Local therapeutic platform prevents postsurgical GBM recurrence by diminishing GICs and reshaping immunosuppressive microenvironment

Domain: chemistry
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
Glioblastoma multiforme (GBM) is characterized by aggressive invasiveness and poor prognosis, and presents a significant clinical challenge due to incomplete surgical resection, poor blood‒brain barrier penetration of therapeutic agents, and inevitable recurrence mainly driven by residual glioma-initiating cells (GICs) and an immunosuppressive microenvironment. Therefore, there is an urgent need to kill remaining GICs and reverse the immunosuppressive tumor microenvironment to prevent postoperative recurrence of GBM. Here, we design an injectable therapeutic platform that targets and kills remaining GICs and reverses the immunosuppressive micro-environment after postoperative resection to prevent the recurrence of GBM. This platform comprises GIC-targeting exosomes that carry siRNA for Notch1 and mitoxantrone to reduce the stemness of GICs and kill residual GICs and glioma cells, respectively, and an immune activator (interleukin-12), which can remodel the immunosuppressive tumor microenvironment, ultimately suppressing postoperative GBM relapse. Our work provides a perspective into the effective inhibition of post resection recurrence of GBM.

Introduction
GBM, the most lethal type of brain tumor with a poor prognosis and a 5-year survival rate of less than 6%1, accounts for approximately 48% of all primary malignant central nervous system tumors2. For patients with GBM, the initial clinical intervention is surgical debulking, and chemoradiotherapy (CRT) is typically administered approximately one month after surgery3–5. This one-month post-operative interval represents a crucial period during which tumor recurrence may be triggered6. GBM recurrence can occur as a result of the difficulty incompletely removing the tumors7; in particular, residual GICs in deep intracranial locations of the brain are highly infiltrative and diffuse, leading to recurrence in almost all GBM patients8. Thus, targeted in situ eradication of residual GICs may be a promising therapeutic strategy against GBM recurrence.

GICs, a special aggressive population of cancer cells with self-renewal ability and multidirectional differentiation ability, are central to the recurrence of malignant glioma and therapeutic resistance9,10. Notch signaling, as an important mediator, is associated with stem cell self-renewal and therapeutic resistance, and evidence has shown that Notch1 is overexpressed in GICs and serves as a critical regulator in maintaining GIC stemness11,12. Therefore, specifically knocking down Notch1 expression via small interfering RNA (siRNA) may be an attractive approach for preventing GBM recurrence. Additionally, evidence has indicated that, compared with the first-line chemotherapy agent temozolomide (TMZ), mitoxantrone (MIT) has approximately 50 times stronger killing effects on GICs13,14, triggering tumor cell immunogenic cell death (ICD) and inducing the maturation of dendritic cells (DCs)15. Codelivery of siNotch1 and MIT may both eliminate GICs and increase the expression of tumor-associated antigens, thus activating immunity. However, the poor stability and targeting of siRNA and MIT are the primary barriers to their application, and suitable vesicles that can be delivered into cells are needed16. Exosomes (exos) are natural nanomembrane vesicles that originate from endocytosis and are released into the extracellular environment following the fusion of multivesicular bodies (MVBs) with the plasma membrane17. Their nanoscale size, low immunogenicity and easy modification make exos excellent candidates as drug carriers18. Therefore, the co-laden exos were further surficially modified with targeted peptides (DVAP peptides have good affinity for GRP78, which is slowly expressed in normal cells and highly expressed in GICs and glioma cells19), with the goal of targeting the remaining GICs and glioma cells.

Another key factor for recurrence is the highly immune inhibitory tumor microenvironment in GBM, which inhibits effective immune surveillance by promoting the release of immune suppressors and altering the infiltration of CD4+ T and CD8+ T cells, DC cells, regulatory T cells (Treg cells) and myeloid-derived suppressor cells (MDSCs)20,21. Interleukin-12 (IL-12), a “master” immune-activating cytokine with potent tumor-suppressive activity, plays a crucial role in cancer immune surveillance22. IL-12 can activate T cells with persistent cytotoxic activity, improve antigen presentation, and reshape endogenous immunosuppressive cells in the tumor microenvironment; thus, it has emerged as an attractive candidate for cancer immunotherapy23. However, systemic IL-12 is poorly tolerated. As a result, a delivery method that increases IL-12 concentrations more selectively in the tumor environment without systemic toxicity is desirable24.

Surgical resection is the initial clinical intervention measure for GBM patients, and the postoperative in situ drug delivery system (PIDDS) can be seamlessly integrated with the operation, filling the postoperative treatment interval, eliminating residual tumor cells, and effectively inhibiting recurrence25,26. In addition, PIDDS provides an effective delivery approach for therapeutic agents, which results in high levels of therapeutic accumulation in the tumor microenvironment, bypassing the blood–brain barrier, thereby reducing side effects27,28. The use of the Gliadel® wafer remains the sole FDA-approved PIDDS for GBM, although it is rarely used clinically due to complications29. Various formulations of intracavity delivery systems have been proposed, among which hydrogels with high biocompatibility and non immunogenicity have exhibited promising potential for use in biomedical applications30.

In this work, we establish a therapeutic platform to serve as a drug reservoir for simultaneous delivery of VEMR (exosomes modified with DVAP targeted peptides carrying MIT and siNotch1 simultaneously, DVAP-Exo/MIT/siNotch1) and IL-12 (IL-12&VEMR@Gel). After intracavity injection, the cargos are released from the prepared formulations in a sustained manner. VEMR target residual GICs and glioma cells in the resection cavity; subsequently, siNotch1 and MIT are released from exos and target to kill the GICs. Furthermore, MIT and IL-12 synergistically activate T cells and reshape the immunosuppressive microenvironment, thereby reducing glioma relapse (Fig. 1). In summary, the regression of glioma relapse following surgery may be achieved using a multifunctional exo-gel delivery system.

Results
Preparation and characterization of VEMR
First, we assessed the viability of exos as carriers for the simultaneous loading of MIT and siNotch1. Exos were isolated from the culture supernatant of RAW264.7 cells via ultracentrifugation, DVAP peptides were modified on exos by click chemistry, and coincubation and transfection reagents (exofect) were performed simultaneously to load MIT and siNotch1 into exos (Fig. 2a). The TEM images in Fig. 2b, c showed that both the empty exos and VEMR exhibited a typical “disc” shape. Nano-particle tracking analysis (NTA) revealed that a peak particle diameter of empty exos was approximately 124 ± 2.55 nm, and the diameter of VEMR expanded to 136 ± 4.23 nm after encapsulation of the two therapeutic agents (Fig. 2d, e). Typical exo markers (CD9, TSG101, and CD63) were detected via Western blotting and enriched in exos (Fig. 2f). TAMRA fluorescent dye was subsequently used to label the DVAP-targeting peptides, and the TAMRA-VE (TAMRA-DVAP-Exo) positivity rate was detected via flow cytometry (FCM) to verify whether the chemical coupling of targeted peptides and exos was successful. The results shown in Fig. 2g revealed that approximately 88.6% of the exos had fluorescent signals, demonstrating successful coupling of DVAP to the exos. These results indicated that the successful extraction of exos and the loading of MIT and siRNA did not notably alter exo morphology or physicochemical properties.

Next, we compared the encapsulation efficiency of MIT among three methods: electroporation, coincubation followed by transfection, and simultaneous coincubation and transfection. As shown in Fig. 2h and Table S1, the loading efficiency of MIT in exos was approximately 21%; thus, we chose the simultaneous coincubation and transfection method for subsequent experiments, and the efficiency of siRNA loading into exos by this method was approximately 72%. Furthermore, we investigated the release of MIT from VEMR at pH 5.5 and 7.4. As shown in Fig. 2i, MIT release occurred faster within the first 12 h but then slowed, and the release rates were approximately 74.16% (pH 5.5) and 52.27% (pH 7.4) at 48 h, indicating that more MIT was released under acidic conditions. One of the primary challenges in the application of siRNAs is their easy degradation by nucleases. To assess the protective effect of exos against siRNAs, the degradation of different samples incubated with RNase A at different timepoints was determined. The results (Fig. 2j) revealed that free siRNAs were mostly degraded at 30 min and completely degraded at 60 min, and the degradation of siRNAs in EMR was similar to that of free siRNAs when Triton X-100 was added; however, siRNAs in EMR without Triton X-100 exhibited little degradation at 120 min, indicating that exos had a good protective effect on siRNAs, which were not easily degraded by nucleases. In summary, these results confirmed the feasibility of coloading MIT and siNotch1 into exos.

Gel and VEMR@Gel were subsequently prepared and characterized (Figs. 2k–m and S1). The results revealed that the gel was liquid at low temperature (4°C) and became solid at 37°C (Fig. 2k), and the viscosity of gel and VEMR@Gel decreased with increasing shear rate (Fig. 2l), which indicated that they experienced shear thinning. In addition, the storage modulus (G’) of gel and VEMR@Gel was much greater than the loss modulus (G”) with increasing temperature, demonstrating that the solid hydrogel gradually formed (Fig. 2m). SEM of gel revealed a porous morphology; after loading with VEMR, fewer pores formed in VEMR@Gel, indicating that VEMR was loaded into the porous structure of the gel, and the hemolysis rate of the gel was 0.58%, which met the requirements of medical materials (Fig. S1). Furthermore, the exos released from the gel were investigated. Exos were labeled with DiI, then loaded into the gel, and the artificial cerebrospinal fluid was used as the release medium. 3D layer scanning was performed via a laser confocal microscope, the results demonstrated that >80% exos could be released from the gel in 24 h (Fig. S2).

In vitro targeting and cellular uptake of VEMR
Immunomagnetic bead sorting was used to obtain U87 GICs, and Western blotting (Fig. S3a) revealed that CD133 and Nestin (stem cell markers), and Notch1 and Hes1 (Notch1 pathway-related proteins) were highly expressed in U87 GICs. Similarly, the immunofluorescence staining results (Fig. S3b) revealed that U87 GICs exhibited elevated expression of CD133 and Notch1. The above results suggested that U87 GICs were successfully sorted.

To investigate the targeting ability of the DVAP peptide, exos and DVAP-exos (VE) were labeled with DiI and subsequently incubated with U87 cells and U87 GICs (GRP78 positive) and HA1800 cells (a normal astrocyte with negative expression of the GRP78 protein). Laser confocal microscopy revealed that the fluorescence intensities of VE and VEMR in U87 cells and U87 GICs were stronger than those in free exos (Fig. 3a–c, f), but their fluorescence signals were weaker when free DVAP targeting peptides were added in advance, indicating that exos were more likely to target glioma cells and GICs after targeted peptide modification and that free DVAP peptides could competitively bind to GRP78 to block VE targeting glioma cells and GICs. Moreover, almost no significant changes in fluorescence signal intensity were detected between the VE and VEMR groups, demonstrating that loading MIT and siRNA had little influence on the targeting ability of the exo delivery system. Next, the cellular uptake of this delivery system in U87 cells and U87 GICs was investigated. As shown in Fig. 3d, e, g–j, almost no fluorescence signals were detected in the PBS, siRNA and MIT groups, whereas few red and green fluorescence signals were detected in the EMR group. Stronger fluorescence signals for both channels were detected in the VEMR group. In other words, free siRNAs were minimally taken up by cells, whereas more siRNAs and MIT loaded into VE were internalized into cells, suggesting that exos modified with DVAP peptides could better promote the cellular uptake of drugs.

To elucidate the cellular uptake mechanism of targeted exos, U87 cells and U87 GICs were treated with various endocytosis inhibitors or maintained at 4°C for incubation. Compared with control group, only a few red fluorescence signals were detected in the groups treated with amiloride (a macropinocytosis inhibitor), dynasore (a dynamin inhibitor) or incubated at 4°C, whereas many red fluorescence signals were detected in the groups incubated with chlorpromazine (a clathrin inhibitor) and M-β-CD (a caveolin inhibitor), suggesting that the cellular uptake mechanism of targeted exos involved endocytosis mediated by dynamin, macropinocytosis and energy-dependent pathways (Fig. 4a, b). Exos endocytosed into cells may participate in the endosome‒lysosome pathway. To investigate whether drugs can escape from lyso/endosomes, we studied the colocalization of siRNAs and MIT with lyso/endosomes. As shown in Fig. 4c, after drugs were internalized into U87 cells at 2 h, the green, red and purple fluorescence exhibited a large amount of overlap. The colocalization coefficient of siRNA and lyso/endosomes was 0.97, and that of MIT and lyso/endosomes was 0.64. However, the overlap fluorescence gradually decreased with increasing time. At 8 h, the colocalization coefficient between siRNA and lyso/endosomes was 0.73, and that between MIT and lyso/endosomes was 0.39. The colocalization of siRNA, MIT and lyso/endosomes was similar in U87 GICs at 2 h and 8 h (Figs. 4d and S4–6), indicating that most of the siRNAs and MIT entered the lyso/endosomes and then escaped from the lyso/endosomes over time.

Synergistic antitumor efficacy of VEMR in vitro
To evaluate the biosafety of exos, HA1800 and U87 cells were exposed to increasing concentrations of exos. As shown in Fig. S7a, b, the cell viability of both HA1800 and U87 remained above 80% even at the highest concentration tested (100 μg/mL), indicating that the exos were safe. In addition, the safety of MIT in different samples was also tested to determine the optimal dosage concentration, and Fig. S7c indicated that as the concentration increased, the viability of U87 cells decreased, and a final concentration of 5 μg/mL was chosen for further studies.

Furthermore, the antitumor effects of VEMR in glioma cells and GICs were investigated in vitro. First, the silencing effects of siNotch1 in U87 GICs were detected at mRNA and protein levels (Fig. 5a–c). Compared with the control group, the mRNA level of Notch1 in U87 GICs treated with DVAP-Exo/siNotch 1 (VER) was reduced by 60%. Similarly, the protein expression level of Notch 1 was significantly downregulated (Fig. 5b, c), which was comparable to that in the positive control group (lipo/siNotch 1), whereas the protein expression level of Notch 1 in free siNotch 1 was not significantly changed, demonstrating that VER could effectively silence the target gene and inhibit the expression of Notch1 in vitro. Furthermore, the proliferation and apoptosis of glioma cells and GICs were assessed through CCK8 and FCM assays to evaluate the therapeutic effects of the exo delivery system in vitro. Cell viability was significantly lower in the VEMR group (Figs. 5d, g, i and S8a) than that in the control group, but no substantial difference in cell survival was observed between the VEMR and VEM groups. However, the cell survival rates of the VER and VEM groups decreased by 18% and 75% in U87 GICs, 37% and 47% in U251 GICs, 46% and 55% in GL261 GICs, and 32% and 65% in CT2A GICs, respectively. Moreover, the cell survival rates in the VEMR group decreased by approximately 94% in U87 GICs, 49% in U251 GICs, 63% in GL261 GICs, and 80% in CT2A GICs, respectively (Figs. 5e, h, j and S8b). One possible reason for these differences is that the expression of Notch1 was low in glioma cells but high in GICs.

An Annexin V-APC/DAPI assay was performed to evaluate the apoptosis of glioma cells and GICs (Figs. 5k, l and S9–S12). In U87 cells (Figs. 5k and S9a), compared with the control group (4.86%), the apoptosis rates in both VE (5.22%) and VER (4.41%) groups did not significantly differ, whereas the apoptosis rates in the VEM and VEMR were 62.73% and 69.44%, respectively. In U87 GICs (Figs. 5l and S9b), compared with the control group (2.03%), the apoptosis rate in the VE group (2.87%) was not significantly different, whereas the apoptosis rates in the VER, VEM and VEMR groups were 8.13%, 35.06% and 64.85%, respectively. The apoptosis rates of U251 cells, GL261 cells, and CT2A cells as well as U251 GICs, GL261 GICs, and CT2A GICs were similar to those of U87 cells and U87 GICs (Figs. S10–S12); these results indicated that the exo delivery system had significant antitumor effects in vitro. Moreover, Western blotting revealed that, compared with control group, VEMR significantly decreased the expression levels of CD133 and Notch1 (Figs. 5f and S13), demonstrating that VEMR significantly reduced the stemness of U87 GICs and GL261 GICs. Taken together, VEMR successfully delivered siNotch1 and MIT into tumor cells to suppress proliferation and promote the apoptosis of GICs and glioma cells, subsequently suppressing the expression of the Notch1 protein to reduce GIC stemness. Additionally, synergistic treatment achieved better results than monotherapy did.

In vivo antitumor efficacy of intratumorally injected IL-12&VEMR
Encouraged by the above results, we further evaluated the therapeutic efficacy of the intratumoral injection of IL-12&VEMR. To establish the GL261 orthotopic glioma model, as depicted in Fig. 6a, luciferase-expressing GL261 (Luc-GL261) cells were implanted into the brains on day 0, and the mice underwent imaging using IVIS to measure glioma growth. On day 12 after being inoculated with Luc-GL261 cells, the mice were allocated into eight groups of different formulations (control, MIT, VER, VEM, EMR, VEMR, IL-12, and IL-12&VEMR). Throughout the therapeutic process, tumor growth and variations in body mass were closely monitored and recorded continually. Within the first three days after each formulation was injected, there was a slight decrease in tumor growth among all treatment groups; however, one week after treatment, compared with the control group, the IL-12&VEMR group exhibited notable tumor suppression (Fig. 6b, c), and the median survival time of mice was significantly prolonged (Fig. 6d). Twenty-one days post-inoculation, mouse brain tumor tissues were harvested and sectioned for hematoxylin-eosin (H&E) staining to facilitate visualization under a microscope. The results revealed that maximal tumor growth suppression was observed in the IL-12&VEMR group among all treatment groups (Fig. 6e, g). TUNEL assays were conducted to detect apoptosis in all the experimental groups, and the green fluorescence signals were the weakest in the control group, but greatest in the IL-12&VEMR group compared with those in other groups (Fig. 6f, h), indicating that treatment with IL-12&VEMR could induce extensive apoptosis of tumor cells. To further assess the biocompatibility, body weight, serum indices (i.e., ALT, AST, BUN, and CRE), and H&E staining of major organs (including heart, liver, spleen, lung, and kidney) were analyzed (Fig. S14). Comparisons between the treatment groups and the control group revealed no significant differences, demonstrating that this exo delivery system had excellent biocompatibility with minimal toxicity.

Therapeutic effects of exo-gel delivery system on glioma relapse model
Surgical operation remains the preferred clinical approach for treating GBM patients, especially in cases involving more advanced tumor stages31. The effectiveness of post surgery treatment options featuring close clinical correlation was evaluated using a personalized multifunctional exo-gel delivery system. To this end, visible gliomas were excised surgically from the mice on the 12th day after they were inoculated with Luc-GL261 cells (Fig. 7a). After surgical removal, mice were randomly assigned to seven groups, and the resection cavities were injected with each hydrogel-mediated treatment (the control group received surgical resection without hydrogel treatment). High signal intensities were observed in the control group and Gel group, whereas only a few were observed in the IL-12&VEMR@Gel group, indicating that IL-12&VEMR@Gel strongly inhibited GBM relapse (Fig. 7b, c). Murine resection cavities injected with EMR@Gel, VEMR@Gel and IL-12@Gel moderately inhibited tumor growth, prolonging the median survival duration to 25, 30 and 64 days, respectively, compared with the 16 days observed in the control group. The IL-12&VEMR@Gel group exhibited notable inhibition of tumor growth, with an extension in survival to 180 days (Fig. 7d). On the 30th day after tumor cell inoculation, the mice whose tumors excised were euthanized, and brain tumor tissues were harvested. H&E staining of tumor-bearing brain sections revealed that the fewest tumors were present in the IL-12&VEMR@Gel group (Figs. 7e and S15a). Additionally, extensive numbers of apoptotic cells were detected in TUNEL-stained tumor-bearing brain slices from the IL-12&VEMR@Gel group (Figs. 7f and S15b).

Based on the above experimental results, the therapeutic effects of this multifunctional exo-gel delivery system on a CT2A orthotopic glioma model were also investigated. Luciferase-expressing CT2A (Luc-CT2A) glioma cells were implanted into the brains on day 0, and the mice underwent imaging using an IVIS to determine glioma growth. On the 6th day after inoculation with Luc-CT2A cells, the brain tumors were surgically removed. Mice were randomly divided into four groups (control, VEMR@Gel, IL-12@Gel, and IL-12&VEMR@Gel), and the multifunctional exo-gel delivery systems were injected into the resection cavity. IL-12&VEMR@Gel significantly inhibited tumor growth and prolonged the survival of the mice (Fig. 7g–i). In summary, these results demonstrated the excellent ability of IL-12&VEMR@Gel to suppress glioma recurrence.

Antitumor immune response of exo-gel delivery system on glioma relapse model
Encouraged by the satisfactory antitumor effects of IL-12&VEMR@Gel, immune activation was assessed in vivo. On the 30th day, the mice were euthanized, and brain tumor tissues were harvested to prepare a single-cell suspension via Percoll cell isolation solution. The immune cells were stained and analyzed using FCM to evaluate the overall changes in immune cells within the tumor microenvironment. Quantitative FCM results (Fig. 8b–f) revealed that after IL-12&VEMR@Gel treatment, the proportions of mature DCs (CD80+CD86+), CD8+ T cells (CD3+CD8+), and activated T cells (CD25+CD69+) in the GBM lesions of tumor-bearing mice significantly increased, whereas the proportions of Treg cells (CD4+Foxp3+) and MDSCs (CD11b+Gr-1+) notably decreased. This finding indicated a broad reversal of the immunosuppressive microenvironment in GBM after injecting IL-12&VEMR@Gel into the intracavity, which is a crucial step in antitumor immunity.

Given that changes in the types and numbers of immune cells in the tumor microenvironment can lead to alterations in cytokine levels, an enzyme-linked immunosorbent assay (ELISA) was used to evaluate the expression of relevant cytokines at the tumor site. After treatment, the IL-12&VEMR@Gel group exhibited the highest levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are closely related to the antitumor effect of CD8+ T cells and the activation of T cells. In contrast, the levels of interleukin-4 (IL-4) and interleukin-10 (IL-10), which are associated with the maintenance of the immunosuppressive characteristics of the tumor microenvironment, were the lowest (Fig. 8g–j). These results further confirm that IL-12&VEMR@Gel comprehensively and thoroughly activated the antitumor immune response in vivo to the brain, resulting in the transformation of GBM from a “cold” tumor to a “hot” tumor.

To explore whether the treatments affected the GICs, the expression of CD133 and Notch1 in the tumor-bearing brains of each group was detected through immunofluorescence and Western blotting. As shown in Fig. S16a, numerous green fluorescence signals were observed in the control and Gel groups, and small amounts of green fluorescence signals appeared in the EM@Gel group, EMR@Gel group, and VEMR@Gel group; the green fluorescence signals were the lowest in the IL-12&VEMR@Gel group. Similarly, red fluorescence signals were significantly attenuated in the IL-12&VEMR@Gel group compared with the control group. Western blotting results shown in Fig. S16b were similar to those mentioned above, and the expression levels of Notch1 and CD133 in the IL-12&VEMR@Gel group were significantly lower. These results indicated that IL-12&VEMR@Gel could significantly inhibit the expression of Notch1 and CD133, which is crucial for inhibiting the recurrence of brain glioma. In conclusion, the injection of a multifunctional exo-hydrogel system into the resection cavity of postoperative GBM mice not only inhibited the stemness of GICs and killed GICs but also reversed the immunosuppressive microenvironment, and thus effectively inhibited GBM recurrence.

