Spatiotemporally engineered tumor-derived extracellular vesicle-based scaffold vaccine for personalized cancer immunotherapy

Domain: biology
Published: 2026-03-23
Editorial outcome: major_revision

Abstract
Personalized vaccines demonstrate remarkable potential in leveraging tumor-specific adaptive immunity for cancer therapy. Nevertheless, current platforms face persistent challenges including premature systemic clearance and imprecise antigen-p resenting cell targeting, culminating in transient and inefficient antitumor immunity. Furthermore, the technical complexity, extended timelines, and prohibitive costs required for tumor-specifEic neoantigen identification continue to impede the clinical translation of personalized cancer vaccines. Here, we report a tumor-derived extracellular vesicle-based scaffold vaccine that elicits robust and durable antitumor immunity for personalized cancer immunotherapy. Following subcutaneous administration, the in situ-formed hydrogel vaccine serves as a sustained reservoir for tumor-derived extracellular vesicle antigens and adjuvants while recruiting antigen-presenting dendritic cells to accumulate within the scaffold. Upon exposure to this antigen-rich depot, immature dendritic cells undergo efficient activation, with subsequently matured dendritic cells migrating to draining lymph nodes, where they induce potent and persistent tumor-specific CD8+ T-cell responses that suppress tumor progression across multiple murine models. Specifically, when using tumor-derived extracellular vesicles isolated from surgically excised tumor tissues, the patient-tailored vaccines demonstrate remarkable efficacy in preventing postoperative recurrence. Our findings validate the robust and durable therapeutic efficacy of this vaccine platform, highlighting its potential as a customizable strategy for personalized cancer immunotherapy.

Introduction
Cancer vaccines represent a paradigm-shifting immunotherapeutic strategy that activates tumor-specific adaptive immunity for precise malignant cell targeting, demonstrating transformative potential to redefine modern oncology frameworks1–4. Personalized cancer vaccines utilize systematically characterized tumor mutanomes to engineer neoantigen-based formulations, which elicit tumor-selective T-cell responses through optimized epitope prioritization5–7. Despite this promise, current personalized cancer vaccines show suboptimal clinical efficacy, with merely three FDA-approved formulations demonstrating limited therapeutic benefits8. These immunogenic limitations stem from rapid mononuclear phagocyte system-mediated clearance of conventional delivery platforms and insufficient antigen accumulation in lymph nodes9. While passive drainage enables partial vaccine component access to lymphatics, inadequate antigen retention and disorganized spatial distribution critically compromise dendritic cell-mediated capture and processing cascades, ultimEately leading to transient T-cell activation and inadequate antitumor immunity10. Moreover, the neoantigen discovery pipeline—encompassing tumor antigen screening, computational prediction, an d experimental validation in individual patients—constitutes a multidimensional, resource-intensive process that critically impedes the clinical translation of personalized cancer vaccines11–13. To overco me these barriers, systematic engineering of neoantigen acquisition coupled with rationally deLsigned antigen-presenting cell -targeted delivery platforms represents imperative to unlock the full clinical potential of this precision immunotherapy.

Extracellular vesicles (EVs), naturally secreted nanoscale vesicles with lipid bilayer architectures, faithfully mirror the molec ular signatures of their parental cells14–16. Tumor-derived extracellular vesicles (TEVs) serve as natural reservoirs of tumor-associated antigens, including neoantigens that evade conventional identification methods17–19. Our protein level validation in prostate cancer surgical specimens revealed preserved antigenic profiles in TEVs, demonstrating equivalent expression levels of prostate-specific antigens (PAP, PSCA, PSMA) between TEVs and matched tumor lysates (Fig. 1, A and B). Furthermore, a protease protection assay confirmed that these antigens are predominantly localized on the surface of TEVs (Supplementary Fig. 1). Mechanistically, TEVs coordinate patient-specific antitumor immunity through intrinsic delivery of individualized neoantigen repertoires6,20,21. Clinically, autologous TEVs could be isolated from surgical specimen, which bypass computational neoantigen prediction through comprehensive antigen payloads, enabling accelerated personalized vaccine manufacturing. TEV-based vaccines thus represent a breakthrough platform in precision immunotherapy by leveraging biomimetic carriers of tumor-specific antigens. However, TEVs are susceptible to rapid clearance by the mononuclear phagocyte system, while their suboptimal size distribution hinders effective lymphatic transport, thereby significantly restricting their therapeutic potential10,22. We therefore propose that spatiotemporal reprogramming of TEVs’ biodistribution to sustain antigen-presenting cell interactions will elicit robust and durable tumor-specific immunity.

Notably, peptide-based supramolecular hydrogels have emerged as a transformative drug delivery platform due to their inherent biosafety, biocompatibility, and multifunctionality23–26. These biomaterials demonstrate exceptional depot functionality by enabling enhanced local drug retention coupled with controlled release kinetics, thus maximizing therapeutic bioavailability at the target site27–29. Our recent work has established their unique suitability for localized delivery of immune modulators, with payload release significantly prolonged through electrostatic interactions30–32 S. Specifically, engineered hydrogel scaffolds can reconstruct lymph-node-mimicking immune niches -S three-dimensional microenvironments where interconnected macroporous networks facilitate immune cell infiltration, interaction, and dissemination33,34. Building on this premise, we propose that hydrogel-based antigen/adjuvant depots, while recruiting dendritic cells (DCs) to accumulate within the scaffold, enable enhanced DC uptake efficiency through sustained exposure to abundanIt payloads. Following maturation, these DCs migrate to lymph nodes to activate T-cell responsesE. This paradigm-shifting strategy overcomes conventional vaccine limitations via spatiotemporal reprogramming of antigen/adjuvant biodistribution, thereby extending DC-antigen engagement duration and amplifying antitumor immunity through coordinated immune modulation.

In this context, we develop a TEVs-based scaffold vaccine for personalized cancer immunotherapy and demonstrated the system’s high potency in eliciting robust and durable immunity against tumor regression (Fig.1C). The vaccine components- TEVs and adjuvant 2'3'-c-di-AM(PS) (CDA), along with the granulocyte-macrophage colony-stimulating factor (GM-CSF) for dendritic cell recruitment - are integrated onto self-assembling FK12 peptide nanofibers (FK F). Following subcutaneous injection, the 12 TCG/FK F complex rapidly transforms into a hydrogel vaccine that serves as a localized depot, substantially extending the retention duration of immunotherapeutic agents. The scaffold facilitates antigen-presenting DCs recruitment and concentration, enabling immature DCs to capture and process TEV antigens with CDA adjuvant support, thereby promoting DC maturation. These antigen-primed mature DCs then migrate to draining lymph nodes, initiating potent tumor-specific CD8+ T-cell activation that confers long-term antitumor protection. Our results demonstrate that the personalized TCG/FK F 12 vaccine effectively suppresses postoperative tumor recurrence and enhances overall survivals in both RM-1 prostate cancer and 4T1 breast cancer models.

Results
Characterization of TCG/FK F hydrogel vaccine
12
To construct a biocompatibility and biodegradability hydrogel scaffold, a self-assembling peptide FKFEFKFEGRGD (FK ) was the synthesized (Supplementary Fig. 2). This amphiphilic molecule is 12 capable of self-assembling into regular and homogeneous nanofibers (FK F) in aqueous solutions, which 12 can subsequently intertwine to form supramolecular hydrogels under physiological conditions (Fig. 2A). We then simulated the in vivo environment by adding counterions (PBS or serum) to the FK F aqueous 12 solution, and found that the originally flowing solution immediately transformed into a self-supporting hydrogel (Fig. 2B and Supplementary Fig. 3A). Rheological tests corroborated this observation, as the storage modulus (G') rapidly exceeded the loss modulus (G'') upon PBS or serum addition (Fig. 2C and Supplementary Fig. 3B), indicative of a phase transition andR enhanced elastic properties.

TEVs were obtained from culture medium supernatants of RM-1 prostate cancer cells to prepare vaccine for subsequent experiments. Transmission electron microscopy revealed that the extracted TEVs showed a characteristic cup-shaped morpholog y (Fig. 2D). The results of nanoflow analysis showed that the particle size distribution of the TEVs was within the 30-160 nm interval (Fig. 2E). Western blot analysis of TEVs compared with RM-1 cell lysates (CLs) demonstrated negligible expression of the contaminant protein Calnexin, TApoa-1 and GM130 in TEVs, alongside positive expression of extracellular vesicle markers Flotillin-1, CD63, and CD9 (Fig. 2F and Supplementary Fig. 4). These findings collectively confirm the successful isolation of TEVs from RM-1 cells. To prepare vaccine preparations, we mixed negatively charged GM-CSF, CDA and TEVs with positively charged FK F. The 12 decrease in the Zeta potential of the solution after loading the vaccine components suggests that the components were successfully condensed on the surface of FK F by electrostatic interaction (Fig. 2G). 12 Meanwhile, confocal microscopy revealed a uniform distribution of TEVs within FK F, confirming their 12 effective incorporation (Fig. 2H). Similar experimental results showed that FK F loaded with vaccine 12 components retained its capacity to form a gel in the presence of PBS (Supplementary Fig. 2, D).

The release of GM-CSF and CDA from the TCG/FK F hydrogel was subsequently investigated in 12 vitro at 37°C. We observed that both components showed a nonlinear sustained slow release (Fig.2I). Within 13 d GM-CSF released 51% while CDA released 80%. This nonlinear release profile is characteristic of therapeutic agents encapsulated within hydrogels, where the release rate decreases over time after an initial abrupt release. We then evaluated the biodegradation of FK F in vivo by subcutaneous 12 injection of FK F solution into the back of C57/BL6 mice. 15 minutes after injection, we euthanized the 12 mice and could observe that the injected FK F solution had formed a well-defined spherical gel. The in 12 situ-formed FK F hydrogel demonstrated gradual biodegradation over 15 days (Supplementary Fig. 5).
12
To further assess the potential of FK F hydrogels as drug reservoirs, we investigated subcutaneous 12 therapeutic release behavior in mice using Cy7-labeled CDA and DIR-labeled TEVs, respectively. The fluorescently labeled CDA and TEVs were mixed with FK F aqueous solution, respectively, and the 12 mixed solution were subsequently injected subcutaneously into mice; free Cy7-labeled CDA and DIR-labeled TEVs were used as controls. In vivo fluorescence imaging revealed that both CDA and TEVs in the FK F hydrogel exhibited strong localized signals at the injection site and negligible accumulation in 12 major organs (liver, spleen, kidney, heart, and lung) (Fig. 2J, K and SuSpplementary Fig. 6). This indicates that the vaccine components were effectively retained locallyS without systemic dissemination. As envisaged, the fluorescence intensity of free CDA and TEVs declined rapidly with time, and fluorescence was barely detectable after 5 days. In contrast, the FK F-treated group still remained strong fluorescent 12 intensity through day 15 (Fig. 2, J and K). These results further demonstrated that the FK F hydrogel can 12 serve as a therapeutic reservoir, significantly exteInding the local release time of the vaccine components.
Moreover, hematoxylin eosin staining of maEjor organs, complete blood counts, serum biochemistry, and cytokine analysis showed no significant differences between TCG/FK12F-treated mice compared to healthy controls (Supplementary Fig. 7). Specifically, serum IL-6 levels were comparable to those in healthy mice (Supplementary Fig. 8), indicating that localized TCG/FK F administration does not induce 12 systemic inflammation and exhibits a favorable safety profile.

