm CAT\nreservation on IL/aC@RBC. Briefly, 109 cells of IL/aC@RBC and nRBC were\nrespectively dispersed in 5 mL of PBS buffer containing 500 μM H O . Then, the\n2 2\nchanges in O concentration were determined in 2 minutes by a dissolved oxygen\n2\nanalyzer. The results manifested that the O production from IL/aC@RBC and nRBC\n2\nexhibited negligible difference, with the amount at 19.83 mg/L and 17.56 mg/L,\nrespectively, demonstrating that the IL-12/aCTLA-4 encapsulation would not disrupt\nthe CAT activity of RBC for O generation (Fig. R12). The O measurement protocol,\n2 2\nincluding the specific amounts of RBCs used and the quantification of O , has been\n2\nsupplemented in the revised supplementary information. In the revised manuscript, we\nhave included Fig. R12 as Fig. 2g and added relevant description.\nFig. R12 In vitro O production from nRBC and IL/aC@RBC. n = 3, mean ± SD.\n2\n- The authors claimed to have used aCTLA-4 but did not indicate its source. They\nmentioned that PD-L1 (B7-H1) (Cat# BE0101) was purchased from BioXcell, although\nthis study did not utilize it. Please clarify the source of aCTLA-4 and the relevance of\nPD-L1 in the context of this study. Besides, were the FITC-aCTLA4 get? Please clarify\naCLTA-4 ELISA detection kit in serum as the kit was quite rare.\nResponse: Thanks for the reviewer’s detailed reminder. We apologize for inadvertently\nmislabeling the commercial information for aCTLA-4 as PD-L1. Thanks for identifying\nthis error. The InVivoMAb anti-mouse CTLA-4 (CD152) antibody (Cat# BE0131, Bio\nX Cell) was used throughout this study. aCTLA-4-FITC was prepared in-house by\nlabeling the aCTLA-4 antibody using Beyotime’s FAST FITC Labeling Kit (Cat#\nP0639M) according to the manufacturer’s protocol. Additionally, the aCTLA-4 ELISA\ndetection kit (Cat# HS1392-Mu) was purchased from Shanghai Hengyuan\nBiotechnology Company. All noted information has been updated in the revised\nsupplementary information.\n- Detailed protocols for labeling both IL-12 and aCTLA-4 should be provided. Please\nalso include spectral data (such as NMR, IR, Mass, GPC, etc.) to confirm labeling and\nmodification. A release study in PBS (pH 7.0) is also needed. Additionally, since\naCTLA-4 has a molecular weight of 150 kDa, how could it be detected in dialysates\nfrom 3 kD MWCO dialysis devices?\nResponse: Thanks for the reviewer’s detailed questions. In our initial submission,\ndetailed protocols for the labeling of IL-12 for IL-12-AMCA have been included in the\nmethods section of the manuscript. In regard to aCTLA-4 labeling for aCTLA-4-FITC,\n100 μg of aCTLA-4 was dissolved in Na CO -NaHCO buffer at pH 9.0 (0.1 M, 100\n2 3 3\nμL). After 10 min of stirring at 4 °C, 1.03 μg of FITC was added and further stirred at\n4 °C for 18 h. Finally, NH Cl (5 M, 1 μL) was added to cease the reaction. Free FITC\n4\nwas removed by ultrafiltration (3.0 kD molecular weight cut off (MWCO), Millipore\nSigma) with the endpoint as determined by the visual clarity of the filtrate. The detailed\nprotocol of aCTLA-4 labeling was supplemented in the methods section of the revised\nmanuscript.\nRegarding spectra data for the labeling and modification verification, both IL-12\nand aCTLA-4 are proteins with structural complexity and large molecular weights of\napproximately 75 kDa and 150 kDa, respectively, thus precluding their fluorescent\nconjugate analysis by NMR, IR, or MALDI-TOF MS. Although the molecular weight\nranges of IL-12-AMCA and aCTLA-4-FITC can be theoretically determined by GPC,\nthe small fluorescent tags are difficult to be detected in the proteins with large molecular\nweights. Moreover, the amount of protein samples required for GPC is quite substantial,\nthus making the technique inadequate to analyze the fluorescent labeling of those\nproteins.\nTo confirm the respective modification of AMCA and FITC on IL-12 and aCTLA-\n4 for IL-12-AMCA and aCTLA-4-FITC, respectively, we conducted UV-Vis absorption\nspectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-\nPAGE) analysis to track the changes of the labeled proteins. As expected, the UV-Vis\nabsorption spectra delineated the distinctive absorption peak of IL-12 (280 nm) and\nAMCA (345 nm) in IL-12-AMCA (Fig. R13a), as well as the characteristic absorption\npeak of aCTLA-4 (280 nm) and FITC (495 nm) in aCTLA-4-FITC (Fig. R13b). More\nimportantly, as presented in the SDS-PAGE gel, the fluorescence signal of FITC was\nalmost colocalized with aCTLA-4 at around 130 kDa (Fig. R14a), along with the\ncolocation of IL-12 and AMCA at approximately 45-55 kDa and 25-28 kDa,\nrespectively (Fig. R14b). Overall, the results substantially verified the successful\nsynthesis of aCTLA-4-FITC and IL-12-AMCA. According to the reviewer’s kind\nadvice, we have respectively included Figs. R13, R14 as Supplementary Fig. 1 in the\nrevised supplementary information.\nFig. R13 a UV-Vis absorption spectra of AMCA, IL-12, and IL-12-AMCA. b UV-Vis\nabsorption spectra of FITC, aCTLA-4, and aCTLA-4-FITC.\nFig. R14 a SDS-PAGE protein analysis and corresponding fluorescence co-localization\nimages of aCTLA-4 and aCTLA-4-FITC. b SDS-PAGE protein analysis and\ncorresponding fluorescence co-localization images of IL-12 and IL-12-AMCA.\nDue to the fact that the pH value of the physiological environment is admittedly\n7.4 instead of 7.0, we have supplemented the release profiles of aCTLA-4 and IL-12\nfrom IL/aC@RBAH at pH 7.4 to mimic the physiological environment. Due to the\nanchorage of IL-12 on RBC, IL-12 exhibited similar dilatory release kinetics at pH 6.5\nand pH 7.4 (Fig. R15), together with slow degradation of the hydrogel independent of\nacidity. Contrarily, the release rate of aCTLA-4 was significantly accelerated at pH 6.5\ncompared to that at pH 7.4, which was possibly attributed to the acidity-induced BaO\n2\ndegradation to trigger H O production for continuous O -mediated pore formation on\n2 2 2\nRBC. However, the pores on RBC were no longer increased at pH 7.4, thus delaying\nthe release kinetics of aCTLA-4 in the neutral environment. Overall, the self-assembled\nIL/aC@RBAH hydrogel exhibited a sequential aCTLA-4/IL-12 release profile in\nmildly acidic and neutral environments. According to the reviewer’s kind advice, we\nhave supplemented relevant description in the revised manuscript and included Fig.