Immunostimulatory hydrogel with synergistic blockage of glutamine metabolism and chemodynamic therapy for postoperative management of glioblastoma

Immunostimulatory hydrogel with synergistic blockage of glutamine metabolism and chemodynamic therapy for postoperative management of glioblastoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Immunostimulatory hydrogel with synergistic blockage of glutamine metabolism and chemodynamic therapy for postoperative management of glioblastoma Junbai Li, Yiran Guo, Tianhe Jiang, Sen Liang, Anhe Wang, Jieling Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4629023/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Glioblastoma multiforme (GBM) stands as one of the most lethal malignant brain tumors affecting the central nervous system. Post-surgery, patients encounter daunting challenges like tumor recurrence, increased intracranial pressure due to cavitation, and constraints linked with immediate postoperative oral chemotherapy. Herein, we construct an injected peptide gel with in situ immunostimulatory functions to harmonize the regulation of glutamine metabolism and chemodynamic therapy in tackling the postoperative obstacles. The methodology entails crafting injectable gel scaffolds with short peptide molecules, incorporating the glutaminase inhibitor CB-839 and copper peptide self-assembled particles (Cu-His NPs) renowned for their chemodynamic therapy (CDT) efficacy. By fine-tuning glutamic acid production via metabolic pathways, our system not only heightens the therapeutic prowess of copper peptide particles in CDT but also escalates intracellular oxidative stress. This dual mechanism culminates in augmented immunogenic cell death (ICD) within glioblastoma multiforme cells and improves a conducive immune microenvironment. Anchored on the tenets of metabolic reprogramming, this treatment strategy showcases substantial promise in significantly curtailing GBM tumor recurrence, prolonging median survival in murine models. Physical sciences/Materials science/Biomaterials/Biomedical materials Biological sciences/Cancer/Cancer therapy Biological sciences/Cancer/Cancer metabolism Short peptide Self-assembly Glutamine metabolism Glioblastoma multiforme Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Glioblastoma multiforme (GBM) is the most aggressive and lethal malignant brain tumor within the central nervous system. 1 Standard clinical management typically involves surgical resection, followed by adjuvant chemotherapy and radiotherapy. 2 , 3 However, the infiltrative nature of GBM often prevents complete surgical removal, and surgery can disrupt the tumor's immune microenvironment, potentially delaying chemotherapy and leading to tumor recurrence. 4 , 5 Additionally, the post-surgical cavity provides space for GBM recurrence. The blood-brain barrier (BBB) further complicates the delivery of drugs and immune cells to the brain. 6 – 8 The malignant growth characteristics of GBM, coupled with the unique postoperative treatment needs, pose significant challenges for drug delivery methods and therapeutic strategies following GBM surgery. 9 , 10 Metabolism plays a pivotal role in the vitality, expansion, invasiveness, and metastatic potential of tumor cells. 11 Unlike normal cells, cancer cells undergo metabolic reprogramming to satisfy their heightened demands for energy and nutrients, inevitably leading to competition for nutrients with coexisting cells. 12 Furthermore, metabolic substrates and products within the tumor environment also impact the functional states of cancer and coexisting cells. Notably, the metabolic reprogramming of tumor cells can facilitate information exchange and crosstalk with immune cells through metabolites, indicating that cellular metabolic processes should not be considered in isolation. 13 Regulating cellular metabolism can influence the functional changes in downstream immune cells and reshape the immune microenvironment, offering a fresh perspective for exploring the interplay between cell metabolism and immunity. 14 , 15 Glutamine plays a critical role in cancer cell metabolism, serving as a key source for energy generation, biosynthesis, and redox balance maintenance. 16 , 17 The regulation of glutamine metabolism in cancer cells also affects the communication and crosstalk with immune cells in the microenvironment through metabolites. 18 , 19 This interaction influences the polarization of tumor-associated macrophages (TAMs) and enhances the activity of T lymphocytes and cytokine expression by depriving cancer cells of essential nutrients like glutamine, thereby improving the efficacy of immunotherapy. 20 , 21 Given the dual significance of suppressing tumor growth and immune modulation, targeting glutamine metabolism has emerged as a key focus for cancer therapy. CB-839, a small molecule glutaminase inhibitor currently in clinical trials (NCT03057600 and NCT03163667), has shown promise. 22 However, clinical outcomes have not met expectations due to tumor heterogeneity, metabolic adaptability, the complex immuno-suppressive microenvironment, and challenges in drug delivery, underscoring the limitations of monotherapy. 23 , 24 Research indicates that inhibiting glutamine catabolism can reduce glutamate levels, a precursor to glutathione (GSH), thereby decreasing GSH content. This reduction impairs the tumor cells' ability to clear intracellular reactive oxygen species (ROS), 25 establishing a link for synergistic dynamic therapy through glutamine modulation. 26 , 27 For deeply situated tissues like GBM in the brain, CDT for ROS delivery is ideal. 28 Copper-based nanomaterials, more efficient and with a broader pH tolerance than iron-based counterparts, are used to generate CDT. 29 , 30 However, excessive free copper ions may lead to severe systemic toxicity, so it is necessary to limit copper ions in time and space, allowing them to be stable in the physiological environment but rapidly released in tumor cells, producing ROS through the Fenton reaction to kill tumors. 30 , 31 In the unique postoperative treatment scenarios of GBM, drug delivery systems must adhere to rigorous temporal and spatial specifications. 32 Injectable peptide hydrogels can fill the irregular cavities left by surgery, physically restricting tumor proliferation, and facilitating a controlled release of therapeutic agents through the modulation of hydrogel degradation. 33 Moreover, endogenous short peptides have excellent biocompatibility and low immunogenicity, making them easy to regulate assembly. 34 This allows for the regulation of carrier degradation time and pH triggering conditions to meet the complex requirements of sustained drug release. In this study, we have developed an innovative postoperative treatment system for GBM. Our approach involves the utilization of an in situ injectable peptide hydrogel that is loaded with the glutaminase inhibitor CB-839 and self-assembling Cu 2+ -peptide coordination nanoparticles (Cu-His NPs) for CDT (Scheme 1 A). As depicted in Scheme 1 B, CB-839 effectively suppresses glutamine production, thereby reducing intracellular GSH levels and enhancing the cytotoxicity of Cu 2+ -mediated CDT. This occurs by diminishing GSH's neutralizing effect on ROS. Moreover, CDT induces a significant increase in ROS production, leading to amplified oxidative stress within tumor cells. Consequently, this triggers immunogenic cell death (ICD) and initiates an immune response to counteract the immunosuppressive tumor microenvironment (Scheme 1 C). 35 – 37 The short peptide molecules, serving as the building blocks of our carrier, exhibit remarkable flexibility and diversity in its assembly. 38 It not only enables the targeted release of Cu 2+ within the tumor region but also allows for control over the mechanical strength and degradation time of the hydrogel carrier, facilitating the sustained release of drugs. Remarkably, our experimental results demonstrate that this immunotherapy strategy, based on metabolic reprogramming, effectively addresses the complex treatment scenarios following GBM surgery. It successfully inhibits GBM recurrence and significantly prolongs overall survival in mice. 2. Results and Discussion 2.1 Synthesis and Characterization of Combo Gel In this study, we successfully synthesized an injectable peptide hydrogel (Combo Gel) loaded with Cu 2+ -peptide nanoparticle (Cu-His NP) complexes and the glutaminase inhibitor CB-839 (Fig. 1 A). Initially, we selected short peptide molecules with positively charged Fmoc-Tyrosine-Tyrosine-Lysine (Fmoc-YYK) and negatively charged Fmoc-Tyrosine-Aspartic acid (Fmoc-YD) to prepare injectable hydrogel scaffolds. 39 , 40 These molecules utilized a variety of intermolecular forces, such as electrostatic and π-π interactions, to assemble with flexibility and diversity. 34 , 41 , 42 The hydrogel scaffold is semi-transparent at room temperature (Figure S1 A), and the scanning electron microscope (SEM) image reveals the common fibrous morphology of the hydrogel at the micro-scale (Figure S1 B). Fourier transform infrared spectroscopy (FT-IR) analysis revealed absorption peaks at 1690 cm − 1 and 1615 cm - 1 , characteristic of the antiparallel β-sheet secondary structure in the peptide hydrogel (Figure S2). 43 Cu 2+ -based CDT nanomaterials have demonstrated promising potential in tumor treatment. However, the targeted release of Cu 2+ and issues related to free Cu 2+ have constrained their advancement. In this study, we coordinated histidine, featuring an imidazole group, with Cu 2+ to develop CDT generators. The histidine, known for its potent membrane-penetrating ability, 44 was used to control the release of Cu 2+ specifically in the tumor microenvironment. Comparing the SEM image of Cu-His NPs in Fig. 1 B, the high-resolution transmission electron microscopy (HRTEM) image of Cu-His NPs in Fig. 1 C, and the HRTEM image of the raw material Fmoc-Histidine-Histidine (Fmoc-HH) in Figure S3, it could be observed that the morphology of Cu-His NPs was distinctly different from that of Fmoc-HH, forming spherical particles with a diameter of approximately 40 nm. HRTEM analysis revealed a lattice spacing of 0.324 nm for the Cu-His NPs, slightly larger than the 0.225 nm spacing of Fmoc-HH, confirming the formation of ligand-bound nanoparticles between Cu 2+ and Fmoc-HH (Figure S4). The FT-IR spectrum of Cu-His NPs and Fmoc-HH revealed that in the presence of Cu 2+ , the stretching vibration of the N-H bond in the histidine imidazole group shifted from 3294 cm − 1 to 3146 cm − 1 , and the absorption peaks for the stretching vibrations of C = O and C-N bonds significantly intensified at 1656 cm − 1 and 1132 cm − 1 , respectively (Figure S5). The shifts and enhancements of these vibrational peaks indicated that Cu 2+ had formed coordination bonds with nitrogen or oxygen atoms within the peptide chain. X-ray photoelectron spectroscopy (XPS) analysis showed peaks for Cu2p, O1s, N1s, and C1s at 933.1, 529.1, 398.1, and 283.1 eV, respectively (Figure S6), with the Cu2p 3/2 and Cu2p 1/2 peaks located at 931.4 and 951.3 eV. The main peaks were separated by a 19.9 eV spin energy, and satellite peaks were observed at 941 and 961 eV, indicating the presence of Cu 2+ in the coordination (Fig. 1 D). 