TCG/FK F activates DCs in situ and enhancing lymph node migration for adaptive immunity
12
To investigate the underlying mechanism of the vaccine, we surgically removed the subcutaneous gel implants after a designated period and performed flow cytometric analysis. By directly comparing the effects of TLs and TEVs on DC activation, we found that in the absence of CDA, TEVs did not markedly enhance the expression of co-stimulatory molecules CD80/CD86 on DCs relative to TLs, however, they induced a significantly higher level of MHC I expression (Supplementary Fig. 9). These data indicate that, although TEVs alone are insufficient to drive full DC maturation, they possess superior antigen-presenting potential compared with TLs. Notably, a significant higher number of DCs were detected in the TCG/FK F group compared to the FK F and CG/FK F groups (Fig. 3A). To further delineate 12 12 12 infiltrating DC subsets, we quantified conventional DCs (cDCs), monocyte-derived DCs (moDCs), and plasmacytoid DCs (pDCs) by flow cytometry. Compared to the FK F group, the CG/FK F formulation 12 12 enhanced the abundance of cDC1s and moDCs (Supplementary Fig. 10), indicating that the adjuvant potentiates the recruitment of these functionally critical DC subsets. Furthermore, the TCG/FK F vaccine 12 significantly increased the percentages of cDC1s, cDC2s, and moDCs within the hydrogel relative to control groups. Meanwhile, the proportion of activated DCs in the TCG/FK12F group was 4-fold and 1.6-fold higher than in the FK12F and CG/FK12F groups, respectively (Fig. 3B). Furthermore, DCs in this group showed significantly elevated expression of the CCR7 receptor (Fig. 3C), a key mediator of lymph node migration. Accordingly, inguinal lymph nodes of TCG/FK F-immunized mice exhibited 2.8-fold 12 and 1.9-fold increases in activated DCs compared to those from FK F- and CG/FK F-treated mice, 12 12 respectively (Fig. 3D). Importantly, TCG/FK 12 F induced significantly higher percentage of CD4+ (21.4%) and CD8+ (16.3%) T cells compare with the FK F (14.2% and 12.5%) and CG/FK F (15.1% and 13.7%) 12 12 treatment groups (Fig. 3E and F).

Furthermore, using an OVA-expressing RM-1 tumor moEdel (Supplementary Fig. 11), we assessed antigen-specific immunity induced by the TEV-based hydrogel vaccine. Flow cytometry revealed that vaccination significantly upregulated MHC class I on D Cs both within the hydrogel and in draining lymph nodes (Fig. 3G, H and Supplementary Fig. 12). This was accompanied by a marked increase in OVA-specific DCs (Fig. 3I), confirming efficient an tigen delivery and antigen-dependent DC activation. In parallel, we observed a substantial expanLsion of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes, and splenocytes from vaccinated mice exhibited increased frequencies of IFN-γ⁺ CD8⁺ T cells upon ex vivo OVA restimulTation (Fig. 3J and K). Together, these data demonstrate that the hydrogel vaccine promotes robust antigen-specific DC activation and primes a functional antigen-specific CD8⁺ T-cell response. Collectively, our results robustly support that TCG/FK12F, as an efficient hydrogel vaccine scaffold system, facilitates DCs recruitment, maturation, and lymph node homing, thereby successfully initiating a potent adaptive anti-tumor immune response (Fig. 3L).

TCG/FK F vaccine elicits robust tumor-specific immunity to suppresses tumors
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To evaluate the therapeutic efficacy of the vaccine initially, we established a subcutaneous tumor model using the murine prostate cancer cell line RM-1. When tumors reached 100-150 mm3, mice were randomized into treatment groups, and the vaccine was injected by subcutaneously into the contralateral side of the tumor to evaluate the TCG/FK F vaccine and its synergistic anti-tumor effect with the immune 12 checkpoint inhibitor aPD-1 (Fig. 4A). The results showed that the TCG/FK 12 F group and the combined treatment group (TCG/FK F+aPD-1) significantly suppressed subcutaneous tumor growth and 12 significantly prolonged the survival time of mice (Fig. 4B and C). In particular, 62.5% of mice in the combined treatment group remained alive at day 60, whereas the aPD-1, /FK F, and ree TCG groups 12 exhibited minimal improvements in survival. To dissect the changes in immune infiltration in the TME after different treatments, tumors were collected ten days after treatment for flow cytometry and cytokine assays. The results show that TCG/FK F vaccine treatment significantly elevated the infiltration of CD8+ 12 T cells and CD8+ IFN-γ+ T cells in TME, which was further enhanced by combining with aPD-1 (Fig. 4, D and E). Likewise, antitumor-associated cytokines were up-regulated in varying proportions (Fig. 4F and Supplementary Fig. 13, A to E). Consistent with the therapeutic outcomes, aPD-1 and CG/FK F 12 treatment alone helped minimally to elevate immune infiltration, while free TCG also failed to demonstrate a significant effect, probably due to its limited in vivo retention.

To assess whether vaccine-activated T cells exhibit antigen spSecificity, splenocytes of mice were harvested 10 days after vaccination and incubated with TEVs (froSm RM-1 cells) for 24 hours. Enzyme-linked immunospot (ELISpot) assay was used to assess Interferon-γ (IFN-γ) secret from mice T cells after exposure to tumor antigens in different groups. Minimal IFN-γ-positive spots were detected in the PBS and CG/FK F groups, indicating that adjuvant alone elicits negligible antigen-specific T-cell activation 12 (Fig. 4G and H). In the TCG treated group, a moIderate number of T cells were activated, and this weak immune response may result from the limiteEd duration of vaccine action. The TCG/FK 12 F treated group exhibited a significantly higher frequency of IFN-γ-positive spots than the other groups, underscoring its superior capacity to activate antigen-specific T cells. Furthermore, to confirm that the therapeutic effect depends on TEV-specific antigens, we generated a control vaccine (T CG/FK F) using TEVs from B16 12 heterologous B16 cells. As shown in Supplementary Fig. 14, this control vaccine lacked significant efficacy, supporting the antigen-specific mechanism of action. These results strongly demonstrate that CD8+ T cells respond to TEVs restimulation in a tumor antigen-specific way. To further delineate the contributions of T-cell subsets to vaccine efficacy, we depleted CD4+ or CD8+ T cells in RM-1 tumor-bearing mice using specific antibodies. Depletion of CD4+ T cells partially impaired the antitumor effect of TCG/FK F, whereas clearance of CD8+ T cells significantly reduced tumor growth inhibition 12 (Supplementary Fig. 15 and Fig. 4I). These results confirm the positive effect of the TCG/FK F vaccine 12 on the tumor immune microenvironment, suggesting that the efficacy of the vaccine is attributable to a robust immune response.

Surviving mice were rechallenged on day 60 by injecting tumor cells into the contralateral flank to assess whether TCG/FK F vaccine established a long-term immune memory (Fig. 4A). Meanwhile, we 12 used untreated naive mice as a control group. As shown in Fig. 4J, naive mice were able to observe significant tumor development after inoculation and continued to grow over time. In contrast, almost no detectable tumors were observed in mice from the previous long-term survival group, suggesting sustained resistance to tumor rechallenge. Meanwhile, these mice maintained a 100% survival rate through day 120 (Fig. 4K). To further investigate the underlying immune mechanisms, we euthanized previously long-surviving mice on day 120 and extracted lymphocytes from the spleen for flow cytometry analysis. We found both CD8+CD44highCD62Lhigh central memory T (T ) cells and cm CD8+CD44highCD62Llow effector memory T (T ) cells were elevated in the TCG/FK F+aPD-1-treated em 12 group of mice compared to naive mice (Supplementary Fig. 16 and Fig. 4L and M). This suggests that the vaccine induced T-cell memory immunity and established long-term immunity to curb tumor recurrence.

TCG/FK F significantly suppresses proliferation in orthotopic pSrostate cancer and metastasis in 12 4T1 breast cancer S
Since prostate cancer is characterized as an immuRnologically cold tumor, we constructed an orthotopic model mouse of prostate cancer to more accurPately replicate the tumor microenvironment and evaluate the efficacy of the vaccine. We subcutaneoNusly injected the vaccine preparations on day five after the in situ injection of RM-1 cells into the mouse prostate. We then assessed the therapeutic efficacy of the vaccine using tumor bioluminescence imaging and by monitoring survival (Fig. 5A). As shown in Fig. 5B and Supplementary Fig. 17, treatmCent with the TCG/FK 12 F vaccine not only significantly inhibited tumor progression but also showedI a further enhanced therapeutic effect when combined with aPD-1 T therapy. Correspondingly, theR two groups exhibited markedly prolonged survival compared to the PBS and CG/FK F groups (FiAg. 5C). The mice were euthanized on day 15 and the tumors were obtained by 12 autopsy. Both groups of mice demonstrated better tumor inhibition similarly (Fig. 5D and Supplementary Fig. 18). Flow cytometry analysis of orthotopic tumor tissues revealed higher infiltration of CD8+ T cells and CD8+ IFN-γ+ T cells in the TCG/FK F and TCG/FK F + aPD-1 groups compared to the other groups 12 12 (Supplementary Fig. 19 and 20). Consistent with this, immunohistochemistry and immunofluorescence analyses revealed increased CD8⁺ T-cell infiltration and a higher abundance of granzyme B⁺ cytotoxic T cells, respectively, in tumors receiving TCG/FK F combined with aPD-1 (Fig. 5E and Supplementary 12 Fig. 21). In addition, immunofluorescence staining revealed elevated PD-1 expression on tumor-infiltrating CD8⁺ T cells following vaccination (Supplementary Fig. 22), suggesting that sustained immune activation induced a state of partial T-cell exhaustion. This finding provides a mechanistic rationale for the enhanced therapeutic benefit achieved upon combination with immune checkpoint blockade. Furthermore, in the OVA-expressing RM-1 orthotopic model, flow cytometry demonstrated a significant increase in H-2Kᵇ–OVA tetramer⁺ CD8⁺ T cells within tumor tissues following TCG/FK F 12 vaccination (Supplementary Fig. 23), indicating robust antigen-specific cytotoxic T-cell activation in the tumor microenvironment. Additionally, cytokine detection in tumor tissues showed a marked elevation of anti-tumor cytokines—including IFN-α, IFN-β, IFN-γ, TNF-α, and IL-6—in these two groups compared to the others (Fig. 5F), underscoring the vaccine’s capacity to modulate the tumor immune microenvironment effectively.