\nR15 as Fig. 3i in the revised supplementary information.\nFig. R15 Cumulative release profiles of aCTLA-4 and IL-12 from IL/aC@RBAH at\npH 7.4 and pH 6.5 within 48 h. n = 3, mean ± SD. P values were calculated via two-\nway ANOVA with Tukey’s post hoc test.\nIn terms of the analytical methods for the release behaviors of IL-12 and aCTLA-\n4, we examined the contents in the leaching solutions of IL/aC@RBAH as reported in\nthe previous studies[1], instead of using the dialysis method. Briefly, a 40-µm cell\nstrainer containing IL/aC@RBAH was embedded in a six-well plate with 5 mL of PBS\nat pH 6.5 and pH 7.4. After 0, 1, 2, 4, 6, 8, 16, 24, and 48 h of incubation, the\nconcentrations of IL-12-AMCA and aCTLA-4-FITC in PBS were respectively detected\nby a microplate reader (Molecular Devices, USA) at the absorption wavelength of 350\nnm and 488 nm. The dialysis method was instead utilized to investigate the in vitro\nrelease of Ba2+ from IL/aC@RBAH, which was also stated in our initial submission.\nReferences:\n[1] Hu, Q. et al. Inhibition of post-surgery tumour recurrence via a hydrogel releasing\nCAR-T cells and anti-PDL1-conjugated platelets. Nat Biomed Eng 5, 1038-1047 (2021).\n- Did the H O produced by BaO hydrolysis and the ROS induced by RT directly\n2 2 2\ndamage tumor tissue? If so, what was the killing efficiency, and how does this compare\nto immune-mediated killing?\nResponse: Thanks for the reviewer’s valuable questions. To differentiate the direct\ntumor damage and immune-mediated killing, we randomly allocated the orthotopic 4T1\ntumor-bearing mice into 6 groups, which were then treated with saline, RT, RT + RBAH,\nIL&aC, RT + IL&aC, and RT + IL/aC@RBAH, respectively. Similar to the modestly\ndirect tumor damage induced by H O production and ROS generation in the RT +\n2 2\nRBAH group, the tumor growth rates in the IL&aC group were also moderately\nsuppressed (Figs. R8a-d), manifesting that simple combination of RT and BaO -\n2\ninduced direct tumor damage or dual cytokines-mediated immunosuppressive\nalleviation could hardly induce effective tumor elimination. Comparatively, the RT +\nIL&aC treatment showed much more prominent tumor inhibition than the RT + RBAH\nand IL&aC groups, possibly attributed to the combined therapeutic strategy of RT and\nimmunotherapy, thus enhancing RT-initiated DAMP release for potentiating antitumor\nimmune responses. Excitingly, the mice in the RT + IL/aC@RBAH group presented the\nuppermost tumor regression with a prolonged lifespan of at least 2 months in about 80%\nof the mice (Fig. R8e), thus evidencing the supreme antitumor efficacy of RT +\nIL/aC@RBAH in the synergy of Ba/O -supplied radiosensitization and aCTLA-4/IL-\n2\n12-mediated multistage immune responses. According to the reviewer’s kind advice,\nwe have supplemented relevant description in the revised manuscript and included Fig.\nR8 as Supplementary Fig. 35 in the revised supplementary information.\nFig. R8 a, b Whole-animal in vivo bioluminescence imaging (a) and corresponding\nbioluminescence quantification (b) of orthotopic 4T1-luciferase (4T1-luc) tumor-\nbearing mice after treated with saline, RT, RT + RBAH, IL&aC, RT + IL&aC, and RT\n+ IL/aC@RBAH, respectively. c, d Average tumor growth curves (c) and individual\ntumor dynamics (d) of orthotopic 4T1 tumor-bearing mice subjected to different\ntreatments. e Survival curves of orthotopic 4T1 tumor-bearing mice subjected to\ndifferent treatments. Data in b-e were presented as mean ± SD. n = 5 biologically\nindependent samples in b-e. Statistical significance was determined using two-way\nANOVA (c).\n- The authors claimed O -mediated pore formation in RBC membranes. Please provide\n2\ndirect evidence to support this claim, rather than relying solely on release data, to\nimprove the rigor of the draft. Additionally, will H O and ROS trigger pore formation\n2 2\non the RBC membrane, and how did the authors design the system to avoid this?\nResponse: Thanks for the reviewer’s constructive suggestions. According to the\nreviewer’s kind advice, we have supplemented the scanning electron microscopy (SEM)\nimages of IL/aC@RBC treated with RT, H O , and BaO in the mildly acidic\n2 2 2\nenvironment. As reported, RT-triggered ROS, including O •−, H O , and ·OH, induce\n2 2 2\ndamage to cellular life processes primarily due to its oxidative properties[1]. However,\nthe IL/aC@RBC exposed to single RT showed negligible pore formation with intact\ncellular morphology (Fig. R16), which signified that RBC membrane could be hardly\nperturbed during ROS generation. Notably, due to the intrinsic catalase (CAT) on\nIL/aC@RBC, H O could be rapidly catalyzed into O (Fig. R12), which made it\n2 2 2\nimpossible to rule out whether H O itself could cause the pore formation on\n2 2\nIL/aC@RBC. Similar to the cells subjected to BaO , the IL/aC@RBC treated with H O\n2 2 2\npresented notable pores with distortions in appearance, which was attributed to CAT-\nmediated O formation from H O . Overall, it could be concluded that the pore\n2 2 2\nformation on IL/aC@RBC was induced by H O -derived O generation. To avoid\n2 2 2\nunnecessary confusion on the claim, the SEM images have been included as\nSupplementary Fig. 11 in the revised supplementary information and relevant\nstatements have also been rectified in the revised manuscript. In the revised manuscript,\nwe have included Fig. R12 as Fig 2g and added relevant description.\nFig. R16 Representative SEM images of IL/aC@RBC exposed to RT, H O , or BaO\n2 2 2\nin a mildly acidic environment (pH 6.5). Scale bar, 2 μm.\nFig. R12 In vitro O production from nRBC and IL/aC@RBC. n = 3, mean ± SD.\n[1] Mittler R. ROS Are Good. Trends Plant Sci 22, 11-19 (2017).\n- The authors claimed that there was a sequence of immune actions by the gel system,\nthat ultimately achieved a butterfly effect, a feature of the paper. Please provide more\nevidence to illustrate this effect, including data on the sequential initiation of the\nimmune response in vivo. Presently, this hypothesis is not consistent with in vivo data.\nThe authors claimed that “rapid release of aCTLA-4 to relieve tumor\nimmunosuppression. Subsequently, IL-12 anchored on RBC, acting as a ‘relay baton’,\nis dilatorily released and interacts with T/NK cells within the TME to induce IFN-γ-\ndependent antitumor immunity”. However, in Figs5i-p, comparing G4 and G5, aCTLA-\n4 alone did not significantly increase T cells, and IL-12 alone also did not significantly\nincrease IFN-γ.