Methods
Cell culture
U87 (CL-0238), U251 (CL-0237) cells were acquired from Procell Life Science and Technology Co., Ltd. (Wuhan, China), GL261 (BNCC341792), Luc-GL261 (BNCC337638), HA1800 cells (BNCC340443) were purchased from Beina Chuanglian Biotechnology Institute (Beijing, China), CT2A (BFN60810497) cells were obtained from Bluefbio Biology Technology Development Co., Ltd. (Shanghai, China), Luc-CT2A cells were constructed by GenCefe Biotech Co., Ltd. (Jiangsu, China), and RAW264.7 cells were generously provided by Jingwen Zhao, Tianjin Medical University General Hospital. All cell lines were authenticated by short tandem repeat (STR) profiling and routinely tested to confirm the absence of mycoplasma contamination. All the cell lines were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Vivacell, Germany) and 1% penicillin-streptomycin (Invitrogen, USA) at 37°C under a 5% CO2 atmosphere.

CD133+ GICs were isolated using magnetic-activated cell sorting (MACS). Following a 7-day culture period, tumor spheres were harvested by centrifugation and then resuspended in serum-free DMEM/F12 medium to adjust the cell concentration (1×105 cells/μL) to minimize serum interference. A mixture containing 200 μL cell suspension, 40 μL FcR blocking reagent, and 40 μL CD133 microbeads was prepared in PBS buffer supplemented with 5% FBS and 2 mM EDTA. After thorough mixing, the mixture was incubated at 4°C for 15 min. The labeled cell suspension was then loaded onto a pre-assembled magnetic separation column. Unbound cells were removed by washing three times with 500 μL buffer. After removing the column from the magnetic field, CD133+ labeled cells (GICs) were obtained and subsequently cultured in stem cell medium consisting of DMEM/F12 supplemented with EGF (20 ng/mL, PeproTech), bFGF (20 ng/mL, PeproTech), and B27 supplement (1:50; Invitrogen, USA). GICs were characterized by Western blotting and immunofluorescence assays.

Isolation and characterization of exos
Exos were isolated from RAW264.7 cell culture supernatant48. In brief, the supernatant was collected and subjected to sequential centrifugation at 2000 × g (20 min) and 10,000 × g (30 min) at 4°C to remove dead cells and cell debris. The supernatant from the previous step was subsequently filtered through a 0.22 μm membrane and ultracentrifuged (Beckman Optima L-100 XP, Beckman Coulter) at 100,000 × g (70 min). Afterward, the pelleted exos were obtained and resuspended in sterilized PBS, and the protein concentration was quantified using a BCA assay and preserved at −80°C for subsequent experiments. The particle size and morphological characteristics were evaluated through NTA (Particle Metrix PMX, Germany) as well as TEM (Hitachi HT7700, Japan). Additionally, the presence of exo-specific markers such as CD9, CD63 and TSG101 was confirmed by western blotting.

Preparation and characterization of the exo delivery system
The exo surfaces were modified with DVAP peptides through the application of click chemistry. First, DBCO-sulfo-NHS ester (Glen Research, VA, USA) was dissolved in DMSO to a concentration of 10 μM, then added to the exos suspension (0.5 mg/mL) prior to incubation at 25°C for 4 h with gentle mixing. Subsequently, extra DBCO-sulfo-NHS was removed by ultrafiltration to obtain DBCO-Exos, after which DVAP peptides modified with 5-azidopentanoic acid (Scilight Biotechnology, Beijing, China) were incubated with them at 4°C overnight. Following the reaction, free DVAP was removed by ultrafiltration to obtain VE. To verify the successful chemical coupling between the targeted peptides and exos, the exos were modified by the same procedure, and the TAMRA-DVAP-Exos were characterized by FCM.

Synthetic siRNAs targeting specific genes were designed based on the sequences described in Table S2, and produced by GenePharma Co. Ltd (Shanghai, China). Transfection of siRNAs into exos was performed using the Exo-fect™ Exosome Transfection Reagent (System Biosciences, CA, USA) according to the manufacturer’s protocol. Briefly, DVAP-Exo, MIT (mitoxantrone dihydrochloride, Med Chem Express, USA) and siRNAs (GenePharma, Shanghai, China) were mixed at a mass ratio of 10:8:1. An appropriate volume of Exo-fect was introduced, and the mixture was incubated at 37°C for 20 min. ExoQuick-TC reagent was subsequently added, and this mixture was allowed to react on ice for 30 min, after which the reaction was stopped. Afterward, the mixture was centrifugated at 12,000 × g for 3 min, and DVAP-Exo/MIT/siRNA (VEMR) was obtained and resuspended in PBS. The particle size and morphological characteristics were assessed through NTA and TEM.

Drug encapsulation efficiency
To determine the content of MIT in the VEMR, a UV–vis spectrophotometer was employed to measure the absorbance at 627 nm. The standard curve of MIT was shown in Fig. S17, and the encapsulation efficiency of MIT was calculated by the following formula: encapsulation efficiency (%) = ME/MI × 100%, where ME represents the mass of MIT encapsulated in the exo delivery system, and MI represents the original mass of MIT. Similarly, siRNAs were labeled with Cy3 (Ex = 530 nm, Em = 570 nm), and the encapsulation efficiency of the siRNA in VEMR was detected via a microplate reader.

Drug stability
To investigate the stability of the siRNA in exos, free siRNA or EMR with or without 0.5% Triton X-100 was coincubated with RNase A at 37°C for a specific duration, and the degradation of the siRNA was analyzed by agarose gel electrophoresis.

The in vitro release profile of MIT from VEMR was further evaluated in PBS at pH 7.4 and 5.5 using a dynamic dialysis method. Adequate volumes of sample solutions were swiftly transferred into dialysis bags with a molecular weight cutoff ranging from 8 to 12 kDa, and subsequently dialyzed with 50 mL of PBS. The released MIT was quantified at 627 nm by a UV‒Vis spectrophotometer at regular intervals.

Targeting and cellular uptake of the exo delivery system
Exos, VE and VEMR were marked with DiI. In short, HA1800 cells, U87 cells and U87 GICs were seeded in confocal dishes, and when the cells density reached approximately 80%, exos, VE and VEMR (5 µg) were added to the dishes and incubated for 4 h. Following incubation, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with DAPI. Subsequently, these cells were observed by CLSM to examine the fluorescence intensity of the DiI-labeled exos. Additionally, to investigate the effect of receptor blocking on targeting in this system, HA1800 cells, U87 cells and U87 GICs were incubated with DVAP peptides for 1 h, VE was added, and the intracellular fluorescence intensity was detected by CLSM.

When the cell density reached approximately 80%, different formulations (PBS, Cy3-labeled siRNA, MIT, EMR, and VEMR) were added for coincubation with U87 cells and U87 GICs for 4 h, after which the cellular uptake of the exo delivery system was observed by CLSM.

To explore the underlying uptake mechanism of this system by U87 cells and U87 GICs, the cells were pre-incubated for 1 h with various endocytic inhibitors (50 μM amiloride hydrochloride, 5 mM methyl-β-cyclodextrin (M-β-CD), 10 μg/mL chlorpromazine and 80 μM dynasore), followed by the addition of DiI-VE and further incubation for 2 h. In addition, to evaluate temperature-dependent uptake, another group of cells was incubated at 4°C for 2 h. The intracellular fluorescence intensity was subsequently detected by CLSM.

Lyso/endosome escape assay
To detect lyso/endosome escape of siRNA and MIT, U87 cells and U87 GICs were seeded and cultured in a confocal dish, and treated with VEMR (Cy3-labeled siRNA) for 2, 4 and 8 h at 37°C. LysoTracker Green was used to mark lyso/endosomes, Hoechst 33342 was used to label nuclei, and lyso/endosomal escape was observed by CLSM.

Cytotoxicity assay
The cytotoxicity of exos and MIT on U87 and HA1800 cells was evaluated using a CCK-8 assay. Briefly, cells were seeded in 96-well plates and treated with various concentrations of exos and formulations for 48 h. Subsequently, CCK-8 reagent was added to each well, followed by incubation for the designated duration. The absorbance at 450 nm was measured using a microplate reader.

Cell proliferation assay
U87 cells, U251 cells, GL261 cells, CT2A cells, U87 GICs, U251 GICs, GL261 GICs, and CT2A GICs were seeded in 96-well plates, and PBS, MIT, VE, VER, VEM, and VEMR were added. After 48 h of incubation, cellular proliferation in different experimental samples was detected by a CCK-8 assay.

Apoptosis assay
The apoptosis of U87 cells, U251 cells, GL261 cells, CT2A cells, U87 GICs, U251 GICs, GL261 GICs, and CT2A GICs was detected by FCM after different treatments. Cells were plated in 6-well plates and treated with different experimental formulas (PBS, MIT, VE, VER, VEM, or VEMR). Following a 36-h incubation period, cells were harvested and stained using an Annexin V-APC/DAPI Apoptosis detection kit according to the manufacture’s protocol, and the cells subsequently assessed by a BD Accuri C6 flow cytometer.

Gene expression assay
After U87 GICs were treated with different samples (PBS, free siNotch1, Lipo/siNotch1, VER) for 48 h, total RNA was extracted utilizing TRIzol reagent and reverse transcribed to cDNA by RTase according to the manufacturer’s guidelines (Tiangen Biochemical Technology Co., Ltd., Beijing, China). Primer sequences used for reverse transcription were provided in Table S3. Afterward, cDNA (1 μL) was added to the PCR system with forward and reverse primers (1 μL), and the reaction volume was supplemented with DEPC-treated water. The amplification process of this reaction system involved heating to 95°C for 15 s, followed by annealing/extension at 60°C for 1 min, which was repeated for a total of 40 cycles. Actin served as a housekeeping gene, and Notch1 expression was estimated via the 2−ΔΔCT method.

Western blotting analysis
RIPA buffer supplemented with phenylmethanesulfonylfluoride (PMSF) (Solarbio Biotechnology, Beijing, China) was used to lyse exos, RAW264.7 cells, U87 cells, U87 GICs, GL261 GICs or mouse brain tumor tissues. Protein samples were denatured by boiling at 100°C for 5 min after mixing with 5× loading buffer. Subsequently, proteins including CD9 (sc-13118, Santa Cruz biotechnology, USA), CD63 (sc-5275, Santa Cruz biotechnology, USA), TSG101 (sc-7964, Santa Cruz biotechnology, USA), Notch1 (10062-2-AP, Proteintech, USA), CD133 (18470-1-AP, Proteintech, USA), Nestin (sc-58813, Santa Cruz biotechnology, USA), and Hes1 (sc-166410, Santa Cruz biotechnology, USA) proteins were separated using 10% SDS‒PAGE. The membranes were immunoblotted overnight at 4°C with the following primary antibodies: anti-CD9 (1:1000), anti-CD63 (1:1000), anti-TSG101 (1:1000), anti-Notch1 (1:200), anti-CD133 (1:200), anti-Nestin (1:200), anti-Hes1 (1:200) and anti-β-actin (1:1000). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature, protein bands were detected via a chemiluminescence reagent and analyzed on a C-DiGit (LI-COR Corporation, USA).

Preparation and characterization of the exo-gel delivery system
Two percent chitosan (CS) acetic acid solution (dissolved in acetic acid solution), 56% β-glycerophosphate (β-GP) sodium solution (dissolved in NaHCO3 solution) and 0.5% gelatin (GA) solution (dissolved in acetic acid solution) were prepared. In accordance with the volume ratio (CS:β-GP:GA = 10:2:0.25), CS and GA were mixed first, and then β-GP was added under stirring conditions; subsequently, the pH was adjusted to 7.4, and a temperature-sensitive hydrogel (gel) was obtained. VEMR and/or recombinant murine IL-12 (Protech Systems Co., Ltd., USA) was added to the gel. The temperature-sensitive properties of the gels were detected at 4°C and 37°C. The morphology of the gel in the freeze-dried state was characterized through SEM, and the rheology of the gel was detected via a rheometer. Moreover, the gel’s biocompatibility was evaluated by a hemolysis test; when the hemolysis rate of a sample is less than 5%, it meets medical material requirements. Moreover, the exos released from the gel were assessed. Exos were labeled with DiI, then loaded into the gel, and the artificial cerebrospinal fluid was used as the release medium. 3D layer scanning was performed via a laser confocal microscope at 0 h and 24 h to obverse the release of exos.

Establishment of the GBM model and antitumor efficacy study in vivo
Male C57BL/6 mice (5–6 weeks old, 18–20 g) were obtained from the China Food and Drug Research Institute and used to establish the GBM model. For the GL261 orthotopic models, a suspension of Luc-GL261 cells (5×105 cells in 10 μL serum-free medium) was stereotactically injected into the right frontal lobe of mice at coordinates 1.0 mm anterior and 2.0 mm lateral to the bregma, and 3.5 mm deep. Following a 12-day period, mice were randomly assigned to eight groups, as follows: PBS, MIT, VE, VER, VEM, VEMR, IL-12, IL-12&VEMR. Subsequently, the mice were subjected to intratumor injection with the formulations (10 μL containing dosages of 3 mg/kg for MIT, 0.375 mg/kg for siRNA, and 50 μg/kg for IL-12) mentioned above. Bioluminescence imaging was carried out on day 12, 16, 18 and 21. On the 21st day, the brain tumor was excised for the TUNEL assay. On day 21, some mice were sacrificed, blood was collected for hematological analysis, and vital organ pathological examinations were conducted to evaluate the in vivo biocompatibility of the delivery system.

Following the successful establishment of the postoperative GBM model, mice were randomized into seven groups at random as follows: control, gel, EM@Gel, EMR@Gel, VEMR@Gel, IL-12@Gel, and IL-12&VEMR@Gel. On day 12, after C57BL/6 mice were inoculated with Luc-GL261 cells, a high-speed ring knife drill was used to remove a tiny proportion of the circle, whose diameter was approximately 4 mm, from the skull of every mouse in the GBM mouse models described above, and surgical resection of the GBM was performed49. Various hydrogels (10 μL containing dosages of 3 mg/kg for MIT, 0.375 mg/kg for siRNA, and 50 μg/kg for IL-12) were subsequently injected into the resection cavity of the mice. Bioluminescence imaging was carried out on day 12, 20, 24 and 30. On the 30th day, the brain tumor bumps were excised for the TUNEL assay. On day 30, some mice were euthanized, and brain tissues were harvested for subsequent H&E staining to detect tumor growth, immunofluorescence, Western blotting and FCM were utilized to detect the expression of CD133 and Notch1, along with the infiltration of immune cells (CD4, CD8, DCs, Treg cells, and MDSCs).

For the CT2A orthotopic models, a Luc-CT2A serum-free cell suspension, containing 3×105 cells in 10 μL, was stereotactically injected into the right frontal lobe of mice (1.0 mm anterior, 2.0 mm lateral to the bregma at 3.5 mm depth) via stereotactic surgery. After the orthotopic models were successfully constructed, the mouse brain tumors were surgically removed on the 6th day, and divided into four groups at random as follows: control, VEMR@Gel, IL-12@Gel, and IL-12&VEMR@Gel. Bioluminescence imaging was carried out on day 6, 9, 12, 15 and 18.

FCM and cytokine analysis
GBM mice were perfused with saline, and the mouse brain tumor tissues were collected. Then, the brain tumor tissues were gently ground on a 70 μm cell filter to separate the tissue. After layering on a discontinuous Percoll gradient (30% Percoll above 70% Percoll), the cell suspension was subjected to centrifugation at 1000 × g for 30 min. The middle white annular layer containing the separated cells was collected, followed by wash with PBS and centrifugation at 500 × g and 4°C for 10 min, and then resuspended in 2% BSA-PBS. The immune cells were stained according to the manufacturer’s instructions and analyzed by FCM assays. The following antibody panels were used: anti-CD3-FITC (E-AB-F1013C, Elabscience), anti-CD4-PE (E-AB-F1097D, Elabscience), and anti-CD8-PercpCy5.5 (E-AB-F1104J, Elabscience) for CD4+/CD8+ T-cell analysis; anti-CD11c-FITC (E-AB-F0991C, Elabscience), anti-CD80-APC (E-AB-F0992E, Elabscience), and anti-CD86-PE (E-AB-F0994D, Elabscience) for DC cell detection; anti-CD3-FITC (E-AB-F1013C, Elabscience), anti-CD25-APC (E-AB-F1102E, Elabscience), and anti-CD69-PE (E-AB-F1187D, Elabscience) for T-cell activation assessment; anti-CD3-PercpCy5.5 (E-AB-F1013J, Elabscience), anti-CD4-FITC (E-AB-F1353C, Elabscience), and anti-Foxp3-PE (E-AB-F1238D, Elabscience) for Treg cell analysis; and anti-CD11b-FITC (E-AB-F1081C, Elabscience) and anti-Gr-1-PE (E-AB-F1120D, Elabscience) for MDSC detection. The secretion levels of IFN-γ (E-EL-M0048c), TNF-α (E-EL-M3063), IL-4 (E-EL-M0043c), and IL-10 (E-EL-M0046c) in brain tumor tissues were determined by ELISA (Elabscience).