Considering that tumor metastasis is a major cause of death in patients with advanced tumors, we evaluated the efficacy of the TCG/FK F vaccine in suppressing metastasis. To monitor tumor metastasis 12 in mice, we used luciferase-labeled 4T1 cells to establish a systemic metastasis model by injecting these cells into the left ventricle of mice (Fig. 5G). We administered vaccinSe and other group treatments on the seventh day after cell injection. Bioluminescence imaging showeSd that by day 15, the PBS and aPD-1 E group developed extensive systemic metastases and progressive mortality, whereas the TCG/FK F and 12 R TCG/FK F + aPD-1 mice displayed significantly attenuated disease progression, with metastatic burden 12 P in the TCG/FK F + aPD-1 group being almost exclusively confined to the lungs (Fig. 5, H and I). After 12 N euthanasia, peripheral blood, spleen, and lung tIissues were collected for analysis. Flow cytometry of peripheral blood revealed a marked increasEe in circulating IFN-γ⁺ CD8⁺ T cells in TCG/FK 12 F-treated L mice (Supplementary Fig. 24A), indicating robust systemic T-cell activation. Consistent with this, ex vivo C restimulation of splenocytes with TEVs induced a significant increase in IFN-γ⁺ CD8⁺ T cells I T (Supplementary Fig. 24B), confirming functional antigen-specific responses. This systemically activated, R tumor-specific immunity further suppressed metastasis, as evidenced by examination of the lungs, which A showed significantly fewer and smaller metastatic nodules in the TCG/FK F and TCG/FK F + aPD-1 12 12 groups compared to the PBS, aPD-1, and TCG alone groups (Fig. 5J, Supplementary Fig. 25).
Collectively, these findings highlight the robust anti-metastatic potential of the TCG/FK F vaccine, 12 underscoring its efficacy not only in local tumor control but also in preventing metastatic spread in a range of cancer models.

Personalized TCG/FK F vaccine effectively prevents tumor recurrence after surgery
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Tumor recurrence is very common after surgery and significantly impacts patients' quality of life and prognosis. Recurrence may occur if surgery fails to completely remove the tumor, leaving behind minimal residual disease (MRD). To simulate this clinical situation, we constructed surgical resection models, including an RM-1 cell-based subcutaneous tumor resection model for prostate cancer and a 4T1 cell-based orthotopic tumor resection model for breast cancer. Mice inoculated with tumors underwent surgical excision after a period of time, leaving a few tumors to simulate residual lesions. The excised tumors were used to extract TEVs to prepare personalized vaccines, which were subsequently infused back into the mice subcutaneously for treatment and monitoring of tumor recurrence (Fig. 6A). We performed relevant characterization of TEVs extracted from tumor tissues and verified the successful preparation of personalized TEVs (Supplementary Fig. 26A to D). As we can see from the RM-1 tumor resection model, although aPD-1 and TCG treatment delayed tumor recurrence in some degree, the anti-tumor effect was very limited and failed to enhance the survival rate of mice (Fig. 6B to D and Supplementary Fig. 27). In contrast, mice treated with the TCG/FK F vaccine demonstrated potent tumor 12 suppression, with a survival rate of 25% at 90 days and up to 62.5% by combining with aPD-1.

We expanded the sample size and further replicated the experimSent to obtain RM-1 tumor tissues from different groups for detection. Transcriptome sequencing anSalysis of the tumor tissue showed that E gene expression upregulated by CG/FK F treatment was relatively limited (Fig. 6E). In contrast, 12 R TCG/FK F vaccine treatment resulted in the expression of a large number of immune-related genes 12 P within the tumor, including Cd8a and Cxcr6, suggesting that TEVs play a key role in the vaccine (Fig. N 6F). This is similarly corroborated by the heat mIaps of gene expression in the three groups (Fig. 6G). E Gene Ontology (GO) and Kyoto EncyclopeEdia of Genes and Genomes (KEGG) enrichment analysies of L genes up-regulated in TCG/FK F showed that most of these genes act on immune-related pathways, such 12 C as T-cell proliferation and activation, cell killing, cytokine response, etc (Fig. 6H). Subsequent immune I T infiltration analysis further disclosed a marked increase in CD8+ T-cell abundance in the TCG/FK F 12 R group compared to both the PBS and CG/FK F groups, indicative of enhanced antitumor immunity (Fig. A 12 6I). Additionally, other anti-tumor immune cells, including DCs, NK cells, and Th1 cells, were also elevated in the TCG/FK F group. Consistently, the TCG/FK F vaccine showed significant efficacy in 12 12 an orthotopic 4T1 breast cancer resection model, significantly inhibiting tumor recurrence (Fig. 6J and K). Accompanying flow cytometry analysis revealed a marked increase in IFN-γ⁺ CD8⁺ T cells (Supplementary Fig. 28), aligning with our earlier results and confirming its role in promoting antitumor T-cell responses. These results suggest that the personalized vaccine significantly inhibits postoperative tumor recurrence, and that this efficacy is enhanced by the combination of immune checkpoint inhibitors.

Methods
Ethics statement
This study adhered to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All the animal experiments were performed in accordance with the animal protocol approved by the Animal Ethics Committee at Shanghai Jiao Tong University (approval number: A2024071). The maximal tumor size permitted by the ethics committee was 2000 mm³, and we confirm that this maximal tumor size was not exceeded in any experiment.

The human prostate tissues collection in this study was approved by the Research Ethics Committee of the Renji Hospital, Shanghai Jiao Tong University School of Medicine (approval number: KY2022-136A). Written informed consent was obtained from all patients prior to the collection and use of human prostate tissue samples.

Study design
The objective of this study was to develop a TEVs-based personalized hydrogel vaccine for cancer immunotherapy. Using multiple murine tumor models, the efficacy of the TCG/FK F vaccine in eliciting 12 a robust antitumor immune response and suppressing tumor progression was validated. All experiments were repeated, with strict control of variables and standardized procedures to ensure consistent results across groups. Mice in the control and experimental groups were age- and sex-matched, and cage randomization was applied during group allocation.

Materials
GM-CSF was purchased from peprotech. CDA (2’3’-c-diAM (PS) (Rp,Rp)) was purchased from InvivoGen. Cy7-labelled CDA was purchased from Biolog Life Science Institute. All of the other chemicals were purchased from Sigma-Aldrich and used without any purification.

Cell lines
The RM-1-luc and 4T1-luc cell lines wCere donated by Renji hospital Shanghai Jiao Tong university school of medicine. The RM-1-luc cell line was cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% peniciRllin/streptomycin (Gibco). The 4T1-luc cell line was cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). All the cells were cultured in a constant temperature incubator at 37 °C with 5% CO supply and 2 routinely tested for mycoplasma.

Animals
Male C57BL/6 and femal BALB/c mice (aged 6-8 weeks; LINGCHANG Biotech) were maintained on a 12-hour light-dark cycle with unrestricted access to food and water.

TLs and TEVs extraction
Tumor tissues were divided into two portions for processing. One portion was homogenized and lysed on ice for 30 minutes in a lysis buffer containing protease inhibitors. The lysate was then centrifuged at 14,000 g for 20 minutes at 4 °C, and the supernatant was collected as TLs. The other portion of tumor tissue was minced and incubated at 37°C for 30 minutes in tissue isolation buffer, prepared with RPMI-1640 medium, collagenase D, and DNAse I. The mixture was filtered through a 70 μm membrane, followed by sequential centrifugation at 2000 g and 10,000 g for 20 minutes each at 4 °C. The final supernatant was filtered through a 0.22 μm membrane, and TEVs were isolated by ultracentrifugation at 120,000 g for 70 minutes twice at 4°C. For tumor cell cultures, the medium was replaced with exosome-free serum medium at an appropriate time before collection. The medium was then centrifuged sequentially at 1,000 g for 10 minutes and at 10,000 g for 20 minutes at 4°C. The supernatant was filtered through a 0.22 μm membrane, and the filtrate was subjected to two rounds of ultracentrifugation at 120,000 g for 70 minutes each at 4°C to isolate TEVs.

Western blot
TLs and TEVs were lysed in RIPA lysis buffer (Beyotime), and protein concentration was quantified by BCA Protein Assay (Thermo Fisher Scientific). Equal amounts of protein were separated using a 10% SDS-PAGE gel and transferred onto 0.45 μm PVDF meRmbranes (Millipore). The membranes were blocked with 5% BSA in TBST and incubated overnPight at 4°C with specific primary antibodies. Afterward, appropriate secondary antibodies were Napplied, followed by detection using an ECL system (Tanon). See the source file for the uncropped scan blots.

Preparation of TCG/FK F
12
The polypeptide sequence FK12 (FKFEFKFEGRGD) was synthesized by peptide synthesizer. Verification of purity and molecular weight by HPLC and MS. To obtain self-assembled FK F, FK12 12 was directly dissolved in deionized water to reach a concentration of 15 mM and aged for 24 h at room temperature. The morphology of nanofibers was confirmed by transmission electron microscopy (Hitachi). The TCG/FK F solution was prepared by adding 50 μg TEVs, 20 μg CDA and 2 μg GM-CSF 12 to the FK F solution. After vortexing, the mixed solution was incubated at 37 ℃ for 30 minutes. For in 12 vivo imaging, CDA-Cy7 and TEVs-DIR was used.

Solution to hydrogel transition behavior
Add 20 μl of 10× PBS to 180 μl of 15 mM FK F or TCG/FK F solution, followed by vortexing. The 12 12 transition of solution to hydrogel was observed by inverted-vial method.

In vivo hydrogel degradation
The FK F solution (15 mM, 200 μl) was injected into the back of C57BL/6 mice. After specified time 12 intervals, the mice were euthanized, and the status of the remaining hydrogel in each mouse was recorded.

Hydrogel release studies
The in vitro release study was conducted at 37 °C in PBS. The release of CDA was analysed using HPLC. The release of GM-CSF was analysed using GM-CSF ELISA kit (Thermo Fisher Scientific). To evaluate the in vivo release of CDA and TEVs, Cy7-labeled CDA and DIR-labeled TEVs were injected subcutaneously into mice by free and TCG/FK F loading, respectively. Fluorescence images were 12 captured at predetermined time points using an IVIS Spectrum imaging system (Perkin Elmer).

In vivo tumor models and treatment
For subcutaneous model, 2.5 × 106 RM-1 cells were inoculated in male C57BL/6 mice with right flank. When the subcutaneous tumor volume reached 100-150 mm3, the tumor-bearing mice were randomly grouped and then treated in different groups. Treatments included i.h. injected with PBS, CG/FK F, TCG 12 or TCG/FK F (50 μg TEVs, 20 μg CDA and 2 μg GM-CSF per mouse, 200 μl) on the contralateral side 12 of the tumor, and intraperitoneal injections of aPD-1 (100 μg per mouse, 100 μl). For the prostate orthotopic model, 1×105 RM-1-luc cells were inoculaPted in male C57BL/6 prostate lobes. For the metastatic model, 1×105 4T1-luc cells were injectedN into female BALB/c through the left ventricle. After I 5 days, Tumor-bearing mice from these two tumor models were randomly divided into five groups and E received different treatments Treatments included i.h. injected with PBS, TCG or TCG/FK F (50 μg 12 TEVs, 20 μg CDA and 2 μg GM-CSFC per mouse, 200 μl), and intraperitoneal injections of aPD-1 (100 μg per mouse, 100 μl). For the breasIt cancer orthotopic model, 1 × 106 4T1-luc cells were inoculated into T the fat pad between the fourthR and fifth nipple in female BALB/c mice. When the tumor volume reached A 300-500 mm3, the tumor-bAearing mice underwent surgical removal of the tumors, with about 10% of the tumor remaining, and were then randomized into five groups to receive different treatments. Treatments included i.h. injected with PBS, TCG or TCG/FK F (50 μg TEVs, 20 μg CDA and 2 μg GM-CSF per 12 mouse, 200 μl) on the contralateral side of the tumor, and intraperitoneal injections of aPD-1 (100 μg per mouse, 100 μl). The tumor size was measured every 2 days. The tumor volume was calculated using the formula V = (major axis) × (minor axis)2/2. For prostate orthotopic and breast metastasis models, tumor progression was monitored by the IVIS spectral imaging system (Perkin Elmer). Mice were euthanized when the subcutaneous tumor volume exceeded 2000 mm3 or the weight loss exceeded 20%. For rechallenge experiment, mice with long-term survival were inoculated with 5×105 RM-1 cells on the opposite flank to develop new tumors.