\nResponse: Thanks for the reviewer’s thorough comments. To elucidate the kinetics of\naCTLA-4 and IL-12 release, we encapsulated both IL-12 and aCTLA-4 within RBCs\nfor IL&aC@RBAH as control. Then, the release behaviors of IL-12 and aCTLA-4 from\nIL&aC@RBAH and IL/aC@RBAH were investigated. As forecasted, almost 40% of\nIL-12 and aCTLA-4 in IL&aC@RBAH were both swiftly released within 48 h (Fig.\nR1), possibly due to the simple diffusion of the mixture of IL-12 and aCTLA-4 from\nthe formed pores on RBCs. Contrarily, IL/aC@RBAH presented a sequential release\nbehavior with rapid release of approximately 40% aCTLA-4 from the oxygen-induced\npores on the RBC membrane within 48 h and slow release of less than 20% IL-12 from\nthe enlarged channels in the crosslinked network of the degrading hydrogel within 48\nh. Collectively, all those results affirmed the sequential aCTLA-4/IL-12 release from\nIL/aC@RBAH, thus building the foundation for the multistage initiation of the immune\nresponses in vivo.\nFig. R1 Release profiles of IL-12 and aCTLA-4 from IL&aC@RBAH and\nIL/aC@RBAH at pH 6.5 within 48 h. n = 3, mean ± SD.\nTo verify the multistage immune responses elicited by the sequential aCTLA-4/IL-\n12 release from IL/aC@RBAH without regard to the potential influence of hydrogels\non the immune evaluation, the orthotopic 4T1-tumor bearing mice were treated with\nsaline, RT, RT + IL-aC (IL-12 prior to aCTLA-4), RT + IL&aC (the mixture of IL-12\nand aCTLA-4), and RT + aC-IL (IL-12 subsequent to aCTLA-4), respectively. Distinct\nfrom the slight upregulation of CD8+ T cells induced by RT-triggered ICD,\nthe\nintratumoral level of those cells was mildly elevated in the RT + IL-aC and RT + IL&aC\ngroups (Figs. R2a, b), demonstrating the modest immune activation in the primary and\nrelay stages. This limitation was likely attributed to the rapid diffusion and loss of IL-\n12 from tumor, which was insufficient to sustain downstream activation during the relay\nstage. Notably, the RT + aC-IL treatment triggered the utmost increase in CD8+ T cells,\nalong with the lowest Treg ratio and the highest NK cell level among all the groups\n(Figs. R2c, d), manifesting the strongest antitumor immune initiation by the sequential\naCTLA-4/IL-12 supply parallel to RT + IL/aC@RBAH. Additionally, the IFN-γ\nproduction in CD8+ T cells, CD4+ T cells, and NK cells in the RT + aC-IL group were\nall at the highest level among all the groups (Figs. R2e-g), further evidencing the most\npotent immune responses through aCTLA-4-mediated primary immunosuppression\nalleviation and relay IL-12-induced immunoactivation. Even more impressive was that\nthe RT + aC-IL treatment likewise elicited the uppermost level of mature DCs, which\nwas nearly 1.44- and 1.27-fold higher than that in the RT + IL-aC and RT + IL&aC\ngroups (Figs. R2h, i). The result further elucidated the most powerful antigen-\npresenting initiation of the sequential aCTLA-4/IL-12 supply in the RT + aC-IL\ntreatment for antitumor immunity through the DC cell-intrinsic IL-12/IFN-γ positive\nfeedback loop.\nFig. R2 a, b Representative flow cytometry plots (a) and quantification (b) of cytotoxic\nT lymphocytes (CD8+ in CD45+CD3+ cells) in orthotopic 4T1 tumors after different\ntreatments. c-g Quantitative analyses of Tregs (CD4+CD25+ in CD45+CD3+ cells) (c)\nand NK cells (CD3-CD49b+ in CD45+ cells) in orthotopic 4T1 tumors after different\ntreatments. e-f Quantification of IFN-γ production by CD8+ T cells (IFN-γ+ in\nCD45+CD3+CD8+ cells) (e), CD4+ T cells (IFN-γ+ in CD45+CD3+CD4+ cells) (f), and\nNK cells (IFN-γ+ in CD45+CD3-CD49b+ cells) (g). h, i Representative flow cytometry\nplots (h) and quantification (i) of mature DC (CD80+CD86+ in CD11c+MHC Ⅱ+ cells)\nin orthotopic 4T1 tumors after different treatments. Data in b-g, i were presented as\nmean ± SD. n = 3 biologically independent samples in b-g, i. P values were calculated\nvia student’s t-test.\nTo explore the impacts of sequential cytokine supply on systemic antitumor\nimmunity, we collected the tumor-draining lymph nodes (TDLNs) of orthotopic 4T1\ntumor-bearing mice subjected to different treatments. Both the levels of CD8+ T cell\ninfiltration and activation were at the peak in the RT + aC-IL group (Figs. R3a-d).\nConcomitantly, the migration of both cDC1 and cDC2 to TDLNs were maximally\nelevated in the RT + aC-IL group, quite different from those in the other groups (Figs.\nR3e, f), further demonstrating the supreme antitumor initiation by the sequential\naCTLA-4/IL-12 supply. Overall, all the results firmly affirmed the virtues of systemic\nmultistage immune responses induced by the sequential aCTLA-4/IL-12 release from\nRT + IL/aC@RBAH. We have included Fig. R1 as Supplementary Fig. 33, Figs. R2b-\ng and Fig. R2i as Figs. 6e-k, Fig. R3c as Supplementary Fig. 34, and Figs. R3d-f as\nFigs. 6l-n. Relevant description has been added in the revised manuscript.\nFig. R3 a Representative flow cytometry plots of cytotoxic T lymphocytes (CD8+ in\nCD45+CD3+ cells) in TDLNs of orthotopic 4T1 tumor-bearing mice post different\ntreatments. b Representative flow cytometry plots of activated CD8+ T lymphocytes\n(CD69+ in CD3+CD8+ cells) in TDLNs of orthotopic 4T1 tumor-bearing mice post\ndifferent treatments. c-f Quantitative analyses of cytotoxic T lymphocytes (CD8+ in\nCD45+CD3+ cells) (c), activated CD8+ T lymphocytes (CD69+ in CD3+CD8+ cells) (d),\ncDC1 (CD103+ in CD11c+MHC Ⅱ+ cells) (e) and cDC2 (CD11b+ in CD11c+MHC Ⅱ+\ncells) (f) in TDLNs of orthotopic 4T1 tumor-bearing mice post different treatments.\nData in c-f were presented as mean ± SD. n = 3 biologically independent samples in c-\nf. P values were calculated via student’s t-test.\nIn regard to the results in Figs. 5j-q (which are Figs. 5i-p in the original manuscript)\nthat there was negligible difference between G4 and G5, it could be attributed to the\nDC cell-intrinsic IL-12/IFN-γ positive feedback loop. It meant that the level of\ncytotoxic T cells and IFN-γ production could be indirectly elevated by aCTLA-4-\nenhanced antigen presentation through DCs, as well as being directly increased by IL-\n12-mediated T cell proliferation and Th1 differentiation in different stages. However,\nneither RT + aC@RBAH nor RT + IL@RBAH could induce effective antitumor\nimmune responses, owing to the insufficient initiation focused on one single stage of\nRIT. In sharp contrast, RT + IL/aC@RBAH could initiate significant upregulation of\nboth cytotoxic T cell infiltration and IFN-γ production through sequential aCTLA-4/IL-\n12 release respectively in the primary stage and relay stage of RIT for boosted antitumor\nimmunity.