Statistics and reproducibility
All statistical analyses were performed using GraphPad Prism 8.0.2 software. The experiments were performed independently, and each group was repeated at least three times. The sample number “n” indicated in the figure legends, and the data were presented as the mean ± SD or SEM. Student’s t test or ANOVA was used for group comparison, a p-value of less than 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Ethics statement
All experimental procedures involving animals were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Tianjin Medical University (TMUaMEC2021001). At no point did we exceed the approved limits for tumor size/burden in our animal experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Given difficulties with CNS penetration via systemic delivery routes, GBM has historically seen a variety of local therapeutic strategies with varying success. The work by Zhu et al builds upon the literature of local therapeutics in GBM by examining the anti-tumoral cytotoxic effects and immunological impact of an intra-tumorally delivered combination therapy involving VEMR and IL-12. Conceptually, the strategy to combine a cytoreductive agent with an immunostimulatory cytokine has sound rationale and their appropriateness as local therapy candidates in these preclinical models is noteworthy with further exploration encouraged. However, some comments should be addressed:\nMajor comments\n-While the in vitro data is thoroughly investigated and lends credence to knockdown of NOTCH and the cytotoxic effectiveness of MIT through their exosome platform, the kinetics and cellular impact of also utilizing IL-12 through this delivery mechanism is unclear. Perhaps the authors could include further in vitro data to defend exosome release and effectiveness of IL-12 through co-culture experiments with T cells to visualize activation and proliferation of Th1 cells.\n-GL261 is mostly considered an outdated syngeneic cell line for glioma. The paper’s findings would be more robust if they included other cell lines such as CT-2A or SB28, which are less immunogenic than GL261.\n-The in vivo results of this treatment strategy need the greatest expansion of data. While histological analysis at day 21 is helpful for examining antitumor efficacy, the authors should include survival curves for transparency in section 2.4 (the non-relapse model) to more thoroughly characterize therapeutic efficacy.\n-It is not clear if the recurrent mouse model with GL261 recapitulates upregulation of GIC similar to human GBM. While they do show increased CD133 and Notch1 on immunofluorescence compared to treated mice, it is unclear how this expression compares to non-relapse models. To justify their rationale for why the relapse model responds more robustly to their therapy, the authors should either clearly cite examples of upregulation of GIC markers post-resection in the literature for this mouse model or provide that data through flow cytometry or western blot of ex vivo tissue.\n-In section 2.5 for the glioma relapse model, the immunological data they provide for seeing increased DCs and T cells in IL-12-treated mice is insufficient to argue that this treatment strategy reverses the immunosuppressive microenvironment. Specific markers from either IHC or flow cytometry to demonstrate increased T cell proliferation (e.g. CD69) or activation (e.g. IFN-y, TNF-a) would be helpful. Furthermore, if the authors wanted to speak further on the immunological impact of combining these tumor-antigen releasing strategies with VEMR and immunostimulation with IL-12, they could do assays to look at formation of effector or memory T cell populations or do rechallenge experiments where they inject new tumors to the contralateral site and observe the behavior of T cell response without further treatment.\n-The authors should include an intratumoral IL-12 only arm to assess if there is any synergistic impact of VEMR that helps with recruitment of immune cells\n-Finally, while it is an excellent immunostimulatory cytokine in many other cancers, IL-12 in GBM has decreased effectiveness in activating T cell responses in situ due to baseline low proportions of infiltrating T cells in GBM. The greatest effectiveness of IL-12 has been seen in combination with other immunomodulators in preclinical studies. Have the authors considered any other immunomodulators in lieu or in combination with IL-12?\nMinor comments:\n-Grammatical revision for better syntactical flow is recommended prior to publication. There are several sentences that linked inappropriately by commas as well as infrequent misuse of certain words, which distract from the narrative.\nOverall, the premise of this paper has good rationale and has potential for robust translational impact. However, the in vivo data requires expansion and the immunological assays are insufficient to justify the claims made in this study.", "reviewer_2": "In this manuscript, the authors developed a new treatment strategy for glioblastoma that targets both glioblastoma (GBM) initiating cells (GICs) and the suppressive tumor microenvironment (TME). The therapeutic agent is exosomes that encapsulated mitoxantrone (MIT) and siRNA for Notch. The peptide dVAP was used to modify the surface of the exosomes to target GRP78 highly expressed in GBM cells and GICs. This therapeutic agent, termed VEMR was shown to deliver MIT ad siRNA to kill GICs. To reverse the suppressive immune TME, VEMR ad IL-12 were encapsulated in hydrogel that was then administered into GBM resection cavity in the GL261 GBM model. This VEMR and IL-12@Gel was most effective at killing residual GICs and reversing the suppressive immune TME, resulting in prevention of tumor relapse and long term animal survival. Although this is a potentially interesting therapeutic approach, the manuscript has a number of major weaknesses as detailed below.\n1. What the authors are proposing is a complex combination of particular agents and molecules and the choice of each was not based on strong rationales or experimentation. For example, GRP78 was used to target with dVAP, but whether GRP78 is indeed highly and selectively expressed on GBM cells or GICs was not presented. MIT was chosen but there could be other chemotherapeutics that can be locally delivered to kill GICs and why particularly MIT is unclear. It felt that the authors just picked up agents and molecules what they like to test and put them together. Scientific basis or rationale is weak.\n2. The authors repeated said that there is one month between GBM surgery and the initiation of chemoradiation and during this one month tumor recurrence occurs. But is this true based on evidence? Reference #28 was cited, but this was not mentioned.\n3. The murine GL261 model abruptly appeared in Figure 6 despite the prior figures focused on human U87 cells didn’t test GL261. There is an irrational logical leap from Figure 5 and Figure 6. Indeed, whether or how GL261 responds to VEMR was not tested or shown. GL261 is a traditional laboratory cell line (so is U87). Does GL261 have GICs that depends on Notch signaling or is GL261 growth driven by its GICs? These questions remain unanswered.\n4. In animal experiments using GL261, IL-12@Gel was not tested as a key control and this is a major problem. GL261 model is well known for its immunogenicity and may respond well to IL-12 monotherapy. Without this, the utility of VEMR and IL-12@Gel cannot be established.\n5. The GBM models that were used herein are U87 and GL261 that are probably not clinically representative. Use of other models such as patient-derived GBM cells or other murine models is highly recommended to validate the results. The authors’ claim that the strategy offers broad clinical applications is certainly not supported by the limited tumor models that were selected.\n6. Figure 8B. What was stained as a marker for DCs? This was unclear even after looking at the Materials and Methods.\n7. Abstract. dVAP and MIT appeared without appropriate explanation and the reader will not understand them.\n8. The phrase “tracking GICs” is probably not appropriate since VEMR targets GICs but not necessarily tracks (chases) them.\n9. For the description of fluorescence experiments, the authors often only referred to the color (red and green, etc), without stating what the color indicated or represented.\n10. Figure 4D seems to have a technical challenge. When cells form spheres and aggregates like U87 GICs, is the method used here able to accurately assess co-localization?\n11. Supplementary Fig 3. What are TP and siNC? Unclear.\n12. Figure 5A-C. Notch1 silencing was not potent particularly in the protein levels. Didn’t this need to be improved?\n13. Figure 5GH needs quantitation of the results.\n14. There are problems in English (language) as I find many areas where the true message was unclear. Use of professional language editing service is highly suggested.", "reviewer_3": "Zhu et al. describe a therapeutic platform that attempts to diminish GIC and reshape the immunosuppressive TME of GBM post-operatively. They propose a post-operative in-situ deliver system targeting both siotch1 and MIT and IL-12 via VAP-targeted exoxomes in a gel. Albeit complexed, the idea is original, however, the text should be re-edited by an English editor and the methods should be revised as proposed below. I recommend a Major revision.\nAbstract- Page 2, line 3:\nThis cell line (U87-MG) was proven as problematic model for GBM. The U87MG glioblastoma (GBM) cell line, widely used in research, has been found to have a DNA profile different from the original tumor, raising concerns about its authenticity (Allen et al., 2016). This misidentification highlights the need for careful validation of cell lines in research. U87MG cells exhibit varied responses to treatments like all-trans retinoic acid, revealing phenotypic nuances that may complicate GBM research (Pokorná et al., 2021). Comparisons between U87-luc2 and U251-RedFLuc models show that they recapitulate different aspects of GBM heterogeneity, with U251-RedFLuc forming more invasive tumors (Schulz et al., 2022).\nFurthermore, commonly used cell lines like U-87 MG grown in serum-containing media do not accurately reflect gene expression profiles of patient tumors. Patient-derived GBM models offer greater diversity in gene expression and better correspond to clinical samples, potentially improving preclinical drug discovery efforts (Ye et al., 2020). Therefore, I propose to re-evaluate the platform on additional clinically-relevant cell lines or patient-derived cells. The authors should identify other GB cells that express GRP78 positive for their purpose.\nPage 1, Line 28-\ndefine VEMR\nIntro- Page 2, Line 21:\nThe sentence is too long. I suggest to split to several clear and short sentences. Besides that, it is incredibly difficult if at all possible, to access GIC surgically.\nPage 3, Line 3:\nDo the authors suggest that GICs play a role only in GBM recurrence? Or are they also the initial source of those tumors? Are those cells a specific subpopulation among cells left post-treatment, or is it referred to as a general name for the remaining cells post-treatment? Please clarify in an objective manner as there are multiple references to cite on this matter.\nLine 5: Cell should be in plural- cells.\nBesides Notch1, what are the GICs additional stemness markers? Why was only this chosen?\nLine 9: TMZ is used because of its ability to cross the BBB, which does not refer to MIT. This should be noted in the text.\nLine 18- “egos” should be “Exos”\nPage 4, Line 4: Define immune cells: microglia, bone-marrow-derived macrophages? DCs?\nLine 19: Gliadel can be given as an FDA-approved PIDDS.\nPage 5, Line 4: It’s unclear what is expected toxicity to healthy adjacent cells in the brain.\nPage 9, Line 1: GFAP is a marker of reactive astrocytes undergoing astrogliosis, not of cancer differentiation. Unless the cells are mostly cancer stem cells differentiating into astrocytes-like cancer cells or originating from astrocytes.\nLine 12: In order to assess the target off-site toxicity, the authors should evaluate what was their targeting of non-glioma and non-GIC brain cells (neurons, astrocytes, oligodendrocytes, and microglia)?\nPage 11, Line 5: Explain and clarify what was the rational behind testing this in such un-physiological conditions.\nPage 13, Line 1: ) “lysosomes were labeled with LysoTracker (green),…” lysotracker marks any acidic pH, therefore, it is not labeling only lysosomes but also endosomes.\nLine 6: Define the nature of HA1800 cells.\nLine 7: define “good safety”. How does this align with clinical demands?\nPage 14, Line 1-6: This section lacks distinction between the aspects referring to the cells’ proliferation rate and their motility (migration).\nLine 10-11: define statistical significance, p values?\nPage 15, Figure 5- Panels G and H are blurry and not very clear.\nPage 16, Line 6: Note that murine GL261 (should be stated in the text that they are murine inoculated into immunocompetent mice as immune profiling is required), unlike the human U87 cell line, this line is known to be far less invasive than actual GBM cells.\nPage 17- Figure 6: quantitative analysis should be performed on the IHC slides following staining of relevant markers: proliferation, apoptosis, immune-profiling, and angiogenesis….and not only H&E and measurement of fluorescence/bioluminescence.\nDiscussion, page 22, lines 3-5: there are several newer treatments approved by the FDA and EMA that can and should be mentioned, such as TTF (optium), immunotherapies/vaccines (gliovac), oncolytic virus, etc...\nLine 7: The ”cold” TME in GBM is there, to begin with, prior to any surgery and treatment. Moreover, this is also a result of the treatments themselves as the radiation and chemo both harm the immune system. It may be worth thinking about how the treatments suggested in the paper can be combined with a general busting of the immune system to improve the outcomes.\nPage 23, Line 5: safety was not addressed in the non-malignant healthy brain tissue.\nPage 24, line 1-6- a much more comprehensive analysis should be done in order to claim that the tumor growth inhibition was achieved by turning the cold TME into a hot one."}, "author_response": "Point-by-point responses to the Reviewers’ comments for Ref:\nNCOMMS-24-61002\nDear reviewers,\nThank you for reviewing our work. The comments and suggestions of the\nreviewers are very helpful in improving the manuscript entitled “A local\ntherapeutic platform prevents postsurgical GBM recurrence by\ndiminishing glioma-initiating cells and reshaping the immunosuppressive\ntumor microenvironment” (Manuscript ID: NCOMMS-24-61002). We\nhave carefully considered the suggestions, and made necessary changes to\nthe manuscript. This response letter has 121 pages and they are also revised\nin the paper accordingly. We believe that our additional experiments,\nrevised analyses, and modifications of the text have substantially improved\nthe manuscript.\nWe focused on making comprehensive modifications in aspects such as the\ncompleteness of experimental data, the representativeness of the model, the\ndepth of mechanism verification, and language expression. We\nsupplemented the in vitro co-culture experiment of IL-12 and T cells to\nverify its effect on T cell activation and Th1 polarization; the more invasive\ncell lines CT2A and U251 were replaced and verified in vivo and in vitro\nto enhance the universality of the research conclusion; the expression of\nNotch1 and CD133 on GICs and tumor tissues, the regulatory mechanism\nof the immune microenvironment and the synergistic effect of drugs were\ndeeply verified by western blot, flow cytometry, etc.; targeted specificity\nanalysis on non-glioma and non-GIC brain cells was also supplemented;\nstandardized the description of fluorescence labeling; improved the citation\nof literature, unified the writing style of the first appearance of acronyms\nespecially in the abstract; supplemented the incomplete quantitative\nstatistics; and embellished the language of the entire text to ensure accurate\nand smooth expression. Meanwhile, we made significant revisions to the\nfigures in the revised manuscript. Figure 5G-J, Figure 6D, Figure 6G,\nFigure 7G-I, Figure 8A-J, Figure S2, Figure S4-6, Figure S8-13, Figure\nS15, Figure S16B were newly added and Figure 1, Figure 3F-J, Figure 4A-\nD, Figure 5A-C, Figure 5K-L, Figure S3A, Figure S7A-B, Figure S14A-E,\nFigure S16A were updated to the revised manuscript.\nWe have addressed all comments in a point-by-point fashion as below. All\nchanges within the manuscript are highlighted in yellow. Overall, we\nbelieve the revised manuscript has substantially improved with a clear and\nsolid plot, supported by more data than the original. We thank all the\nreviewers for their attention to detail and kind and informative comments.\nWe believe the revised manuscript now meets the high standards expected\nby Nature Communications and comprehensively describes our research\nfindings.\nResponse to Reviewer's comments:\nCritiques From Reviewer #1:\nThank you for taking the time to read and provide valuable comments on\nour manuscript. We appreciate your positive comments on our work and\nthe significance of our findings. We have carefully considered your\nsuggestions and made the necessary changes to the manuscript. In addition,\nwe have added more experiments based on your suggestions, which are\nimportant for us to further improve our study. As a result of these revisions,\nwe believe that the overall structure of the manuscript and the organization\nof its contents have been significantly improved. Thank you again for your\nattention and valuable feedback on our study, and we look forward to\ncontinuing to communicate with you and improve our work.\nCritique #1:\nWhile the in vitro data is thoroughly investigated and lends credence to\nknockdown of NOTCH and the cytotoxic effectiveness of MIT through their\nexosome platform, the kinetics and cellular impact of also utilizing IL-12\nthrough this delivery mechanism is unclear. Perhaps the authors could\ninclude further in vitro data to defend exosome release and effectiveness of\nIL-12 through co-culture experiments with T cells to visualize activation\nand proliferation of Th1 cells.\nResponse #1:\nThank you for your in-depth questioning of the dynamics and cellular\neffects of IL-12 in the exosome platform. Regarding the characterization\nof exosome release from the gel, we supplemented in vitro release\nexperiments as follows. Exosomes were labeled with CM-DiI, and then\nloaded into the gel. Using artificial cerebrospinal fluid as the release\nmedium, and 3D layer scanning was carried out with a laser confocal\nmicroscope at 0 h and 24 h, respectively. The results demonstrated that >80%\nexosomes could be released from the gel in 24h.\nFig. 1. The release of CM-DiI labeled exosomes from the hydrogel in the CSF. (A) 3D\nlayer scanning of exosome-loaded hydrogel at 0 h and 24 h. (B) Quantification of the\nfluorescence intensity in (A).\nFurthermore, we have carried out supplementary experiments on this issue\nand verified the in vitro effectiveness of IL-12 through the co-culture\nmodel of IL-12&VEMR and T cells. CD25+CD69+ is the classic marker of\nT cell activation. The proportion (%) of CD25+CD69+/CD3+T cells reflects\nthe activation level of T cells. The proportion of activated T cells in the\ncontrol group, IL-12 group and IL-12&VEMR group was 23.45%, 35.55%\nand 49.86% respectively. IFNγ+ proportion mirrors the magnitude of the\nTh1 immune response. The proportion (%) of CD4+IFNγ+/CD3+T cell were\nused to reflects the proliferation of Th1 cells. The proportion of Th1 cells\nin the control group, IL-12 group and IL-12&VEMR group was 1.99%,\n4.79% and 7.40% respectively. The results confirmed that IL-12&VEMR\nsignificantly promoted T cell activation, and Th1 proliferation.\nFig. 2. The proportion of (A) activated T cells and (B) Th1 cells when IL-12 or IL-\n12&VEMR co-cultured with T cells in vitro.\nIn conclusion, we have successfully demonstrated that exosomes could be\nreleased from the hydrogel, and IL-12 as well as IL-12 & VEMR could\neffectively activate T cells and promote the proliferation of Th1 cells.\nCritique #2:\nGL261 is mostly considered an outdated syngeneic cell line for glioma. The\npaper’s findings would be more robust if they included other cell lines such\nas CT-2A or SB28, which are less immunogenic than GL261.\nResponse #2:\nThank you for your professional advice on model selection. We understand\nthe limitations of GL261 as a traditional homologous model. Therefore, we\nfurther verified the therapeutic effect of the IL-12&VEMR@Gel on CT2A\n(mouse GBM cell line with immunogenicity lower than GL261) in vitro\nand in vivo. The relevant data have significantly enhanced the universality\nof the research conclusions. As showed in Section 2.5 (Figure 7G-I),\nluciferase-expressing CT2A (Luc-CT2A) were implanted into brains on\nday 0, and mice underwent imaging using IVIS to determine the glioma\ngrowth. On the 6th day after inoculation with Luc-CT2A cells, the brain\ntumor was surgically removed. Subsequently, VEMR@Gel, IL-12@Gel,\nIL-12&VEMR@Gel were injected into the resection cavity of the mice.\nThe fluorescence intensity of the Control group continued to increase and\nreached 6.7×108 on day 18, while the fluorescence intensity in the\nVEMR@Gel, IL-12@Gel and IL-12&VEMR@Gel group were much\nlower than that in the Control group. It is particularly worth noting that the\nfluorescence intensity of IL-12&VEMR@Gel group was only 8.8×106.\nThe results indicated that IL-12&VEMR@Gel significantly inhibited\ntumor growth and prolonged the survival of mice, demonstrating the\nexcellent ability of IL-12&VEMR@Gel in suppressing glioma recurrence.\nFig. 3. (A) Luminescence images of orthotopic Luc-CT2A GBM tumor-bearing mice\nfollowing different treatments monitored on day 6, 9, 12, 15 and 18. (B) Quantified\nsignal intensity of (A). (C) Survival analysis of orthotopic Luc-CT2A GBM tumor-\nbearing mice in each group.\nCritique #3:\nThe in vivo results of this treatment strategy need the greatest expansion\nof data. While histological analysis at day 21 is helpful for examining\nantitumor efficacy, the authors should include survival curves for\ntransparency in section 2.4 (the non-relapse model) to more thoroughly\ncharacterize therapeutic efficacy.\nResponse #3:\nThank you for your suggestion on the integrity of the in vivo experimental\ndata. We fully agree that the survival curve is a key indicator for evaluating\nthe efficacy of anti-tumor treatment. The survival analysis of the non-\nrecurrence model has been supplemented in Section 2.4, Figure 6D. The\nresults showed that the survival period of mice in the VEMR group was 63\ndays, which was superior to that of the other agent groups (MIT, VEM,\nVER, EMR). Mice in the IL-12 group also received relatively effective\ntreatment, with the survival period prolonged to 79 days. The combined\ntreatment of IL-12 and VEMR (IL-12&VEMR group) had the most\npronounced effect, extending the survival of mice to 83 days, indicating\nthat the IL-12&VEMR regimen exhibited the optimal therapeutic efficacy\ncompared to all other groups. IL-12&VEMR group significantly prolonged\nthe median survival time of mice. The above results showed that the\ncombination of VEMR and IL-12 yielded the most significant therapeutic\neffect.\nFig. 4. (A) Luminescence images of orthotopic Luc-GL261 GBM tumor-bearing mice\nfollowing different treatments monitored on day 12, 16, 18 and 21. (B) Quantified\nsignal intensity of (A). (C) Survival analysis of mice in each group.\nCritique #4:\nIt is not clear if the recurrent mouse model with GL261 recapitulates\nupregulation of GIC similar to human GBM. While they do show increased\nCD133 and Notch1 on immunofluorescence compared to treated mice, it\nis unclear how this expression compares to non-relapse models. To justify\ntheir rationale for why the relapse model responds more robustly to their\ntherapy, the authors should either clearly cite examples of upregulation of\nGIC markers post-resection in the literature for this mouse model or\nprovide that data through flow cytometry or western blot of ex vivo tissue.\nResponse #4:\nThank you for your rigorous questioning of the expression of GIC markers\nin the GL261 recurrence model. Through literature verification and\nsupplementary experimental data, we explain the upregulation\ncharacteristics and therapeutic response mechanism of GIC as follows:\nYi et al. indicated that GL261 GIC exhibited higher invasiveness and\ntumor-forming ability, and was more similar to the histological\ncharacteristics of human GBM [1].