45 , 46 Electron probe microanalysis (EPMA) further confirmed the uniform distribution of Cu 2+ throughout the sample, indicating that the Cu-His NPs were formed by the self-assembly of Cu 2+ and Fmoc-HH (Fig. 1 E). This hydrogel scaffold was loaded with the CDT generator Cu-His NPs and the glutaminase inhibitor CB-839. SEM images revealed that the Cu-His NPs were uniformly dispersed within the hydrogel's fibrous matrix (Fig. 1 F), and the formulation demonstrated superior injectability (Fig. 1 G). Rheological testing indicated that Combo Gel's viscosity significantly decreased with an increase in shear rate from 0.1 to 100 s - 1 , showcasing its pronounced shear-thinning behavior (Figure S7). Given the unique mechanical characteristics of brain tissue, with a modulus of approximately 1–2 kPa, any mechanical discrepancy between implanted materials and the brain could potentially induce epilepsy or other undesirable immune reactions. 47 Therefore, gel scaffolds with varying mechanical strengths were fabricated by adjusting the assembly of short peptide molecules. Select the gel scaffold matching the mechanical properties of brain tissue (Fig. 1 H), and verify the biocompatibility of the gel scaffold (Fig. 1 I). 2.2 In Vitro Anti-Tumor Studies of Combo Gel: Next, we investigated the in vitro antitumor efficacy of Combo Gel. The Cu 2+ was capable of undergoing a redox reaction with glutathione (GSH), resulting in the formation of Cu + and oxidized glutathione (GSSG). When equal mass ratios of Cu-His NPs and GSH were combined in an aqueous solution, a rapid emergence of purplish-red fluorescence was observed under UV light (Figure S8A), indicating the occurrence of a redox reaction. 48 – 50 Over time, this fluorescence gradually faded, confirming the progression of the reaction. The fluorescence and emission spectra of the mixtures were further confirmed using a fluorescence spectrophotometer (Figure S8B). SEM analysis showed a significant morphological change in the Cu-His NPs after only 5 minutes of reaction, with the nanoparticles losing their spherical integrity (Fig. 2 A), suggesting an extremely rapid reaction kinetics. Post-reaction, the supernatant was collected and the lyophilized samples were analyzed by FT-IR (Figure S9), high-resolution mass spectrometry (HRMS) (Figure S10), and hydrogen nuclear magnetic resonance ( 1 H NMR) (Figure S11), all confirming the formation of GSSG. 51 These findings confirmed that Cu-His NPs degraded in the presence of GSH, undergoing a redox reaction to produce Cu + and GSSG. Methylene blue (MB), known for its characteristic UV-absorption peak, can be degraded by hydroxyl radicals (·OH). Figure 2 B illustrates that the UV absorption peaks of MB reacted with H 2 O 2 alone or with a mixture of Cu-His NPs and GSH showed minimal changes after 4 hours. However, significant changes in the UV absorption peaks were observed when MB, H 2 O 2 , Cu-His NPs, and GSH were all present, suggesting that the Fenton-like reaction of Cu 2+ with H 2 O 2 in the presence of GSH can initiate a cascade reaction to produce ·OH. The rate of ·OH production in the absence of GSH was significantly lower compared to when GSH was added, as observed after mixing MB, H 2 O 2 , and Cu-His NPs (Figure S12). This further supports the notion that Cu-His NPs can more effectively generate ·OH under GSH influence, targeting tumor cell destruction. Figure S13 showed that the rate of ·OH production in the presence of Cu-His NPs and H 2 O 2 gradually slowed down as the reaction time increased with GSH. We further investigated the synergistic tumor cell-killing effects of Cu-His NPs combined with CB-839 in vitro. The Cell Counting Kit-8 (CCK-8) assay was utilized to assess the cell inhibition rates of the GL261 GBM cell line following 24 hours of treatment with Cu-His NPs, CB-839 alone, or their combination (Fig. 2 C). The findings indicated that the cell inhibition rate with CB-839 and Cu-His NPs co-cultured with tumor cells was markedly higher than that of single-agent treatments. At a Combo group drug concentration of 10 µg/mL, the inhibition rate after 24 hours reached 27%, exhibiting strong cytotoxicity. As the drug concentration increased, the cytotoxicity of the combined treatment against cancer cells was also significantly enhanced. Live/dead cell staining, after culturing at a drug concentration of 10 µg/mL for 12 hours, yielded the same conclusion: the synergistic effect of Cu-His NPs and CB-839 amplified the cytotoxicity against GL261 glioma cells in vitro, with a significantly higher cell inhibition rate than monotherapy (Fig. 2 F). We immersed hydrogels loaded with Cu-His NPs (Cu-His Gel) and CB-839 (CB-839 Gel) separately in phosphate-buffered saline (PBS) and incubated at 37°C. The release concentrations were monitored using high-performance liquid chromatography (HPLC). The release profiles revealed that by day 5, the release amounts of Cu-His NPs and CB-839 were 57% and 49%, respectively, with the release rate subsequently decreasing. By day 20, approximately 80% of both drugs had been released (Fig. 2 D). Both agents followed zero-order release kinetics, suggesting that the hydrogel scaffold effectively sustained drug release, extending the retention time in the mouse brain. The drug release rate was found to be nearly concurrent with the hydrogel volume loss, indicating that release was primarily dependent on hydrogel degradation. To further evaluate the in vivo release behavior, Combo Gel was injected into the brains of mice, and the Cy5-labeled hydrogel signal was quantitatively monitored at set time points using an in vivo imaging system (IVIS) (Fig. 2 E and 2 G). The injected hydrogel rapidly underwent the solution-to-gel transition within the mouse cranium, and this swift in situ gelation effectively prevented rapid drug release. Continuous monitoring indicated that the gel remained in the mice for approximately two weeks, with no significant fluorescent signals detected in other major organs or blood (Figure S14), and the mice's body weight remained unaffected (Figure S15). The mice's major organs were harvested for H&E staining, showing no notable differences compared to healthy mice, which suggested that the gel formulation possessed excellent biocompatibility (Figure S16). Previous experiments demonstrated that Cu-His NPs emitted purplish-red fluorescence upon interaction with GSH. After a 2-hour incubation, confocal microscopy revealed intracellular red fluorescence, indicating that the Cu-His NPs had been internalized and initiated their reaction with GSH (Figure S17A). By 8 hours, the red fluorescence within the cells had nearly vanished, suggesting the completion of the Fenton-like reaction of Cu-His NPs (Figure S17B). To further visualize the intracellular drug release process of Cu-His NPs, lysosomes were labeled with Lysotracker Red and co-localized with Cy5-labeled Cu-His NPs, which exhibited blue fluorescence, and the nucleus. After 4 hours of incubation, the fluorescence of Cu-His NPs almost entirely overlapped with that of the lysosomes. The overlapping region displayed yellow fluorescence, indicating that the Cu-His NPs were sequestered in lysosomes following endocytosis, and some had escaped into the cytoplasm (Fig. 3 A and Figure S18). These findings confirmed that Cu-His NPs could undergo lysosomal escape post-cell entry, releasing the drug into the cytoplasm and enhancing its intracellular efficacy. The mechanism of intracellular escape by Cu-His NPs likely involved synergistic effects of histidine properties within the specific environment of tumor cells. 52 Histidine's protonation in the lysosome's acidic milieu and its subsequent charge change may have facilitated its interaction with the lysosomal membrane and promoted escape. Additionally, histidine's transition from hydrophobic to hydrophilic properties, along with its proton sponge effect, facilitated the efficient uptake and swift drug release of Cu-His NPs. These effects collectively promote the efficient uptake of Cu-His NPs within cells, enhance the bioavailability of Cu 2+ , and enhance anti-tumor effects. 53 , 54 ROS in excess exerted a complex impact on tumor cells, disrupting redox balance and inducing damage or death. This triggered ICD, releasing DAMPs that activated antigen-presenting cells (APCs), including dendritic cells, and stimulated the immune system. GSH was critical for countering ROS-induced oxidative stress; its levels inversely correlated with ROS, and reduced GSH could lead to ROS accumulation and cell death. Our study assessed the effects of different formulations on GSH and GSSG levels in GL261 cells. Both CB-839 and Cu-His NPs individually reduced GSH levels to 19.0 and 11.7 µM, respectively. However, the synergistic effect of CB-839 and Cu-His NPs more significantly blocked both the synthesis of new GSH and the depletion of pre-existing GSH, with an effective concentration of just 8.2 µM (Fig. 3 B). Subsequently, ROS production in tumor cells post-treatment was monitored using DCFH-DA staining. The Combo group exhibited the highest ROS production, with a marked increase in fluorescence intensity (Fig. 3 D). In addition, DAMPs associated with ICD, such as surface calreticulin (CRT), high mobility group box 1 protein (HMGB1), and ATP, were released during cell death. 55 ATP emitted "find-me" signals, promoting the phagocytosis of apoptotic cells and triggering specific anti-tumor immune responses. 56 CRT served as an "eat-me" signal, enhancing the phagocytosis of apoptotic cells by dendritic cells or their precursors, thereby providing antigens and promoting DC maturation and function. 