Flow cytometry
Lymphocytes were extracted from peripheral blood, gel, lymph node, spleen, or tumor tissue, respectively, to prepare single-cell suspensions. Cells were stained with fluorescence-labeled antibodies following the manufacturer’s instructions. The samples were then analyzed on a LSRFortessa instrument (BD Biosciences), and data were analysed using FlowJo. Antibodies against CD45 (30-F11), CD3 (17A2), CD11c (N418), CD80 (16-10A1), CD86 (GL-1), CD197 (4B12), CD4 (RM4-5), CD8α(53-6.7), IFN-γ (XMG1.2), CD44 (IM7) and CD62L (MEL-14) were purchased from Biolegend.

Enyzme-linked immunospot assay
Splenocytes from RM-1 tumor-bearing mice were collected for restimulation with TEVs. 10 days after i.h. injected with PBS, CG/FK F, TCG or TCG/FK F, spleens were collected and spleen cells were 12 12 extracted. Splenocytes (1 × 105) were stimulated for 24 h with 1 μg/well TEVs (from RM-1 cells). Cells were subsequently assayed for IFN-γ by Elispot kit (Mabtech) and spot counts using instrument (BD Biosciences).

In vivo T-cell depletion
To deplete CD4+ T and CD8+ T cells, tumor-bearing m ice were i.p. injected with anti-CD4 (clone GK1.5, BioXcell), anti-CD8-α (clone 2.43, BioXcell) or isotype (RatIgG1, BioXcell) antibodies. Antibodies were administered with an initial dose of 400 mg 1 day prior to treatment, followed by 200 mg every 3 days. Flow cytometry analysis of peripheral bloLod mononuclear cells confirmed the depletion of CD4+ T cells and CD8+ T cells.

Transcriptome data analysis
Differential gene expression analysis was performed in R (v4.4.1) using the DESeq2 (v1.44.0) package, with genes considered significantly differentially expressed at an adjusted p value < 0.05. Functional enrichment analyses were conducted using clusterProfiler (v4.12.6). Visualization of differentially expressed genes was generated using ggplot2 (v3.5.1) for volcano plots and pheatmap (v1.0.12) for hierarchical clustering heatmaps. Immune cell infiltration was estimated from bulk RNA-seq data using the CIBERSORT algorithm, which infers the relative abundance of immune cell subset within the tumor microenvironment.

Statistics and Reproducibility
Statistical analysis was conducted using GraphPad Prism 9. Data are presented as mean ± SEM. Statistical significance between two groups was determined using two-tailed unpaired t-tests. Statistical significance between multiple groups was determined using one-way ANOVA. Survival analysis was performed with Kaplan–Meier curves and evaluated using two-sided log-rank (Mantel–Cox) tests. No statistical method was used to predetermine sample size. The Investigators were not blinded to allocation during experiments and outcome assessment.