\nFigure Redacted\nFig. 5. GICs were enriched in GL261-NS and expressed stem cell markers CD133 (red),\nNestin (green) and Olig2 (red) [1].\nFurther, to verify whether GIC was upregulated in the recurrence model,\nwe characterized the expression of CD133 and Notch1 in the GL261\nrecurrent tumors by immunofluorescence and western blot (Section 2.5,\nFigure S16). As shown in Figure S16A, numerous green fluorescence\nsignals were observed in Control group and Gel group, a small amount of\ngreen fluorescence signals appeared in the EM@Gel group, EMR@Gel\ngroup, VEMR@Gel group. Similarly, significantly attenuated red\nfluorescence signals were detected in mice treated with IL-\n12&VEMR@Gel in contrast to those in Control group. And the results of\nwestern blotting in Figure S16B were similar to those mentioned above.\nThese results indicated that CD133 and Notch1 are both highly expressed\nin recurrent GBM, and IL-12&VEMR@Gel could significantly inhibit the\nexpressions of Notch1 and CD133. In the cellular level, western blotting\nanalysis also revealed that VEMR significantly decreased the expression\nlevels of CD133 and Notch1 than that in control group (Figure S13),\ndemonstrating VEMR significantly reduced the stemness of GL261 GICs.\nFig. 6. The expression of CD133 and Notch1 in tumor-bearing brain tissues of\nrecurrence model. (A) Immunofluorescence analysis of Notch1 and CD133 expressions\nin tumor-bearing brain tissues. (B) Western blot analysis of Notch1 and CD133\nexpressions in tumor-bearing brain tissues.\nFig. 7. Western blotting analysis of Notch1 and CD133 expressions in GL261 GICs\nincubated with PBS, VE, VER, VEM and VEMR for 48 h.\nIt is worth emphasizing that the superior therapeutic efficacy of the\npostoperative resection model compared to the primary model can be\nattributed to several key factors, including the tumor microenvironment,\ncharacteristics of residual cells, and drug half-life. Firstly, the primary\nmodel is characterized by a more pronounced immunosuppressive\nmicroenvironment. This intense immunosuppression may significantly\nundermine the synergistic effects of immunotherapy and chemotherapy\ndrugs, thereby limiting their overall therapeutic impact. In contrast, the\npostoperative resection model presents a distinct advantage. Following\nsurgical resection, only a small number of unresectable tumor cells and\nstem cells remain. The use of gel to uniformly fill the cavity wound surface\nis a crucial aspect. This gel encapsulates IL-12 and exosomes, enabling\nlong-acting, sustained-release of these therapeutic agents. This sustained-\nrelease mechanism is particularly beneficial as it allows for a continuous\nand controlled delivery of the active substances over an extended period.\nOn the other hand, in the primary model, IL-12 and exosomes are directly\ninjected into the tumor. This method lacks the sustained-release capability\noffered by the gel in the postoperative resection model. As a result, the\ntherapeutic agents are not able to exert their effects over a prolonged period,\nwhich may contribute to the less effective treatment outcomes observed in\nthe primary model.\nReference:\n[1] YI L, ZHOU C, WANG B, et al. Implantation of GL261 neurospheres into\nC57/BL6 mice: a more reliable syngeneic graft model for research on glioma-\ninitiating cells[J]. Int J Oncol, 2013,43(2):477-484.\nCritique #5:\nIn section 2.5 for the glioma relapse model, the immunological data they\nprovide for seeing increased DCs and T cells in IL-12-treated mice is\ninsufficient to argue that this treatment strategy reverses the\nimmunosuppressive microenvironment. Specific markers from either IHC\nor flow cytometry to demonstrate increased T cell proliferation (e.g. CD69)\nor activation (e.g. IFN-y, TNF-a) would be helpful. Furthermore, if the\nauthors wanted to speak further on the immunological impact of combining\nthese tumor-antigen releasing strategies with VEMR and\nimmunostimulation with IL-12, they could do assays to look at formation\nof effector or memory T cell populations or do rechallenge experiments\nwhere they inject new tumors to the contralateral site and observe the\nbehavior of T cell response without further treatment.\nResponse #5:\nThank you for the detailed suggestions on the verification of the\nimmunological mechanism. We fully agree that the existing data need to\nbe combined with more specific T-cell activation markers and immune\nmemory verification. In response to your suggestions, we conducted\nsupplementary experiments. On the 30th day, brain tumor tissues were\nharvested to prepare a single cell suspension via the percoll cell isolation\nsolution, T cell activation markers (CD25+CD69+) were stained and\nanalyzed by flow cytometry. Given that changes of immune cells in the\ntumor microenvironment, which can lead to alterations in cytokine levels,\nthus the levels of the cytokines IFN-γ, TNF-α, IL-4 and IL-10 at the tumor\nsite were quantified by ELISA. The relevant results have been added to\nSection 2.5, Figure 8D, and Figure G-J.\nAs shown in Figure 8D, compared with the gel group (Gel), there was no\nsignificant difference in the proportion of CD25+CD69+ T cells between\nthe control group and the gel group; the proportions of CD25+CD69+\nT cells in the EM@Gel group, EMR@Gel group, VEMR@Gel group, IL-\n12@Gel group and IL-12&VEMR@Gel group were significantly higher\nthan those in the control group. The increase in the proportion of double-\npositive cells indicates that these treatments may exert immunomodulatory\neffects by enhancing T cell activation.\nIFN-γ is predominantly secreted by Th1 cells and NK cells and serves as\nthe signature cytokine of Th1-type immunity. It enhances macrophage\nphagocytosis and microbicidal activity, accelerates dendritic-cell\nmaturation, up-regulates MHC-molecule expression, and thereby\npotentiates cell-mediated immunity. Its concentration thus mirrors the\nmagnitude of the Th1 immune response. TNF-α, chiefly secreted by\nmacrophages and T cells, is a pivotal pro-inflammatory cytokine that\ninitiates the inflammatory cascade, activates immune effector cells, and\npotentiates anti-tumor immunity. IL-10 is a potent immunosuppressive\ncytokine produced by Treg and Th2 cells, macrophages, and other cell\ntypes. It restrains the activation of Th1 cells, macrophages, and dendritic\ncells, reduce the release of pro-inflammatory factors, and maintain immune\nbalance. Increased IL-10 expression is a hallmark of immunosuppressive\nmicroenvironments. In our research, as shown in Figure 8G-J, compared\nwith the control group, the concentration of IFN-γ, TNF-α, IL-4, and IL-\n10 in the Gel group and EM@Gel group did not show any significant\nchange. The levels of IFN-γ and TNF-α were significantly increased in\nVEMR@Gel, IL-12@Gel, IL-12&VEMR@Gel group, whereas IL-4 and\nIL-10 were reduced to varying extents.\nFig. 8. (A) Quantification results of the proportion of T cell activation. (B-E) ELISA\nquantification results of IFN-γ (B), TNF-α (C), IL-4 (D), and IL-10 (E) in tumor-\nbearing brain issues.\nIn addition, in response to the reviewers' suggestions, we conducted a\ntumor re-challenge experiment to verify the immune memory and the\nability of anti-tumor persistence response. Luc-GL261 cells were re-\ninjection in situ to the mice in IL-12 group and IL-12&VEMR group. 20\ndays after re-injection, the mice were euthanized, the effector memory T\ncells and central memory T cells in the brain, superficial cervical lymph\nnodes (SLN), and deep cervical lymph nodes (DLN) were analyzed by flow\ncytometry. The proportion of memory T cells in the IL-12@Gel group and\nIL-12&VEMR@Gel group was significantly higher than that in the control\ngroup. The results showed that the treatment with IL-12 and VEMR can\ninduce immune memory and inhibit tumor occurrence.\nFig. 9. The effector memory T cells and central memory T cells in the brain (A), SLN\n(B), DLN (C) in Luc-GL261 tumor re-challenge model.\nCritique #6:\nThe authors should include an intratumoral IL-12 only arm to assess if\nthere is any synergistic impact of VEMR that helps with recruitment of\nimmune cells.\nResponse #6:\nThank you for the valuable suggestions on the grouping of animal\nexperiments. We understand the reviewers’ concerns regarding the high\nimmunogenicity of GL261. Therefore, in the supplementary experiment, a\nnew group of IL-12 monotherapy was added to verify the single effect of\nIL-12 and the synergistic value of VEMR. The relevant results have been\nadded to Section 2.4 (Figure 6) and Section 2.5 (Figure 7). Notably,\nalthough the therapeutic efficacies of IL-12 and IL-12&VEMR were\ncomparable in the primary-tumor model, in the postoperative resection\nmodel, IL-12&VEMR@Gel exhibited a markedly amplified antitumor\neffect than of IL-12@Gel in both GL261 and CT2A recurrent model,\nunderscoring the pivotal contribution of GICs to recurrence. Our results\nfurther confirmed that IL-12-mediated immune activation alone is\ninsufficient; thus, direct and specific elimination of GICs becomes\nindispensable for GBM recurrence, as follow:\nIn the primary model (Section 2.4, Figure 6), luciferase-expressing GL261\n(Luc-GL261) glioma cells were implanted into the brains of mice on day\n0. Subsequently, intracranial tumor growth was monitored using IVIS\nimaging. On day 12 post-implantation, mice were randomly assigned to\neight distinct treatment groups: Control, MIT, VER, VEM, EMR, VEMR,\nIL-12, and IL-12&VEMR. Bioluminescence signals from intracranial\ntumors were measured on Day 12, 16, 18, and 21. The results revealed that\nthe signal intensity in the Control group increased significantly over time,\nreaching approximately 1.4×108. In contrast, while the signals in the\nmonotherapy groups (MIT, VEM, VER, EMR) were somewhat attenuated,\nthey remained relatively strong. The VEMR group exhibited a slightly\nstronger signal compared to the IL-12 and IL-12&VEMR groups. The\nfluorescence signal in the IL-12 group was 3.0×106, indicating that IL-12\nalone has a certain anti-tumor effect, but the anti-tumor effect would be\nenhanced when combined with VEMR. The IL-12&VEMR group\ndemonstrated a significant prolongation of the median survival time of\nmice. Moreover, the median survival time in the IL-12&VEMR group was\nsuperior to that in the IL-12 and VEMR groups. At 21 days post-\nimplantation, brain tumor tissues were harvested from the mice.\nSubsequently, brain slices were stained with hematoxylin-eosin (H&E) to\nfacilitate microscopic visualization. The results showed that the IL-\n12&VEMR group exhibited the most significant tumor growth suppression\namong all treatment groups. Additionally, a TUNEL assay was conducted\nto detect apoptosis in all experimental groups. The green fluorescence\nsignals, indicative of apoptotic cells, were minimal in the Control group\nbut most abundant in the IL-12&VEMR group compared to other groups.\nThis finding suggests that treatment with IL-12&VEMR effectively\ninduces extensive apoptosis of tumor cells.\nFig. 10. In vivo therapeutic therapy of different treatments on GBM. (A) Timeline\nschematic of the animal experiment. (B) Luminescence images of orthotopic Luc-\nGL261 GBM tumor-bearing mice following different treatments monitored on day 12,\n16, 18, and 21. (C) Quantified signal intensity of (B). (D) Survival analysis of mice in\neach group. (E) Representative H&E images of the orthotopic GBM tumors after the\nindicated treatments. (F) TUNEL staining of brain tissue sections obtained from mice\ntumors after treatments. (G) Quantification of tumor area of (E). (H) Fluorescence\nquantification of (F).\nIn the postoperative resection model (Section 2.5, Figure 7), visible\ngliomas were surgically excised from mice on the 12th day after\ninoculation with Luc-GL261 cells. Following surgery, mice were randomly\nassigned to seven groups, and the resection cavities were injection with\neach hydrogel-mediated treatment (Gel, EM@Gel, EMR@Gel,\nVEMR@Gel, IL-12@Gel, IL-12&VEMR@Gel), and the control group\nreceived surgery resection without hydrogel treatment. Bioluminescence\nsignals were monitored over time (Day 12, 20, 24, 30) and showed that\nsignals in the Control and Gel groups continued to increase. In contrast, the\nfluorescence signals of the EM@Gel, EMR@Gel, VEMR@Gel, and IL-\n12@Gel groups were attenuated to varying degrees, with IL-12@Gel\nshowing a signal intensity of 1.5×106, slightly higher than that of the IL-\n12&VEMR@Gel group (3.3×104). However, the survival outcomes were\nstrikingly different. Mice in the VEMR@Gel group had a survival period\nof 36 days, while those in the IL-12@Gel group survived for 123 days.\nNotably, 60% of the mice in the IL-12&VEMR@Gel group were still alive\nat 180 days, highlighting the significant therapeutic advantage of the\ncombined treatment. On the 30th day post-inoculation, mice with excised\ntumors were euthanized, and brain tumor tissues were harvested. H&E\nstaining of tumor-bearing brain sections revealed that the IL-\n12&VEMR@Gel group had the fewest tumors among all groups.\nAdditionally, TUNEL staining of tumor-bearing brain slices showed\nextensive apoptotic cells in the IL-12&VEMR@Gel group, indicating\neffective tumor cell apoptosis.\nFig. 11. In vivo therapy of exo-gel delivery system. (A) Timeline schematic of the\nanimal experiment. (B) Luminescence images of orthotopic Luc-GL261 GBM tumor-\nbearing mice following different treatments monitored on day 12, 20, 24 and 30. (C)\nQuantified signal intensity of (B). (D) Survival analysis of mice in each group. (E)\nRepresentative H&E images of the orthotopic GBM tumors after the indicated\ntreatments. (F) TUNEL staining of brain tissue sections obtained from mice tumors\nafter treatments. (G) Luminescence images of orthotopic Luc-CT2A GBM tumor-\nbearing mice following different treatments monitored on day 6, 9, 12, 15 and 18. (H)\nQuantified signal intensity of (G). (I) Survival analysis of orthotopic Luc-CT2A GBM\ntumor-bearing mice in each group.\nMoreover, the anti-tumor immune response was evaluated in vivo. On the\n30th day, the mice were euthanized, brain tumor tissues were harvested to\nprepare a single cell suspension via the percoll cell isolation solution, the\nimmune cells were stained and analyzed by FCM in order to evaluate the\noverall changes of immune cells within the tumor microenvironment.\nQuantitative FCM results showed that after IL-12&VEMR@Gel treatment,\nthe proportions of mature DC cells (CD80+CD86+), CD8+ T cells\n(CD3+CD8+), and the activation of T cells (CD25+CD69+) in the GBM\nlesions of tumor-bearing mice were significantly increased, whereas the\nproportions of Treg cells (CD4+Foxp3+) and MDSCs (CD11b+Gr-1+) were\nnotably reduced. This indicated a broad reversal of the immunosuppressive\nmicroenvironment in GBM after injecting IL-12&VEMR@Gel into\nintracavity, which was a crucial step to exert an antitumor immunity. Given\nthat changes in the types and numbers of immune cells in the tumor\nmicroenvironment, which can lead to alterations in cytokine levels,\nenzyme-linked immunosorbent assay (ELISA) was used to evaluate the\nexpression of relevant cytokines at the tumor site. After treatments, the IL-\n12&VEMR@Gel group induced the highest levels of interferon-γ (IFN-γ)\nand tumor necrosis factor-α (TNF-α), which were closely related to the\nanti-tumor effect of CD8+ T cells and the activation of T cells. In contrast,\nthe levels of interleukin-4 (IL-4) and interleukin-10 (IL-10), which are\nassociated with the maintenance of the immunosuppressive characteristics\nof the tumor microenvironment, were the lowest. These results further\nconfirmed that IL-12&VEMR@Gel comprehensively and thoroughly\nactivated the anti-tumor immune response within the brain, transforming\nGBM from a “cold” tumor to a “hot” tumor.\nTaken together, the combination of IL-12 and VEMR is more conducive to\nrecruiting immune cells, thereby exerting anti-tumor immune effects and\ninhibiting the recurrence of GBM.\nFigure 12. The evaluation of antitumor immune response in GL261 postresection\nglioma-bearing brain tissue. (A) Schematic diagram of the experimental design. (B)\nRepresentative FCM contour plots of T cells in each group and quantification of CD8+\nT cells. (C-F) Quantification results of the proportion of DC cells (C), T cell activation\n(D), Treg cells (E), and MDSCs (F) in tumor-bearing brain issues of each group. (G-J)\nELISA quantification results of IFN-γ (G), TNF-α (H), IL-4 (I), and IL-10 (J) in tumor-\nbearing brain issues.\nCritique #7:\nFinally, while it is an excellent immunostimulatory cytokine in many other\ncancers, IL-12 in GBM has decreased effectiveness in activating T cell\nresponses in situ due to baseline low proportions of infiltrating T cells in\nGBM. The greatest effectiveness of IL-12 has been seen in combination\nwith other immunomodulators in preclinical studies. Have the authors\nconsidered any other immunomodulators in lieu or in combination with IL-\n12?\nResponse #7:\nThank you for your profound insight into the limitations of IL-12 in GBM\nimmunotherapy. The suggestion of combined immunomodulators you put\nforward is of great value.\nAlthough T-cell infiltration is relatively low in the immune\nmicroenvironment of GBM, IL-12, as a “master regulatory factor”\ncytokine, its immune activation effect is not limited to the T-cell population.\nInstead, it reshapes the immune microenvironment through multiple\nmechanisms and enhances the anti-tumor immune response: (1) IL-12 is a\nkey regulator of NK cell activation, which can upregulate the expression\nof activating receptors (e.g., NKG2D) on NK cells, enhancing their\ncytotoxicity against tumor cells [1-4]. (2) IL-12-induced Th1 polarization\nincreases the production of IFN-γ, which can directly inhibit GBM cell\nproliferation and enhance the sensitivity of tumor cells to immune attack\n[5-7]. (3) IL-12 can upregulate the expression of CD25 and CD69 on CTLs,\npromoting T cell activation and proliferation [1,8-9]. (4) IL-12 can reprogram\nM2-type immunosuppressive macrophages into M1-type pro-\ninflammatory phenotypes, enhancing their antigen presentation and\nphagocytic capabilities, and improving the immunosuppressive\nmicroenvironment [10]. (5) IL-12 can suppress GBM angiogenesis [11], a key\nprocess for tumor growth and progression.\nIn our research, the immune cells were stained and analyzed by FCM to\nevaluate the overall changes of immune cells within the tumor\nmicroenvironment in the recurrence model. Quantitative FCM results\nrevealed that after IL-12&VEMR@Gel treatment, the proportions of\nmature DCs (CD80+CD86+), CD8+ T cells (CD3+CD8+), and activated T\ncells (CD25+CD69+) in the GBM lesions of tumor-bearing mice\nsignificantly increased, whereas the proportions of Treg cells (CD4+Foxp3+)\nand MDSCs (CD11b+Gr-1+) notably decreased. This finding indicated a\nbroad reversal of the immunosuppressive microenvironment in GBM after\ninjecting IL-12&VEMR@Gel into the intracavity, which is a crucial step\nin antitumor immunity. Given that changes in the types and numbers of\nimmune cells in the tumor microenvironment can lead to alterations in\ncytokine levels, an enzyme-linked immunosorbent assay (ELISA) was\nused to evaluate the expression of relevant cytokines at the tumor site. After\ntreatment, the IL-12&VEMR@Gel group exhibited the highest levels of\ninterferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are\nclosely related to the antitumor effect of CD8+ T cells and the activation of\nT cells. In contrast, the levels of interleukin-4 (IL-4) and interleukin-10\n(IL-10), which are associated with the maintenance of the\nimmunosuppressive characteristics of the tumor microenvironment, were\nthe lowest. These results further confirm that IL-12&VEMR@Gel\ncomprehensively and thoroughly activated the antitumor immune response\nwithin the brain, resulting in the transformation of GBM from a “cold”\ntumor to a “hot” tumor.\nFigure 13. The evaluation of antitumor immune response in GL261 postresection\nglioma-bearing brain tissue. (A) Schematic diagram of the experimental design. (B)\nRepresentative FCM contour plots of T cells in each group and quantification of CD8+\nT cells. (C-F) Quantification results of the proportion of DC cells (C), T cell activation\n(D), Treg cells (E), and MDSCs (F) in tumor-bearing brain issues of each group. (G-J)\nELISA quantification results of IFN-γ (G), TNF-α (H), IL-4 (I), and IL-10 (J) in tumor-\nbearing brain issues.\nWe acknowledge that preclinical studies have shown enhanced\neffectiveness of IL-12 when combined with other immunomodulators (e.g.,\nanti-PD-1) [12,13]. For example, Chen et al.[12] demonstrated that combining\nNano-IL-12 (i.v.) with anti-PD-1 antibodies (i.p.) enhances anti-tumor\nimmunity. However, most of these studies focus on primary tumor, while\nour study specifically targets recurrent GBM—a setting with distinct\nbiological features. Herein, following your recommendation, we further\ncombined the widely used immune checkpoint inhibitor anti-PD-1 in\nCT2A postoperative resection recurrence model. The results showed that\nthe combination of anti-PD-1 (IL-12&VEMR&PD-1@Gel) did not\nsignificantly inhibit tumor growth compared with IL-12&VEMR@Gel.\nFig. 14. (A) Luminescence images of orthotopic Luc-CT2A GBM tumor-bearing mice\nfollowing different treatments monitored on day 6, 9, 12, 15 and 18. (B) Quantified\nsignal intensity of (A). (C) Survival analysis of orthotopic Luc-CT2A GBM tumor-\nbearing mice in each group.\nWe hypothesized that this lack of synergy is due to the unique\nmicroenvironment of recurrent GBM: (1) GICs are pivotal drivers of\nrecurrent GBM. Their inherently low expression of MHC-I and co-\nstimulatory molecules renders them invisible to cytotoxic T cells,\nconferring natural resistance to adaptive immunity [14]. IL-12 breaks this\nevasion by unleashing multifaceted antitumor programs. (2) The IL-\n12&VEMR@Gel group achieved almost complete tumor clearance\nespecially in the GL261 post-treatment recurrence model, only IL-\n12&VEMR@Gel can fully activate anti-tumor immunity and achieve\nexcellent therapeutic effects by employing the “elimination of GICs and\nreshaping the microenvironment” dual mechanism. At this point,\ncombining other regulators no longer provides additional therapeutic space;\ninstead, it would increase the complexity of the drug combination and the\ntreatment cost, thereby burdening the patients more and being unfavorable\nfor clinical application.\nAs previously mentioned, GICs play a critical role in recurrent GBM,\nwhich are resistant to conventional therapies and are the main source of\ntumor recurrence [15,16]. These suggested that modulating immunity alone\nis insufficient to inhibit recurrent GBM, while targeting and eliminating\nGICs is essential. Thus, we combined IL-12 with VEMR (a strategy\ntargeting both glioma cells and GICs, inhibiting GICs stemness and killing\nGICs/glioma cells). The results showed remarkable synergistic effects. The\nfewest tumors were present and the longest survival time were monitored\nin the IL-12&VEMR@Gel group compared with IL-12@Gel group and\nVEMR@Gel group. These suggested that IL-12 has already been\ncombined with VEMR to inhibit recurrence by employing the “elimination\nof GICs and reshaping the microenvironment” dual mechanism.\nIn summary, our study has two key advantages. First, we fully utilized the\nmulti-faceted mechanisms of IL-12 (activating NK/CTL cells, inducing\nTh1 polarization, reprogramming macrophages, inhibiting angiogenesis)\nto overcome the low baseline T cell infiltration in GBM, and experimental\nresults confirmed its effective immunostimulatory effects. Second, we\nfocused on the unmet clinical need of recurrent GBM, recognizing that\nGICs are the core driver of recurrence. By combining IL-12 with VEMR\n(targeting GICs), we addressed both the immunosuppressive\nmicroenvironment and the root cause of recurrence (GICs), achieving\nsuperior anti-tumor efficacy compared to monotherapies. This strategy\nprovides a novel and promising approach for treating recurrent GBM.\nReferences:\n[1] GlLASSMAN CR, MATHIHARAN YK, JUDE KM, et al. Structural basis for IL-\n12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus\nNK cells[J]. Cell, 2021, 184(4):983-999.e24.