57 , 58 HMGB1 is bound to pattern recognition receptors (PRRs) on bone marrow cells, activating immune signaling pathways and further amplifying immune responses. 59 The experimental results revealed that the Combo group significantly increased the expression of DAMP signaling molecules in tumor cells compared to the single drug treatment group (Figs. 3 C, 3 E, and 3 F). The Combo therapy not only enhanced ICD by promoting the release of DAMPs from GBM cells but also amplified the immune response, suggesting a potential for long-lasting anti-tumor immunity and a promising approach for cancer therapy. 2.3 In vivo anti-tumor studies of Combo Gel: The infiltrative growth of GBM often results in indistinct tumor boundaries, making complete surgical removal challenging and leading to frequent post-surgical recurrence. A primary GBM model was successfully established using Luc-GL261 cells (Fig. 4 A, 4 B, and Figure S19). Mice with similar tumor growth were selected and underwent resection on the seventh day after inoculation with Luc-GL261 cells (Fig. 4 C). The mice were treated with either PBS, blank Fmoc-YD/YYK hydrogel (Blank Gel), CB-839 hydrogel (CB-839 Gel), Cu-His NPs hydrogel (Cu-His Gel), free CB-839 and Cu-His NPs (Free Combo), or CB-839 and Cu-His NPs hydrogel (Combo Gel). Tumor recurrence was tracked by monitoring bioluminescent signals, and the mice's body weights and survival rates were recorded throughout the study. The PBS-treated mice experienced rapid GBM recurrence and subsequent weight loss due to the absence of drug intervention. The Free Combo group initially lost weight, likely due to the rapid release of a high drug dose. In contrast, the CB-839 Gel, Cu-His Gel, and Combo Gel groups showed minimal weight changes, suggesting that drug-loaded hydrogels effectively suppressed GBM growth without significant toxic side effects (Fig. 4 D). Post-surgical mice with bioluminescence intensity between 10^6–10^7 were selected for surgery, and the success of the procedure was confirmed by re-monitoring bioluminescence on day 8. Tumor bioluminescence was monitored every 7 days until the PBS group's mouse count dropped below three. The Combo Gel group exhibited the most pronounced inhibitory effect on GBM growth, with the lowest luciferase intensity across all monitoring points (Fig. 4 E and 4 G). The Kaplan-Meier survival curves revealed that the median survival time for the Combo Gel-treated group was significantly extended to 48 days, surpassing the 27 days observed in the PBS group, 28 days in the Blank Gel group, 33 days in the CB-839 Gel group, 35 days in the Cu-His Gel group, and 30 days in the Free Combo group (Fig. 4 F). Figure 4 H displays H&E-stained brain tissue sections from mice two weeks post-surgical resection and in situ administration of the various treatments. In comparison to the control group, mice treated with Combo Gel exhibited the smallest tumor area, suggesting its superior efficacy in preventing tumor recurrence. In summary, the experimental results demonstrated that the Combo Gel treatment, which integrates glutamine metabolism regulation with CDT, provided outstanding therapeutic outcomes. It effectively suppressed the recurrence of GBM tumors, extended the median survival of mice, and induced systemic anti-tumor immunity in vivo. 2.4 In vivo Tumor Immune Effect Study of Combo Gel The tumor-killing process of ROS-induced ICD is closely related to the production of related DAMPs, and HMGB1 and CRT are typical biomarkers associated with DAMPs. 60 Fig. 5 A demonstrated that after two weeks of in situ administration of each group of gel biomaterials following surgical resection, the Combo Gel group exhibited the highest fluorescence signal intensity for CRT and HMGB1 in the brain. Notably, the expression of HMGB1 (green fluorescence) was significantly upregulated in the Combo Gel group, effectively promoting the maturation of DC cells in vivo. When comparing the fluorescence signals among the CB-839 Gel, Cu-His Gel, and Combo Gel groups, the CB-839 Gel group showed lower DAMP expression than the other two groups, suggesting that the enhanced ICD effect was primarily due to the excessive ROS production by Cu-His NPs within tumor cells. Symbiotic cells contribute to the development and metastasis of GBM through metabolic interactions, including substrates and products. Metabolic competition for nutrients between tumor cells and immune cells can significantly influence antitumor immunotherapy. 61 Inhibition of GLS1 in tumor cells by the glutaminase inhibitor CB-839 not only altered the metabolic pattern of GBM but also helped reverse the overconsumption of glutamine in the tumor environment, providing nutrients for immune cells to more effectively perform their tumor-killing function. Cellular metabolism regulation not only inhibits tumor cell proliferation and survival but also impacts immune cell function and state. In this system, reprogramming metabolic processes breaks the communication network between the metabolic cycle and downstream immune microenvironment, and affects the activity and function of immune cells, discovering and eliminating residual tumor cells after surgery. CB-839 is a small molecule inhibitor of GLS1, which has been proven in vitro to block the significant consumption of glutamine by tumor cells. When the content of glutamine increases, it reduces the competition pressure between immune cells and tumor cells for glutamine, promotes T cell proliferation and cytokine production, and affects macrophage polarization. Initially, we assessed the polarization of TAM using flow cytometry (FCM) analysis. The findings revealed a notable increase in the proportion of M1-type TAM and a marginal rise in M2-type TAM percentages in both the PBS and Combo Gel groups (Fig. 5 B, 5 C, 5 G, and 5 H). Further analysis indicated that the M1/M2 TAM ratio rose to 23.6 ± 1.0 and 23.1 ± 1.5 in the CB-839 Gel and Combo Gel groups, respectively, which was 4.6 times greater than that observed in the PBS group (5.0 ± 0.4) (Fig. 5 D). The experimental outcomes suggested that CB-839's modulation of the glutamine metabolic pathway could induce the polarization of M2-type TAM to M1-type TAM, potentially reshaping the tumor immune microenvironment. As antigen-presenting cells, the activation of dendritic cells (DCs) is a pivotal step in initiating T-cell responses and establishing anti-tumor immunity. Encouragingly, the Combo Gel group exhibited a significantly higher percentage of activated DCs. In locally recurrent tumor tissues, the DC cell count in the Combo Gel group increased 12-fold compared to the PBS group (Fig. 5 E and 5 I), indicating a reduction in the immunosuppressive microenvironment post-treatment. CD8 + T cells, upon activation, differentiate into cytotoxic T lymphocytes (CTLs) that specifically target and eliminate tumor cells, forming the cornerstone of the adaptive immune system. The presence of CD8 + T cells is crucial for preventing tumor recurrence. To confirm whether Combo Gel effectively activated and recruited CD8 + T cells, we conducted flow cytometry and immunofluorescence analyses on tumor tissues. As anticipated, the Combo Gel-treated tumors showed a marked increase in CD8 + T cell infiltration compared to the PBS group (Fig. 5 F and 5 J). Interestingly, the Cu-His Gel group also significantly attracted CD8 + T cells around the resection site (Fig. 5 K). However, the effect was notably diminished in the Free Combo group, which lacked the gel scaffold, suggesting that the sustained drug release in the Combo Gel group facilitated T-cell infiltration, effectively reshaped the tumor immune microenvironment, and achieved long-term local immune modulation. We collected and analyzed mouse brain tissue homogenates for tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and interleukin-10 (IL-10) using enzyme-linked immunosorbent assay (ELISA) in each treatment group. The cytokine concentrations in the Combo Gel group were significantly different from those in other treatment groups. The Combo Gel group exhibited increased levels of pro-inflammatory cytokines TNF-α, IFN-γ, and IL-6 (Fig. 5 L, 5 M, and 5 N), while the concentration of the anti-inflammatory cytokine IL-10 was reduced (Fig. 5 O). TNF-α, primarily secreted by activated macrophages, can directly kill tumor cells. The Combo Gel group showed a marked increase in TNF-α expression to 2076.9 ± 29.4 pg/mL, which was 1.4 times higher than the PBS group. IFN-γ, known to promote antigen processing and delivery, inhibit tumor cell growth, and enhance systemic anti-tumor immunotherapy, was expressed at 765.3 ± 25.1 pg/mL in the Combo Gel group, a 2.4-fold increase over the PBS group. IL-6 expression in the Combo Gel group was 2293.0 ± 29.4 pg/mL, which was 2.4 times and 2.0 times higher than that in the PBS group (943.7 ± 18.0 pg/mL) and the Blank Gel group (1161.6 ± 27.1 pg/mL), respectively, while IL-10 expression was lower than in the PBS group. The above experiments indicate that Combo Gel not only increases the expression of pro-inflammatory cytokines but also inhibits anti-inflammatory cytokines, which help regulate the function of immune cells. In summary, immunotherapy based on glutamine metabolic regulation synergized with CDT to enhance T-cell infiltration, promote TAM polarization, and elicit an immune response in mice. 3 Conclusions In conclusion, we have devised an immunotherapy approach centered on in situ peptide gel injection to synergistically enhance CDT by means of metabolic reprogramming tailored to the intricate demands of post-operative treatment for GBM. Initially, the in situ injection hydrogel was synthesized by orchestrating the assembly of short peptide molecules to fill the irregular cavity resulting from surgical resection. The sustained release of drugs post-implantation bolstered the therapeutic efficacy, successfully inhibiting GBM recurrence. We harnessed the self-assembly coordination of Cu 2+ with Fmoc-HH to fabricate ROS-generating Cu-His NPs. These nanoparticles were engineering to respond to the tumor-specific microenvironment, deplete GSH and H 2 O 2 within tumor cells, generate ROS, and disrupt the redox balance of the tumor cells. By inhibiting glutaminase activity through CB-839, GSH generation is curtailed, thereby synergistically amplifying oxidative stress and ultimately enhancing the ICD effect. Our experimental findings evinced that by modulating the glutamine metabolic pathway, we could influence the downstream immune microenvironment, augmenting T-cell infiltration and macrophage activation, and eliciting an immune response in mice, thus effecting long-term modulation of the local immune microenvironment. These results underscore the high effectiveness of combined gels as a postoperative GBM treatment system, underscoring its considerable clinical application potential. Declarations Acknowledgments: This work was financially supported by the National Nature Science Foundation of China (Grant Nos. 22193031; 22072155; 22277121; 22172174; 22307117; 21961142022). The National Key R&D Plan of China (Grant No. 2023YFC3904601). The Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB0520300). Chinese Major Program for National Key Research and Development Project (Grant No.2020YFA0112603). Open Funding Project of the State Key Laboratory of Biochemical Engineering & Key Laboratory of Biopharmaceutical Preparation and Delivery (No. 2023KF-06). We express our gratitude to Yiwen Zhang, Haoyu You, and Guihong Lu for their expert guidance in modeling recurrent GBM. References Wei, W. et al. Glioblastoma Multiforme (GBM): An overview of current therapies and mechanisms of resistance. Pharmacol Res. 171 , 105780-105780 (2021). Chris, M., Meera, N., A, M. S. & Puneet, P. 