\n- Local injection of IL-2 and aCTLA-4 plus RT should be included as a control.\nResponse: Thanks for the reviewer’s valuable advice. Following the reviewer’s kind\nsuggestions, local injection of IL-12 and aCTLA-4 plus RT (RT + IL&aC) have been\nset as a control for comprehensive evaluation in the therapeutic efficacy of RT +\nIL/aC@RBAH. The orthotopic 4T1 tumor-bearing mice were randomly allocated into\n6 groups and respectively treated with saline, RT, RT + RBAH, IL&aC, RT + IL&aC,\nand RT + IL/aC@RBAH. Compared to the slight tumor inhibition in the RT group, the\ntumor volumes in the RT +RBAH and IL&aC groups were moderately suppressed (Figs.\nR8a-d), elucidating the inadequate antitumor efficacy of the sole Ba/O -supplied\n2\nradiosensitization by RBAH or the single antitumor immune responses induced by\nIL&aC. Comparatively, the RT + IL&aC treatment showed much more prominent\ntumor inhibition than the RT + RBAH and IL&aC groups, possibly ascribed to the\ncombined therapeutic strategy of RT and immunotherapy. Notably, the mice in the RT\n+ IL/aC@RBAH group presented the utmost tumor regression with a prolonged\nlifetime of at least 2 months in about 80% of the mice (Fig. R8e), thus substantiating\nthe supreme antitumor efficacy of RT + IL/aC@RBAH-induced multistage RIT.\nAccording to the reviewer’s kind advice, we have supplemented relevant description in\nthe revised manuscript and included Fig. R8 as Supplementary Fig. 35 in the revised\nsupplementary information.\nFig. R8 a, b Whole-animal in vivo bioluminescence imaging (a) and corresponding\nbioluminescence quantification (b) of orthotopic 4T1-luciferase (4T1-luc) tumor-\nbearing mice after treated with saline, RT, RT + RBAH, IL&aC, RT + IL&aC, and RT\n+ IL/aC@RBAH, respectively. c, d Average tumor growth curves (c) and individual\ntumor dynamics (d) of orthotopic 4T1 tumor-bearing mice subjected to different\ntreatments. e Survival curves of orthotopic 4T1 tumor-bearing mice subjected to\ndifferent treatments. Data in b-e were presented as mean ± SD. n = 5 biologically\nindependent samples in b-e. P values were calculated via two-way ANOVA with\nTukey’s post hoc test.\n- Will local RT treatment and/or ROS damage the gel structure? If so, how could cargo\nburst release be avoided?\nResponse: Thanks for the reviewer’s constructive questions. Mechanistically, the stable\nnetwork structure of the gel is constructed on the covalent cross-linking between Ba2+\nand Alg, which is inherently not a ROS-sensitive linkage. Moreover, the RT treatment\ncould not induce the pore formation on RBC membrane (Fig. R16), suggesting minimal\nimpact on the aCTLA-4 release kinetics from IL/aC@RBAH. To validate the\nassumption, the release profiles of aCTLA-4 and IL-12 from IL/aC@RBAH were\ninvestigated. Similar to those in the mildly acidic environment (pH 6.5) without the RT\ntreatment, aCTLA-4 and IL-12 in IL/aC@RBAH at pH 6.5 with the RT treatment\nlikewise showed sequential release behaviors within 48 h (Fig. R17). Notably, nearly\n40% of aCTLA-4 was rapidly released from IL/aC@RBAH within 48 h with or without\nthe RT treatment. Besides, IL-12 in IL/aC@RBAH with or without the RT treatment\nalso showed a similar release behavior, thus evidencing the effective anchorage on RBC,\nas well as the excellent stability of the hydrogel under RT irradiation. Consequently, it\ncould be concluded that the RT treatment could not disrupt the aCTLA-4 release\nthrough the O -induced pores on RBC membrane while showing negligible impact on\n2\nthe cross-linked network of the hydrogel, thereby controllably ensuring sequential drug\nrelease without the potential risk of burst IL-12 leakage.\nFig. R17 Release profiles of aCTLA-4 and IL-12 from IL/aC@RBAH at pH 6.5 with\nor without the RT treatment. n = 3, mean ± SD. P values were calculated via two-way\nANOVA with Tukey’s post hoc test.\n- In Fig. 2b, compared to nRBC, what proteins (MWCO 25 kDa) were lost in RBC?\nIt\nis necessary to add a set of IL/aC@RBC for comparison.\nResponse: Thanks for the reviewer’s detailed comments. In Fig. 2b, the lost protein at\nMWCO 25 kDa in RBC was probably carbonic anhydrase, which would be degraded\ninto small molecular weight proteins or polypeptides during the “alternate hypotonic\nand hypertonic dialysis” process. Notably, the process would maintain the integrity for\ndrug encapsulation capacity, as well as the CAT bioactivity at around 250 kDa for O\n2\nproduction ability. Following the suggestions proposed by the reviewer, we have\nsupplemented a set of IL/aC@RBC in the SDS-PAGE analysis for comparison (Fig.\nR18). The result presented that the characteristic protein bands of aCTLA-4 at ~55 kDa\nand ~25 kDa in IL/aC@RBC, along with the distinct protein bands of IL-12 at ~25 kDa\nand ~50 kDa, verifying the co-encapsulation of aCTLA-4 and IL-12 in IL/aC@RBC.\nIn the revised manuscript, we have included Fig. R18 as Fig. 2b and added relevant\ndescription.\nFig. R18 SDS-PAGE protein analysis of nRBC, RBC, IL/aC@RBC, IL-12, and\naCTLA-4. Marker indicates the molecular weight in kilodaltons (kDa).\n- Please provide high-resolution TEM images of the BaO products (Fig. S5). It is\n2\ndifficult to conclude that a shortened bamboo-like structure exists for BaO here, as\n2\nwell as the particle size distribution.\nResponse: Thanks for the reviewer’s valuable critiques. Following the reviewer’s kind\nsuggestions, we have provided high-resolution TEM images of the BaO products to\n2\nconfirm the shortened bamboo-like structure of BaO at feeding mass ratios of\n2 2 2\nhave reanalyzed the length of BaO products in the high-resolution TEM images, as\n2\ndetermined to be around 140 nm (Fig. R19b). According to the reviewer’s kind\nsuggestions, we have supplemented the high-resolution TEM images and\ncorresponding particle size distribution of bamboo-like BaO in length as Fig. 2i and\ndescription in the revised manuscript.