\n[2] RADEMACHER MJ, Cruz A, Faber M, Oldham RAA, Wang D, Medin JA,\nSchloemer NJ. Sarcoma IL-12 overexpression facilitates NK cell\nimmunomodulation[J]. Sci Rep, 2021, 11(1):8321.\n[3] ZHANG C, ZHANG J, NIU J, et al. Interleukin-12 improves cytotoxicity of natural\nkiller cells via upregulated expression of NKG2D[J]. Hum Immunol, 2008,\n69(8):490-500.\n[4] HUANG Y, LEI Y, ZHANG H, et al. MicroRNA regulation of STAT4 protein\nexpression: rapid and sensitive modulation of IL-12 signaling in human natural\nkiller cells[J]. Blood, 2011, 118(26):6793-6802.\n[5] CHIOCCA EA, YU JS, LUKAS RV, et al. Regulatable interleukin-12 gene therapy\nin patients with recurrent high-grade glioma: Results of a phase 1 trial[J]. Sci Transl\nMed, 2019, 11(505):eaaw5680.\n[6] HEWITT SL, BAILEY D, ZIELINSKI J, et al. Intratumoral IL12 mRNA Therapy\nPromotes TH1 Transformation of the Tumor Microenvironment[J]. Clin Cancer\nRes, 2020, 26(23):6284-6298.\n[7] GRIFKA-WALK HM, Giles DA, SEGAL BM. IL-12-polarized Th1 cells produce\nGM-CSF and induce EAE independent of IL-23[J]. Eur J Immunol, 2015,\n45(10):2780-2786.\n[8] PARK J, PARK SA, KIM YS, et al. Intratumoral IL-12 delivery via mesenchymal\nstem cells combined with PD-1 blockade leads to long-term antitumor immunity\nin a mouse glioblastoma model[J]. Biomed Pharmacother, 2024, 173:115790.\n[9] XUE D, MOON B, LIAO J, et al. A tumor-specific pro-IL-12 activates preexisting\ncytotoxic T cells to control established tumors[J]. Sci Immunol, 2022,\n7(67):eabi6899.\n[10] SOUSA F, LEE H, ALMEIDA M, et al. Immunostimulatory nanoparticles\ndelivering cytokines as a novel cancer nanoadjuvant to empower glioblastoma\nimmunotherapy[J]. Drug Deliv Transl Res, 2024, 14(10):2655-2667.\n[11] LASEK W, ZAGOZDZON R, JAKOBISIAK M. Interleukin 12: still a promising\ncandidate for tumor immunotherapy?[J] Cancer Immunol Immunother, 2014,\n63(5):419-435.\n[12] CHEN P, YANG W, NAGAOKA K, et al. An IL-12-Based Nanocytokine Safely\nPotentiates Anticancer Immunity through Spatiotemporal Control of Inflammation\nto Eradicate Advanced Cold Tumors[J]. Adv Sci (Weinh), 2023, 10(10):e2205139.\n[13] SAHA D, MARTUZA RL, RABKIN SD. Macrophage Polarization Contributes to\nGlioblastoma Eradication by Combination Immunovirotherapy and Immune\nCheckpoint Blockade[J]. Cancer Cell, 2017, 32(2):253-267.\n[14] BURSTER T, GARTNER F, BULACH C, et al. Regulation of MHC I Molecules\nin Glioblastoma Cells and the Sensitizing of NK Cells[J]. Pharmaceuticals (Basel),\n2021, 14(3):236.\n[15] JOEL M, MUGHAL AA, GRIEG Z, et al. Targeting PBK/TOPK decreases growth\nand survival of glioma initiating cells in vitro and attenuates tumor growth in\nvivo[J]. Mol Cancer, 2015, 14:121.\n[16] Yang W, Li Y, Gao R, et al. MHC class I dysfunction of glioma stem cells escapes\nfrom CTL-mediated immune response via activation of Wnt/β-catenin signaling\npathway[J]. Oncogene, 2020, 39(5):1098-1111.\nCritique #8:\nGrammatical revision for better syntactical flow is recommended prior to\npublication. There are several sentences that linked inappropriately by\ncommas as well as infrequent misuse of certain words, which distract from\nthe narrative.\nResponse #8:\nThank you very much for your valuable suggestion on the language\nexpression of our manuscript. In response to your comment, we have\ncompleted a comprehensive language revision through the Springer Nature\nAuthor Service (SNAS) provided on the official website. We have carefully\nexamined the sentence logic and grammatical structure of the manuscript\nparagraph by paragraph, with a particular focus on identifying and\naddressing issues such as the splitting of long sentences and the connection\nof subordinate clauses that might affect the smoothness of reading. We also\ncarefully checked each word that might be ambiguous or used\ninappropriately. All revisions have been integrated into the revised\nmanuscript. We believe these adjustments have significantly improved the\nreadability and professionalism of the text, and we sincerely hope the\nrevised version meets the requirements of the journal.\nCritiques From Reviewer #2:\nThank you for reviewing our manuscript and providing valuable feedback.\nWe take your comments very seriously and have taken your suggestions\ninto account. We have revised the manuscript to meet your requirements\nbetter. We hope you will give us more support and encouragement after\nreviewing the revised manuscript. Thank you again for your valuable time\nand comments, and we look forward to your continued support.\nCritique #1:\nWhat the authors are proposing is a complex combination of particular\nagents and molecules and the choice of each was not based on strong\nrationales or experimentation. For example, GRP78 was used to target\nwith dVAP, but whether GRP78 is indeed highly and selectively expressed\non GBM cells or GICs was not presented. MIT was chosen but there could\nbe other chemotherapeutics that can be locally delivered to kill GICs and\nwhy particularly MIT is unclear. It felt that the authors just picked up\nagents and molecules what they like to test and put them together. Scientific\nbasis or rationale is weak.\nResponse #1:\nWe sincerely appreciate your meticulous review and insightful comments\non our manuscript. We apologize for any lack of clarity in the Abstract and\nIntroduction regarding the selection rationales of the agents used in our\nresearch. However, we would like to emphasize that these choices were not\narbitrary but based on careful consideration and prior knowledge.\n(1) Regarding the use of GRP78 targeted by DVAP:\nGRP78, a specific cancer cell-surface marker, is implicated in cancer cells\nproliferation, apoptosis resistance, metastasis and drug resistance. Beyond\nits intracellular functions, GRP78’s presence on the cell surface and its role\nin the tumor microenvironment underscore its potential as a therapeutic\ntarget. There were numerous reports indicating that GRP78 protein\noverexpressed on both glioma cells and GICs [1,2]. Recent studies have\nexplored innovative strategies to target GRP78, including small molecule\ninhibitors [3], monoclonal antibodies [4], and chimeric antigen receptor\n(CAR) T cell therapy [5], showing significant potential in glioma treatment.\nTo provide a more intuitive demonstration, we characterized the expression\nlevels of GRP78 through Western blot, the results showed that all four\nglioma cell lines (CT2A, GL261, U251, U87) exhibited high expression of\nGRP78.\nFig. 15. The expression levels of GRP78 in HEK293T, CT2A, GL261, U251 and U87.\nDVAP (DSDNDTDRDVDADP) is a stable peptide synthesized from 7 D-amino\nacids possessing high binding affinity to GRP78 [6], the binding affinity (K)\ni\nis 1.23 nmol/L. The Arg in DVAP formed electronic interaction with\nGlu566 and hydrogen bond with Tyr570 and Asn563 of GRP78 [1]. In our\nresearch, we investigated the targeting ability of DVAP-exosome (VE) in\nU87 cells, U87 GICs (GRP78 positive) and HA1800 cells (GRP78\nnegative), proving that the fluorescence signals of VE on normal cells were\nsignificantly lower than that on tumor cells. This result suggested that when\nthe system targeted GRP78, non-specific damage to healthy brain tissue\nmay be significantly reduced.\nFig. 16. (A-C) In vitro cellular targeting of exos, VE, VEMR, DVAP peptides & VE in\nHA1800 cells (A), U87 cells (B) and U87 GICs (C). (D) Quantification of targeting in\nHA1800 cells, U87 cells and U87 GICs.\n(2) Regarding the choice of mitoxantrone (MIT):\nTemozolomide (TMZ) is the primary chemotherapeutic agent for GBM,\nbut at least 50% of the patients develop resistance to treatment [7, 8].\nAcquired resistance to TMZ seems to be the main reason for the short\nmedian survival of GBM patients. TMZ, a DNA methylating agent, exerts\nits effect by methylating the guanine base, interfering with the DNA\nreplication and repair processes [9]. However, tumor cells can easily\ndevelop resistance to TMZ by upregulating DNA repair mechanisms, such\nas the expression of O6-methylguanine DNA methyltransferase (MGMT),\nwhich is the most well-known resistance mechanism [10,11]. In contrast, the\nunique structure of MIT enables it to incorporate into the DNA double\nstrands, inhibiting the activity of topoisomerase II, thereby hindering the\nfunction of DNA [12]. This mechanism of action is not easily affected by the\ncommon drug resistance mechanisms of tumor cells. Especially when\ndealing with tumor cells that are resistant to TMZ, it can effectively\novercome the MGMT-mediated resistance problem and continuously exert\nthe effect of killing tumor cells.\nAnd the second reason, evidences demonstrated that MIT exerts cytotoxic\neffects on GICs approximately 50-fold greater than TMZ and surpasses the\nefficacy of other agents such as trifluoperazine and pyrvinium pamoate [13],\nwhich is more applicable to the recurrence model. Moreover, MIT can\ninduce GBM to expose calreticulin (CRT) and release ATP [14], activating\ndendritic cell antigen presentation, forming a “chemotherapy-immunity”\nsynergy with IL-12, while the ICD effect of TMZ is significantly weaker.\n(3) Regarding the choice of siNotch1:\nNotch signaling, as an important mediator, is associated with self-renewal\nand therapeutic resistance of stem cells, and evidence has been found that\nNotch1 is overexpressed in GICs, which has a critical role in maintaining\nGICs stemness [15]. The stemness of GICs can lead to immune escape. This\nis because GICs express low levels of major histocompatibility complex\nclass I molecules and co-stimulatory molecules, which reduces their\nrecognition and killing by immune cells [16]. By knocking down Notch1 via\nsmall interfering RNA (siRNA), we aimed to disrupt the stemness of GICs,\nthereby enhancing their susceptibility to chemotherapy and immune-\nmediated killing.\nIn summary, the combination of these agents was carefully designed.\nTargeting GRP78 with DVAP allows for specific delivery of drugs to both\nglioma cells and GICs. MIT was chosen for its superior efficacy against\nGICs compared to TMZ. Knocking down Notch1 helps to eliminate the\nimmune-evasive property of GICs by reducing their stemness. The overall\ntreatment strategy achieved a significant improvement in inhibiting the\ngrowth and recurrence of GBM in our research.\nReference:\n[1] RAN D, MAO J, SHEN Q, et al. GRP78 enabled micelle-based glioma targeted\ndrug delivery[J]. J Control Release, 2017, 255:120-131.\n[2] KANG BR, YANG SH, CHUNG BR, et al. Cell surface GRP78 as a biomarker and\ntarget for suppressing glioma cells[J]. Sci Rep, 2016, 6:34922.\n[3] GURUSINGHE KRDSNS, MISHRA A, MISHRA S. Glucose-regulated protein 78\nsubstrate-binding domain alters its conformation upon EGCG inhibitor binding to\nnucleotide-binding domain: Molecular dynamics studies[J]. Sci Rep, 2018,\n8(1):5487.\n[4] LIU R, LI X, GAO W, et al. Monoclonal antibody against cell surface GRP78 as a\nnovel agent in suppressing PI3K/AKT signaling, tumor growth, and metastasis[J].\nClin Cancer Res, 2013, 19(24):6802-6811.\n[5] WANG S, WEI W, YUAN Y, et al. Chimeric antigen receptor T cells targeting cell\nsurface GRP78 efficiently kill glioblastoma and cancer stem cells[J]. J Transl Med,\n2023, 21(1):493.\n[6] WANG X, MENG N, WANG S, et al. Non-immunogenic, low-toxicity and\neffective glioma targeting MTI-31 liposomes[J]. J Control Release, 2019, 316:381-\n392.\n[7] JEZIERZANSKI M, NAFALSKA N, STOPYRA M, et al. Temozolomide (TMZ)\nin the Treatment of Glioblastoma Multiforme-A Literature Review and Clinical\nOutcomes[J]. Curr Oncol, 2024, 31(7):3994-4002.\n[8] LEE SY. Temozolomide resistance in glioblastoma multiforme[J]. Genes Dis, 2016,\n3(3):198-210.\n[9] ZHANG J, STEVENS MF, BRADSHAW TD. Temozolomide: mechanisms of\naction, repair and resistance[J]. Curr Mol Pharmacol, 2012, 5(1):102-114.\n[10] YIN J, WANG X, GE X, et al. Hypoxanthine phosphoribosyl transferase 1\nmetabolizes temozolomide to activate AMPK for driving chemoresistance of\nglioblastomas[J]. Nat Commun, 2023, 14(1):5913.\n[11] LIN K, ZOU C, HUBBARD A, et al. Multiplexed epigenetic memory editing using\nCRISPR off sensitizes glioblastoma to chemotherapy[J]. Neuro Oncol, 2025,\n27(6):1443-1457.\n[12] EVISON BJ, SLEEBS BE, WATSON KG, et al. Mitoxantrone, More than Just\nAnother Topoisomerase II Poison[J]. Med Res Rev, 2016, 36(2):248-99.\n[13] DATTA S, SEARS T, CORTOPASSI G, et al. Repurposing FDA approved drugs\ninhibiting mitochondrial function for targeting glioma-stem like cells[J]. Biomed\nPharmacother, 2021, 133:111058.\n[14] ZHANG J, CHEN C, LI A, et al. Immunostimulant hydrogel for the inhibition of\nmalignant glioma relapse post-resection[J]. Nat Nanotechnol, 2021, 16(5):538-548.\n[15] YI L, ZHOU X, LI T, et al. Notch1 signaling pathway promotes invasion, self-\nrenewal and growth of glioma initiating cells via modulating chemokine system\nCXCL12/CXCR4[J]. J Exp Clin Cancer Res, 2019, 38(1):339.\n[16] YANG W, LI Y, GAO R, et al. MHC class I dysfunction of glioma stem cells\nescapes from CTL-mediated immune response via activation of Wnt/β-catenin\nsignaling pathway[J]. Oncogene, 2020, 39(5):1098-1111.\nCritique #2:\nThe authors repeated said that there is one month between GBM surgery\nand the initiation of chemoradiation and during this one month tumor\nrecurrence occurs. But is this true based on evidence? Reference #28 was\ncited, but this was not mentioned.\nResponse #2:\nThank you for your rigorous questioning of the description of the\nrecurrence time window in the text. Regarding the clinical evidence of\ntumor recurrence within one month after GBM surgery, we re-examined\nthe literature citations and clinical data, and we have updated the new\nreferences “Injectable nanomedicine hydrogel for local chemotherapy of\nglioblastoma after surgical resection”, namely Reference [6] in the revised\nmanuscript. The description in the text is “As chemoradiation can have an\nimpact on the wound healing process, GBM patients generally follow the\nstandard radio- and chemotherapy regimen several weeks after surgery,\nonce the wound has healed”.\nReference:\nBASTIANCICH C, BIANCO J, VANVARENBERG K, et al. Injectable nanomedicine\nhydrogel for local chemotherapy of glioblastoma after surgical resection [J]. J Control\nRelease, 2017, 264: 45-54.\nCritique #3:\nThe murine GL261 model abruptly appeared in Figure 6 despite the prior\nfigures focused on human U87 cells didn’t test GL261. There is an\nirrational logical leap from Figure 5 and Figure 6. Indeed, whether or how\nGL261 responds to VEMR was not tested or shown. GL261 is a traditional\nlaboratory cell line (so is U87). Does GL261 have GICs that depends on\nNotch signaling or is GL261 growth driven by its GICs? These questions\nremain unanswered.\nResponse #3:\nThank you for your rigorous questioning of the logic introduced to the\nGL261 model in Figure 6. We understand that the transition from human\nU87 cells to the murine GL261 model requires clearer transitional\ninstructions. As a human GBM cell line, U87 is suitable for in vitro\nmechanism research, but lacks an immune microenvironment. GL261, as\na GBM cell line derived from C57BL/6 mice, can be used to construct in\nsitu tumor models in immune-active mice for conducting comprehensive\nimmunological analysis of IL-12&VEMR@Gel. In the revised manuscript,\nwe supplemented the cytotoxicity of VEMR on GL261 and GL261 GICs\nthrough CCK8 and apoptosis in vitro. The relevant results have been added\nto Section 2.3 (Figure 5G-H and Figure S11). GL261 and GL261 GIC were\ntreated with different agents. The cell viability was significantly reduced\nin VEMR group compared to control group, but no substantial variation in\ncell survival rate was observed between VEMR and VEM groups. However,\nthe cell survival rate of VER and VEM groups decreased to 52.7% and 45.7%\nin GL261 GICs. Meanwhile, VEMR group decreased by to 36.5% in\nGL261 GICs respectively. Further, we detected the apoptosis of GL261 and\nGL261 GIC by Annexin-APC/DAPI flow cytometry and conducted\nquantitative statistics on early apoptosis and late apoptosis. The results\nshowed similar trends. For GL261, there was no significant difference in\nthe apoptosis rate among the VE group, VER group and the Control group,\nwhile the apoptosis rate of the VER group in GL261 GICs was significantly\nincreased. The apoptosis rates of the VEMR group in both GL261 and\nGL261 GIC were higher than that of MIT group and VEM group. The\npossible reason was that Notch1 was low expression in glioma cells but\nhigh expression in GICs.\nFig. 17. (A-B) Cell viability of GL261 cells (A) and GL261 GICs (B).\nFig. 18. (A-B) Cell apoptosis of GL261 cells (A) and GL261 GICs (B) incubated with\nPBS, MIT, VE, VER, VEM and VEMR for 36 h. (C-D) Quantification of (A) and (B).\nIn addition, GL261 glioblastoma cells harbor GICs that relied on Notch\nsignaling for survival and self-renewal. Multiple studies have shown that\nGICs play a key role in the growth and invasion of glioblastoma. For\ninstance, Yi et al. found that GL261 nerve spheres are more invasive in\nvivo than adherent GL261 cells, and exhibit stronger tumor growth ability\nin homologous mouse models, indicating that GICs plays an important role\nin the growth of GL261 cells[1].\nReference:\n[1] YI L, ZHOU C, WANG B, et al. Implantation of GL261 neurospheres into\nC57/BL6 mice: a more reliable syngeneic graft model for research on glioma-\ninitiating cells[J]. Int J Oncol, 2013, 43(2):477-484.\nCritique #4:\nIn animal experiments using GL261, IL-12@Gel was not tested as a key\ncontrol and this is a major problem. GL261 model is well known for its\nimmunogenicity and may respond well to IL-12 monotherapy. Without this,\nthe utility of VEMR and IL-12@Gel cannot be established.\nResponse #4:\nThank you for pointing out the issue of cell selection. We understand\nthe reviewers’ concerns regarding the high immunogenicity of GL261.\nTherefore, in the supplementary experiment, a new group of IL-12\nmonotherapy was added to verify the single effect of IL-12 and the\nsynergistic value of VEMR. The relevant results have been added to\nSection 2.4 (Figure 6) and Section 2.5 (Figure 7). Notably,\nalthough the therapeutic efficacies of IL-12 and IL-12&VEMR were\ncomparable in the primary-tumor model, in the postoperative resection\nmodel, IL-12&VEMR@Gel exhibited a markedly amplified antitumor\neffect than of IL-12@Gel in both GL261 and CT2A recurrent model,\nunderscoring the pivotal contribution of GICs to recurrence. Our results\nfurther confirmed that IL-12-mediated immune activation alone is\ninsufficient; thus, direct and specific elimination of GICs becomes\nindispensable for GBM recurrence, as follow:\nIn the primary model (Section 2.4, Figure 6), luciferase-expressing GL261\n(Luc-GL261) glioma cells were implanted into the brains of mice on day\n0. Subsequently, intracranial tumor growth was monitored using IVIS\nimaging. On day 12 post-implantation, mice were randomly assigned to\neight distinct treatment groups: Control, MIT, VER, VEM, EMR, VEMR,\nIL-12, and IL-12&VEMR. Bioluminescence signals from intracranial\ntumors were measured on Day 12, 16, 18, and 21. The results revealed that\nthe signal intensity in the Control group increased significantly over time,\nreaching approximately 1.4×108. In contrast, while the signals in the\nmonotherapy groups (MIT, VEM, VER, EMR) were somewhat attenuated,\nthey remained relatively strong. The VEMR group exhibited a slightly\nstronger signal compared to the IL-12 and IL-12&VEMR groups. The\nfluorescence signal in the IL-12 group was 3.0×106, indicating that IL-12\nalone has a certain anti-tumor effect, but would be enhanced when\ncombined with VEMR. The IL-12&VEMR group demonstrated a\nsignificant prolongation of the median survival time of mice. Moreover,\nthe median survival time in the IL-12&VEMR group was superior to that\nin the IL-12 and VEMR groups. At 21 days post-implantation, brain tumor\ntissues were harvested from the mice. Subsequently, brain slices were\nstained with hematoxylin-eosin (H&E) to facilitate microscopic\nvisualization. The results showed that the IL-12&VEMR group exhibited\nthe most significant tumor growth suppression among all treatment groups.\nAdditionally, a TUNEL assay was conducted to detect apoptosis in all\nexperimental groups. The green fluorescence signals, indicative of\napoptotic cells, were minimal in the Control group but most abundant in\nthe IL-12&VEMR group compared to other groups. This finding suggests\nthat treatment with IL-12&VEMR effectively induces extensive apoptosis\nof tumor cells.\nFig. 19. In vivo therapeutic therapy of different treatments on GBM. (A) Timeline\nschematic of the animal experiment. (B) Luminescence images of orthotopic Luc-\nGL261 GBM tumor-bearing mice following different treatments monitored on day 12,\n16, 18, and 21. (C) Quantified signal intensity of (B). (D) Survival analysis of mice in\neach group. (E) Representative H&E images of the orthotopic GBM tumors after the\nindicated treatments. (F) TUNEL staining of brain tissue sections obtained from mice\ntumors after treatments. (G) Quantification of tumor area of (E). (H) Fluorescence\nquantification of (F).\nIn the postoperative resection model (Section 2.5, Figure 7), visible\ngliomas were surgically excised from mice on the 12th day after\ninoculation with Luc-GL261 cells. Following surgery, mice were randomly\nassigned to seven groups, and the resection cavities were injection with\neach hydrogel-mediated treatment (Gel, EM@Gel, EMR@Gel,\nVEMR@Gel, IL-12@Gel, IL-12&VEMR@Gel), and the control group\nreceived surgery resection without hydrogel treatment. Bioluminescence\nsignals were monitored over time (Day 12, 20, 24, 30) and showed that\nsignals in the Control and Gel groups continued to increase. In contrast, the\nfluorescence signals of the EM@Gel, EMR@Gel, VEMR@Gel, and IL-\n12@Gel groups were attenuated to varying degrees, with IL-12@Gel\nshowing a signal intensity of 1.5×106, slightly higher than that of the IL-\n12&VEMR@Gel group (3.3×104). However, the survival outcomes were\nstrikingly different. Mice in the VEMR@Gel group had a survival period\nof 36 days, while those in the IL-12@Gel group survived for 123 days.\nNotably, 60% of the mice in the IL-12&VEMR@Gel group were still alive\nat 180 days, highlighting the significant therapeutic advantage of the\ncombined treatment. On the 30th day post-inoculation, mice with excised\ntumors were euthanized, and brain tumor tissues were harvested. H&E\nstaining of tumor-bearing brain sections revealed that the IL-\n12&VEMR@Gel group had the fewest tumors among all groups.\nAdditionally, TUNEL staining of tumor-bearing brain slices showed\nextensive apoptotic cells in the IL-12&VEMR@Gel group, indicating\neffective tumor cell apoptosis. In summary, these results demonstrated the\nexcellent ability of IL-12&VEMR@Gel to suppress glioma recurrence.\nFig. 20. In vivo therapy of exo-gel delivery system. (A) Timeline schematic of the\nanimal experiment. (B) Luminescence images of orthotopic Luc-GL261 GBM tumor-\nbearing mice following different treatments monitored on day 12, 20, 24 and 30. (C)\nQuantified signal intensity of (B). (D) Survival analysis of mice in each group. (E)\nRepresentative H&E images of the orthotopic GBM tumors after the indicated\ntreatments. (F) TUNEL staining of brain tissue sections obtained from mice tumors\nafter treatments. (G) Luminescence images of orthotopic Luc-CT2A GBM tumor-\nbearing mice following different treatments monitored on day 6, 9, 12, 15 and 18. (H)\nQuantified signal intensity of (G). (I) Survival analysis of orthotopic Luc-CT2A GBM\ntumor-bearing mice in each group.\nCritique #5:\nThe GBM models that were used herein are U87 and GL261 that are\nprobably not clinically representative. Use of other models such as patient-\nderived GBM cells or other murine models is highly recommended to\nvalidate the results. The authors’ claim that the strategy offers broad\nclinical applications is certainly not supported by the limited tumor models\nthat were selected.\nResponse #5:\nThank you for your profound insights into the clinical representativeness\nof the GBM model in this study. We fully agree with the limitations of the\nU87 and GL261 models in terms of genetic heterogeneity. Therefore, we\nspecifically used the highly invasive U251 (human GBM cell line) and\nCT2A (mouse GBM cell line) for supplementary experiments to verify the\nuniversality of the platform. We evaluated the cytotoxicity of VEMR on\nCT2A, CT2A GIC, U251, and U251 GIC using CCK8 and apoptosis assays.\nThe results all demonstrated the same anti-tumor effect as that of U87 cells\nand U87 GIC, as shown in Section 2.3 (Figure 5I-J, Figure S8, Figure S10\nand Figure S12).