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Covalently triggered self-assembly of peptide-based nanodrugs for cancer theranostics. iScience. 26 (2023). Xiaoli, F. et al. Mitochondria-associated ER stress evokes immunogenic cell death through the ROS-PERK-eIF2a pathway under PTT/CDT combined therapy. Acta Biomater. 160 , 211-224 (2023). Bianbian, Z. et al. Perylene-Mediated Electron Leakage in Respiratory Chain to Trigger Endogenous ROS Burst for Hypoxic Cancer Chemo-Immunotherapy. Adv Sci. 10 , e2204498-e2204498 (2022). Zhaoting, L. et al. Fluorine assembly nanocluster breaks the shackles of immunosuppression to turn the cold tumor hot. PNAS. 117 (2020). Li, Q. et al. Biogenic sensors based on dipeptide assemblies. Matter 5 , 3643-3658 (2022). Yang, H. et al. Electrostatic-Driven Lamination and Untwisting of b-Sheet Assemblies. ACS nano. 10 , 880-888 (2016). Toksoz, S., Mammadov, R., Tekinay, A. B. & Guler, M. O. Electrostatic effects on nanofiber formation of self-assembling peptide amphiphiles. 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A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J. 31 , 1062-1079 (2012). Apetoh, L. et al. Toll-like receptor 4–dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med. 13 , 1050-1059 (2007). Chen, G. et al. Glutamine Antagonist Synergizes with Electrodynamic Therapy to Induce Tumor Regression and Systemic Antitumor Immunity. (2022). Carrer, A. & Wellen, K. E. Metabolism and epigenetics: a link cancer cells exploit. Curr Opin Biotechnol. 34 , 23-29 (2015). Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files SupportingInformation.docx Scheme1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4629023","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":321653773,"identity":"1006f580-2987-4f68-822c-8d6cffb7e98b","order_by":0,"name":"Junbai Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYBACxhnMDQc+GJCmhbHh4AyStDBIMDYw85Ckg3l2Y+NhmwI7ef7+A4wffjDY5RF22JyDDYdzDJINZ9xIYJbsYUguJqxlRiJIy4EEhhsMDNIMDAcSG4jSYgHUIn/+APNv4rUwALUAERuRtgD9crAH6JeNNxLbLIEMwloMZzcf/vDjj5283PnDh2/8qLAjQgtCBSOQSUycyhOhZhSMglEwCkY6AAAYiT9KX2iT+wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9575-3125","institution":"Institute of Chemistry, Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Junbai","middleName":"","lastName":"Li","suffix":""},{"id":321653774,"identity":"130d3af6-f6a1-458a-9ff6-a27dec37619a","order_by":1,"name":"Yiran Guo","email":"","orcid":"","institution":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Yiran","middleName":"","lastName":"Guo","suffix":""},{"id":321653775,"identity":"d470bf0e-19e5-4ee8-8df4-f8a18247eca5","order_by":2,"name":"Tianhe Jiang","email":"","orcid":"","institution":"Institute of Process Engineering, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tianhe","middleName":"","lastName":"Jiang","suffix":""},{"id":321653776,"identity":"afada60c-52f2-441c-8e08-4d9fd3775c20","order_by":3,"name":"Sen Liang","email":"","orcid":"","institution":"Institute of Process Engineering, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sen","middleName":"","lastName":"Liang","suffix":""},{"id":321653777,"identity":"28b19893-4fc6-4ae9-b1b8-76f59594273f","order_by":4,"name":"Anhe Wang","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Anhe","middleName":"","lastName":"Wang","suffix":""},{"id":321653778,"identity":"3c10e3f8-05d1-4a8d-9f54-16ac8d4351e6","order_by":5,"name":"Jieling Li","email":"","orcid":"","institution":"Institute of Process Engineering","correspondingAuthor":false,"prefix":"","firstName":"Jieling","middleName":"","lastName":"Li","suffix":""},{"id":321653779,"identity":"1eec4460-44c5-4cc1-9ddb-8d6fcda376e0","order_by":6,"name":"Qi Li","email":"","orcid":"https://orcid.org/0000-0001-9764-4539","institution":"Institute of Process Engineering, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Li","suffix":""},{"id":321653780,"identity":"4b32d8a2-c82a-47ad-a8b1-d4f0d4dbd1bc","order_by":7,"name":"Jian Yin","email":"","orcid":"https://orcid.org/0000-0002-2284-1666","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Yin","suffix":""},{"id":321653781,"identity":"c3d0dbec-5d68-4448-839f-63bbc5f1baf2","order_by":8,"name":"Shuo Bai","email":"","orcid":"https://orcid.org/0000-0001-5360-3399","institution":"Institute of Process Engineering, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Bai","suffix":""},{"id":321653782,"identity":"0bf43b4b-70eb-42b9-867c-0328d517f68e","order_by":9,"name":"Yi Jia","email":"","orcid":"https://orcid.org/0000-0001-9812-667X","institution":"Institute of Chemistry, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Jia","suffix":""}],"badges":[],"createdAt":"2024-06-24 09:25:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4629023/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4629023/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59608677,"identity":"52ce0bc2-2e02-419e-ad22-a6c7155da20b","added_by":"auto","created_at":"2024-07-03 19:13:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2081499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the injectable hydrogel Combo Gel. \u003c/strong\u003e(A) Schematic illustration of Combo Gel preparation. (B) SEM image of Cu-His NPs. (C) HRTEM image of Cu-His NPs. (D) XRD spectra of Cu-His NPs and Fmoc-HH, with black arrows indicating the positions of satellite peaks. (E) The COMPO images and elemental distribution images of Cu and N for Cu-His NPs detected by EPMA. (F) SEM image of Combo Gel. (G) Combo Gel, when injected through a 22-gauge needle into water, immediately forms a stable gel. (H) Amplitude sweep curves of Fmoc-YD/YYK hydrogels at various concentrations at 37°C. (I) Biocompatibility of Fmoc-YD/YYK hydrogels at different concentrations compared to a positive control group. Fmoc-YD/YYK refers to a 1:1 mixture of Fmoc-YD and Fmoc-YYK, n = 3.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/9d1575d82f14fc0f0ccf6a9a.png"},{"id":59608682,"identity":"7792c880-0868-4b34-b07e-84ecb0bfdd14","added_by":"auto","created_at":"2024-07-03 19:13:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1305227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CDT mechanism of Cu-His NPs and the synergistic cytotoxic effects of Cu-His NPs and CB-839 in vitro, along with the hydrogel's capacity to extend the local retention and release of drugs.\u003c/strong\u003e (A) SEM images of Cu-His NPs mixed with GSH at various time intervals. (B) The degradation of MB after a 4-hour reaction confirms the Fenton-like reaction between Cu\u003csup\u003e+\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, producing ·OH. (C) The inhibitory effects of Cu-His NPs and CB-839 on the GL261 cell line were assessed after 24 hours of incubation, n = 3. (D) The cumulative release profiles of Cu-His NPs and CB-839 from the hydrogel, n = 3. (E) The degradation of the hydrogel in the brains of mice was quantitatively analyzed, n = 5. (F) Live/dead staining of GL261 cells after 12 hours of exposure to Cu-His NPs and CB-839, with green fluorescence representing live cells stained with calcein AM and red fluorescence indicating dead cells stained with propidium iodide (PI). (G) Fluorescence images of the hydrogel retention in the brains of mice at specific time points were captured using an IVIS system. The hydrogel was labeled with Cy5 for tracking. Data are presented as the mean ± S.D, n = 5.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/c862debf54328c0cbc722447.png"},{"id":59608675,"identity":"4ff10c6f-02be-4a2e-aa18-b89194b4a1f5","added_by":"auto","created_at":"2024-07-03 19:13:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":895829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular uptake of Cu-His NPs and the augmentation of ROS generation and ICD effects through synergistic therapy.\u003c/strong\u003e (A) Confocal microscopy images of GL261 cells post 2-hour Cu-His NPs treatment, with nuclei stained blue by DAPI, lysosomes stained green with Lysotracker, and Cy5-labeled Cu-His NPs fluorescing red. (B) Quantification of intracellular GSH and GSSG levels in GBM cells following a 24-hour exposure to the agents. (C) Measurement of intracellular ATP levels in GBM cells after 24 hours of treatment. (D) ROS staining fluorescence in GBM cells after a 4-hour incubation with the agents, was visualized with DAPI-stained nuclei in blue and DCFH-DA-stained ROS in green. (E) Release of CRT and (F) HMGB1 from GBM cells after 24 hours of treatment with the agents. Data are presented as the mean ± S.D. (n = 3; one-way ANOVA, Tukey's multiple comparisons test, ns: not significant, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/a2562cc3cec45b2978dbd324.png"},{"id":59608678,"identity":"0ada7bbf-9b21-45a0-82a1-07c0b848c483","added_by":"auto","created_at":"2024-07-03 19:13:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1964018,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-tumor recurrence efficacy of different treatments in an orthotopic GBM model following resection. (A) Schematic of the animal study timeline. (B) T2-weighted MRI images of orthotopic GBM mice with Luc-GL261 at various time points (n = 3). The tumor mass is outlined in orange. (C) Surgical debulking of mice with orthotopic Luc-GL261 cells on day 7 post-tumor implantation. The GBM mass is outlined in orange. (D) Post-treatment changes in mouse body weight, (E) tumor bioluminescence over time, and (F) mouse survival curves. (G) Temporal changes in bioluminescence signals from Luc-GL261 tumors across treatment groups (partial data). Data are expressed as mean ± SEM (n = 6; one-way ANOVA, Tukey's multiple comparisons test, ***P \u0026lt; 0.001). (H) Two weeks following surgical resection and in situ drug treatment, brain slices from mice were obtained for histological analysis using H\u0026amp;E staining.