Data Availability
The RNA sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1330898 [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1330898]. All other data supporting the findings of this study are included in the main article, supplementary information or source data file. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors provide an impressive account of tumor-derived EV (TEV) serving as a scaffold-based personalized antitumor vaccine that elicits robust and durable antitumor immunity. The studies were performed in mice bearing a prostate tumor or a breast tumor. The vaccine is a scaffold (in situ formed hydrogen gel) which incorporates TEV carrying tumor antigens, adjuvant (CDA), GM-CSF [TCG/FK12F} and anti-PD1 Ab. (The components are held attached to FK12F by electrostatic interactions, and TEV are evenly dispersed throughout the scaffold. The manuscript reports results of various experiments addressing DC activation in situ, antigen presentation to T cells, the generation of tumor-specific immunity that suppressed tumor growth prostate cancer and blocked metastasis in 4T1 breast cancer. The experimental designs are presented as informative figures, and the experiments comparing variously composed vaccines are carefully controlled. The authors’ experiments are remarkably successful, given the background of little success of anti-cancer vaccines in general, as discussed by the authors in the Introduction.\n\nThere are a number of questions and concerns. First, the comparison of the TL vaccine and TEV vaccine is not presented. TL and TEV were only compared in WBs, which tells us that the tumor antigens were present in the vesicle either in the lumen or both in the lumen and on the vesicle surface. It would be important to know definitively whether these target antigens are present on the vesicle surface. Next, entry of DC into the hydrogen scaffold and DC activation were evaluated by flow cytometry (Fig 3). What recruits DCs to hydrogels? And what type of DCs are recruited?? The increased % of CD11c+, CD80+CD86+, CCR7+ DCs are significant in TCG/FK12F hydrogels. This does not tell us anything about activation state of these infiltrating DCs. This is a critical issue, because it has been shown that TEV suppress activation and differentiation of DC. Specifically, both expression of APM components in DC and the ability of DCs to present Ags to T cells were reported to be suppressed by TEV in a variety of tumor settings. The same argument can be made about T cells: activity of CD8+ and CD4+ T cells has been widely reported to be suppressed by TEV. It is surprising and needs to be explained how TEVs in hydrogels do not exercise their immunosuppressive activities and instead appear to activate DC and facilitate Ag presentation to T cells. Thus, the statement that “hydrogen vaccine system facilitates the recruitment, maturation and lymph node homing of DC” is not supported by the presented data. Further, while the vaccine suppressed tumor growth and prolonged the survival time (Fig. 4), the evidence of antigen specific reactivity, rests on Fig 4G, H without annotation as to which antigen was tested in ELISPOT. The fact that depletion of eripheral T cells in vaccinated mice promoted tumor growth only means that T cells but not necessarily antigen-specific T cells are modulating the responses to TEV antigens. Studies aiming to show infiltration of vaccinated mice by IFNγ+ CD8+ T cells are supported by flow cytometry and should be shown by quantitative immunohistochemistry to be convincing. Studies shown in Fig 5. illustrate survival and suppressed proliferation of orthotopic tumors or lung metastases formation in 4T1 breast cancer model by vaccinated mice without really linking these vaccination results to mechanisms that could rationalize and justify such dramatic responses. The same concern applies to the data in Fig 6.\n\nOverall, it is difficult to accept the presented data for the following reasons: it is very well documented and widely known that TEV are strongly immunosuppressive and are especially deadly for activated CD8+ T cells. While TEV carry immunostimulatory proteins, they always carry an excess of immunosuppressive molecules. All immune cells (T, B, NK, MØ and DC) are suppressed in the TME, and TEV made and released in large quantities are largely responsible for this suppression. Thus, the ability of a vaccine to overcome this tumor-induced suppression is difficult to accept without an extremely convincing explanation of how a hydrophobic gel incorporating the vaccine components facilitates immune activity and eliminates immunoinhibitory activity that prevails locally and systemically in subjects with cancer. I am not persuaded that TEV embedded in the hydrogel lose their immunosuppressive properties to favor activation of anti-tumor immune responses.", "reviewer_2": "The manuscript presents a cancer vaccine platform utilizing a tumor-derived extracellular vesicle (TEV)-based hydrogel, which aims to enhance immunogenicity and prevent postoperative recurrence. The hydrogel improves vaccine retention, facilitates spatiotemporal control over antigen and adjuvant delivery, and promotes dendritic cell (DC) recruitment and activation, leading to CD8⁺ T-cell anti-tumour responses. The authors also demonstrate that this system can be combined with anti-PD-1 therapy to significantly suppress tumor growth, improve survival, and generate durable immune memory, preventing recurrence in both orthotopic and metastatic models. However, there are a few critical aspects that need to be addressed before publication.\n\nMajor comments\n1) TEVs were isolated by ultracentrifugation at 120,000 xg for 70 minutes twice at 4°C. Unlike gradient density centrifugation, ultracentrifugation alone carries a significant risk of co-isolating protein aggregates, lipoproteins, apoptotic bodies, and cellular debris, which may interfere with downstream analyses. The authors demonstrated negligible expression of the contaminant protein GM130 in TEVs, but this is not sufficient to prove the absence of non-exosomal components. It would be more convincing if the authors could provide additional negative markers to confirm the purity of the TEVs.\n2) The authors mentioned that TCG/FK12F induced a significantly higher percentage of antigen-specific CD4+ and CD8+ T cells compared with the FK12F and CG/FK12F treatment groups (Fig. 3, E and F). However, these T cell populations were gated only by CD45/CD3/CD4/CD8 expression, which is not sufficient to distinguish bystander T cells from antigen-specific T cells.\n3) The local versus systemic biodistribution is not thoroughly assessed. Besides the subcutaneous injection site, what is the biodistribution profile of TCG/FK12F in other organs?\n4) Figures 4D-E showed that TCG/FK12F vaccine treatment significantly increased infiltration of CD8+ T cells and CD8+ IFN-γ+ T cells in the TME. Have the authors investigated whether these populations of CD8+ T cells are antigen-specific?\n5) Figure 6I showed immune infiltration analysis in PBS, CG/FK12F, and TCG/FK12F treated groups. How were different immune subsets determined? Are there predetermined markers for each subset?\n6) There is no information about the methods used for transcriptome sequencing analysis.\n7) The authors mentioned that pro-tumor cells, such as Tregs, were slightly downregulated in the TCG/FK12F group (Fig. 6I). However, the difference does not appear to be substantial and seems marginal to me. Some TEVs may also be immunosuppressive. Furthermore, have the authors validated these findings using other methods?\n8) It appears that the proposed strategy is specifically optimized for postoperative adjuvant therapy to prevent recurrence. Given that TEVs are autologous, how long does it take to produce EVs from tumour samples in sufficient quantity to treat patients? Can you comment on the TEV yield?\n\nMinor comments\n1) 1) Figures 1A-B demonstrate equivalent expression levels of prostate-specific antigens (PAP, PSCA, PSMA) between TEVs and matched tumor lysates. Since the authors only show a representative Western blot, I wonder how strong this correlation is across a cohort of patients.\n2) In the subcutaneous therapeutic release experiments (Fig. 1 J-K), fluorescently labeled CDA and TEVs were each mixed with FK12F aqueous solution, and the mixtures were subsequently injected subcutaneously into mice. Did the encapsulation of EVs occur before or after the injection? How long does the encapsulation process take?\n3) Does the granzyme B signal colocalize with CD8 T cells in the immunofluorescent images (Fig. 5E and Fig. S10)?\n4) Can the authors comment on how this platform can overcome the highly heterogeneous antigen profiles present in some patients?", "reviewer_3": "In this manuscript, the authors presented a novel tumor-derived extracellular vesicle-based vaccine integrated into peptide-based supramolecular hydrogels. The vaccine demonstrates the ability to form in situ hydrogels that effectively recruit dendritic cells for subsequent T cell priming. Comprehensive evaluation across multiple tumor models revealed significant tumor growth inhibition, with mechanistic studies demonstrating CD8+ T cell-dependent anti-tumor immunity. This well-designed and executed study makes a substantial contribution to the development of personalized cancer vaccines, offering important insights into extracellular vesicle-based immunotherapy strategies.\n\nSpecific comments,\n1. In Figure 3A, the authors claim that DCs were activated by TCG/FK12F. The authors should provide the DC activation data for TEV stimulation alone. The authors claimed that the vaccine induced “antigen-specific CD4+ (21.4%) and CD8+ (16.3%) T cells compare with the FK12F (14.2% and 12.5%) and CG/FK12F (15.1% and 13.7%) treatment groups (Fig. 3, E and F)”. The data from Figure 3E and F can not support this conclusion, as the elevated CD4/8 T cell population may be induced from non-specific expansion. The increase of CD4/8 population may be antigen-specific T cells, but the results in Figure E and F did not directly show antigen specific T cell population.\n2. In Figure 4G and H, the authors showed the ELISPOT results for three mice in each group. The authors should provide the data for all the mice in supplementary information. The authors tested the synergistic anti-tumor effect with the immune checkpoint inhibitor aPD-1. Did the authors test whether the RM-1 cells express substantial PD-L1 or the vaccine boosted T cells infiltrated in tumors express high levels of PD-1?\n3. In Figure 5. The authors should provide the quantified data for tumor fluorescence values for each treatment mice group and indicate comparative analysis of the tumor inhibition efficacy.\n4. In Figure 4F and 5F, the authors report the elevated cytokines produced in tumor in TCG/FK12F+aPD-1 group. Did the authors test the cytokine levels in serum? It will be a safety concern if persistently elevated IL-6 was induced by this vaccine.\n5. In Figure 2B and C, PBS was used for the rheological tests. What if the PBS is replaced with serum or cell lysis?\n6. The authors claimed that the therapeutic efficacy was TEV specific, which was distinct from conventional EV vaccines loaded with shared tumor antigens. Did the authors test whether the EVs from unrelated tumor cell lines, or components from EVs, could induce any anti-tumor immune responses or tumor inhibition?\n7. Line 489: Splenocytes (1 × 105) were stimulated for 24 h with 1 g/well TEVs (from RM-1 cells). The concentration of TEVs should be re-checked.\n8. The reference formats were not consistent and should be carefully re-edited."}, "author_response": "We appreciate the reviewers for their critical and constructive comments, which we have taken seriously and addressed in full detail in this point-by-point response. For clarity, the comments by the reviewers are in black, with our point-by-point responses in blue and changes to the manuscript in red. We sincerely thank the reviewers as their comments and suggestions have greatly helped us improve the quality of our manuscript. We hope the revised version is now suitable for publication in Nature Communications.\n\nReviewer #1\nIn this manuscript, the authors provide an impressive account of tumor-derived EV (TEV) serving as a scaffold-based personalized antitumor vaccine that elicits robust and durable antitumor immunity. The studies were performed in mice bearing a prostate tumor or a breast tumor. The vaccine is a scaffold (in situ formed hydrogen gel) which incorporates TEV carrying tumor antigens, adjuvant (CDA), GM-CSF [TCG/FK F] and anti-PD1 Ab. The components are held attached to FK F by 12 12 electrostatic interactions, and TEV are evenly dispersed throughout the scaffold. The manuscript reports results of various experiments addressing DC activation in situ, antigen presentation to T cells, the generation of tumor-specific immunity that suppressed tumor growth prostate cancer and blocked metastasis in 4T1 breast cancer. The experimental designs are presented as informative figures, and the experiments comparing variously composed vaccines are carefully controlled. The authors’ experiments are remarkably successful, given the background of little success of anti-cancer vaccines in general, as discussed by the authors in the Introduction. There are a number of questions and concerns.\nResponse: We thank the reviewer for his/her time to assess our manuscript and appreciate the positive comments.\n1. The comparison of the TL vaccine and TEV vaccine is not presented. TL and TEV were only compared in WBs, which tells us that the tumor antigens were present in the vesicle either in the lumen or both in the lumen and on the vesicle surface. It would be important to know definitively whether these target antigens are present on the vesicle surface.