\nFor CT2A and CT2A GIC, the cell viability of VEMR group was\nsignificantly reduced to 18.92% and 19.85%, respectively, compared to\ncontrol group. The cell viability of CT2A was no significant difference in\nthe VER group, while the cell viability of CT2A GIC was significantly\nreduced to 67.8%. And the MIT and VEM can significantly reduce cell\nviability in both CT2A and CT2A GIC. The apoptosis rate shows the same\ntrend. We conducted a statistical analysis of early apoptosis and late\napoptosis. The apoptosis rates of VEMR were 78.5% and 77.6% in CT2A\nand CT2A GIC respectively. VER did not induce obvious apoptosis in\nCT2A cells, but the proportion of apoptotic cells significantly increased in\nCT2A GIC.\nFor U251 and U251 GIC, the cell viability of VEMR group was\nsignificantly reduced to 4.7% and 51% respectively, and the MIT and\nVEM also can significantly inhibit cell viability. The VE group did not\nshow any significant differences in both U251 and U251 GIC. The cell\nviability of U251 was no significant difference in the VER group, while\nthe cell viability of U251 GIC was significantly reduced to 63%. The\napoptosis result showed the same trend. The apoptosis rates of VEMR were\n54.9% and 62.2% in U251 and U251 GIC, respectively.\nFig. 21. (A-D) Cell viability of CT2A cells (A), CT2A GICs (B), U251 cells (C) and\nU251GICs (D).\nFig. 22. (A-D) Cell apoptosis of CT2A cells (A), CT2A GICs (B), U251 cells (C), U251\nGICs (D) incubated with PBS, MIT, VE, VER, VEM and VEMR for 36 h. (E-H)\nQuantification of (A-D).\nFurthermore, we established an in situ glioblastoma model in mice with\nCT2A, and investigated the therapeutic effect of the IL-12&VEMR@Gel.\nThe relevant experimental methods and results have been added to Section\n2.5 (Figure 7G-I). We investigated the therapeutic effect of this\nmultifunctional exo-gel delivery system on CT2A orthotopic glioma model.\nLuciferase-expressing CT2A (Luc-CT2A) glioma cells were implanted\ninto brains on day 0, and mice underwent imaging using IVIS to determine\nthe glioma growth. On the 6th day after inoculation with Luc-CT2A cells,\nthe brain tumor was surgically removed. The mice were randomly divided\ninto four groups, and a multifunctional exo-gel delivery system was\ninjected into the resection cavity. The results showed that the\nbioluminescence signals in the Control group continued to increase to\n6.7×108, while the signals in the VEMR@Gel (4.3×107) and IL-12@Gel\n(3.9×107) groups were significantly weaker than those in the Control group,\nshowing a certain degree of inhibition. Notably, the fluorescence signal of\nthe IL-12&VEMR@Gel group was weakened to 8.8×106, indicating that\nIL-12&VEMR@Gel can significantly inhibit tumor growth, and prolong\nthe survival of mice. Taken together, these results demonstrated the\nexcellent ability of IL-12&VEMR@Gel in suppressing glioma recurrence.\nFig. 23. (A) Luminescence images of orthotopic Luc-CT2A GBM tumor-bearing mice\nfollowing different treatments monitored on day 6, 9, 12, 15 and 18. (B) Quantified\nsignal intensity of (A). (C) Survival analysis of orthotopic Luc-CT2A GBM tumor-\nbearing mice in each group.\nCritique #6:\nFigure 8B. What was stained as a marker for DCs? This was unclear even\nafter looking at the Materials and Methods.\nResponse #6:\nThank you for your attention to the details of the experiment. In the original\nmanuscript, DCs were stained with CD11c and then imaged using laser\nscanning confocal microscopy. However, to ensure greater accuracy, in the\nrevised manuscript, we re-performed the verification using flow cytometry.\nBriefly, brain tumor tissues were harvested to prepare a single cell\nsuspension via the percoll cell isolation solution, DCs was stained with\nCD11c-FITC, CD80-APC, CD86-PE and analyzed by flow cytometry.\nCompared with the control group, the proportion of DC cells in the Gel\ngroup showed no significant difference. In the EM@Gel group, EMR@Gel\ngroup, VEMR@Gel group, IL-12@Gel group and IL12&VEMR@Gel\ngroup, the proportion of DC cells increased to varying degrees. Especially\nin the IL-12&VEMR@Gel group, the proportion of DC cells increased to\n10.3%. The results showed that combined treatment can promote the\nproliferation of DC cells.\nIn the “Materials and Methods”, we have already provided a detailed\ndescription in the revised manuscript (Section 4.15) as follow: “The\nfollowing antibody panels were used: ... anti-CD11c-FITC, anti-CD80-\nAPC, and anti-CD86-PE for DC cell detection”.\nFig. 24. Immunofluorescence of DC cells in tumor-bearing brain tissues. (In the original\nmanuscript)\nFig. 25. Quantification results of the proportion of DCs in tumor-bearing brain issues.\n(In the revised manuscript)\nCritique #7:\nAbstract. dVAP and MIT appeared without appropriate explanation and\nthe reader will not understand them.\nResponse #7:\nThanks for the reviewer’s suggestions. We have already made the\nnecessary additions in the abstract. DVAP is a targeted peptides which have\na good affinity with GRP78, and is used to specifically bind to glioma cells\nand GICs; MIT, the abbreviation of mitoxantrone, is a chemotherapy agent\nwhich has a strong killing effect on GICs and glioma cells. The revised\nabstract statement is as follows: “This platform comprises GIC-targeting\nexosomes that carry siNotch1 and mitoxantrone to reduce the stemness of\nGICs and kill residual GICs and glioma cells, respectively, and an immune\nactivator (interleukin-12), which can remodel the immunosuppressive\nmicroenvironment, ultimately suppressing postoperative GBM relapse.”\nCritique #8:\nThe phrase “tracking GICs” is probably not appropriate since VEMR\ntargets GICs but not necessarily tracks (chases) them.\nResponse #8:\nWe would like to thank the reviewer for his/her suggestion. For a more\nrigorous expression, we delete the expression of \"tracking GICs\", as follow:\n“GBM recurrence can occur as a result of the difficulty incompletely\nremoving the tumors; in particular, residual GICs in deep intracranial\nlocations of the brain are highly infiltrative and diffuse, leading to\nrecurrence in almost all GBM patients. Thus, targeted in situ eradication of\nresidual GICs may be a promising therapeutic strategy against GBM\nrecurrence.” (Section 1, Paragraph 1, Line 22)\nCritique #9:\nFor the description of fluorescence experiments, the authors often only\nreferred to the color (red and green, etc), without stating what the color\nindicated or represented.\nResponse #9:\nThank you for your correction regarding the rigor of the description of the\nfluorescence experiment. We fully agree that the biological significance of\nfluorescence labeling needs to be clearly explained. We have added the\nfollowing details:\nFigure 3A-B: Exo was labeled with DiI (red), cell nucleus was labeled with\nDAPI (blue).\nFigure 3C: Exo was labeled with DiI (red).\nFigure 3D-E: Cell nucleus was labeled with DAPI (blue), siNotch1 was\nlabeled with Cy3 (red), and MIT was green fluorescence.\nFigure 4A-B: VE was labeled with DiI (red), cell nucleus was labeled with\nlabeled with hoechst 33342 (blue).\nFigure 4C-D: Cell nucleus was labeled with hoechst 33342 (blue),\nlysosomes were labeled with LysoTracker (green), siNotch1 was labeled\nwith Cy3 (red), and MIT was purple fluorescence.\nCritique #10:\nFigure 4D seems to have a technical challenge. When cells form spheres\nand aggregates like U87 GICs, is the method used here able to accurately\nassess co-localization?\nResponse #10:\nThank you for your attention and questioning regarding the experimental\nmethod in Figure 4D. We understand your concerns about the accuracy of\nthe co-localization assessment when cells form spheres and aggregates, but\nwe believe that the method used in this study can accurately evaluate co-\nlocalization. We employed confocal laser scanning microscopy (CLSM)\ncombined with fluorescence labeling techniques to observe and analyze the\nco-localization situation. CLSM has the capabilities of high resolution and\ndeep imaging, enabling clear imaging of different fluorescently labeled\nmolecules within cells. Even when cells form spheres and aggregates, by\nadjusting the microscope’s focal length, different layers of cells can be\nscanned to obtain detailed spatial information, thereby accurately\ndetermining the co-localization situation of different molecules. In order to\nmake the results more persuasive, we used the Z-stack layer scanning\nfunction of the confocal microscope to perform continuous axial (Z-axis)\nsectioning of the spheres or aggregated structures, and allows imaging of\ndifferent depth cell layers separately. The results showed that a clear\nfluorescence distribution was observed in all different cell layers, and most\nof the siRNA and MIT entered the lyso/endosomes and then escaped from\nthe lyso/endosomes over time.\nFig. 26. (A-C) Z-stack layer scanning images of U87 GICs incubated with VEMR for\n2h (A), 4h (B) and 8 h (C). (D-F) Z-stack layer scanning images of GL261 GICs\nincubated with VEMR for 2h (D), 4h (E) and 8 h (F).\nCritique #11:\nSupplementary Fig 3. What are TP and siNC? Unclear.\nResponse 11:\nThank you for pointing out the omissions in the term descriptions in\nSupplementary Figure 3. We made supplements in the revised manuscript\n(Figure S7). TP is the abbreviation of targeted peptide, that is DVAP, In the\nrevised version, we replaced “TP” with “DVAP”; siNC is siRNA of\nnegative control, used to exclude nonspecific effects.\nFig. 27. (A) CCK8 of Exo with different concentration in HA1800 cells. (B) CCK8 of\nExo with different concentration in U87 cells. (C) CCK8 of MIT in different samples\n(MIT, EMR, VEMR) with different concentration in U87 cells.\nCritique #12:\nFigure 5A-C. Notch1 silencing was not potent particularly in the protein\nlevels. Didn’t this need to be improved?\nResponse #12:\nThank you for suggestions. We acknowledge that the reviewers’ concerns\nare reasonable. This data does indeed require further improvement and\nvalidation. We improved the experimental conditions and re-characterized\nthe silencing of siRNA in the protein level. The results showed that Notch\n1 protein expression level was significantly down-regulated to ~21.3% in\nVER (***P＜0.001) and the new image has been replaced to Figure 5B-C.\nFig. 28. (A) Western blotting analysis of Notch1 expression in U87 GICs incubated\nwith PBS, siNotch1, Lipo/siNotch1 and VER for 48 h. (B) Quantification of Notch1\nexpression in (A).\nCritique #13:\nFigure 5GH needs quantitation of the results.\nResponse #13:\nThanks for the reviewer’s suggestions. We further conducted a quantitative\nanalysis of the scratch assay. The scratch images at 0h, 12h, and 24h were\nprocessed using the Image J. The cell migration rate was calculated by\nmeasuring the width of the scratches. Compared with the PBS group, U87\ncells in VE and VER group were almost completely confluent, while VER\ngroup weakened the healing of U87 GICs, VEM and VEMR group\nsignificantly reduced the “wound” healing rate of U87 cells and U87 GICs,\nsignifying that siRNA in VER did not affect U87 cells migration but\naffected U87 GICs migration, MIT combined with siRNA can enhance the\ninhibitory influence of VEMR on the migration of U87 cells and U87 GICs.\nFig. 29. (A) Scratch assay of U87 cells incubated with PBS, MIT, VE, VER, VEM and\nVEMR for 0h, 12h and 24 h. (B-C) Quantification of relative migration distance for\n12h (B) and 24 h (C). (D) Scratch assay of U87 GICs incubated with PBS, MIT, VE,\nVER, VEM and VEMR for 0h, 12h and 24 h. (E-F) Quantification of relative migration\ndistance for 12h (E) and 24 h (F).\nCritique #14:\nThere are problems in English (language) as I find many areas where the\ntrue message was unclear. Use of professional language editing service is\nhighly suggested.\nResponse #14:\nThank you very much for your valuable suggestion on the language\nexpression of our manuscript. In response to your comment, we have\nstrictly followed the journal’s guidelines and completed a comprehensive\nlanguage revision through the Springer Nature Author Service (SNAS)\nprovided on the official website for proper English language, grammar,\npunctuation, spelling, and overall style. All revisions have been integrated\ninto the revised manuscript. We believe these adjustments have\nsignificantly improved the readability and professionalism of the text, and\nwe sincerely hope the revised version meets the requirements of the journal.\nCritiques From Reviewer #3:\nThank you for your meticulous review of the paper and the valuable\nsuggestions you provided. Your suggestions have provided us with an\nimportant direction for further improving the research content and\nenhancing the quality of the paper. We will carefully consider each\nsuggestion, meticulously sort out and supplement relevant data, optimize\nthe logical structure of the discussion, in order to ensure the rigor and\nscientificity of the paper. Once again, thank you for your professional\nguidance and assistance.\nCritique #1:\nAbstract- Page 2, line 3:\nThis cell line (U87-MG) was proven as problematic model for GBM. The\nU87MG glioblastoma (GBM) cell line, widely used in research, has been\nfound to have a DNA profile different from the original tumor, raising\nconcerns about its authenticity (Allen et al., 2016). This misidentification\nhighlights the need for careful validation of cell lines in research. U87MG\ncells exhibit varied responses to treatments like all-trans retinoic acid,\nrevealing phenotypic nuances that may complicate GBM research (Pokorn\ná et al., 2021). Comparisons between U87-luc2 and U251-RedFLuc\nmodels show that they recapitulate different aspects of GBM heterogeneity,\nwith U251-RedFLuc forming more invasive tumors (Schulz et al., 2022).\nFurthermore, commonly used cell lines like U-87 MG grown in serum-\ncontaining media do not accurately reflect gene expression profiles of\npatient tumors. Patient-derived GBM models offer greater diversity in gene\nexpression and better correspond to clinical samples, potentially\nimproving preclinical drug discovery efforts (Ye et al., 2020). Therefore, I\npropose to re-evaluate the platform on additional clinically-relevant cell\nlines or patient-derived cells. The authors should identify other GB cells\nthat express GRP78 positive for their purpose.\nResponse #1:\nThank you for your rigorous consideration of the authenticity of the cell\nline model. We fully recognize the significance of the consistency between\nthe cell model and clinical samples. In response to your suggestion, highly\ninvasive U251 and CT2A cell lines were specially introduced for\nsupplementary experiments. We evaluated the cytotoxicity of VEMR on\nCT2A, CT2A GIC, U251, and U251 GIC using CCK8 and apoptosis assays.\nThe relevant results are supplemented to Section 2.3 (Figure 5I-J, Figure\nS8, Figure S10 and Figure S12).\nFor CT2A and CT2A GIC, the cell viability of VEMR group was\nsignificantly reduced to 18.92% and 19.85% respectively compared to\ncontrol group. The cell viability of CT2A was no significant difference in\nthe VER group, while the cell viability of CT2A GIC was significantly\nreduced to 67.8%. And the MIT and VEM can significantly reduce cell\nviability in both CT2A and CT2A GIC. The apoptosis result showed the\nsame trend. We conducted a statistical analysis of early apoptosis and late\napoptosis. The apoptosis rates of VEMR were 78.5% and 77.6% in CT2A\nand CT2A GIC respectively. VER did not induce obvious apoptosis in\nCT2A cells, but the proportion of apoptotic cells significantly increased in\nCT2A GIC.\nFor U251 and U251 GIC, the cell viability of VEMR group was\nsignificantly reduced to 4.7% and 51%, respectively, and the MIT and\nVEM also can significantly inhibit cell viability. The VE group did not\nshow any significant differences in both U251 and U251 GIC. The cell\nviability of U251 was no significant difference in the VER group, while\nthe cell viability of U251 GIC was significantly reduced to 63%. The\napoptosis rate shows the same trend. The apoptosis rates of VEMR were\n54.9% and 62.2% in U251 and U251 GIC respectively.\nFig. 30. (A-D) Cell viability of CT2A cells (A), CT2A GICs (B), U251 cells (C) and\nU251GICs (D).\nFig. 31. (A-D) Cell apoptosis of CT2A cells (A), CT2A GICs (B), U251 cells (C), U251\nGICs (D) incubated with PBS, MIT, VE, VER, VEM and VEMR for 36 h. (E-H)\nQuantification of (A-D).\nFurthermore, we established an in situ glioblastoma model in mice with\nCT2A, and investigated the therapeutic effect of the IL-12&VEMR@Gel.\nThe relevant experimental methods and results have been added to Section\n2.5 (Figure 7G-I). We investigated the therapeutic effect of this\nmultifunctional exo-gel delivery system on CT2A orthotopic glioma model.\nLuciferase-expressing CT2A (Luc-CT2A) glioma cells were implanted\ninto brains on day 0, and mice underwent imaging using IVIS to determine\nthe glioma growth. On the 6th day after inoculation with Luc-CT2A cells,\nthe brain tumor was surgically removed. The mice were randomly divided\ninto four groups, and a multifunctional exo-gel delivery system was\ninjected into the resection cavity. The results showed that the\nbioluminescence signals in the Control group continued to increase to\n6.7×108, while the signals in the VEMR@Gel (4.3×107) and IL-12@Gel\n(3.9×107) groups were significantly weaker than those in the Control group,\nshowing a certain degree of inhibition. Notably, the fluorescence signal of\nthe IL-12&VEMR@Gel group was weakened to 8.8×106, indicating that\nIL-12&VEMR@Gel can significantly inhibit tumor growth, and prolong\nthe survival of mice. Taken together, these results demonstrated the\nexcellent ability of IL-12&VEMR@Gel in suppressing glioma recurrence.\nFig. 32. (A) Luminescence images of orthotopic Luc-CT2A GBM tumor-bearing mice\nfollowing different treatments monitored on day 6, 9, 12, 15 and 18. (B) Quantified\nsignal intensity of (A). (C) Survival analysis of orthotopic Luc-CT2A GBM tumor-\nbearing mice in each group.\nIn response to the “The authors should identify other GB cells that express\nGRP78 positive for their purpose” raised by the reviewer. There were\nnumerous reports indicating that GRP78 protein overexpressed on both\nglioma cells and GICs [1-3]. To provide a more intuitive demonstration, we\ncharacterized the expression levels of GRP78 through Western blot, the\nresults showed that all four glioma cell lines (CT2A, GL261, U251, U87)\nexhibited high expression of GRP78.\nFig. 33. The expression levels of GRP78 in HEK293T, CT2A, GL261, U251 and U87.\nReference:\n[1] RAN D, MAO J, SHEN Q, et al. GRP78 enabled micelle-based glioma targeted\ndrug delivery[J]. J Control Release, 2017, 255:120-131.\n[2] KANG BR, YANG SH, CHUNG BR, et al. Cell surface GRP78 as a biomarker and\ntarget for suppressing glioma cells[J]. Sci Rep, 2016, 6:34922.\n[3] YANG Y, LI W, ZHAO Y, et al. GRP78 in Glioma Progression and Therapy:\nImplications for Targeted Approaches[J]. Biomedicines, 2025, 13(2):382.\nCritique #2:\nPage 1, Line 28- define VEMR.\nResponse #2:\nThank you for your suggestion. VEMR is the abbreviation of “DVAP-\nExo/MIT/siNotch1”, exosomes modified with DVAP targeted peptides\ncarried MIT and siNotch1 simultaneously. We have already made the\nnecessary additions to the revised manuscript.\nNotch1 is overexpressed in GICs, which has a critical role in maintaining\nGICs stemness. Specifically knocking down Notch1 via siRNA is an\nattractive approach against GBM recurrence. MIT is a potential\nchemotherapy drug based on its highly efficient killing effect on GIC and\nits ability to induce immunogenic cell death (ICD). Co-delivering siNotch1\nand MIT may both inhibit GBM recurrence and increase the tumor-\nassociated antigens, thus activating immunity. The co-laden exosomes\nwere further surficial modified with targeted peptides (DVAP peptides have\na good affinity with GRP78, which is low expressed in normal cells and\nhigh expressed in GIC cells and glioma cells), aiming to target remaining\nGICs and glioma cells.\nCritique #3:\nIntro- Page 2, Line 21: The sentence is too long. I suggest to split to several\nclear and short sentences. Besides that, it is incredibly difficult if at all\npossible, to access GIC surgically.\nResponse #3:\nThank you for your valuable suggestions. We have split the overly long\nsentences into clearer and shorter ones to improve readability, as follows:\n“For patients with GBM, the initial clinical intervention is surgical\ndebulking, and chemoradiotherapy (CRT) is typically administered\napproximately one month after surgery. This one-month post-operative\ninterval represents a crucial period during which tumor recurrence often\noccurs. GBM recurrence can occur as a result of the difficulty incompletely\nremoving the tumors; in particular, residual GICs in deep intracranial\nlocations of the brain are highly infiltrative and diffuse, leading to\nrecurrence in almost all GBM patients. Thus, targeted in situ eradication of\nresidual GICs may be a promising therapeutic strategy against GBM\nrecurrence.” (Section 1, Paragraph 1, Line 19)\nCritique #4:\nPage 3, Line 3: Do the authors suggest that GICs play a role only in GBM\nrecurrence? Or are they also the initial source of those tumors? Are those\ncells a specific subpopulation among cells left post-treatment, or is it\nreferred to as a general name for the remaining cells post-treatment?\nPlease clarify in an objective manner as there are multiple references to\ncite on this matter.\nResponse #4:\nWe sincerely appreciate your insightful questions. Regarding the role of\nGICs (glioma stem cells) in GBM and the definition of cell subsets after\ntreatment, existing research evidence shows that they have the dual\nattributes of tumor-origin cells and recurrence-driven cells[1-4]. GICs\nexhibit robust self-renewal and multilineage differentiation potential,\nthereby generating the tumor’s cellular heterogeneity. Moreover, their\nintrinsic resistance to conventional therapies enables them to persist after\ntreatment and drive recurrence. In our study, while we focus on recurrence-\nrelated responses, we acknowledge GICs’ dual role in initiation and\nrecurrence. Moreover, GIC is a distinct subpopulation, defined by\nfunctional properties and biomarkers, and they represent a minority but\nhighly aggressive subset of remaining cells, not a general term for all\nresidual cells. The relevant descriptions have been supplemented in the\nrevised manuscript.\nIn the primary tumor, glioma cells account for a significant proportion.\nFollowing surgical resection, only a small number of unresectable tumor\ncells and stem cells remain, in this condition, the role of GIC in tumor\nrecurrence is more worthy of attention than that of glioma cells.\nReference:\n[1] DIGREGORIO M, LOMBARD A, LUMAPAT PN, et al. Relevance of\nTranslation Initiation in Diffuse Glioma Biology and its Therapeutic Potential[J].\nCells, 2019, 8(12):1542.\n[2] HOU J, UEJIMA T, TANAKA M, et al. EVA1-antibody drug conjugate is a new\ntherapeutic strategy for eliminating glioblastoma-initiating cells[J]. Neuro Oncol,\n2025, 27(3):682-694.\n[3] JOEL M, MUGHAL AA, GRIEG Z, et al. Targeting PBK/TOPK decreases\ngrowth and survival of glioma initiating cells in vitro and attenuates tumor growth\nin vivo[J]. Mol Cancer, 2015, 14:121.\n[4] WILLEMS E, LOMBARD A, DEDOBBELEER M, et al. The Unexpected Roles\nof Aurora A Kinase in Gliobastoma Recurrences[J]. Target Oncol, 2017, 12(1):11-\n18.\nCritique #5:\nLine 5: Cell should be in plural- cells. Besides Notch1, what are the GICs\nadditional stemness markers? Why was only this chosen?\nResponse #5:\nThank you very much for your valuable suggestions. We have immediately\nconducted a systematic review of the entire text, carefully checked all the\nexpressions involving “cell”, identified the errors in the use of singular and\nplural forms due to negligence, and have corrected them all to the correct\nplural form “cells”, which conforms to the expression norms of academic\nEnglish.\nAdditionally, thank you for your in-depth questions regarding the selection\nof GIC markers. Several studies showed that, in addition to Notch1,\nGICs also exhibit several other stemness markers, including CD133,\nNestin, BMI1, OLIG2, Oct4, Sox2, CD44, etc[1-2]. We fully recognized the\nsignificance of comprehensively characterizing the stemness\ncharacteristics of GICs. Therefore, in our research, U87 GICs were\nobtained by immunomagnetic bead sorting method, the expression of\nCD133, Nestin, Notch1 and Hes1 were detected by western blot (Section\n2.2, Figure S3). When verifying the therapeutic effect of GIC, in addition\nto Notch1, we also focused on detecting the expression level of CD133 to\nconduct multi-dimensional validation of the inhibitory effects of VEMR\nand IL-12&VEMR@Gel on the stemness of GICs (Figure 5F, Figure S13,\nFigure S16).