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/fc6c94d2f0a7b590a5950aad.png"},{"id":59608679,"identity":"d268acbf-159e-4d28-9798-fc74a11af6fa","added_by":"auto","created_at":"2024-07-03 19:13:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1643016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivate anti-tumor immunity post-treatment with different agents.\u003c/strong\u003e (A) Immunofluorescence analysis of HMGB1 and CRT. (B) Flow cytometry analysis of M1-type TAMs, (C) M2-type TAMs, (D) the M1/M2 TAM ratio, (E) mature DCs, and (F) CD8 T cells in mouse brain tissue two weeks post-resection and in situ drug delivery (n = 3). Flow cytometry quantification of infiltrating (G) M1-type TAMs, (H) M2-type TAMs, (I) mature DCs, and (J) CD8 T cells in brain tissue across groups. (K) Immunofluorescence analysis of CD8 T cell infiltration in mouse tumor tissue. ELISA quantification of (L) TNF-a, (M) IFN-g, (N) IL-6, and (O) IL-10 in mouse brain tissue homogenates for each group (n = 5). Data are expressed as mean ± S.D. (one-way ANOVA with Tukey's multiple comparisons test, ns: not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/ef0c6d75a5f1e14266db1544.png"},{"id":61578199,"identity":"14062a29-7be4-4915-98b6-161bdc5904ae","added_by":"auto","created_at":"2024-08-01 12:49:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9885391,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/fe5239a2-c208-4587-90f7-582750b4ccbc.pdf"},{"id":59608992,"identity":"c84527e2-582b-4f34-a45b-8266dc706217","added_by":"auto","created_at":"2024-07-03 19:21:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30348469,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/7532ef2f6591a13db7785c06.docx"},{"id":59608676,"identity":"6041f0ef-c690-4fd9-a72b-2d2205c6bba9","added_by":"auto","created_at":"2024-07-03 19:13:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":681307,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4629023/v1/a39c8518d75a6a805a187ebe.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Immunostimulatory hydrogel with synergistic blockage of glutamine metabolism and chemodynamic therapy for postoperative management of glioblastoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlioblastoma multiforme (GBM) is the most aggressive and lethal malignant brain tumor within the central nervous system.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Standard clinical management typically involves surgical resection, followed by adjuvant chemotherapy and radiotherapy.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e However, the infiltrative nature of GBM often prevents complete surgical removal, and surgery can disrupt the tumor's immune microenvironment, potentially delaying chemotherapy and leading to tumor recurrence.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Additionally, the post-surgical cavity provides space for GBM recurrence. The blood-brain barrier (BBB) further complicates the delivery of drugs and immune cells to the brain.\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The malignant growth characteristics of GBM, coupled with the unique postoperative treatment needs, pose significant challenges for drug delivery methods and therapeutic strategies following GBM surgery.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMetabolism plays a pivotal role in the vitality, expansion, invasiveness, and metastatic potential of tumor cells.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Unlike normal cells, cancer cells undergo metabolic reprogramming to satisfy their heightened demands for energy and nutrients, inevitably leading to competition for nutrients with coexisting cells.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Furthermore, metabolic substrates and products within the tumor environment also impact the functional states of cancer and coexisting cells. Notably, the metabolic reprogramming of tumor cells can facilitate information exchange and crosstalk with immune cells through metabolites, indicating that cellular metabolic processes should not be considered in isolation.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Regulating cellular metabolism can influence the functional changes in downstream immune cells and reshape the immune microenvironment, offering a fresh perspective for exploring the interplay between cell metabolism and immunity.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eGlutamine plays a critical role in cancer cell metabolism, serving as a key source for energy generation, biosynthesis, and redox balance maintenance.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The regulation of glutamine metabolism in cancer cells also affects the communication and crosstalk with immune cells in the microenvironment through metabolites.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e This interaction influences the polarization of tumor-associated macrophages (TAMs) and enhances the activity of T lymphocytes and cytokine expression by depriving cancer cells of essential nutrients like glutamine, thereby improving the efficacy of immunotherapy. \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Given the dual significance of suppressing tumor growth and immune modulation, targeting glutamine metabolism has emerged as a key focus for cancer therapy. CB-839, a small molecule glutaminase inhibitor currently in clinical trials (NCT03057600 and NCT03163667), has shown promise.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e However, clinical outcomes have not met expectations due to tumor heterogeneity, metabolic adaptability, the complex immuno-suppressive microenvironment, and challenges in drug delivery, underscoring the limitations of monotherapy.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Research indicates that inhibiting glutamine catabolism can reduce glutamate levels, a precursor to glutathione (GSH), thereby decreasing GSH content. This reduction impairs the tumor cells' ability to clear intracellular reactive oxygen species (ROS),\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e establishing a link for synergistic dynamic therapy through glutamine modulation.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFor deeply situated tissues like GBM in the brain, CDT for ROS delivery is ideal.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Copper-based nanomaterials, more efficient and with a broader pH tolerance than iron-based counterparts, are used to generate CDT.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e However, excessive free copper ions may lead to severe systemic toxicity, so it is necessary to limit copper ions in time and space, allowing them to be stable in the physiological environment but rapidly released in tumor cells, producing ROS through the Fenton reaction to kill tumors.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the unique postoperative treatment scenarios of GBM, drug delivery systems must adhere to rigorous temporal and spatial specifications.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Injectable peptide hydrogels can fill the irregular cavities left by surgery, physically restricting tumor proliferation, and facilitating a controlled release of therapeutic agents through the modulation of hydrogel degradation.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Moreover, endogenous short peptides have excellent biocompatibility and low immunogenicity, making them easy to regulate assembly.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e This allows for the regulation of carrier degradation time and pH triggering conditions to meet the complex requirements of sustained drug release.\u003c/p\u003e \u003cp\u003eIn this study, we have developed an innovative postoperative treatment system for GBM. Our approach involves the utilization of an in situ injectable peptide hydrogel that is loaded with the glutaminase inhibitor CB-839 and self-assembling Cu\u003csup\u003e2+\u003c/sup\u003e-peptide coordination nanoparticles (Cu-His NPs) for CDT (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, CB-839 effectively suppresses glutamine production, thereby reducing intracellular GSH levels and enhancing the cytotoxicity of Cu\u003csup\u003e2+\u003c/sup\u003e-mediated CDT. This occurs by diminishing GSH's neutralizing effect on ROS. Moreover, CDT induces a significant increase in ROS production, leading to amplified oxidative stress within tumor cells. Consequently, this triggers immunogenic cell death (ICD) and initiates an immune response to counteract the immunosuppressive tumor microenvironment (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e The short peptide molecules, serving as the building blocks of our carrier, exhibit remarkable flexibility and diversity in its assembly.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e It not only enables the targeted release of Cu\u003csup\u003e2+\u003c/sup\u003e within the tumor region but also allows for control over the mechanical strength and degradation time of the hydrogel carrier, facilitating the sustained release of drugs. Remarkably, our experimental results demonstrate that this immunotherapy strategy, based on metabolic reprogramming, effectively addresses the complex treatment scenarios following GBM surgery. It successfully inhibits GBM recurrence and significantly prolongs overall survival in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Synthesis and Characterization of Combo Gel\u003c/h2\u003e \u003cp\u003eIn this study, we successfully synthesized an injectable peptide hydrogel (Combo Gel) loaded with Cu\u003csup\u003e2+\u003c/sup\u003e-peptide nanoparticle (Cu-His NP) complexes and the glutaminase inhibitor CB-839 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Initially, we selected short peptide molecules with positively charged Fmoc-Tyrosine-Tyrosine-Lysine (Fmoc-YYK) and negatively charged Fmoc-Tyrosine-Aspartic acid (Fmoc-YD) to prepare injectable hydrogel scaffolds.