\nFig. R19 a High-resolution TEM images of BaO . Scale bars, 100 nm and 20 nm. b\n2\n- The authors should indicate the gel position in the PA images. Additionally, will the\nintertumoral-injected gels with cells affect PA imaging? RBC is also rich in O and\n2\nsonification will help O release from cell mimics.\nstudy was mainly utilized to observe the saturated O (sO ) content in the tumors,\n2 2\nthereby evaluating the oxygen production capability of IL/aC@RBAH. Due to the weak\nnear-infrared absorption properties of the gel, it fails to generate detectable signals in\nPA imaging to indicate its own position. Consequently, the position of the implanted\nhydrogel would not affect the results of PA imaging. To exclude the potential\nbackground interference of the hydrogel or intrinsic O in the RBC, four groups,\n2\nincluding BAH (the hydrogel without RBC), RBC, RB (the mixture of RBC and BaO ),\n2\nand RBAH, were set for PA imaging. As hypothesized, indiscernible sO level change\n2\nwas observed in tumors after local injection of BAH (Fig. R20). Notably, the\nintratumoral sO contents were perceptibly elevated in the RBC group, possibly due to\n2\nthe intrinsic O released from the cells. Compared to the sO levels in the RBC group,\n2 2\nthose in the RB and RBAH groups were significantly elevated, which might be\noriginated from the hydrolysis of BaO in the acidic environment to produce H O for\n2 2 2\nin situ O production. Notably, the sO levels in the RBAH were moderately lower than\n2 2\nthose in the RB group, possibly due to the reduced reactive surface areas between H O\n2 2\nand CAT induced by the 3D crosslinked network of the hydrogel. However, the\ncrosslinked skeleton of RBAH could be conducive to continuous O generation within\n2\n48 h (Fig. 4k) for effective hypoxia alleviation (Fig. 4l).\nFig. R20 (a) Representative PA images and (b) corresponding quantification of sO\n2\nlevels in 4T1 tumors before and after the treatments of BAH, RBC, RB, and RBAH. n\n= 3, mean ± SD. P values were calculated via student’s t-test.\n- In Figs. 4l-m, 5f, and 9a, the dense tumor cell nuclei (DAPI staining) showed no signs\nof reduced tumor cell proliferation following RT+IL/aC@RBAH treatment.\nAdditionally, the HE staining appeared to be collected from the tumor margin.\nResponse: Thanks for the reviewer’s meticulous remarks. DAPI, as a DNA-specific\nfluorescent dye primarily used for nuclear localization, cannot serve as a reliable\nindicator of tumor cell apoptosis or proliferation[1]. Given the experimental differences\namong tumor hypoxia alleviation (Fig. 4l), DNA damage (Fig. 4m) and antitumor T\ncell infiltration (Fig. 9a), the samples were respectively collected at 24 h post different\ntreatments, when the tumor nuclei (DAPI staining) reasonably maintained\nmorphological integrity for cell localization. In our study, Ki67 and TUNEL staining\nmethods, as well-validated utilized techniques, were applied to visualize tumor cell\nproliferation and apoptosis following different treatments. The results showed the\nweakest green fluorescence of Ki67 in the 4T1 tumors (Fig. 5h) and the highest red\nsignal of TUNEL in the Panc02 tumors (Fig. 8h) after the RT + IL/aC@RBAH\ntreatment. Both of the findings could evidence the significantly reduced tumor cell\nproliferation following the RT + IL/aC@RBAH treatment. Moreover, the H&E staining\nimages in Fig. 5f and Fig. 8f were collected from the representative intratumoral\nregions rather than the margins of tumors in different groups. To enhance the\nrepresentativeness and reliability of the data, we have replaced the original localized\nH&E-stained analysis with the results encompassing larger tumor areas. As presented\nin Figs. R21 and R22, the tumors in the RT + IL/aC@RBAH group exhibited massive\nnuclear fragmentation with loss of cytoplasmic boundaries in both of the 4T1 and\nPanc02 models, collectively demonstrating prominent necrosis in the tumor tissues.\nOverall, all these findings comprehensively affirmed the excellent antitumor efficacy\nof the synergistic therapeutic strategy of RT + IL/aC@RBAH-induced multistage RIT.\nAccording to the reviewer’s kind advice, we have respectively included Fig. R21 and\nFig. R22 as Figs. 5f, g and Figs. 8f, g in the revised manuscript.\nFig. R21 a, b H&E staining images (a) and corresponding necrosis analysis (b) of\northotopic 4T1 tumors subjected to different treatments. Scale bar, 500 μm. G1, Saline;\nG2, RT; G3, RT + RBAH; G4, RT + aC@RBAH; G5, RT + IL@RBAH; G6, RT +\nIL/aC@RBAH. n = 3, mean ± SD. P values were calculated via student’s t-test.\nFig. R22 a, b H&E staining images (a) and corresponding necrosis analysis (b) of\nsubcutaneous Panc02 tumors after different treatments. Scale bar, 500 μm. G1, Saline;\nG2, RT; G3, RT + RBAH; G4, RT + aC@RBAH; G5, RT + IL@RBAH; G6, RT +\nIL/aC@RBAH. n = 3, mean ± SD. P values were calculated via student’s t-test.\nReferences:\n[1] Mabrouk, R. et al. DAPI (4’,6-diamidino-2-phenylindole) stains compact amyloid\nplaques. J Alzheimers Dis 88, 949-955 (2022).\n- What were the encapsulation efficiency and loading capacity of IL-12 and aCTLA4?\nResponse: Thanks for the reviewer’s detailed questions. The loading capacities of IL-\n12 and aCTLA-4 in IL/aC@RBC were 4.79 μg/108 cells and 2.88 μg/108 cells, with\ncorresponding encapsulation efficiency at 22.54% and 11.52%, respectively (Fig. R23).\nRelevant details have been incorporated into the revised manuscript. We have\nsupplemented relevant description in the revised manuscript and included Fig. R23 as\nSupplementary Fig. 3 in the revised supplementary information.\nFig. R23 a, b Standard curves of aCTLA-4-FITC at 340 nm (a) and IL-12-AMCA at\n485 nm (b). c UV-Vis absorption spectrum of IL/aC@RBC. aCTLA-4 and IL-12 in\nIL/aC@RBC were labeled with FITC and AMCA, respectively.\n- For transcriptome analysis, a PBS group is needed as a control.