\nResponse: We thank the reviewer for raising this important and insightful point. We fully agree that clarifying whether tumor antigens are located on the surface of TEVs or confined within their lumen is critical for understanding their immunogenic potential, as surface-exposed antigens are directly accessible to antigen-presenting cells. To address this, we performed a protease protection assay and analyzed tumor antigen levels by western blotting after treating TEVs with proteinase K (PK), or PK plus Triton X-100. The results showed that the majority of PAP, PSCA, and PSMA signals were lost after PK treatment (fig. S1), indicating that these antigens are largely surface-exposed. The residual signal was abolished only after Triton X-100 permeabilization, suggesting a small luminal component. These results confirmed that tumor antigens are predominantly localized on the surface of TEVs. We have added the corresponding results in the Introduction on page 2.\nPrevious studies have demonstrated that TEVs elicit more potent antitumor immune activation than tumor lysates by providing enriched tumor-associated antigens and promoting cross-presentation1–3. In the revised manuscript, we performed additional experiments comparing blank gel (FK F), the TL 12 vaccine (LG/FK F, FK F + TL + GM-CSF), and the TEV vaccine (TG/FK F, FK F + TEV + GM- 12 12 12 12 CSF) in the absence of CDA. As shown in the new data (fig. S9), TEV vaccine failed to markedly enhance the expression of co-stimulatory molecules CD80/CD86 on infiltrating DCs compared with TL vaccine. This finding consistent with reports that TEV may inhibit DC activation in the absence of adjuvant. Importantly, TEV vaccine significantly upregulated MHC class I expression on DCs relative to TL vaccine, suggesting an enhanced antigen presentation potential even without CDA. They emphasize that while CDA is required to fully overcome TEV-mediated DC suppression and induce co-stimulatory molecule upregulation, TEVs still provide superior antigen cross-presentation capacity compared to TLs. These results have been added into the revised Results section on page 8.\nReferences:\n1. Rao, Q. et al. Tumor-derived exosomes elicit tumor suppression in murine hepatocellular carcinoma models and humans in vitro. Hepatol. Baltim. Md 64, 456–472 (2016).\n2. Liu, H. et al. Co-delivery of tumor-derived exosomes with alpha-galactosylceramide on dendritic cell-based immunotherapy for glioblastoma. Cancer Lett. 411, 182–190 (2017).\n3. Gu, X., Erb, U., Büchler, M. W. & Zöller, M. Improved vaccine efficacy of tumor exosome compared to tumor lysate loaded dendritic cells in mice. Int. J. Cancer 136, E74-84 (2015).\nAdded: Page 2\nOur protein level validation in prostate cancer surgical specimens revealed preserved antigenic profiles in TEVs, demonstrating equivalent expression levels of prostate-specific antigens (PAP, PSCA, PSMA) between TEVs and matched tumor lysates (Fig. 1, A and B). Furthermore, a protease protection assay confirmed that these antigens are predominantly localized on the surface of TEVs (fig. S1).\nfig. S1. Protease protection assay analyzing the indicated antigens (PSMA, PSCA, PAP) in TEVs. TEVs extracted from human prostate cancer tissue was detected by western blotting after being treated with proteinase K (PK), or PK plus Triton X-100.\nPage 8\nBy directly comparing the effects of TLs and TEVs on DC activation, we found that in the absence of CDA, TEVs did not markedly enhance the expression of co-stimulatory molecules CD80/CD86 on DCs relative to TLs, however, they induced a significantly higher level of MHC I expression (fig. S9). These data indicate that, although TEVs alone are insufficient to drive full DC maturation, they possess superior antigen-presenting potential compared with TLs.\nfig. S9. Effects of TL and TEV vaccines on DC stimulation. A, Representative flow cytometry plots of CD80+CD86+ DCs in different treatment groups: LG/FK F (FK F + TL + GM-CSF) and TG/FK F 12 12 12 (FK F + TEV + GM-CSF) (n = 5). B, Quantification of the percentage of CD80+CD86+ DCs. C, 12 Representative histograms of MHC class I mean fluorescence intensity (MFI) on DCs. D, Quantification of MHC class I MFI in DCs. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001.\n2. What recruits DCs to hydrogels? And what type of DCs are recruited?\nResponse: We sincerely appreciate the reviewer’s insightful questions. In our vaccine system, GM- CSF serves as the key chemotactic cytokine responsible for recruiting DCs to the hydrogel vaccine. It establishes a potent cytokine gradient that directs circulating myeloid cells to the local microenvironment. Mechanistically, GM-CSF upregulates adhesion molecules and chemokine receptors on these cells4, while simultaneously providing critical survival and maintenance signals, thereby promoting DC retention and sustaining a high density of antigen-presenting cells for immune activation5. This is consistent with established literature demonstrating that local GM-CSF delivery promotes myelopoiesis and the subsequent recruitment and differentiation of myeloid progenitors into dendritic cells6–8.\nTo characterize the DC subsets recruited into the hydrogels, we performed subset-specific flow cytometry analysis (fig. S10). Vaccination with TCG/FK F markedly increased the frequencies of 12 cDC1, cDC2, and moDCs relative to the FK F or CG/FK F groups, whereas pDC levels were 12 12 unchanged. Notably, these enriched subsets represent the major professional antigen-presenting DC populations, with cDC1s being specialized for cross-presentation to CD8⁺ T cells. Their coordinated expansion indicates that the TCG/FK F vaccine fosters an APC-enriched microenvironment 12 conducive to efficient T-cell priming, underpinning the observed enhancement of antitumor immunity. These findings have been added into the revised manuscript on page 8.\nReferences:\n4. Yi, M. et al. Targeting cytokine and chemokine signaling pathways for cancer therapy. Signal Transduct. Target. Ther. 9, 176 (2024).\n5. Soiffer, R. J., Kooshesh, K. A. & Ho, V. Whole tumor cell vaccines engineered to secrete GM-CSF (GVAX). ImmunoMedicine 1, e1025 (2021).\n6. Kumar, A., Taghi Khani, A., Sanchez Ortiz, A. & Swaminathan, S. GM-CSF: A Double-Edged Sword in Cancer Immunotherapy. Front. Immunol. 13, 901277 (2022).\n7. Inaba, K. et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176, 1693–1702 (1992).\n8. Liu, J., Zhang, X., Cheng, Y. & Cao, X. Dendritic cell migration in inflammation and immunity. Cell. Mol. Immunol. 18, 2461–2471 (2021).\nAdded: Page 8\nTo further delineate infiltrating DC subsets, we quantified conventional DCs (cDCs), monocyte- derived DCs (moDCs), and plasmacytoid DCs (pDCs) by flow cytometry. Compared to the FK F 12 group, the CG/FK F formulation enhanced the abundance of cDC1s and moDCs (fig. S10), indicating 12 that the adjuvant potentiates the recruitment of these functionally critical DC subsets. Furthermore, the TCG/FK F vaccine significantly increased the percentages of cDC1s, cDC2s, and moDCs within the 12 hydrogel relative to control groups.\nfig. S10. Analysis of DC subsets in the hydrogel post-vaccination. A, Quantification by flow cytometry of cDC1, (B) cDC2, (C) moDC and (D) pDC populations within the hydrogel (n = 5). Data are mean ± SEM. Statistical significance was determined using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001.\n3. The increased % of CD11c+, CD80+CD86+, CCR7+ DCs are significant in TCG/FK F hydrogels. 12 This does not tell us the activation state of these infiltrating DCs. It has been shown that TEV suppress activation and differentiation of DC. The same argument can be made about T cells: activity of CD8+ and CD4+ T cells has been widely reported to be suppressed by TEV. It is surprising and needs to be explained how TEVs in hydrogels do not exercise their immunosuppressive activities and instead appear to activate DC and facilitate Ag presentation to T cells. Thus, the statement that “hydrogel vaccine system facilitates the recruitment, maturation and lymph node homing of DC” is not supported by the presented data.\nResponse: We fully agree with the reviewer on the importance of this issue. Accordingly, we performed supplementary experiments to directly evaluate DC activation and antigen-presentation capacity. Consistent with reports that TEVs alone can partially suppress DC function, our data indicate that TEVs without adjuvant induce only modest DC activation (fig. S9). However, this limited activation is substantially enhanced when TEVs are co-delivered with the STING agonist CDA within the FK12F hydrogel, resulting in strong upregulation of the co-stimulatory molecules CD80/CD86 (Fig. 3B). Thus, CDA is indispensable for counteracting the suboptimal immunogenicity of TEVs and fully augmenting the stimulatory profile of DCs. In parallel, our new data demonstrate that the TCG/FK12F vaccine significantly upregulated MHC class I on DCs, both within the hydrogel and in draining lymph nodes (Fig. 3G, H and fig. S12). Specifically, in an OVA-expressing tumor model (fig. S11), TEV-based vaccination markedly increased antigen-specific DCs (Fig. 3I), confirming antigen delivery and subsequent DC activation in an antigen-dependent manner. Correspondingly, vaccination induced a substantial increase in SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in the draining lymph nodes (Fig. 3J and K). These findings align with existing literature that positions TEVs as a potent antigenic source capable of eliciting antitumor responses through enhanced cross-presentation and T- cell priming9–12.\nFurthermore, our biodistribution analysis demonstrates effective local confinement of TEVs at the injection site, with minimal systemic dissemination (Fig. 2J and fig. S6). This localized retention likely minimizes the risk of TEV-mediated systemic T-cell suppression reported in other studies. Taken together, our data confirm that the vaccine induces antigen-specific DC activation, restoring their capacity for efficient antigen cross-presentation and T-cell priming. These results robustly support our original conclusion that the hydrogel vaccine system facilitates DC recruitment, maturation, and lymph-node homing. Please find the corresponding updates on pages 7 and 8 of the revised manuscript.\nReferences:\n9. Prakash, A., Gates, T., Zhao, X., Wangmo, D. & Subramanian, S. Tumor-derived extracellular vesicles in the colorectal cancer immune environment and immunotherapy. Pharmacol. Ther. 241, 108332 (2023).\n10. Whiteside, T. L., Diergaarde, B. & Hong, C.-S. Tumor-Derived Exosomes (TEX) and Their Role in Immuno-Oncology. Int. J. Mol. Sci. 22, 6234 (2021).\n11. Peng, Z. et al. Tumor-Derived Extracellular Vesicles Enable Tumor Tropism Chemo-Genetherapy for Local Immune Activation in Triple-Negative Breast Cancer. ACS Nano 18, 30943–30956 (2024).\n12. Liang, K. et al. Biologically Self-Assembled Tumor Cell-Derived Cancer Nanovaccines as an All- in-One Platform for Cancer Immunotherapy. ACS Nano 18, 6702–6717 (2024).\nAdded: Page 8\nFurthermore, using an OVA-expressing RM-1 tumor model (fig. S11), we assessed antigen-specific immunity induced by the TEV-based hydrogel vaccine. Flow cytometry revealed that vaccination significantly upregulated MHC class I on DCs both within the hydrogel and in draining lymph nodes (Fig. 3G, H and fig. S12). This was accompanied by a marked increase in OVA-specific DCs (Fig. 3I), confirming efficient antigen delivery and antigen-dependent DC activation. In parallel, we observed a substantial expansion of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes, and splenocytes from vaccinated mice exhibited increased frequencies of IFN-γ⁺ CD8⁺ T cells upon ex vivo OVA restimulation (Fig. 3J and K). Together, these data demonstrate that the hydrogel vaccine promotes robust antigen-specific DC activation and primes a functional antigen-specific CD8⁺ T-cell response.\nFig. 3. TCG/FK F vaccine recruits and activates DCs in situ and promotes their lymph node 12 migration. A-C, Quantification using flow cytometry gating of DCs (A), CD80+CD86+ DCs (B) and CCR7+ DCs (C) within the hydrogel in mice (n = 5). D-F, Quantification using flow cytometry gating of (D) CD80+CD86+ DCs, (E) CD4+ T cells and (F) CD8+ T cells within the inguinal lymph nodes in mice (n = 5). G, MHC class I mean fluorescence intensity (MFI) in gel-infiltrating DCs (n = 5). H, MHC class I MFI in DCs from draining lymph nodes (n = 5). I, Percentage of OVA antigen-presenting DCs determined by flow cytometry (n = 5). J, Frequency of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes (n = 5). K, Percentage of IFN-γ-producing CD8⁺ T cells in splenocytes following ex vivo OVA peptide restimulation (n = 5). L, Schematic depicting the mechanism by which the TCG/FK F vaccine recruits and activates DCs in situ, thereby promoting the activation of tumor- 12 specific T cells within the lymph nodes. Data are mean ± SEM. Statistical significance in A-K was determined using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001.\nPage7\nIn vivo fluorescence imaging revealed that both CDA and TEVs in the FK F hydrogel exhibited strong 12 localized signals at the injection site and negligible accumulation in major organs (liver, spleen, kidney, heart, and lung) (Fig. 2J, K and fig. S6). This indicates that the vaccine components were effectively retained locally without systemic dissemination.\nfig. S6. Biodistribution of vaccine components. Representative ex vivo fluorescence images of major organs (liver, heart, spleen, lung, kidney) collected at the indicated time points following subcutaneous injection of (A) DIR-labeled TEVs or (B) Cy7-labeled CDA within the TCG/FK F formulation (n = 12 3).\n4. While the vaccine suppressed tumor growth and prolonged the survival time (Fig. 4), the evidence of antigen specific reactivity, rests on Fig 4G, H without annotation as to which antigen was tested in ELISPOT.