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e These molecules utilized a variety of intermolecular forces, such as electrostatic and π-π interactions, to assemble with flexibility and diversity.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e The hydrogel scaffold is semi-transparent at room temperature (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), and the scanning electron microscope (SEM) image reveals the common fibrous morphology of the hydrogel at the micro-scale (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Fourier transform infrared spectroscopy (FT-IR) analysis revealed absorption peaks at 1690 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1615 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, characteristic of the antiparallel β-sheet secondary structure in the peptide hydrogel (Figure S2).\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e-based CDT nanomaterials have demonstrated promising potential in tumor treatment. However, the targeted release of Cu\u003csup\u003e2+\u003c/sup\u003e and issues related to free Cu\u003csup\u003e2+\u003c/sup\u003e have constrained their advancement. In this study, we coordinated histidine, featuring an imidazole group, with Cu\u003csup\u003e2+\u003c/sup\u003e to develop CDT generators. The histidine, known for its potent membrane-penetrating ability, \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e was used to control the release of Cu\u003csup\u003e2+\u003c/sup\u003e specifically in the tumor microenvironment. Comparing the SEM image of Cu-His NPs in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the high-resolution transmission electron microscopy (HRTEM) image of Cu-His NPs in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, and the HRTEM image of the raw material Fmoc-Histidine-Histidine (Fmoc-HH) in Figure S3, it could be observed that the morphology of Cu-His NPs was distinctly different from that of Fmoc-HH, forming spherical particles with a diameter of approximately 40 nm. HRTEM analysis revealed a lattice spacing of 0.324 nm for the Cu-His NPs, slightly larger than the 0.225 nm spacing of Fmoc-HH, confirming the formation of ligand-bound nanoparticles between Cu\u003csup\u003e2+\u003c/sup\u003e and Fmoc-HH (Figure S4). The FT-IR spectrum of Cu-His NPs and Fmoc-HH revealed that in the presence of Cu\u003csup\u003e2+\u003c/sup\u003e, the stretching vibration of the N-H bond in the histidine imidazole group shifted from 3294 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 3146 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the absorption peaks for the stretching vibrations of C\u0026thinsp;=\u0026thinsp;O and C-N bonds significantly intensified at 1656 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1132 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Figure S5). The shifts and enhancements of these vibrational peaks indicated that Cu\u003csup\u003e2+\u003c/sup\u003e had formed coordination bonds with nitrogen or oxygen atoms within the peptide chain. X-ray photoelectron spectroscopy (XPS) analysis showed peaks for Cu2p, O1s, N1s, and C1s at 933.1, 529.1, 398.1, and 283.1 eV, respectively (Figure S6), with the Cu2p\u003csub\u003e3/2\u003c/sub\u003e and Cu2p\u003csub\u003e1/2\u003c/sub\u003e peaks located at 931.4 and 951.3 eV. The main peaks were separated by a 19.9 eV spin energy, and satellite peaks were observed at 941 and 961 eV, indicating the presence of Cu\u003csup\u003e2+\u003c/sup\u003e in the coordination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Electron probe microanalysis (EPMA) further confirmed the uniform distribution of Cu\u003csup\u003e2+\u003c/sup\u003e throughout the sample, indicating that the Cu-His NPs were formed by the self-assembly of Cu\u003csup\u003e2+\u003c/sup\u003e and Fmoc-HH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThis hydrogel scaffold was loaded with the CDT generator Cu-His NPs and the glutaminase inhibitor CB-839. SEM images revealed that the Cu-His NPs were uniformly dispersed within the hydrogel's fibrous matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), and the formulation demonstrated superior injectability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Rheological testing indicated that Combo Gel's viscosity significantly decreased with an increase in shear rate from 0.1 to 100 s\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, showcasing its pronounced shear-thinning behavior (Figure S7). Given the unique mechanical characteristics of brain tissue, with a modulus of approximately 1\u0026ndash;2 kPa, any mechanical discrepancy between implanted materials and the brain could potentially induce epilepsy or other undesirable immune reactions. \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e Therefore, gel scaffolds with varying mechanical strengths were fabricated by adjusting the assembly of short peptide molecules. Select the gel scaffold matching the mechanical properties of brain tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), and verify the biocompatibility of the gel scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 In Vitro Anti-Tumor Studies of Combo Gel:\u003c/h2\u003e \u003cp\u003eNext, we investigated the in vitro antitumor efficacy of Combo Gel. The Cu\u003csup\u003e2+\u003c/sup\u003e was capable of undergoing a redox reaction with glutathione (GSH), resulting in the formation of Cu\u003csup\u003e+\u003c/sup\u003e and oxidized glutathione (GSSG). When equal mass ratios of Cu-His NPs and GSH were combined in an aqueous solution, a rapid emergence of purplish-red fluorescence was observed under UV light (Figure S8A), indicating the occurrence of a redox reaction. \u003csup\u003e\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Over time, this fluorescence gradually faded, confirming the progression of the reaction. The fluorescence and emission spectra of the mixtures were further confirmed using a fluorescence spectrophotometer (Figure S8B). SEM analysis showed a significant morphological change in the Cu-His NPs after only 5 minutes of reaction, with the nanoparticles losing their spherical integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting an extremely rapid reaction kinetics. Post-reaction, the supernatant was collected and the lyophilized samples were analyzed by FT-IR (Figure S9), high-resolution mass spectrometry (HRMS) (Figure S10), and hydrogen nuclear magnetic resonance (\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR) (Figure S11), all confirming the formation of GSSG. \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e These findings confirmed that Cu-His NPs degraded in the presence of GSH, undergoing a redox reaction to produce Cu\u003csup\u003e+\u003c/sup\u003e and GSSG. Methylene blue (MB), known for its characteristic UV-absorption peak, can be degraded by hydroxyl radicals (\u0026middot;OH). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB illustrates that the UV absorption peaks of MB reacted with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e alone or with a mixture of Cu-His NPs and GSH showed minimal changes after 4 hours. However, significant changes in the UV absorption peaks were observed when MB, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, Cu-His NPs, and GSH were all present, suggesting that the Fenton-like reaction of Cu\u003csup\u003e2+\u003c/sup\u003e with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the presence of GSH can initiate a cascade reaction to produce \u0026middot;OH. The rate of \u0026middot;OH production in the absence of GSH was significantly lower compared to when GSH was added, as observed after mixing MB, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and Cu-His NPs (Figure S12). This further supports the notion that Cu-His NPs can more effectively generate \u0026middot;OH under GSH influence, targeting tumor cell destruction. Figure S13 showed that the rate of \u0026middot;OH production in the presence of Cu-His NPs and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e gradually slowed down as the reaction time increased with GSH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further investigated the synergistic tumor cell-killing effects of Cu-His NPs combined with CB-839 in vitro. The Cell Counting Kit-8 (CCK-8) assay was utilized to assess the cell inhibition rates of the GL261 GBM cell line following 24 hours of treatment with Cu-His NPs, CB-839 alone, or their combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The findings indicated that the cell inhibition rate with CB-839 and Cu-His NPs co-cultured with tumor cells was markedly higher than that of single-agent treatments. At a Combo group drug concentration of 10 \u0026micro;g/mL, the inhibition rate after 24 hours reached 27%, exhibiting strong cytotoxicity. As the drug concentration increased, the cytotoxicity of the combined treatment against cancer cells was also significantly enhanced. Live/dead cell staining, after culturing at a drug concentration of 10 \u0026micro;g/mL for 12 hours, yielded the same conclusion: the synergistic effect of Cu-His NPs and CB-839 amplified the cytotoxicity against GL261 glioma cells in vitro, with a significantly higher cell inhibition rate than monotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eWe immersed hydrogels loaded with Cu-His NPs (Cu-His Gel) and CB-839 (CB-839 Gel) separately in phosphate-buffered saline (PBS) and incubated at 37\u0026deg;C. The release concentrations were monitored using high-performance liquid chromatography (HPLC). The release profiles revealed that by day 5, the release amounts of Cu-His NPs and CB-839 were 57% and 49%, respectively, with the release rate subsequently decreasing. By day 20, approximately 80% of both drugs had been released (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Both agents followed zero-order release kinetics, suggesting that the hydrogel scaffold effectively sustained drug release, extending the retention time in the mouse brain. The drug release rate was found to be nearly concurrent with the hydrogel volume loss, indicating that release was primarily dependent on hydrogel degradation. To further evaluate the in vivo release behavior, Combo Gel was injected into the brains of mice, and the Cy5-labeled hydrogel signal was quantitatively monitored at set time points using an in vivo imaging system (IVIS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The injected hydrogel rapidly underwent the solution-to-gel transition within the mouse cranium, and this swift in situ gelation effectively prevented rapid drug release. Continuous monitoring indicated that the gel remained in the mice for approximately two weeks, with no significant fluorescent signals detected in other major organs or blood (Figure S14), and the mice's body weight remained unaffected (Figure S15). The mice's major organs were harvested for H\u0026amp;E staining, showing no notable differences compared to healthy mice, which suggested that the gel formulation possessed excellent biocompatibility (Figure S16).