\nResponse: Thanks for the reviewer’s constructive suggestions. Given that saline is\nmore commonly used as a control in animal experiments, we administered saline rather\nthan PBS into tumors as a control. Compared with the saline group, there were 1071\ndifferentially expressed genes (DEGs) with 849 upregulated ones and 222\ndownregulated ones in the RT + IL/aC@RBAH group (Figs. R24a, b). The DEGs in\nthe RT + IL/aC@RBAH group, as shown by the Kyoto Encyclopedia of Genes and\nGenomes (KEGG) enrichment analysis, were enriched in antitumor immune response-\nassociated pathways, including cytokine-cytokine receptor interaction, NOD-like\nreceptor, TNF, MAPK, B cell receptor, NF-κB, natural killer cell-mediated cytotoxicity,\nand Toll-like receptor signaling pathway (Fig. R24c). Furthermore, Gene Ontology\n(GO) enrichment analysis manifested that the upregulated genes in the RT +\nIL/aC@RBAH group were mainly involved in response to IFN-γ, B cell activation,\nlymphocyte-mediated immunity, T cell proliferation, cell activation involved in\nimmune response, and lymphocyte proliferation (Fig. R24d). Notably, the multistage\nRIT initiated by RT + IL/aC@RBAH could activate antitumor immune effect-related\nsignaling pathways, such as C-type lectin receptor signaling pathway, natural killer cell-\nmediated cytotoxicity, TNF signaling pathway, and NOD-like receptor signaling\npathway (Fig. R24e), as evidenced by the Gene Set Enrichment Analysis (GESA).\nOverall, all the above results elucidated that the RT + IL/aC@RBAH treatment could\nreinvigorate antitumor immune responses for effective tumor regression. In the revised\nmanuscript, we have included Fig. R24 as Fig. 7a, c and Supplementary Figs. 37, 38\nand supplemented relevant discussion.\nFig. R24 a Volcano plot of DEGs between the RT + IL/aC@RBAH and control groups.\nb Heat map analysis of DEGs in orthotopic 4T1 tumors after different treatments. c\nKEGG enrichment analysis of DEGs between the RT + IL/aC@RBAH and control\ngroups. d GO enrichment analysis of DEGs between the RT + IL/aC@RBAH and control\ngroups. BP, biological process; CC, cellular component; MF, molecular function.\ne GSEA analysis of signaling pathways including C-type receptor, nature killer\nmediated cytotoxicity, TNF, and NOD-like receptor after the treatment of RT +\nIL/aC@RBAH."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The revised manuscript presents several significant advancements:\n1. Sequential Immune Activation: The authors demonstrate a clear sequential release profile of aCTLA-4 (40% within 48 h) followed by IL-12 (<20% over 48 h) from the hydrogel, enabling multistage immune modulation. This is supported by enhanced CD8+ T cell infiltration, reduced Treg ratios, and elevated IFN-γ production in the RT+aC-IL group.\n2. Ba/O₂ Dual Compensation: The hydrogel’s ability to generate O₂ via CAT-catalyzed H2O2 decomposition and sustain hypoxia alleviation is well-validated, partially addressing a key limitation of radiotherapy.\n3. Long-Term Biosafety: Comprehensive toxicity studies and biodistribution data confirm minimal systemic leakage of BaO₂ or RBCs, with no significant organ damage over 2 months.\nHowever, the authors have the following points to consider further:\n1. Inadequate Comparison to Current Standards: No comparison to anti-PD1/L1 therapies; Limited data on how the hydrogel outperforms conventional CTLA-4/IL-12 combotherapy.\n2. Expand Toxicity Discussion: Include potential immune cell exhaustion risks from prolonged IL-12 exposure.\n3. Mechanistic Evidence: While Fig. R16 (Supplementary Fig. 11) shows O₂-induced RBC pore formation, direct visualization of pore dynamics (e.g., time-lapse microscopy) would strengthen the mechanism.\n4. Overly Complex System with Limited Clinical Feasibility: The multi-component hydrogel system (RBCs + BaO₂ + Alginate + dual cytokines) introduces significant manufacturing and quality control challenges that undermine translational potential.\nWhile the authors demonstrate proof-of-concept in mice, scaling up engineered RBC production while maintaining batch-to-batch consistency for human trials remains unaddressed. The requirement for intratumoral injection further limits applicability to deep-seated or metastatic tumors.\nThese comments don't invalidate the work but highlight areas requiring substantial strengthening before the approach could be considered competitive with existing radioimmunotherapy strategies. The authors should address these limitations through additional experiments and more balanced discussion of clinical constraints.", "reviewer_3": "I have no further comments", "reviewer_4": "They have addressed the previous reviews."}, "author_response": "Detailed responses to the reviewers’ comments\nReviewer #2:\nThe revised manuscript presents several significant advancements:\n1. Sequential Immune Activation: The authors demonstrate a clear sequential release\nprofile of aCTLA-4 (40% within 48 h) followed by IL-12 (<20% over 48 h) from the\nhydrogel, enabling multistage immune modulation. This is supported by enhanced\nCD8+ T cell infiltration, reduced Treg ratios, and elevated IFN-γ production in the\nRT+aC-IL group.\n2. Ba/O₂ Dual Compensation: The hydrogel’s ability to generate O₂ via CAT-catalyzed\nH O decomposition and sustain hypoxia alleviation is well-validated, partially\n2 2\naddressing a key limitation of radiotherapy.\n3. Long-Term Biosafety: Comprehensive toxicity studies and biodistribution data\nconfirm minimal systemic leakage of BaO₂ or RBCs, with no significant organ damage\nover 2 months.\nResponse: Many thanks for the reviewer’s endorsement of our work.\nHowever, the authors have the following points to consider further:\n1. Inadequate Comparison to Current Standards: No comparison to anti-PD1/L1\ntherapies; Limited data on how the hydrogel outperforms conventional CTLA-4/IL-12\ncombotherapy.\nResponse: Thanks for the reviewer’s valuable comments. Following the reviewer’s\nkind suggestions, the respective administrations of anti-PD-1 (aPD-1) and anti-PD-L1\n(aPD-L1) in combination with RT were included as standard-of-care (SoC) control\ngroups for a comprehensive evaluation of the therapeutic efficacy of RT +\nIL/aC@RBAH. The orthotopic 4T1 tumor-bearing mice were randomly allocated into\n4 groups and treated with saline, RT + aPD-1, RT + aPD-L1, and RT + IL/aC@RBAH.