\nHowever, our decision to use Notch1 as the pivotal therapeutic target and\nto silence it with siRNA was driven by the following scientific evidence:\nNotch1 is a key regulatory factor for the self-renewal of GIC. The\noverexpression of Notch1 is directly associated with the recurrence and\ntreatment resistance of GBM, rather than merely serving as a superficial\nmarker. The Notch signaling pathway, which serves as the hub of the “stem\ncell maintenance network” in GIC, also interacts with other stemness\npathways (such as Hedgehog pathway, Wnt/β-catenin pathway,\nPI3K/AKT/mTOR pathway, etc) to enhance GIC survival and resistance to\napoptosis[3]. Therefore, Notch1’s role in regulating the GIC niche and\npromoting tumor progression positioned it as both a marker and a\ntherapeutic target, whereas many other markers serve primarily as\nphenotypic identifiers without such active regulatory functions in GIC\nmaintenance and tumorigenesis. Fan et al. indicated that Notch blockade\nreduced neurosphere growth and clonogenicity, and the putative CSC\nmarkers CD133, Nestin, BMI1, and OLIG2 were reduced following Notch\nblockade[4].\nFig. 34. (A) Western blot analysis the expressions of CD133, Nestin, Notch1 and Hes\n1 in U87 cells and U87 GIC. (B) Immunofluorescence staining analysis the expressions\nof CD133 and Notch1 in U87 GIC.\nFig. 35. (A) Immunofluorescence analysis of Notch1 and CD133 expressions in tumor-\nbearing brain tissues. (B) Western blot analysis of Notch1 and CD133 expressions in\ntumor-bearing brain tissues. (C-D) Western blotting analysis of Notch1 and CD133\nexpressions in U87 GICs (C) and GL261 GICs (D) incubated with PBS, VE, VER,\nVEM and VEMR.\nReference:\n[1] HAI L, ZHANG C, LI T, et al. Notch1 is a prognostic factor that is distinctly\nactivated in the classical and proneural subtype of glioblastoma and that promotes\nglioma cell survival via the NF-κB(p65) pathway[J]. Cell Death Dis, 2018,\n9(2):158.\n[2] HAI L, LIU P, YU S, et al. Jagged1 is Clinically Prognostic and Promotes Invasion\nof Glioma-Initiating Cells by Activating NF-κB(p65) Signaling[J]. Cell Physiol\nBiochem, 2018, 51(6):2925-2937.\n[3] MEURETTE O, MEHLEN P. Notch Signaling in the Tumor Microenvironment[J].\nCancer Cell, 2018, 34(4):536-548.\n[4] FAN X, KHAKI L, ZHU TS, et al. NOTCH pathway blockade depletes CD133-\npositive glioblastoma cells and inhibits growth of tumor neurospheres and\nxenografts[J]. Stem Cells, 2010, 28(1):5-16.\nCritique #6:\nLine 9: TMZ is used because of its ability to cross the BBB, which does not\nrefer to MIT. This should be noted in the text.\nResponse #6:\nThank you for your detailed suggestions on the basis of drug selection. We\nfully agree that TMZ has become one of the standard drugs for\nchemotherapy of GBM due to its penetration ability of the blood-brain\nbarrier (BBB). However, we chose MIT not because of the ability to cross\nthe blood-brain barrier, because we administer drugs in situ. We chose MIT\nis a potential chemotherapy drug based on its highly efficient killing effect\non GIC and its ability to induce immunogenic cell death (ICD).\nMechanistically, MIT can induce GICs to expose calreticulin (CRT) and\nrelease ATP, activating dendritic cell antigen presentation, forming a\n“chemotherapy-immunity” synergy with IL-12, while the ICD effect of\nTMZ is significantly weaker.\nCritique #7:\nLine 18- “egos” should be “Exos”\nResponse #7:\nThanks for the reviewer’s correction. We have changed “egos” to “Exos”.\nWe have meticulously reviewed the manuscript, corrected all grammatical\nerrors and spelling mistakes, and are confident that the revised version now\nfully meets the journal’s requirements.\nCritique #8:\nPage 4, Line 4: Define immune cells: microglia, bone-marrow-derived\nmacrophages? DCs?\nResponse #8:\nThank you for your correction. We have supplemented the definition of\nimmune cells and the description of their characteristics in GBM on Line\n3-6 of Page 4, as follow: “Another key factor for recurrence is the highly\nimmune inhibitory tumor microenvironment in GBM, which inhibits\neffective immune surveillance by promoting the release of immune\nsuppressors and altering the infiltration of CD4+ T and CD8+ T cells, DC\ncells, regulatory T cells (Treg cells) and myeloid-derived suppressor cells\n(MDSCs)”.\nCritique #9:\nLine 19: Gliadel can be given as an FDA-approved PIDDS.\nResponse #9:\nThank you for your suggestion. We have supplemented the following\ndescription of Gliadel as an FDA-approved Local Drug Delivery System\n(PIDDS) in the revised manuscript (Line 20-21 of Page 4), as follow: “The\nuse of the Gliadel® wafer remains the sole FDA-approved PIDDS for\nGBM, although it is rarely used clinically due to complications.”\nCritique #10:\nPage 5, Line 4: It’s unclear what is expected toxicity to healthy adjacent\ncells in the brain.\nResponse #10:\nThanks for the reviewers’ comments. Regarding this, we would like to\nmake the following responses: Firstly, we investigated the targeting ability\nof DVAP-exosome (VE) in U87 cells, U87 GICs (GRP78 positive) and\nHA1800 cells (GRP78 negative), proving that the fluorescence signals of\nVE on normal cells was significantly lower than that on tumor cells. This\nresult suggested that when the system targets GRP78, non-specific damage\nto healthy brain tissue may be significantly reduced.\nFurther, we also evaluated the targeting of non-glioma and non-GIC brain\ncells (neurons, astrocytes, oligodendrocytes, microglia). We compared the\nuptake of Exo, VE, and VEMR in HT22, BV2, HMC3 and MO3.13.\nExosomes were labeled with CM-DiI and then incubated with different cell\nlines. Among the three types of cells, there was no statistically significant\ndifference in the fluorescence intensity of the four groups.\nMoreover, the survival time of mice in the IL-12&VEMR@Gel treatment\ngroup was prolonged longer than 180 days in GL261 model (Figure 7D),\nand no neurological dysfunction symptoms (such as dyskinesia, epilepsy,\netc.) occurred, indirectly reflecting the limited damage to the brain\nparenchyma caused by local administration.\nFig. 36. (A-B) In vitro cellular targeting of Exo, VE, VEMR, DVAP peptides+VE in\nHA1800 cells (A) and U87 cells (B). (C) Quantification of targeting in HA1800 cells\nand U87 cells.\nFig. 37. (A-D) In vitro cellular targeting of Exo, VE, VEMR, DVAP peptides & VE in\nHT22 cells (A), BV2 cells (B), HMC3 cells (C), MO3.13 cells (D), U87 cells (E) and\nU87 GIC (F). (G) Quantification of targeting in different cells.\nCritique #11:\nPage 9, Line 1: GFAP is a marker of reactive astrocytes undergoing\nastrogliosis, not of cancer differentiation. Unless the cells are mostly\ncancer stem cells differentiating into astrocytes-like cancer cells or\noriginating from astrocytes.\nResponse #11:\nThank you for the reviewer’s meticulous correction of the definition of the\nGFAP marker, the viewpoint is extremely crucial. We then conducted a\nfurther review of the literatures and found that our statement was not\nprecise. Our intention was to illustrate the stem cell characteristics. Based\non your suggestion, we have deleted the WB results related to GFAP, as\nfollow (Section 2.2, Figure S3):\nFig. 38. Western blot analysis the expressions of CD133, Nestin, Notch1 and Hes1 in\nU87 cells and U87 GIC.\nThe results showed that CD133 and Nestin (stem cell markers), Notch1\nand Hes1 (Notch1 pathway-related proteins) were highly expressed in U87\nGICs.\nCritique #12:\nLine 12: In order to assess the target off-site toxicity, the authors should\nevaluate what was their targeting of non-glioma and non-GIC brain cells\n(neurons, astrocytes, oligodendrocytes, and microglia)?\nResponse #12:\nWe sincerely appreciate the reviewer’s in-depth consideration of the\ntargeted specificity and safety of this research. Your suggestion for\nevaluating the targeting of non-glioma and non-GIC brain cells (neurons,\nastrocytes, oligodendrocytes, microglia) is of great significance. We have\ncompleted the relevant verification through supplementary experiments.\nHT22 is a type of mouse hippocampal neuron cell line, which is frequently\nutilized for studying the functions and characteristics of neurons. BV2, a\ntype of mouse microglial cell line, is an innate immune cell of the central\nnervous system. HMC3 is a type of human microglial cell line. Compared\nto the BV2 cells derived from mice, HMC3 is better able to reflect the\nbiological characteristics of human microglial cells. MO3.13 is a cell line\nassociated with human oligodendrocytes. We compared the uptake of Exo,\nVE, and VEMR in HT22, BV2, HMC3 and MO3.13. Exosomes were\nlabeled with CM-DiI and then incubated with different cell lines.\nFurthermore, we used targeting peptide DVAP to block GRP78. Among the\nthree types of cells, there was no statistically significant difference in the\nfluorescence intensity of the four groups.In conclusion, the exo system we\nconstructed had almost no targeting effect on non-glioma and non-GIC\nbrain cells (neurons, astrocytes, oligodendrocytes, microglia).\nFig. 39. (A-D) In vitro cellular targeting of Exo, VE, VEMR, DVAP peptides & VE in\nHT22 cells (A), BV2 cells (B), HMC3 cells (C), MO3.13 cells (D), U87 cells (E) and\nU87 GIC (F). (G) Quantification of targeting in different cells.\nCritique #13:\nPage 11, Line 5: Explain and clarify what was the rational behind testing\nthis in such un-physiological conditions.\nResponse #13:\nWe appreciate the reviewer’s question regarding the experimental\nconditions. At 4℃, the low temperature inhibits the activity of cellular\nmetabolic enzymes, blocking the energy supply, thereby specifically\ninhibiting the energy-dependent endocytic pathway. If the cell uptake\nsignificantly decreases at 4℃, it indicates that the uptake process is energy-\ndependent and belongs to active endocytosis rather than passive diffusion.\nIn order to investigate the effect of energy on cell uptake, one group of cells\nwas taken at 4℃ to form a contrast with the physiological condition of\n37℃. Although 4℃ is not a physiological temperature, the purpose of this\nexperiment is for mechanism research rather than simulating the in vivo\nenvironment. Therefore, it is scientifically reasonable. Furthermore, in the\nresearch on the cell uptake mechanism in the literatures, this experimental\ncondition is commonly used.\nFig. 40. (A-B) After treating U87 cells (A) and U87 GICs (B) with different endocytosis\ninhibitors (4℃, amiloride, dynasore, chlorpromazine, M-β-CD), the confocal laser\nscanning microscope (CLSM) images of their co-culture with VE for 2 h were obtained.\nExos were labeled with DiI (red), and cell nucleus was labeled with hoechst 33342\n(blue).\nCritique #14:\nPage 13, Line 1:“lysosomes were labeled with LysoTracker (green),…”\nlysotracker marks any acidic pH, therefore, it is not labeling only\nlysosomes but also endosomes.\nResponse #14:\nThank you for your meticulous review of the experimental details. The\nquestion you raised regarding the range of LysoTracker labeling is\nextremely crucial. LysoTracker is a fluorescent dye that selectively stains\nacidic compartments within living cells. The pH of the early endosomes is\nclose to neutral, while the pH of the late endosomes is close to that of\nlysosomes (late endosomes pH 5.0-6.0, lysosomes pH 4.5-5.0). Therefore,\ntheoretically, lysotracker should be able to label the late endosomes and\nlysosomes. We used LysoTracker to label the acidic compartments, aiming\nto track the distribution of drugs within the cells. However, we failed to\nclearly distinguish between endosomes and lysosomes, which was an\noversight in the experimental description. Thus, we change the expression\nof “lysosomes” to “lyso/endosomes”.\nAlthough the LysoTracker labeling range covers endosomes and lysosomes,\nwe inferred the escape path of the MIT and siRNA through the analysis of\nthe time course. High colocalization of siRNA/MIT with LysoTracker at 2\nh, suggesting that the drugs have entered the acidic compartments. The\ncolocalization coefficient significantly decreased at 8 h, and the\nfluorescence signal diffused from the acidic compartments to the\ncytoplasm (Figure 4D). This dynamic change conforms to the temporal\ncharacteristics of “endosome-lysosome escape”. The results indicated that\nmost of the siRNA and MIT entered the lyso/endosomes and then escaped\nfrom the lyso/endosomes over time.\nFig. 41. CLSM images of U87 cells (A) and U87 GICs (B) incubated with VEMR for\n2h, 4h and 8 h. Cell nucleus was labeled with hoechst 33342 (blue), lyso/endosomes\nwere labeled with LysoTracker (green), siNotch1 was labeled with Cy3 (red), and MIT\nwas purple fluorescence.\nCritique #15:\nLine 6: Define the nature of HA1800 cells.\nResponse #15:\nThank you for the reminder. HA1800 cells are derived from human normal\nastrocytes, the most abundant glial cells in the central nervous system\n(CNS). As a \"normal\" astrocyte line, HA1800 cells are theoretically non-\ntransformed, meaning they retain physiological functions without\noncogenic modifications. The primary purpose of selecting HA1800 cells\nin this study was to serve as a negative control, which was used to verify\nthe targeting specificity and assess the safety of the formulation.\nCritique #16:\nLine 7: define “good safety”. How does this align with clinical demands?\nResponse #16:\nThank you for your attention to the safety and clinical translational value\nof this research. We fully agree that good safety is the foundation for\nclinical application. In order to express more precisely, we change “good\nsafety” to “good biocompatibility”. In our research, we verified the\ncompatibility of IL-12&VEMR@Gel through multi-dimensional\nexperiments. (1) The hemolysis rate of the hydrogel was 0.58% (Section\n2.1, Figure S1C), which was much lower than the 5% standard for medical\nmaterials, indicating its excellent blood compatibility. Moreover, no local\ninflammation was observed, which is in line with the strict requirements\nfor material safety in local postoperative administration. (2) In Section 2.4,\nFigure S14, we conducted a comprehensive analysis of the body weight,\nserum biochemical serum indexes (ALT, AST, BUN, CRE), and major\norgans (heart, liver, spleen, lung, kidney) of the mice in the treatment group.\nThe results showed that compared with the control group, none of the\ntreatment groups caused significant weight loss or liver/kidney function\ndamage. H&E staining further indicated that there were no obvious\npathological changes in the organ tissues, confirming the overall safety of\nthis platform. The safety in clinical applications still needs to be further\nverified.\nFig. 42. Hemolysis test of hydrogel.\nFig. 43. Biocompatibility assessment. (A) Mouse body weight changes during different\ntreatments. (B) Serum biochemistry markers including AST, ALT, CRE, BUN. (C)\nH&E staining of major organs (heart, liver, spleen, lung and kidney) obtained from\ndifferent groups (PBS, MIT, VER, VEM, VEMR, IL-12&VEMR) after treatment.\nCritique #17:\nPage 14, Line 1-6: This section lacks distinction between the aspects\nreferring to the cells’ proliferation rate and their motility (migration).\nResponse #17:\nThank you for bringing this crucial issue to our attention. The concern you\nraised regarding “how to distinguish whether wound healing in the cell\nscratch assay depends on cell proliferation or migration” is indeed an area\nwhere our experimental design was not comprehensive enough. The cell\nscratch assay, as a commonly used method for evaluating cell migration\nability, its results may indeed be interfered with by cell proliferation. Your\nprofessional insight has helped us identify this defect. Meanwhile, apart\nfrom the reasons you mentioned, we also realized that since GICs are\nusually in a semi-adherent state, using cell scratch assays to characterize\nthe migration of GICs is not entirely reliable. After much consideration, we\nhave decided to delete the data related to cell scratch experiments. And cell\nproliferation is demonstrated through CCK8 assays.\nCritique #18:\nLine 10-11: define statistical significance, p values?\nResponse #18:\nThank you for pointing out the need to clarify statistical significance and\np-value definitions. All statistical analyses were performed using\nGraphPad Prism 8 software. Experiments were independently repeated a\nminimum of three times (n = 3 for in vitro assays; n = 5 for animal cohorts),\nand the data are presented as the mean ± SD or SEM. Comparisons between\ntwo groups were made using Student’s t-test, while differences among\nmultiple groups were assessed by one-way ANOVA. A p-value of less than\n0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p <\n0.001, ****p < 0.0001). We have now explicitly defined these in the Section\n4.16 to ensure transparency.\nCritique #19:\nPage 15, Figure 5- Panels G and H are blurry and not very clear.\nResponse #19:\nThank you for pointing out the clarity issues of Figures 5G and H. We have\nreprocessed the original data of this figure to ensure that the image\nresolution and contrast meet the publication standards. The scratch images\nat 0h, 12h, and 24h were processed using the Image J. The cell migration\nrate was calculated by measuring the width of the scratches. However, we\nrealized that since GICs are usually in a semi-adherent state, using cell\nscratch assays to characterize the migration of GICs is not entirely reliable.\nAfter much consideration, we have decided to delete the data related to cell\nscratch experiments.\nFig. 44. (A) Scratch assay of U87 cells incubated with PBS, MIT, VE, VER, VEM and\nVEMR for 0h, 12h and 24 h. (B-C) Quantification of relative migration distance for\n12h (B) and 24 h (C). (D) Scratch assay of U87 GICs incubated with PBS, MIT, VE,\nVER, VEM and VEMR for 0h, 12h and 24 h. (E-F) Quantification of relative migration\ndistance for 12h (E) and 24 h (F).\nCritique #20:\nPage 16, Line 6: Note that murine GL261 (should be stated in the text that\nthey are murine inoculated into immunocompetent mice as immune\nprofiling is required), unlike the human U87 cell line, this line is known to\nbe far less invasive than actual GBM cells.\nResponse #20:\nThank you to the reviewers for pointing out the limitations of the GL261\nmodel in simulating the invasiveness of human GBM. Based on this\nrecommendation, we revalidated the therapeutic effect of IL-\n12&VEMR@Gel using a CT2A mouse glioma cell line (derived from\nC57BL/6 mice, significantly more invasive than GL261) that is closer to\nthe clinical GBM characteristics. The relevant data have been\nsupplemented to the Section 2.5 (Figure 7G-I). We investigated the\ntherapeutic effect of this multifunctional exo-gel delivery system on CT2A\northotopic glioma model. Luciferase-expressing CT2A (Luc -CT2A)\nglioma cells were implanted into brains on day 0, and mice underwent\nimaging using IVIS to determine the glioma growth. On the 6th day after\ninoculation with Luc-CT2A cells, the brain tumor was surgically removed.\nThe mice were randomly divided into four groups, and a multifunctional\nexo-gel delivery system was injected into the resection cavity. The results\nshowed that IL-12&VEMR@Gel significantly inhibited tumor growth and\nprolonged the survival of mice. Taken together, these results demonstrated\nthe excellent ability of IL-12&VEMR@Gel in suppressing glioma\nrecurrence.\nFig. 45. (A) Luminescence images of orthotopic Luc-CT2A GBM tumor-bearing mice\nfollowing different treatments monitored on day 6, 9, 12, 15 and 18. (B) Quantified\nsignal intensity of (A). (C) Survival analysis of orthotopic Luc-CT2A GBM tumor-\nbearing mice in each group.\nCritique #21:\nPage 17- Figure 6: quantitative analysis should be performed on the IHC\nslides following staining of relevant markers: proliferation, apoptosis,\nimmune-profiling, and angiogenesis … .and not only H&E and\nmeasurement of fluorescence/bioluminescence.\nResponse #21:\nThank you for your suggestions on the depth of immunohistochemical\nanalysis. We fully agree with the importance of quantitative analysis for\nmechanism elucidation and conducted quantitative analysis of H&E and\nTUNEL staining. The relevant results have been supplemented to Section\n2.5, Figure S15. H&E staining results showed that there was no significant\ndifference in the proportion of tumor-bearing brain regions between the\nControl group and the Gel group, and the proportions in the EM@Gel,\nEMR@Gel and VEMR@Gel groups decreased further significantly.\nNotably, the fewest tumors were displayed in the IL-12&VEMR@Gel\ngroup among all groups (Figure 7E, Figure S15A). Additionally, extensive\nap extensive\napoptotic cells were detected in TUNEL-stained tumor-bearing brain slices\nof IL-12&VEMR@Gel group compared with other groups (Figure 7F,\nFigure S15B).\nFig. 46. (A) Representative H&E images of the orthotopic GBM tumors after the\nindicated treatments. (B) TUNEL staining of brain tissue sections obtained from mice\ntumors after treatments. (C) Quantification of tumor area in H&E images. (D)\nFluorescence quantification of TUNEL assay.\nCritique #22:\nDiscussion, page 22, lines 3-5: there are several newer treatments\napproved by the FDA and EMA that can and should be mentioned, such as\nTTF (optium), immunotherapies/vaccines (gliovac), oncolytic virus, etc...\nResponse #22:\nThank you for your valuable suggestions. We have added in the Discussion\nthe latest approved treatments by the FDA and EMA, as follow: “The\nstandard clinical therapeutic approach for GBM involves surgical removal\nof tumors, after which TMZ treatment and/or radiation therapy are\nadministered following a several-week recovery interval. In addition,\nemerging adjunctive therapies such as tumor electrical field therapy (TTF),\nGliovac™, and oncolytic viruses are used to treat GBM. Nevertheless,\nincomplete surgical removal of the tumor, resistance to chemotherapy and\nradiotherapy, poor patient compliance and limited targeting ability for\npostoperative microscopic residual TTF lesions, the small scale of clinical\ntrials for Gliovac™, and systemic inflammatory toxicities of oncolytic\nviruses all contribute to the difficulty in curing GBM.” (Page 16, Line 21-\n26，Page 17, Line ,1-3)\nWe believe that compared with these emerging therapies, our approach\noffers several decisive advantages. (1) TTF interferes with the mitosis of\ntumor cells through alternating electric fields, offering a non-invasive\nmodality with negligible systemic toxicity [1]. However, the limitations of\nTTF include the need to wear the device continuously (for at least 18 hours\nper day), poor patient compliance, and limited targeting ability for\npostoperative microscopic residual lesions. Our approach is free from such\ncompliance burdens and is designed to reach and eradicate residual disease\nthat TTF cannot effectively target. (2) Gliovac (an anti-GBM vaccines in\nclinical trial) can induce specific T-cell responses by loading tumor-\nassociated antigens (TAAs) [2,3]. However, profound inter-patient\nheterogeneity prevents any single vaccine from covering all cases. Our\nsystem circumvents this limitation because it does not rely on predefined\nantigens; it not only induces T cell infiltration but also reduces M2-type\nmacrophages and inhibitory cells such as Tregs. Compared with vaccines,\nit is more adaptable to the immunosuppressive microenvironment of GBM.\n(3) Oncolytic viruses (such as DNX-2401, for diffuse intrinsic pontine\nglioma in clinical trial) selectively lyse tumor cells and trigger antitumor\nimmunity, but viral replication can provoke systemic inflammatory\ntoxicities [4,5]. In contrast, our system is designed for local release, thereby\nvirtually eliminating the risk of systemic side effects.\nReferences:\n[1] KHAGI S, KOTECHA R, GATSON NTN, et al. Recent advances in Tumor\nTreating Fields (TTFields) therapy for glioblastoma[J]. Oncologist, 2025,\n30(2):oyae227.\n[2] SCHIJNS VE, PRETTO C, DEVILLERS L, et al. First clinical results of a\npersonalized immunotherapeutic vaccine against recurrent, incompletely resected,\ntreatment-resistant glioblastoma multiforme (GBM) tumors, based on combined\nallo- and auto-immune tumor reactivity[J]. Vaccine, 2015, 33(23):2690-2696.