\u003c/p\u003e \u003cp\u003ePrevious experiments demonstrated that Cu-His NPs emitted purplish-red fluorescence upon interaction with GSH. After a 2-hour incubation, confocal microscopy revealed intracellular red fluorescence, indicating that the Cu-His NPs had been internalized and initiated their reaction with GSH (Figure S17A). By 8 hours, the red fluorescence within the cells had nearly vanished, suggesting the completion of the Fenton-like reaction of Cu-His NPs (Figure S17B). To further visualize the intracellular drug release process of Cu-His NPs, lysosomes were labeled with Lysotracker Red and co-localized with Cy5-labeled Cu-His NPs, which exhibited blue fluorescence, and the nucleus. After 4 hours of incubation, the fluorescence of Cu-His NPs almost entirely overlapped with that of the lysosomes. The overlapping region displayed yellow fluorescence, indicating that the Cu-His NPs were sequestered in lysosomes following endocytosis, and some had escaped into the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Figure S18). These findings confirmed that Cu-His NPs could undergo lysosomal escape post-cell entry, releasing the drug into the cytoplasm and enhancing its intracellular efficacy. The mechanism of intracellular escape by Cu-His NPs likely involved synergistic effects of histidine properties within the specific environment of tumor cells. \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e Histidine's protonation in the lysosome's acidic milieu and its subsequent charge change may have facilitated its interaction with the lysosomal membrane and promoted escape. Additionally, histidine's transition from hydrophobic to hydrophilic properties, along with its proton sponge effect, facilitated the efficient uptake and swift drug release of Cu-His NPs. These effects collectively promote the efficient uptake of Cu-His NPs within cells, enhance the bioavailability of Cu\u003csup\u003e2+\u003c/sup\u003e, and enhance anti-tumor effects. \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eROS in excess exerted a complex impact on tumor cells, disrupting redox balance and inducing damage or death. This triggered ICD, releasing DAMPs that activated antigen-presenting cells (APCs), including dendritic cells, and stimulated the immune system. GSH was critical for countering ROS-induced oxidative stress; its levels inversely correlated with ROS, and reduced GSH could lead to ROS accumulation and cell death. Our study assessed the effects of different formulations on GSH and GSSG levels in GL261 cells. Both CB-839 and Cu-His NPs individually reduced GSH levels to 19.0 and 11.7 \u0026micro;M, respectively. However, the synergistic effect of CB-839 and Cu-His NPs more significantly blocked both the synthesis of new GSH and the depletion of pre-existing GSH, with an effective concentration of just 8.2 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Subsequently, ROS production in tumor cells post-treatment was monitored using DCFH-DA staining. The Combo group exhibited the highest ROS production, with a marked increase in fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, DAMPs associated with ICD, such as surface calreticulin (CRT), high mobility group box 1 protein (HMGB1), and ATP, were released during cell death. \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e ATP emitted \"find-me\" signals, promoting the phagocytosis of apoptotic cells and triggering specific anti-tumor immune responses. \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e CRT served as an \"eat-me\" signal, enhancing the phagocytosis of apoptotic cells by dendritic cells or their precursors, thereby providing antigens and promoting DC maturation and function. \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e HMGB1 is bound to pattern recognition receptors (PRRs) on bone marrow cells, activating immune signaling pathways and further amplifying immune responses. \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e The experimental results revealed that the Combo group significantly increased the expression of DAMP signaling molecules in tumor cells compared to the single drug treatment group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The Combo therapy not only enhanced ICD by promoting the release of DAMPs from GBM cells but also amplified the immune response, suggesting a potential for long-lasting anti-tumor immunity and a promising approach for cancer therapy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 In vivo anti-tumor studies of Combo Gel:\u003c/h2\u003e \u003cp\u003eThe infiltrative growth of GBM often results in indistinct tumor boundaries, making complete surgical removal challenging and leading to frequent post-surgical recurrence. A primary GBM model was successfully established using Luc-GL261 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, and Figure S19). Mice with similar tumor growth were selected and underwent resection on the seventh day after inoculation with Luc-GL261 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The mice were treated with either PBS, blank Fmoc-YD/YYK hydrogel (Blank Gel), CB-839 hydrogel (CB-839 Gel), Cu-His NPs hydrogel (Cu-His Gel), free CB-839 and Cu-His NPs (Free Combo), or CB-839 and Cu-His NPs hydrogel (Combo Gel). Tumor recurrence was tracked by monitoring bioluminescent signals, and the mice's body weights and survival rates were recorded throughout the study. The PBS-treated mice experienced rapid GBM recurrence and subsequent weight loss due to the absence of drug intervention. The Free Combo group initially lost weight, likely due to the rapid release of a high drug dose. In contrast, the CB-839 Gel, Cu-His Gel, and Combo Gel groups showed minimal weight changes, suggesting that drug-loaded hydrogels effectively suppressed GBM growth without significant toxic side effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Post-surgical mice with bioluminescence intensity between 10^6\u0026ndash;10^7 were selected for surgery, and the success of the procedure was confirmed by re-monitoring bioluminescence on day 8. Tumor bioluminescence was monitored every 7 days until the PBS group's mouse count dropped below three. The Combo Gel group exhibited the most pronounced inhibitory effect on GBM growth, with the lowest luciferase intensity across all monitoring points (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). The Kaplan-Meier survival curves revealed that the median survival time for the Combo Gel-treated group was significantly extended to 48 days, surpassing the 27 days observed in the PBS group, 28 days in the Blank Gel group, 33 days in the CB-839 Gel group, 35 days in the Cu-His Gel group, and 30 days in the Free Combo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH displays H\u0026amp;E-stained brain tissue sections from mice two weeks post-surgical resection and in situ administration of the various treatments. In comparison to the control group, mice treated with Combo Gel exhibited the smallest tumor area, suggesting its superior efficacy in preventing tumor recurrence. In summary, the experimental results demonstrated that the Combo Gel treatment, which integrates glutamine metabolism regulation with CDT, provided outstanding therapeutic outcomes. It effectively suppressed the recurrence of GBM tumors, extended the median survival of mice, and induced systemic anti-tumor immunity in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 In vivo Tumor Immune Effect Study of Combo Gel\u003c/h2\u003e \u003cp\u003eThe tumor-killing process of ROS-induced ICD is closely related to the production of related DAMPs, and HMGB1 and CRT are typical biomarkers associated with DAMPs.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA demonstrated that after two weeks of in situ administration of each group of gel biomaterials following surgical resection, the Combo Gel group exhibited the highest fluorescence signal intensity for CRT and HMGB1 in the brain. Notably, the expression of HMGB1 (green fluorescence) was significantly upregulated in the Combo Gel group, effectively promoting the maturation of DC cells in vivo. When comparing the fluorescence signals among the CB-839 Gel, Cu-His Gel, and Combo Gel groups, the CB-839 Gel group showed lower DAMP expression than the other two groups, suggesting that the enhanced ICD effect was primarily due to the excessive ROS production by Cu-His NPs within tumor cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSymbiotic cells contribute to the development and metastasis of GBM through metabolic interactions, including substrates and products. Metabolic competition for nutrients between tumor cells and immune cells can significantly influence antitumor immunotherapy. \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e Inhibition of GLS1 in tumor cells by the glutaminase inhibitor CB-839 not only altered the metabolic pattern of GBM but also helped reverse the overconsumption of glutamine in the tumor environment, providing nutrients for immune cells to more effectively perform their tumor-killing function. Cellular metabolism regulation not only inhibits tumor cell proliferation and survival but also impacts immune cell function and state. In this system, reprogramming metabolic processes breaks the communication network between the metabolic cycle and downstream immune microenvironment, and affects the activity and function of immune cells, discovering and eliminating residual tumor cells after surgery.