\nDistinct from the rapid tumor progression in the saline group, the tumor volumes in the\nRT + aPD-1 and RT + aPD-L1 groups were moderately suppressed (Figs. R1a-c),\nindicating the deficient antitumor efficacy of RT combined with immune checkpoint\nblockade (ICB) therapy. Intriguingly, the RT + IL/aC@RBAH treatment showed much\nmore prominent tumor inhibition than the RT + aPD-1 and RT + aPD-L1 groups, thus\nillustrating the superior antitumor efficacy of RT + IL/aC@RBAH-mediated multistage\nRIT over RT combined with ICB therapy. To further elucidate the underlying immune\nmechanisms, we compared the immune effects of RT + IL/aC@RBAH with RT + aPD-\n1 and RT + aPD-L1 therapies by assessing immune cell populations in orthotopic 4T1\ntumor-bearing mice after different treatments. Relative to the moderate changes in\nimmune cell levels observed with RT + aPD-1 and RT + aPD-L1, RT + IL/aC@RBAH\nmarkedly increased CD8⁺ T cells and NK cells infiltration while reducing intratumoral\nTregs, thereby enhancing the effector-to-suppressor balance in the tumor\nmicroenvironment (TME) (Figs. R1d-f). Taken together, these results indicate that RT\nsynergized with the sequential release of IL-12 and aCTLA-4 is superior over its\ncombination with anti-PD-1/PD-L1 in priming and amplifying antitumor immunity and\nin reshaping the TME toward an effector-dominant state that favors immune-mediated\nantitumor efficacy.\nFig. R1 (a, b) Whole-animal in vivo bioluminescence imaging (a) and corresponding\nbioluminescence quantification (b) of orthotopic 4T1-luciferase (4T1-luc) tumor-\nbearing mice after treated with saline, RT + aPD-1, RT + aPD-L1, and RT +\nIL/aC@RBAH, respectively. c Average tumor growth curves of orthotopic 4T1 tumor-\nbearing mice subjected to different treatments. d-f Quantitative analyses of cytotoxic T\nlymphocytes (CD8+ in CD45+CD3+ cells) (d), NK cells (CD3-CD49b+ in CD45+ cells)\n(e) and Tregs (CD4+CD25+ in CD45+CD3+ cells) (f) in orthotopic 4T1 tumors after\ndifferent treatments. Data in b-f were presented as mean ± SD. n = 5 biologically\nindependent samples in b, c. n = 3 biologically independent samples in d-f. Statistical\nsignificance was determined using two-way ANOVA (b, c). Statistical significance was\ndetermined using student’s t-test in (d-f).\nNotably, our initial submission demonstrated that RT + IL/aC@RBAH achieved\nsuperior therapeutic efficacy over conventional aCTLA-4/IL-12 combination therapy,\nas presented in Supplementary Fig. 35. Herein, to further elucidate how the hydrogel\noutperformed conventional aCTLA-4/IL-12 combination therapy, we investigated the\nimmune cell populations in orthotopic 4T1 tumor-bearing mice exposed to different\ntreatments. Briefly, the orthotopic 4T1-tumor bearing mice were randomly divided into\n3 groups and then respectively treated with saline, RT + IL&aC (the mixture of IL-12\nand aCTLA-4), and RT + IL/aC@RBAH. In contrast to the modest increases in\nintratumoral CD8+ T cells and NK cells induced by RT + IL&aC, a marked\naccumulation of both effector populations was instigated by RT + IL/aC@RBAH (Figs.\nR2a, b). Concordantly, Tregs were reduced to the lowest levels, approximately 0.46-\nfold relative to those in the RT + IL&aC group (Fig. R2c), thereby sharpening the\nintratumoral effector-to-suppressor balance. These findings were aligned with\nhydrogel-enabled sequential release of aCTLA-4 followed by IL-12, producing early\nrelief of checkpoint and Treg constraints and later reinforcement of immune cell-\nmediated antitumor activities. To unveil whether these local changes would propagate\nto systemic priming, we analyzed tumor-draining lymph nodes (TDLNs) from\northotopic 4T1 tumor-bearing mice across different groups. The migration of both\ncDC1 and cDC2 to TDLNs was highest in the RT + IL/aC@RBAH group, exceeding\nthat in the RT + IL&aC group (Figs. R2d, e), consistent with enhanced antigen transport\nand cross-priming, thus providing a mechanistic basis for the superior initiation of\nantitumor immunity conferred by the sequential release of aCTLA-4 and IL-12 from\nthe hydrogel. We have included Fig. R1 as Supplementary Fig. 38, Fig. R2 as\nSupplementary Fig. 36. Relevant description has been added in the revised manuscript.\nFig. R2 (a-c) Quantification of cytotoxic T lymphocytes (CD8+ in CD45+CD3+ cells)\n(a), NK cells (CD3-CD49b+ in CD45+ cells) (b) and Tregs (CD4+CD25+ in CD45+CD3+\ncells) (c) in orthotopic 4T1 tumors after different treatments. d, e Quantitative analyses\nof cDC1 (CD103+ in CD11c+MHC Ⅱ+ cells) (d) and cDC2 (CD11b+ in CD11c+MHC\nⅡ+ cells) (e) in TDLNs of orthotopic 4T1 tumor-bearing mice post different treatments.\nData in a-e were presented as mean ± SD. n = 3 biologically independent samples in a-\ne. Statistical significance was determined using student’s t-test.\n2. Expand Toxicity Discussion: Include potential immune cell exhaustion risks from\nprolonged IL-12 exposure.\nResponse: Thanks for the reviewer’s constructive advice. Short-term antigenic or\ninflammatory exposure prompts immune cells to undergo memory differentiation,\nwhereas persistent stimulation drives them toward an exhausted phenotype. Immune\ncell exhaustion is characterized by diminished effector function and increased\ninhibitory receptor expression, which is associated with detrimental drug resistance[1, 2].\nStudies have shown that key cytokines driving immune cell exhaustion in vivo include\nIL-10, IFN-I, TGF-β, and IL-23, while IL-12 often acts to reverse or alleviate the\nexhaustion in the majority of settings[3-6]. Despite its generally restorative role in the\nimmune recovery, sustained intratumoral IL-12 exposure at a high level from the\nhydrogel might paradoxically promote immune cell exhaustion, accompanied by\nheightened metabolic demand and increased susceptibility to activation-induced\ndysfunction. Nevertheless, the critical driving factor of exhaustion remains persistent\nantigen exposure[7]. By rapidly suppressing tumor growth and thereby markedly\nreducing the antigen exposure of intratumoral immune cells, RT + IL/aC@RBAH is\nexpected to prevent immune cell exhaustion, thereby outweighing any potential IL-12-\nassociated pro-exhaustion effect. In the revised manuscript, we explicitly\nacknowledged the possibility of immune cell exhaustion under sustained IL-12, and\ndiscussed that limited dose intensity and exposure duration could reduce this risk.\nRelevant description has been added in the revised manuscript.\nReferences:\n[1] McLane, L. M. et al. CD8 T cell exhaustion during chronic viral infection and cancer.\nAnnu Rev Immunol 37, 457-495 (2019).\n[2] Chow, A. et al. Clinical implications of T cell exhaustion for cancer immunotherapy.\nNat Rev Clin Oncol 19, 775-790 (2022).