\nResponse: We thank the reviewer for raising this important point regarding the antigen used in the ELISPOT assay. To address this, we confirm that the antigenic stimulus was TEVs isolated from RM- 1 cells, as described in the Methods. Using whole TEVs allows us to present a broad spectrum of tumor-associated antigens, capitalizing on their natural antigenic diversity. The revised manuscript now explicitly states this in the Results section on page 23.\nRevised: Page 23\nSplenocytes from RM-1 tumor-bearing mice were collected for restimulation with TEVs. 10 days after i.h. injected with PBS, CG/FK F, TCG or TCG/FK F, spleens were collected and spleen cells were 12 12 extracted. Splenocytes (1 × 105) were stimulated for 24 h with 1 μg/well TEVs (from RM-1 cells). Cells were subsequently assayed for IFN-γ by Elispot kit (Mabtech) and spot counts using instrument (BD Biosciences).\n5. The fact that depletion of eripheral T cells in vaccinated mice promoted tumor growth only means that T cells but not necessarily antigen-specific T cells are modulating the responses to TEV antigens.\nResponse: We thank the reviewer for this insightful comment on the need to demonstrate antigen specificity. In direct response, we conducted new experiments using an OVA-expressing RM-1 tumor model. As shown in the newly added data (fig. S11), vaccination with TCG/FK12F induced a significant expansion of OVA-specific DCs and (SIINFEKL–H-2Kᵇ tetramer⁺) CD8⁺ T cells in the draining lymph nodes (Fig. 3I and J). Furthermore, ex vivo restimulation of splenocytes with OVA peptide markedly increased the frequency of IFN-γ⁺ CD8⁺ T cells (Fig. 3K).\nThese results provide direct evidence that the antitumor efficacy of our vaccine is mediated by antigen- specific CD8⁺ T-cell responses. We have incorporated these findings into the revised Results section (Page 8) and included Fig. 3I-K with detailed legends to enhance clarity. We appreciate the reviewer's suggestion, which has strengthened the mechanistic link between vaccination and antigen-specific T- cell-mediated tumor control.\nAdded: Page 8\nFurthermore, using an OVA-expressing RM-1 tumor model (fig. S11), we assessed antigen-specific immunity induced by the TEV-based hydrogel vaccine. Flow cytometry revealed that vaccination significantly upregulated MHC class I on DCs both within the hydrogel and in draining lymph nodes (Fig. 3G, H and fig. S12). This was accompanied by a marked increase in OVA-specific DCs (Fig. 3I), confirming efficient antigen delivery and antigen-dependent DC activation. In parallel, we observed a substantial expansion of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes, and splenocytes from vaccinated mice exhibited increased frequencies of IFN-γ⁺ CD8⁺ T cells upon ex vivo OVA restimulation (Fig. 3J and K). Together, these data demonstrate that the hydrogel vaccine promotes robust antigen-specific DC activation and primes a functional antigen-specific CD8⁺ T-cell response.\nFig. 3. TCG/FK F vaccine recruits and activates DCs in situ and promotes their lymph node 12 migration. A-C, Quantification using flow cytometry gating of DCs (A), CD80+CD86+ DCs (B) and CCR7+ DCs (C) within the hydrogel in mice (n = 5). D-F, Quantification using flow cytometry gating of (D) CD80+CD86+ DCs, (E) CD4+ T cells and (F) CD8+ T cells within the inguinal lymph nodes in mice (n = 5). G, MHC class I mean fluorescence intensity (MFI) in gel-infiltrating DCs (n = 5). H, MHC class I MFI in DCs from draining lymph nodes (n = 5). I, Percentage of OVA antigen-presenting DCs determined by flow cytometry (n = 5). J, Frequency of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes (n = 5). K, Percentage of IFN-γ-producing CD8⁺ T cells in splenocytes following ex vivo OVA peptide restimulation (n = 5). L, Schematic depicting the mechanism by which the TCG/FK F vaccine recruits and activates DCs in situ, thereby promoting the activation of tumor- 12 specific T cells within the lymph nodes. Data are mean ± SEM. Statistical significance in A-K was determined using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001.\n6. Studies aiming to show infiltration of vaccinated mice by IFNγ+ CD8+ T cells are supported by flow cytometry and should be shown by quantitative immunohistochemistry to be convincing.\nResponse: We thank the reviewer for this valuable suggestion. We agree that spatial visualization of IFN-γ⁺ CD8⁺ T-cell infiltration would be informative. However, as IFN-γ is a rapidly secreted cytokine rather than a stably retained intracellular protein, it diffuses readily in tissue and is not reliably retained for specific detection by immunohistochemistry or immunofluorescence. Our attempts at immunohistochemical detection in tumor sections yielded diffuse, non-specific signals that could not be reliably quantified.\nTo provide complementary spatial and functional evidence, we therefore employed alternative approaches. We measured IFN-γ levels in tumor homogenates by ELISA, which showed a significant increase in the TCG/FK12F group in both subcutaneous and orthotopic models (Fig. 4F and 5F). Moreover, in the orthotopic model, immunofluorescence staining revealed a marked increase in granzyme B⁺ CD8⁺ T cells within treated tumors (fig. S21), indicating enhanced local cytotoxic activity. These data, together with our earlier flow cytometry results, offer compelling evidence of functional CD8⁺ T-cell activation and intratumoral recruitment following vaccination. The relevant findings have been added to page 13 of the revised manuscript.\nAdded: Page 13\nFlow cytometry analysis of orthotopic tumor tissues revealed higher infiltration of CD8+ T cells and CD8+ IFN-γ+ T cells in the TCG/FK F and TCG/FK F + aPD-1 groups compared to the other groups 12 12 (fig. S19 and 20). Consistent with this, immunohistochemistry and immunofluorescence analyses revealed increased CD8⁺ T-cell infiltration and a higher abundance of granzyme B⁺ cytotoxic T cells, respectively, in tumors receiving TCG/FK F combined with aPD-1 (Fig. 5E and fig. S21). 12\nfig. S21. Granzyme B immunofluorescence of RM-1 orthotopic tumors in different treatment groups.\n7. Studies shown in Fig 5. illustrate survival and suppressed proliferation of orthotopic tumors or lung metastases formation in 4T1 breast cancer model by vaccinated mice without really linking these vaccination results to mechanisms that could rationalize and justify such dramatic responses. The same concern applies to the data in Fig 6.\nResponse: We thank the reviewer for highlighting the need for mechanistic data to support the therapeutic efficacy observed in Figs. 5 and 6. As described in the original manuscript, we have already performed comprehensive immune analyses to reveal the mechanism of action. In the orthotopic tumor model, immunohistochemistry, immunofluorescence (Fig. 5E), and ELISA (Fig. 5D) revealed enhanced CD8⁺ T-cell infiltration, elevated production of IFN-γ and granzyme B, and remodeling of the tumor immune microenvironment. In the postsurgical personalized model, bulk RNA-seq of residual tumors demonstrated upregulation of immune activation pathways following vaccination (Fig. 6E–I).\nTo directly address the reviewer’s point and further solidify these mechanistic links, we have now performed additional experiments in the revised manuscript:\n1) Antigen-specific T-cell priming. In the OVA-expressing RM-1 orthotopic model, SIINFEKL– H-2Kᵇ tetramer analysis revealed a significant increase in tumor-infiltrating, antigen-specific CD8⁺ T cells following vaccination (fig. S23), confirming efficient antigen-specific priming.\n2) Systemic immune activation. In the 4T1 lung metastasis model, flow cytometry of peripheral blood and splenocytes showed a marked elevation in IFN-γ⁺ CD8⁺ T cells (fig. S24), indicating robust systemic immune activation.\n3) Effector response in the postsurgical model. In the 4T1 resection model, flow cytometric analysis of tumor-infiltrating lymphocytes demonstrated a significant increase in IFN-γ⁺ CD8⁺ T cells in vaccinated mice (fig. S28), correlating with improved survival and reduced recurrence.\nCollectively, the data establish a clear mechanistic pathway from vaccine administration to antigen- specific CD8⁺ T-cell activation and subsequent local/systemic immune responses, ultimately underpinning the therapeutic benefits. Corresponding updates are provided in the Results and Supplementary Material. All new results have been incorporated into the revised Results and Supplementary Figures.\nAdded: Page 13\nFurthermore, in the OVA-expressing RM-1 orthotopic model, flow cytometry demonstrated a significant increase in H-2Kᵇ–OVA tetramer⁺ CD8⁺ T cells within tumor tissues following TCG/FK F 12 vaccination (Fig. S23), indicating robust antigen-specific cytotoxic T-cell activation in the tumor microenvironment.\nPage 15\nAfter euthanasia, peripheral blood, spleen, and lung tissues were collected for analysis. Flow cytometry of peripheral blood revealed a marked increase in circulating IFN-γ⁺ CD8⁺ T cells in TCG/FK F- 12 treated mice (fig. S24A), indicating robust systemic T-cell activation. Consistent with this, ex vivo restimulation of splenocytes with TEVs induced a significant increase in IFN-γ⁺ CD8⁺ T cells (fig. S24B), confirming functional antigen-specific responses. This systemically activated, tumor-specific immunity further suppressed metastasis, as evidenced by examination of the lungs, which showed significantly fewer and smaller metastatic nodules in the TCG/FK F and TCG/FK F + aPD-1 groups 12 12 compared to the PBS, aPD-1, and TCG alone groups (Fig. 5J, fig. S25).\nPage 18\nConsistently, the TCG/FK F vaccine showed significant efficacy in an orthotopic 4T1 breast cancer 12 resection model, significantly inhibiting tumor recurrence (Fig. 6J and K). Accompanying flow cytometry analysis revealed a marked increase in IFN-γ⁺ CD8⁺ T cells (fig. S28), aligning with our earlier results and confirming its role in promoting antitumor T-cell responses.\nfig. S23. Quantification of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in orthotopic tumor in different treatment groups (n = 5). Data are mean ± SEM. Statistical significance was determined using one- way ANOVA. ***p < 0.001 and ****p< 0.0001.\nfig. S24. Flow cytometric analysis of systemic immune responses in the 4T1 metastatic tumor model. A, Quantification of IFN-γ⁺ CD8⁺ T cells in peripheral blood in different treatment groups (n = 5). B, Quantification of IFN-γ⁺ CD8⁺ T cells in splenocytes after ex vivo restimulation with TEVs (n = 5). Data are mean ± SEM. Statistical significance was determined using one-way ANOVA. **p < 0.01; ***p < 0.001 and ****p< 0.0001.\nfig. S28. Quantification of IFN-γ⁺ CD8⁺ T cells across treatment groups in 4T1 breast cancer orthotopic resection model (n = 5). Data are mean ± SEM. Statistical significance was determined using one-way ANOVA. ****p< 0.0001.\n\nReviewer #2\nThe manuscript presents a cancer vaccine platform utilizing a tumor-derived extracellular vesicle (TEV)-based hydrogel, which aims to enhance immunogenicity and prevent postoperative recurrence. The hydrogel improves vaccine retention, facilitates spatiotemporal control over antigen and adjuvant delivery, and promotes dendritic cell (DC) recruitment and activation, leading to CD8⁺ T-cell anti- tumour responses. The authors also demonstrate that this system can be combined with anti-PD-1 therapy to significantly suppress tumor growth, improve survival, and generate durable immune memory, preventing recurrence in both orthotopic and metastatic models. However, there are a few critical aspects that need to be addressed before publication.\nResponse: We thank the reviewer for his/her accurate evaluation of our manuscript and appreciate the constructive comments.\nMajor comments\n1. TEVs were isolated by ultracentrifugation at 120,000×g for 70 minutes twice at 4℃. Unlike gradient density centrifugation, ultracentrifugation alone carries a significant risk of co-isolating protein aggregates, lipoproteins, apoptotic bodies, and cellular debris, which may interfere with downstream analyses. The authors demonstrated negligible expression of the contaminant protein GM130 in TEVs, but this is not sufficient to prove the absence of non-exosomal components. It would be more convincing if the authors could provide additional negative markers to confirm the purity of the TEVs.\nResponse: We thank the reviewer for this valuable suggestion, which has strengthened the rigor of our TEV characterization. In the revised manuscript, we have supplemented the Western blot analysis with key negative markers to more thoroughly assess TEV purity. Specifically, we now include calnexin (an endoplasmic reticulum marker) to exclude cellular contamination, and ApoA-1 (a major apolipoprotein) to rule out co-isolated lipoproteins. Both markers were undetectable in our TEV preparations, confirming that our ultracentrifugation protocol yields vesicles with minimal contamination from intracellular components or lipoproteins. These data provide stronger evidence for the purity of our TEV preparations. Please see the corresponding updates on page 5 and fig. S4 of the revised manuscript.\nRevised: Page 5\nWestern blot analysis of TEVs compared with RM-1 cell lysates (CLs) demonstrated negligible expression of the contaminant protein Calnexin, Apoa-1 and GM130 in TEVs, alongside positive expression of extracellular vesicle markers Flotillin-1, CD63, and CD9 (Fig. 2F and fig. S4).\nfig. S4. Assessment of TEV purity using negative markers. Western blot analysis of Calnexin and ApoA-1 in cell lysates (CLs) versus isolated TEVs, demonstrating the absence of these negative markers in TEV fractions.\n2. The authors mentioned that TCG/FK F induced a significantly higher percentage of antigen- 12 specific CD4+ and CD8+ T cells compared with the FK F and CG/FK F treatment groups (Fig. 3, E 12 12 and F). However, these T cell populations were gated only by CD45/CD3/CD4/CD8 expression, which is not sufficient to distinguish bystander T cells from antigen-specific T cells.