\u003c/p\u003e \u003cp\u003eCB-839 is a small molecule inhibitor of GLS1, which has been proven in vitro to block the significant consumption of glutamine by tumor cells. When the content of glutamine increases, it reduces the competition pressure between immune cells and tumor cells for glutamine, promotes T cell proliferation and cytokine production, and affects macrophage polarization. Initially, we assessed the polarization of TAM using flow cytometry (FCM) analysis. The findings revealed a notable increase in the proportion of M1-type TAM and a marginal rise in M2-type TAM percentages in both the PBS and Combo Gel groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Further analysis indicated that the M1/M2 TAM ratio rose to 23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 and 23.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 in the CB-839 Gel and Combo Gel groups, respectively, which was 4.6 times greater than that observed in the PBS group (5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The experimental outcomes suggested that CB-839's modulation of the glutamine metabolic pathway could induce the polarization of M2-type TAM to M1-type TAM, potentially reshaping the tumor immune microenvironment.\u003c/p\u003e \u003cp\u003eAs antigen-presenting cells, the activation of dendritic cells (DCs) is a pivotal step in initiating T-cell responses and establishing anti-tumor immunity. Encouragingly, the Combo Gel group exhibited a significantly higher percentage of activated DCs. In locally recurrent tumor tissues, the DC cell count in the Combo Gel group increased 12-fold compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), indicating a reduction in the immunosuppressive microenvironment post-treatment. CD8\u003csup\u003e+\u003c/sup\u003e T cells, upon activation, differentiate into cytotoxic T lymphocytes (CTLs) that specifically target and eliminate tumor cells, forming the cornerstone of the adaptive immune system. The presence of CD8\u003csup\u003e+\u003c/sup\u003e T cells is crucial for preventing tumor recurrence. To confirm whether Combo Gel effectively activated and recruited CD8\u003csup\u003e+\u003c/sup\u003e T cells, we conducted flow cytometry and immunofluorescence analyses on tumor tissues. As anticipated, the Combo Gel-treated tumors showed a marked increase in CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Interestingly, the Cu-His Gel group also significantly attracted CD8\u003csup\u003e+\u003c/sup\u003e T cells around the resection site (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). However, the effect was notably diminished in the Free Combo group, which lacked the gel scaffold, suggesting that the sustained drug release in the Combo Gel group facilitated T-cell infiltration, effectively reshaped the tumor immune microenvironment, and achieved long-term local immune modulation.\u003c/p\u003e \u003cp\u003eWe collected and analyzed mouse brain tissue homogenates for tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and interleukin-10 (IL-10) using enzyme-linked immunosorbent assay (ELISA) in each treatment group. The cytokine concentrations in the Combo Gel group were significantly different from those in other treatment groups. The Combo Gel group exhibited increased levels of pro-inflammatory cytokines TNF-α, IFN-γ, and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN), while the concentration of the anti-inflammatory cytokine IL-10 was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). TNF-α, primarily secreted by activated macrophages, can directly kill tumor cells. The Combo Gel group showed a marked increase in TNF-α expression to 2076.9\u0026thinsp;\u0026plusmn;\u0026thinsp;29.4 pg/mL, which was 1.4 times higher than the PBS group. IFN-γ, known to promote antigen processing and delivery, inhibit tumor cell growth, and enhance systemic anti-tumor immunotherapy, was expressed at 765.3\u0026thinsp;\u0026plusmn;\u0026thinsp;25.1 pg/mL in the Combo Gel group, a 2.4-fold increase over the PBS group. IL-6 expression in the Combo Gel group was 2293.0\u0026thinsp;\u0026plusmn;\u0026thinsp;29.4 pg/mL, which was 2.4 times and 2.0 times higher than that in the PBS group (943.7\u0026thinsp;\u0026plusmn;\u0026thinsp;18.0 pg/mL) and the Blank Gel group (1161.6\u0026thinsp;\u0026plusmn;\u0026thinsp;27.1 pg/mL), respectively, while IL-10 expression was lower than in the PBS group. The above experiments indicate that Combo Gel not only increases the expression of pro-inflammatory cytokines but also inhibits anti-inflammatory cytokines, which help regulate the function of immune cells. In summary, immunotherapy based on glutamine metabolic regulation synergized with CDT to enhance T-cell infiltration, promote TAM polarization, and elicit an immune response in mice.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Conclusions","content":"\u003cp\u003eIn conclusion, we have devised an immunotherapy approach centered on in situ peptide gel injection to synergistically enhance CDT by means of metabolic reprogramming tailored to the intricate demands of post-operative treatment for GBM. Initially, the in situ injection hydrogel was synthesized by orchestrating the assembly of short peptide molecules to fill the irregular cavity resulting from surgical resection. The sustained release of drugs post-implantation bolstered the therapeutic efficacy, successfully inhibiting GBM recurrence. We harnessed the self-assembly coordination of Cu\u003csup\u003e2+\u003c/sup\u003e with Fmoc-HH to fabricate ROS-generating Cu-His NPs. These nanoparticles were engineering to respond to the tumor-specific microenvironment, deplete GSH and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e within tumor cells, generate ROS, and disrupt the redox balance of the tumor cells. By inhibiting glutaminase activity through CB-839, GSH generation is curtailed, thereby synergistically amplifying oxidative stress and ultimately enhancing the ICD effect. Our experimental findings evinced that by modulating the glutamine metabolic pathway, we could influence the downstream immune microenvironment, augmenting T-cell infiltration and macrophage activation, and eliciting an immune response in mice, thus effecting long-term modulation of the local immune microenvironment. These results underscore the high effectiveness of combined gels as a postoperative GBM treatment system, underscoring its considerable clinical application potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the National Nature Science Foundation of China (Grant Nos. 22193031; 22072155; 22277121; 22172174; 22307117; 21961142022). The National Key R\u0026amp;D Plan of China (Grant No. 2023YFC3904601). The Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB0520300). Chinese Major Program for National Key Research and Development Project (Grant No.2020YFA0112603). Open Funding Project of the State Key Laboratory of Biochemical Engineering \u0026amp; Key Laboratory of Biopharmaceutical Preparation and Delivery (No. 2023KF-06). We express our gratitude to Yiwen Zhang, Haoyu You, and Guihong Lu for their expert guidance in modeling recurrent GBM.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWei, W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Glioblastoma Multiforme (GBM): An overview of current therapies and mechanisms of resistance. \u003cem\u003ePharmacol Res.\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 105780-105780 (2021).\u003c/li\u003e\n \u003cli\u003eChris, M., Meera, N., A, M. S. \u0026amp; Puneet, P. Glioblastoma: clinical presentation, diagnosis, and management. \u003cem\u003eBMJ.\u003c/em\u003e \u003cstrong\u003e374\u003c/strong\u003e, n1560-n1560 (2021).\u003c/li\u003e\n \u003cli\u003eRupesh, K., Yazmin, O., A, K. A. \u0026amp; S, A. M. 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Metabolism and epigenetics: a link cancer cells exploit. \u003cem\u003eCurr Opin Biotechnol.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 23-29 (2015).\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Short peptide, Self-assembly, Glutamine metabolism, Glioblastoma multiforme","lastPublishedDoi":"10.21203/rs.3.rs-4629023/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4629023/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlioblastoma multiforme (GBM) stands as one of the most lethal malignant brain tumors affecting the central nervous system. Post-surgery, patients encounter daunting challenges like tumor recurrence, increased intracranial pressure due to cavitation, and constraints linked with immediate postoperative oral chemotherapy. Herein, we construct an injected peptide gel with in situ immunostimulatory functions to harmonize the regulation of glutamine metabolism and chemodynamic therapy in tackling the postoperative obstacles. The methodology entails crafting injectable gel scaffolds with short peptide molecules, incorporating the glutaminase inhibitor CB-839 and copper peptide self-assembled particles (Cu-His NPs) renowned for their chemodynamic therapy (CDT) efficacy. By fine-tuning glutamic acid production via metabolic pathways, our system not only heightens the therapeutic prowess of copper peptide particles in CDT but also escalates intracellular oxidative stress. This dual mechanism culminates in augmented immunogenic cell death (ICD) within glioblastoma multiforme cells and improves a conducive immune microenvironment. Anchored on the tenets of metabolic reprogramming, this treatment strategy showcases substantial promise in significantly curtailing GBM tumor recurrence, prolonging median survival in murine models.\u003c/p\u003e","manuscriptTitle":"Immunostimulatory hydrogel with synergistic blockage of glutamine metabolism and chemodynamic therapy for postoperative management of glioblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 19:13:11","doi":"10.21203/rs.3.rs-4629023/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e53026f-a916-47f6-a7b3-1812814aeb38","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34005443,"name":"Physical sciences/Materials science/Biomaterials/Biomedical materials"},{"id":34005444,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":34005445,"name":"Biological sciences/Cancer/Cancer metabolism"}],"tags":[],"updatedAt":"2024-08-01T12:41:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 19:13:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4629023","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4629023","identity":"rs-4629023","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}