\n[3] Baessler, A. et al. T Cell Exhaustion. Annu Rev Immunol 42, 179-206 (2024).\n[4] Sumida, T. S. et al. Type I interferon transcriptional network regulates expression of\ncoinhibitory receptors in human T cells. Nat Immunol 23, 632-642 (2022).\n[5] Wertheimer, T. et al. IL-23 stabilizes an effector Treg cell program in the tumor\nmicroenvironment. Nat Immunol 25, 512-524 (2024).\n[6] Kang, S. et al. Engineered IL-7 synergizes with IL-12 immunotherapy to prevent T\ncell exhaustion and promote memory without exacerbating toxicity. Sci Adv 9,\neadh9879 (2023).\n[7] Zebley, C. C. et al. T cell dysfunction and therapeutic intervention in cancer. Nat\nImmunol 25, 1344-1354 (2024).\n3. Mechanistic Evidence: While Fig. R16 (Supplementary Fig. 11) shows O₂-induced\nRBC pore formation, direct visualization of pore dynamics (e.g., time-lapse microscopy)\nwould strengthen the mechanism.\nResponse: Thanks for the reviewer’s valuable suggestion. Owing to the small size\n(approximately 5 nm) and rapid pore formation kinetics of RBC, it is challenging to\ndirectly and dynamically monitor the pore dynamics. Herein, time-lapse microscopy\nwas employed to observe the pore dynamics in real-time under an acidic environment\nwith BaO . Toward this, DiD-labeled RBC were cocultured with BaO in medium\n2 2\ncontaining Sulforhodamine B (SRB), a cell-impermeable dye that stains the basic\nproteins upon plasma membrane permeabilization, and subjected to time-lapse\nmicroscopy (Fig. R3). As anticipated, DiD-labeled RBC co-cultured with BaO₂ in an\nacidic milieu turned SRB-positive within 3 minutes, in agreement with the SEM image\nin Supplementary Fig. 11. Collectively, all these results evidence that the acidity-\ninduced hydrolysis of BaO yields H O , which is promptly decomposed by RBC-\n2 2 2\nresident catalase to O , thereby inducing pore formation on the RBC membrane and\n2\nenabling SRB entry.\nFig. R3. Live cell time-lapse imaging of DiD-labeled RBC (blue) cocultured with BaO\n2\nin acidic medium containing SRB (red). DID, a cell membrane fluorescent dye. SRB, a\ncell-impermeable dye. Scale bars: upper, 10 μm; lower, 5 μm.\n4. Overly Complex System with Limited Clinical Feasibility: The multi-component\nhydrogel system (RBCs + BaO₂ + Alginate + dual cytokines) introduces significant\nmanufacturing and quality control challenges that undermine translational potential.\nWhile the authors demonstrate proof-of-concept in mice, scaling up engineered RBC\nproduction while maintaining batch-to-batch consistency for human trials remains\nunaddressed. The requirement for intratumoral injection further limits applicability to\ndeep-seated or metastatic tumors.\nResponse: Thanks for the reviewer’s insightful advice. Notwithstanding the potent\nimmunotherapeutic efficacy elicited by our designed multicomponent hydrogel system\nacross multiple mouse tumor models, its clinical translation faces several challenges.\nForemost among these considerations is the quality control during manufacturing.\nAlthough the preparation of engineered RBC is relatively complex, several companies\nhave already advanced such products (e.g., RTX-224, RTX-240, etc.) into clinical\ndevelopment, suggesting a degree of feasibility for large-scale production of\nIL/aC@RBC[1, 2]. Nevertheless, the additional encapsulation of immunomodulatory\nfactors within RBC further increases manufacturing complexity and quality-control\nchallenges for clinical application of IL/aC@RBC. Furthermore, beyond the concerns\nregarding the stability and batch-to-batch consistency of BaO nanoparticles during\n2\nlarge-scale production, the formation of highly uniform cell-encapsulating hydrogels in\npatients requires in-depth exploration integrating chemical technologies with\nbiosynthetic techniques. Apart from the quality control issues discussed above, the\nintratumoral injection route somewhat limits the application of IL/aC@RBAH in deep-\nseated or metastatic tumors. Future efforts should focus on developing an in vivo\nplatform for in situ tumor-site assembly of intravenously injected nanogels into a bulk\nhydrogel or combining interventional therapeutic techniques with stereotactic\nradiotherapy to expand the therapeutic applicability of multistage RIT elicited by RT +\nIL/aC@RBAH. Relevant translational discussion has been added in the revised\nmanuscript.\nReferences:\n[1] McArdel, S. L. et al. Anti-tumor effects of RTX-240: an engineered red blood cell\nexpressing 4-1BB ligand and interleukin-15. Cancer Immunol Immunother 70, 2701-\n2719 (2021).\n[2] Zhang, X. et al. Engineered red blood cells as an off-the-shelf allogeneic anti-tumor\ntherapeutic. Nat Commun 12, 2637 (2021).\nThese comments don’t invalidate the work but highlight areas requiring substantial\nstrengthening before the approach could be considered competitive with existing\nradioimmunotherapy strategies. The authors should address these limitations through\nadditional experiments and more balanced discussion of clinical constraints.\nResponse: Thanks for the reviewer’s positive assessment and insightful comments. In\nthe revised manuscript, we substantially strengthened the manuscript by adding head-\nto-head comparisons of anti-PD-1/PD-L1 and conventional aCTLA-4/IL-12 therapies\nwith multistage RIT elicited by RT + IL/aC@RBAH, incorporating time-lapse\nvisualization of RBC pore formation, and expanding discussions on potential immune\ncell exhaustion risks. Moreover, we provided a balanced discussion of clinical\nconstraints, including manufacturing, quality control and delivery feasibility. We also\noutlined strategies to expand the applicability of RT + IL/aC@RBAH, strengthening its\ncompetitiveness against existing radioimmunotherapy. We believe that the proposed\nsupplementary experiments and discussions will meaningfully deepen the study,\nthereby reinforcing its scientific rigor and enhancing its overall impact.\nReviewer #3:\nI have no further comments.\nResponse: Thanks for the reviewer’s thoughtful review and support of our manuscript.\nReviewer #4:\nThey have addressed the previous reviews.\nResponse: Thanks sincerely for the reviewer’s considerate review and endorsement of\nour manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_2": "I am sorry for the repeated comments. For the revised manuscript, I have no other revision comments."}, "author_response": ""}]

Decision letter