\n[3] ZHAO S, QU Y, SUN Z, et al. Glioblastoma Cell Lysate and Adjuvant\nNanovaccines via Strategic Vaccination Completely Regress Established Murine\nTumors[J]. Adv Healthc Mater, 2025, 14(14):e2500911.\n[4] PHILBRICK B, ADAMSON DC. DNX-2401: an investigational drug for the\ntreatment of recurrent glioblastoma[J]. Expert Opin Investig Drugs, 2019,\n28(12):1041-1049.\n[5] NASSIRI F, PATIL V, YEFET LS, et al. Oncolytic DNX-2401 virotherapy plus\npembrolizumab in recurrent glioblastoma: a phase 1/2 trial[J]. Nat Med, 2023,\n29(6):1370-1378.\nCritique #23:\nLine 7: The ”cold” TME in GBM is there, to begin with, prior to any\nsurgery and treatment. Moreover, this is also a result of the treatments\nthemselves as the radiation and chemo both harm the immune system. It\nmay be worth thinking about how the treatments suggested in the paper\ncan be combined with a general busting of the immune system to improve\nthe outcomes.\nResponse #23:\nThank you to the reviewers for pointing out the inherent attributes of “cold\nTME” in GBM and the immunosuppressive issues of traditional treatments.\nThe core innovation of this study lies precisely in breaking through the\nimmunosuppressive limitations of traditional radiotherapy and\nchemotherapy through the dual strategy of local immune activation and\ntumor stem cell targeting. Locally delivered IL-12 could activate tumor\nimmunity, while MIT induced immunogenic cell death (ICD), and the two\nsynergistically reverse “cold TME”. Mechanically, the damage-related\nmolecular patterns (DAMPs) released by MIT form a positive feedback\nwith the IL-12-activated Th1-type immune response, rather than the\nimmunosuppression of traditional chemotherapy.\nAvailable data suggest that IL-12&VEMR@Gel can reshape the local\nimmune microenvironment of the surgical remnant. Immunofluorescence\nshowed that the infiltration of CD4+ T cells, CD8+ T cells and mature DC\ncells in the brain tumor tissues of the IL-12&VEMR@Gel treatment group\nwas significantly higher than that of the control group.\nFig. 47. (A) The CD4+ T cells、CD8+ T cells、DC cells in tumor-bearing brain tissues.\n(C-E) Quantification of CD4+ T cells (B), CD8+ T cells (C), DC cells (D).\nMoreover, quantitative FCM results (Figure 8B-F) showed that after IL-\n12&VEMR@Gel treatment, the proportions of mature DC cells\n(CD80+CD86+), CD8+ T cells (CD3+CD8+), and the activation of T cells\n(CD25+CD69+) in the GBM lesions of tumor-bearing mice were\nsignificantly increased, whereas the proportions of Treg cells (CD4+Foxp3+)\nand MDSCs (CD11b+Gr-1+) were notably reduced. In conclusion, the IL-\n12&VEMR@Gel system has initially improved the inherent and\nchemotherapy-induced “cold” TME defect of GBM through the synergistic\neffect of ICD and IL-12.\nFigure 48. The evaluation of antitumor immune response in GL261 postresection\nglioma-bearing brain tissue. (A) Schematic diagram of the experimental design. (B)\nRepresentative FCM contour plots of T cells in each group and quantification of CD8+\nT cells. (C-F) Quantification results of the proportion of DC cells (C), T cell activation\n(D), Treg cells (E), and MDSCs (F) in tumor-bearing brain issues of each group.\nCritique #24:\nPage 23, Line 5: safety was not addressed in the non-malignant healthy\nbrain tissue.\nResponse #24:\nThanks for the reviewers’ comments. Regarding this, we would like to\nmake the following responses: Firstly, we investigated the targeting ability\nof DVAP-exosome (VE) in U87 cells, U87 GICs (GRP78 positive) and\nHA1800 cells (GRP78 negative), proving that the fluorescence signals of\nVE on normal cells was significantly lower than that on tumor cells. This\nresult suggests that when the system targets GRP78, non-specific damage\nto healthy brain tissue may be significantly reduced. Further, we also\nevaluated the targeting of non-glioma and non-GIC brain cells (neurons,\nastrocytes, oligodendrocytes, microglia). We compared the uptake of Exo,\nVE, and VEMR in HT22, BV2, HMC3 and MO3.13. Exosomes were\nlabeled with CM-DiI and then incubated with different cell lines. Among\nthe three types of cells, there was no statistically significant difference in\nthe fluorescence intensity of the four groups.\nMoreover, the survival time of mice in the IL-12&VEMR@Gel treatment\ngroup was prolonged longer than 180 days in GL261 model (Figure 7D),\nand no neurological dysfunction symptoms (such as dyskinesia, epilepsy,\netc.) occurred, indirectly reflecting the limited damage to the brain\nparenchyma caused by local administration.\nFig. 49. (A-B) In vitro cellular targeting of Exo, VE, VEMR, DVAP peptides+VE in\nHA1800 cells (A) and U87 cells (B). (C) Quantification of targeting in HA1800 cells\nand U87 cells.\nFig. 50. (A-D) In vitro cellular targeting of Exo, VE, VEMR, DVAP peptides & VE in\nHT22 cells (A), BV2 cells (B), HMC3 cells (C), MO3.13 cells (D), U87 cells (E) and\nU87 GIC (F). (G) Quantification of targeting in different cells.\nCritique #25:\nPage 24, line 1-6- a much more comprehensive analysis should be done in\norder to claim that the tumor growth inhibition was achieved by turning\nthe cold TME into a hot one.\nResponse #25:\nThank you for your meticulous review and valuable suggestions on our\nresearch. Your suggestion that the mechanism verification of “cold TME\ntransforming into hot TME” requires a more comprehensive analysis of\nimmune cells is crucial for deepening the mechanism explanation in this\narticle and we fully concur with this perspective. On the 30th day, the mice\nwere euthanized, brain tumor tissues were harvested to prepare a single cell\nsuspension via the percoll cell isolation solution, the immune cells were\nstained and analyzed by FCM in order to evaluate the overall changes of\nimmune cells within the tumor microenvironment, including Treg cells\n(CD4+FoxP3+), MDSCs (CD11b+Gr-1+), DC (CD80+CD86+), effector T\ncells (CD45+CD3+CD8+), T cell activation (CD25+CD69+). Given that\nchanges in the types and numbers of immune cells in the tumor\nmicroenvironment, which can lead to alterations in cytokine levels,\nenzyme-linked immunosorbent assay (ELISA) was used to evaluate the\nexpression of relevant cytokines at the tumor site, including IFN-γ, TNF-\nα, IL-4 and IL-10. The relevant results have been supplemented to Section\n2.5, Figure 8.\nIFN-γ is predominantly secreted by Th1 cells and NK cells and serves as\nthe signature cytokine of Th1-type immunity. It enhances macrophage\nphagocytosis and microbicidal activity, accelerates dendritic-cell\nmaturation, up-regulates MHC-molecule expression, and thereby\npotentiates cell-mediated immunity. Its concentration thus mirrors the\nmagnitude of the Th1 immune response. TNF-α, chiefly secreted by\nmacrophages and T cells, is a pivotal pro-inflammatory cytokine that\ninitiates the inflammatory cascade, activates immune effector cells, and\npotentiates anti-tumor immunity. IL-10 is a potent immunosuppressive\ncytokine produced by Treg and Th2 cells, macrophages, and other cell\ntypes. It restrains the activation of Th1 cells, macrophages, and dendritic\ncells, reduce the release of pro-inflammatory factors, and maintain immune\nbalance. Increased IL-10 expression is a hallmark of immunosuppressive\nmicroenvironments. In our research, compared with the control group, the\nconcentration of IFN-γ, TNF-α, IL-4, and IL-10in the Gel group and\nEM@Gel group did not show any significant change. The levels of IFN-γ\nand TNF-α were significantly increased in VEMR@Gel, IL-12@Gel, IL-\n12&VEMR@Gel group, whereas IL-4 and IL-10 were reduced to varying\nextents. Notably, the IL-12&VEMR@Gel group induced the highest levels\nof IFN-γ and TNF-α, while the levels of interleukin-4 (IL-4) and\ninterleukin-10 (IL-10) were the lowest.\nThe proportions of CD4+ T cells and CD8+ T cells in different treatment\ngroups were detected by flow cytometry. The proportions of CD4+ T cells\nin different treatment groups (Control, Gel, EM@Gel, EMR@Gel,\nVEMR@Gel, IL-12@Gel, IL-12&VEMR@Gel) were 7.83%, 5.76%,\n9.18%, 11.88%, 13.36%, 12.91%, 16.18% respectively, while the\nproportions of CD8+ T cells were 10.58%, 11.17%, 22.36%, 22.75%,\n24.27%, 26.37%, 37.16%. Compared with the control group, the\nproportions of CD4+ T cells and CD8+T cells in the Gel group showed no\nsignificant changes. In the other group, the proportions of CD4+T cells and\nCD8+T cells all increased. Among them, the proportion of CD4+ T cells\nand CD8+ T cells in the IL-12&VEMR@Gel group increased significantly.\nThis indicates that the combined treatments of IL-12 with VEMR can\npromote the proliferation of CD4+ T cells and CD8+ T cells.\nCompared with the control group, the proportion of DC cells in the Gel\ngroup showed no significant difference. In the EM@Gel group, EMR@Gel\ngroup, VEMR@Gel group, IL-12@Gel group and IL12&VEMR@Gel\ngroup, the proportion of DC cells increased to varying degrees. Especially\nin the IL-12&VEMR@Gel group, the proportion of DC cells increased to\n10.3%. The results showed that combined treatment can promote the\nproliferation of DC cells.\nCD25+CD69+ is the classic marker of T cell activation. Compared with the\ngel group (Gel), there was no significant difference in the proportion of\nCD25+CD69+ T cells between the control group and the gel group; the\nproportions of CD25+CD69+ T cells in the EM@Gel group, EMR@Gel\ngroup, VEMR@Gel group, IL-12@Gel group and IL-12&VEMR@Gel\ngroup were significantly higher than those in the control group. The\nincrease in the proportion of double-positive cells indicates that these\ntreatments may exert immunomodulatory effects by enhancing T cell\nactivation.\nRegulatory T cells (Treg) are a subset of T cells with immunosuppressive\nfunctions, which can promote tumor progression by suppressing the anti-\ntumor immune response in the tumor microenvironment. Therefore, by\nmarking FoxP3, it is possible to specifically identify and quantify Treg\ncells. Compared with the gel group (Gel), there was no significant\ndifference in the proportion of Treg between the control group and the gel\ngroup. In the EM@Gel group, EMR@Gel group, VEMR@Gel group, IL-\n12@Gel group and IL-12&VEMR@Gel group, the proportion of Treg\ngradually decreases. IL-12&VEMR@Gel group demonstrated the lowest\nlevel, which was 3.4%.\nMyeloid-derived suppressor cells (MDSC) is also a type of cell population\nwith significant immunosuppressive functions. The combined labeling of\nCD11b and Gr-1 can effectively distinguish MDSC from mature myeloid\ncells and other immune cells, and is currently a standardized method for\nidentifying MDSC. Similar to Treg cells, combined administration of IL-\n12 and VEMR can significantly reduce the proportion of MDSC.\nTo sum up, after IL-12&VEMR@Gel treatment, the proportions of mature\nDC cells (CD80+CD86+), CD8+ T cells, and the activation of T cells\n(CD25+CD69+) in the GBM lesions of tumor-bearing mice were\nsignificantly increased, whereas the proportions of Treg cells (CD4+Foxp3+)\nand MDSCs (CD11b+Gr-1+) were notably reduced.\nThese results further confirm that IL-12&VEMR@Gel comprehensively\nand thoroughly activated the anti-tumor immune response within the brain,\ntransforming GBM from a “cold” tumor to a “hot” tumor.\nFigure 51. The evaluation of antitumor immune response in GL261 postresection\nglioma-bearing brain tissue. (A) Schematic diagram of the experimental design. (B)\nRepresentative FCM contour plots of T cells in each group and quantification of CD8+\nT cells. (C-F) Quantification results of the proportion of DC cells (C), T cell activation\n(D), Treg cells (E), and MDSCs (F) in tumor-bearing brain issues of each group. (G-J)\nELISA quantification results of IFN-γ (G), TNF-α (H), IL-4 (I), and IL-10 (J) in tumor-\nbearing brain issues.\nThe above is our point-by-point response to the reviewer's comments.We\nsincerely appreciate the dedicated work of the editors and reviewers and\nhope that the changes will be recognised. Once again, thank you very much\nfor your comments and suggestions. If you have any questions, you can\ncontact me at any time at your convenience.\nMany thanks!\nBest Regards,\nYours truly,\nProf. Huining He\nTianjin Key Laboratory on Technologies Enabling Development of\nClinical Therapeutics and Diagnostics, International Joint Laboratory of\nOcular Diseases, School of Pharmacy, School of Biomedical Engineering\nand Technology, Tianjin Medical University.\nChinaAddress: No. 22, Meteorological Bureau Road, Heping District,\nTianjin 300070, P.R. China\nTel/Fax: +86-22-83336658;\nE-mail: hehuining@tmu.edu.cn"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a thorough job of addressing the previous concerns. As it stands, the manuscript is substantially improved and should be considered for publication.", "reviewer_2": "This revised manuscript has been improved due to new data derived from additional experiments that have conducted during the revision. However, I still note some remaining weaknesses as listed below.\n1. “This one-month post-operative interval represents a crucial period during which tumor recurrence often occurs.” This statement is not true. GBM recurrence typically occurs about 8-10 months after the initial surgery and after post-operative chemoradiation therapy. Relapse can occur earlier but rarely happens within the first one month.\n2. Exos release from the gel was shown in Figure S2. However, I have not found that the authors showed whether IL-12 encapsulation into the gel was feasible and IL-12 can be released from the gel. Since IL-12@gel was effective in suppressing GBM growth (Figure 7), apparently gel encapsulation of IL-12 was feasible, but it would be nice to show the basis for this.\n3. What was exactly the IL-12 that was used in this work? Recombinant mouse IL-12? This should be clarified in the Materials and Methods section.\n4. In the Result description of Figure 6D and G, the authors did not recognize that survival and tumor suppression were comparable in the IL-12 and IL-12&VEMR groups.\n5. The results description of Figure 7 (Section 2.5) perhaps should be split into two paragraphs for the GL261 part and the CT2A part.\n6. I suggest that description of Figure 8 could be a separate section, 2.6.\n7. Also consider re-structuring section 2.1, where the second paragraph is very lengthy. The part starting with “Gel and VEMR@Gel were subsequently prepared…” could be a new paragraph.\n8. Abstract. siNotch1 should be siRNA for Notch1.\n9. What siNC is in Figure S7 was not explained in the figure legends.", "reviewer_3": "The authors addressed properly and thorouly all the concerns."}, "author_response": "Point-by-point responses to the Reviewers’ comments for Ref:\nNCOMMS-24-61002B\nDear reviewers,\nWe would like to express our sincere gratitude to all reviewers for their\nrigorous, professional, and constructive comments on our revised\nmanuscript. Your valuable feedback has been instrumental in refining the\nquality, accuracy, and clarity of our work, and we greatly appreciate the\ntime and effort you have dedicated to evaluating our study.\nFirst, we are particularly encouraged by Reviewer #1 and Reviewer #3’s\nrecognition that we have addressed previous concerns thoroughly and that\nthe manuscript has been substantially improved to be considered for\npublication. Additionally, we have carefully reviewed each of Reviewer\n#2’s 9 points and have taken targeted actions to address them, including\ncorrecting inaccurate statements, supplementing key experimental\nevidence, clarifying reagent details, optimizing result structure, and\nrefining terminology. Below, we provide a point-by-point response along\nwith explanations of the revisions made to the manuscript.\nOnce again, we thank all reviewers for their invaluable contributions to\nstrengthening our study. We believe the revised manuscript now meets the\nhigh standards of the journal, and we look forward to your further\nevaluation.\nResponse to Reviewer's comments:\nWe would like to express our sincere gratitude to the reviewers for their\ntime and constructive feedback. We greatly appreciate their approval of\nour manuscript and their thoughtful comments, which have been invaluable\nin improving the quality of our work.\nCritiques From Reviewer #1:\nThe authors have done a thorough job of addressing the previous concerns.\nAs it stands, the manuscript is substantially improved and should be\nconsidered for publication.\nResponse:\nThank you for your positive feedback and for acknowledging the\nimprovements made to the manuscript. We are pleased to hear that the\nrevised version meets your expectations and are grateful for your\nrecommendation for publication.\nCritiques From Reviewer #2:\nCritique #1:\n\"This one-month post-operative interval represents a crucial period during\nwhich tumor recurrence often occurs,\" This statement is not true. GBM\nrecurrence typicaly occurs about 8-10 months after the initial surgery and\nafter post-operative chemoradiation therapy. Relapse can occur earlier but\nrarely happens within the first one month.\nResponse #1:\nThank you for your meticulous review of the manuscript. We have revised\nthe statement “This one-month post-operative interval represents a crucial\nperiod during which tumor recurrence often occurs” to “This one-month\npost-operative interval represents a crucial period during which tumor\nrecurrence may be triggered” (Page 2, Line 23-24, Section 1).\nWe also have reviewed recent clinical studies and systematic reviews. We\nagreed that 8~10 month period after surgery is the peak time for GBM\nrecurrence, and also a large number of clinical studies have shown that\nGBM can also exhibit rapid early progression (REP) in the early\npostoperative period (≤1 month), which has a significant impact on\nprognosis. Merkel et al. found that in 61 newly diagnosed GBM patients,\n59% developed new enhancement foci or residual tumor progression\napproximately 24 days (within one month) after surgery[1]. Waqar et al.\nreviewed and pointed out that approximately half of the GBM patients\nexperienced macroscopic visible REP between surgery and radiotherapy\nand chemotherapy, and this was significantly associated with a decline in\nsurvival[2]. These all indicated that the early progression of microscopic\nresidual tumor cells could eventually drive clinical recurrence in the post-\noperative period.\nReference:\n[1] MERKEL A, SOELDNER D, WENDL C, et al. Early postoperative tumor\nprogression predicts clinical outcome in glioblastoma-implication for clinical\ntrials[J]. J Neurooncol, 2017, 132(2):249-254.\n[2] WAQAR M, TRIFILETTI DM, MCBAIN C, et al. Early Therapeutic Interventions\nfor Newly Diagnosed Glioblastoma: Rationale and Review of the Literature[J].\nCurr Oncol Rep, 2022, 24(3):311-324.\nCritique #2:\nExos release from the gel was shown in figure S2. However, I have not\nfound that the authors showed whether IL-12 encapsulation into the gel\nwas feasible and IL-12 can be released from the gel. Since IL-12@gel was\neffective in suppressing GBM growth (figure 7), apparently gel\nencapsulation of IL-12 was feasible, but it would be nice to show the basis\nfor this.\nResponse #2:\nWe appreciate you pointing out this missing experimental basis of IL-12\nencapsulation into the gel and its release behavior in the manuscript.\nFor IL-12 encapsulation feasibility, as described in the revised manuscript,\nduring preparation, we first mixed the hydrogel matrix at room temperature\nto maintain its liquid state, then added IL-12 to the liquid gel. Since no\nphase transition occurs at room temperature, IL-12 is uniformly dispersed\nwithin the liquid matrix without precipitation or leakage. Upon transfer to\n37°C, the gel solidifies instantly, trapping all IL-12 within its network\nstructure, without loss. Figure 2K can prove the viewpoint that all materials\ncan be polymerized and form gels.\nFigure 1. The temperature sensitive properties of Gel at 4 ℃ and 37 ℃.\nFor characterization of IL-12 in vitro release behavior, we supplemented\nin vitro release experiments as follows. IL-12 was marked with Cy3 and\nloaded into the gel. Using artificial cerebrospinal fluid as the release\nmedium, and 3D layer scanning was carried out with a laser confocal\nmicroscope at 0 h and 24 h, respectively. The fluorescence quantitative\nresults demonstrated that ~86% IL-12 could be released from the gel in 24h,\nwhich was consistent with the release results of the exosomes.\nFigure 2. The release of IL-12-Cy3 from the hydrogel in the CSF. (A) 3D layer scanning of IL-12-\nloaded hydrogel at 0 h and 24 h. (B) Quantification of the fluorescence intensity in (A).\nCritique #3:\nWhat was exactly the IL-12 that was used in this work? Recombinant\nmouse 1L-12? This should be clarified in the Materials and Methods\nsection.\nResponse #3:\nThank you for pointing out the lack of clarity regarding the IL-12 used in\nour study. To address this clearly, we have supplemented the details of the\nIL-12 used in all related experiments in the revised \"Materials and\nMethods\" section. The IL-12 utilized was recombinant murine IL-12\n(Protech Systems Co., Ltd., USA).\nCritique #4:\nIn the Result description of Figure 6D and G, the authors did not recognize\nthat survival and tumor suppression were comparable in the IL-12 and IL-\n12&VEMR groups.\nResponse #4:\nThank you very much for your valuable comment on the result description\nof Figure 6D and G. After receiving your comment, we immediately\nprovided additional explanations for the results.. Notably, although the\ntherapeutic efficacies of IL-12 and IL-12&VEMR were comparable in the\nprimary-tumor model, in the postoperative resection model, IL-\n12&VEMR@Gel exhibited a markedly amplified antitumor effect than of\nIL-12@Gel in both GL261 and CT2A recurrent model, underscoring the\npivotal contribution of GICs to recurrence. Our results further confirmed\nthat IL-12-mediated immune activation alone is insufficient; thus, direct\nand specific elimination of GICs becomes indispensable for GBM\nrecurrence.\nCritique #5:\nThe results description of Fiqure 7 (Section 2.5) perhaps should be split\ninto two paragraphs for the GL261 part and the CT2A part.\nResponse #5:\nThank you for this constructive suggestion. We have now divided the\noriginal single paragraph describing Figure 7 (Section 2.5) into two\nseparate paragraphs: the first focuses on the GL261 data and the second on\nthe CT2A data.\nCritique #6:\nI suggest that description of Figure 8 could be a separate section 2.6.\nResponse #6:\nThank you very much for your valuable suggestions. We have made\nadjustments to the structure of the manuscript according to your\nsuggestions.\nCritique #7:\nAlso consider re-structuring section 2.1, where the second paragraph is\nvery lengthy, The part starting with \"Gel and VEMR@Gel were\nsubsequently prepared...\" could be a new paragraph.\nResponse #7:\nThank you for this helpful suggestion. We have re-structured Section 2.1\nby splitting the original lengthy second paragraph. The text starting with\n“Gel and VEMR@Gel were subsequently prepared …” now begins a new\nparagraph.\nCritique #8:\nAbstract. siNotch1 should be siRNA for Notch1.\nResponse #8:\nThank you for pointing this out. We have corrected “siNotch1” to\n“siRNA for Notch1” in the Abstract.\nCritique #9:\nWhat siNC is in Figure S7 was not explained in the figure legends.\nResponse #9:\nThank you very much for your careful review of the manuscript. In\nresponse to the question you raised, we have supplemented the legend of\nFigure S7. siNC is a siRNA of negative control.\nCritiques From Reviewer #3:\nThe authors addressed properly and thorouly all the concerns.\nResponse:\nThank you very much for your positive feedback. We are pleased that all\nconcerns have been properly and thoroughly addressed to your satisfaction.\nThe above is our point-by-point response to the reviewer's comments.We\nsincerely appreciate the dedicated work of the editors and reviewers and\nhope that the changes will be recognized. Once again, thank you very much\nfor your comments and suggestions. If you have any questions, you can\ncontact me at any time at your convenience.\nMany thanks!\nBest Regards,\nYours truly,\nProf. Huining He\nTianjin Key Laboratory on Technologies Enabling Development of\nClinical Therapeutics and Diagnostics, International Joint Laboratory of\nOcular Diseases, School of Pharmacy, School of Biomedical Engineering\nand Technology, Tianjin Medical University.\nChinaAddress: No. 22, Meteorological Bureau Road, Heping District,\nTianjin 300070, P.R. China\nTel/Fax: +86-22-83336658;\nE-mail: hehuining@tmu.edu.cn"}]

Decision letter