\nResponse: We appreciate the reviewer’s comment on the need for more definitive evidence of antigen specificity beyond standard surface marker gating. In response, a vaccine was prepared using TEVs isolated from OVA-expressing RM-1 cells, and experiments were repeated in the orthotopic model. The new results show a significant elevation of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes of TCG/FK12F-treated mice. Additionally, ex vivo restimulation with OVA protein induced a marked increase in IFN-γ⁺ CD8⁺ T cells. These data directly demonstrate antigen-specific CD8⁺ T-cell activation and function, providing mechanistic support for the observations in Fig. 3E and F. The corresponding data have been added to page 8 of the revised manuscript (Fig. 3I–K).\nAdded: Page 8\nFurthermore, using an OVA-expressing RM-1 tumor model (fig. S11), we assessed antigen-specific immunity induced by the TEV-based hydrogel vaccine. Flow cytometry revealed that vaccination significantly upregulated MHC class I on DCs both within the hydrogel and in draining lymph nodes (Fig. 3G, H and fig. S12). This was accompanied by a marked increase in OVA-specific DCs (Fig. 3I), confirming efficient antigen delivery and antigen-dependent DC activation. In parallel, we observed a substantial expansion of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes, and splenocytes from vaccinated mice exhibited increased frequencies of IFN-γ⁺ CD8⁺ T cells upon ex vivo OVA restimulation (Fig. 3J and K). Together, these data demonstrate that the hydrogel vaccine promotes robust antigen-specific DC activation and primes a functional antigen-specific CD8⁺ T-cell response.\nfig. S11. A, Fluorescence microscopy image of OVA-expressing RM-1 cells (OVA fused with GFP), confirming stable expression of OVA. Scale bar, 100 μm. B, Western blot analysis of OVA protein expression in OVA-RM-1 cells and corresponding TEVs.\nFig. 3. TCG/FK F vaccine recruits and activates DCs in situ and promotes their lymph node 12 migration. A-C, Quantification using flow cytometry gating of DCs (A), CD80+CD86+ DCs (B) and CCR7+ DCs (C) within the hydrogel in mice (n = 5). D-F, Quantification using flow cytometry gating of (D) CD80+CD86+ DCs, (E) CD4+ T cells and (F) CD8+ T cells within the inguinal lymph nodes in mice (n = 5). G, MHC class I mean fluorescence intensity (MFI) in gel-infiltrating DCs (n = 5). H, MHC class I MFI in DCs from draining lymph nodes (n = 5). I, Percentage of OVA antigen-presenting DCs determined by flow cytometry (n = 5). J, Frequency of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes (n = 5). K, Percentage of IFN-γ-producing CD8⁺ T cells in splenocytes following ex vivo OVA peptide restimulation (n = 5). L, Schematic depicting the mechanism by which the TCG/FK F vaccine recruits and activates DCs in situ, thereby promoting the activation of tumor- 12 specific T cells within the lymph nodes. Data are mean ± SEM. Statistical significance in A-K was determined using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001.\n3. The local versus systemic biodistribution is not thoroughly assessed. Besides the subcutaneous injection site, what is the biodistribution profile of TCG/FK F in other organs?\nAdded: Page7\nThe fluorescently labeled CDA and TEVs were mixed with FK F aqueous solution, respectively, and 12 the mixed solution were subsequently injected subcutaneously into mice; free Cy7-labeled CDA and DIR-labeled TEVs were used as controls. In vivo fluorescence imaging revealed that both CDA and TEVs in the FK F hydrogel exhibited strong localized signals at the injection site and negligible 12 accumulation in major organs (liver, spleen, kidney, heart, and lung) (Fig. 2J, K and fig. S6). This indicates that the vaccine components were effectively retained locally without systemic dissemination.\nfig. S6. Biodistribution of vaccine components. Representative ex vivo fluorescence images of major organs (liver, heart, spleen, lung, kidney) collected at the indicated time points following subcutaneous injection of (A) DIR-labeled TEVs or (B) Cy7-labeled CDA within the TCG/FK F formulation (n = 12 3).\n4. Figures 4D-E showed that TCG/FK F vaccine treatment significantly increased infiltration of CD8+ 12 T cells and CD8+ IFN-γ+ T cells in the TME. Have the authors investigated whether these populations of CD8+ T cells are antigen-specific?\nResponse: We appreciate the reviewer for raising this important point, as confirming antigen specificity is fundamental to elucidating the mechanism of vaccine-induced tumor control. Initially, direct measurement of tumor-infiltrating, antigen-specific CD8⁺ T cells was not feasible due to the lack of commercially available tetramers for the relevant RM-1 tumor antigen. Therefore, in our original submission, we employed an Enzyme-linked Immunospot (ELISpot) assay as an alternative to validate antigen-specific T-cell activation induced by the TCG/FK F vaccine following ex 12 vivo restimulation with TEV antigen. These data were presented in Figures 4G and 4H.\nTo directly address the reviewer's concern, we have now performed additional experiments. We prepared the TCG/FK F vaccine using TEVs isolated from RM-1 cells engineered to express 12 ovalbumin (OVA). Using this model in an orthotopic RM-1 tumor setting, we observed a significant increase in SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells within the tumors of TCG/FK F-treated mice 12 compared to controls. This result provides direct evidence of vaccine-primed, antigen-specific CD8⁺ T-cell infiltration. These new findings have been incorporated into the revised manuscript on page 13.\nAdded: Page 13\nFurthermore, in the OVA-expressing RM-1 orthotopic model, flow cytometry demonstrated a significant increase in H-2Kᵇ–OVA tetramer⁺ CD8⁺ T cells within tumor tissues following TCG/FK F 12 vaccination (Fig. S23), indicating robust antigen-specific cytotoxic T-cell activation in the tumor microenvironment.\nfig. S23. Quantification of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in orthotopic tumor in different treatment groups (n = 5). Data are mean ± SEM. Statistical significance was determined using one-way ANOVA. ***p < 0.001 and ****p< 0.0001.\n5. Figure 6I showed immune infiltration analysis in PBS, CG/FK F, and TCG/FK F treated groups. 12 12 How were different immune subsets determined? Are there predetermined markers for each subset?\nResponse:\nWe thank the reviewer for this important point. As described, the relative proportions of various immune cell subsets were estimated computationally using CIBERSORT analysis based on our bulk RNA-seq data. CIBERSORT is a widely validated deconvolution algorithm that infers cell-type abundances from bulk tissue transcriptomes by leveraging reference gene expression signatures13,14. Specifically, the algorithm compares the gene expression profile of each sample to a predefined signature matrix—the LM22 matrix for human data or a mouse-specific matrix (e.g., MICE) for murine data—which contains marker genes characteristic of distinct immune cell types. Through this comparison, CIBERSORT quantifies the relative abundance of populations such as T cells, B cells, NK cells, macrophages, and dendritic cells. Thus, the identification of each immune subset is driven by these well-established marker gene sets, enabling a systematic and quantitative assessment of immune infiltration across our experimental groups. We hope this clarifies the methodological basis for the immune cell profiling in our study.\nReferences:\n13. Newman, A. M. et al. Robust enumeration of cell subsets from tissue expression profiles. Nat. Methods 12, 453–457 (2015).\n14. Chen, Z. et al. Inference of immune cell composition on the expression profiles of mouse tissue. Sci. Rep. 7, 40508 (2017).\n6. There is no information about the methods used for transcriptome sequencing analysis.\nResponse: We thank the reviewer for bringing this issue to our attention, which has been addressed. Briefly, gene expression count data were imported into R (v4.4.1) for downstream analyses. Differentially expressed genes (DEGs) were identified using the DESeq2 (v1.44.0) package, with an adjusted p value < 0.05 considered significant. Functional enrichment analysis was performed using clusterProfiler (v4.12.6). Immune cell infiltration was estimated from bulk RNA-seq data using the CIBERSORT (v0.1.0) algorithm and visualization was generated using ggplot2 (v3.5.1) for volcano plots and pheatmap (v1.0.12) for heatmaps. These methodological details have been incorporated into the revised Methods section.\nAdded: Page 24\nTranscriptome data analysis\nDifferential gene expression analysis was performed in R (v4.4.1) using the DESeq2 (v1.44.0) package, with genes considered significantly differentially expressed at an adjusted p value < 0.05. Functional enrichment analyses were conducted using clusterProfiler (v4.12.6). Visualization of differentially expressed genes was generated using ggplot2 (v3.5.1) for volcano plots and pheatmap (v1.0.12) for hierarchical clustering heatmaps. Immune cell infiltration was estimated from bulk RNA-seq data using the CIBERSORT algorithm, which infers the relative abundance of immune cell subsets within the tumor microenvironment.\n7. The authors mentioned that pro-tumor cells, such as Tregs, were slightly downregulated in the TCG/FK F group (Fig. 6I). However, the difference does not appear to be substantial and seems 12 marginal to me. Some TEVs may also be immunosuppressive. Furthermore, have the authors validated these findings using other methods?\nResponse: We thank the reviewer for bringing this point to our attention. Following your suggestion, we have carefully re-examined the CIBERSORT immune infiltration data. We agree that the Treg levels in the TCG/FK F group showed only a marginal downward trend and did not reach statistical 12 significance. We apologize for the overstatement in the original manuscript and have removed the corresponding claim regarding Treg downregulation in the revised version.\nAs the observed change was limited and not statistically supported, we did not pursue further experimental validation for this specific point. We sincerely appreciate the reviewer’s vigilance, which has helped ensure the accuracy and appropriate interpretation of our data.\nRevised: Page 18\nSubsequent immune infiltration analysis further disclosed a marked increase in CD8+ T-cell abundance in the TCG/FK F group compared to both the PBS and CG/FK F groups, indicative of enhanced 12 12 antitumor immunity (Fig. 6I). Additionally, other anti-tumor immune cells, including DCs, NK cells, and Th1 cells, were also elevated in the TCG/FK F group. 12\n8. It appears that the proposed strategy is specifically optimized for postoperative adjuvant therapy to prevent recurrence. Given that TEVs are autologous, how long does it take to produce EVs from tumour samples in sufficient quantity to treat patients? Can you comment on the TEV yield?\nResponse: We thank the reviewer for raising this critical and clinically pertinent question. Indeed, the translational potential of an autologous TEV-based vaccine hinges on two key practical factors: the production timeline and the achievable yield. In our current preclinical study, TEVs were successfully isolated from both cultured tumor cells and ex vivo mouse tumor tissues. Under laboratory conditions, the ultracentrifugation-based isolation process can typically be completed within one day. We recognize, however, that scaling this process for clinical application will require the development of robust, large-scale, and GMP-compliant manufacturing protocols.\nRegarding yield, the TEVs obtained from ex vivo mouse tumors in this study were sufficient for vaccine formulation and efficacy testing in our experimental models. While we have not yet conducted analyses at a human scale, it is important to note that TEV isolation technology is rapidly advancing. Methods such as improved ultracentrifugation, size-exclusion chromatography, and microfluidic platforms continue to enhance both yield and purity. Furthermore, the expansion of patient-derived tumor cells or organoids ex vivo presents a promising avenue to amplify TEV production for clinical use.\nTo address this point directly, we have added a paragraph in the revised Discussion section acknowledging these practical challenges and emphasizing that future work will focus on optimizing scalable, GMP-compatible manufacturing processes. We are grateful to the reviewer for this insightful comment, which has helped us clarify a vital translational aspect of our research.\nAdded: Page 19\nThe successful translation of this approach to the clinic, nevertheless, depends on several critical steps: comprehensive safety evaluation, precise dose optimization, the development of scalable GMP- compatible manufacturing protocols to ensure sufficient TEV yield, and a plan for ongoing therapeutic monitoring.\nMinor comments:\n1. Figures 1A-B demonstrate equivalent expression levels of prostate-specific antigens (PAP, PSCA, PSMA) between TEVs and matched tumor lysates. Since the authors only show a representative Western blot, I wonder how strong this correlation is across a cohort of patients.\nResponse: We thank the reviewer for this important comment. The target antigens (PAP, PSCA, PSMA) were selected based on their established relevance in prostate cancer. In our original study, Western blot analysis of TEVs and matched tumor lysates from three independent patients confirmed the consistent expression of all three antigens across samples. We acknowledge that extending this analysis to a larger patient cohort was beyond the scope of the current study due to practical constraints. In future work, we plan to employ multi-omics approaches on paired tumor tissues and TEVs to more comprehensively investigate antigen correlation and heterogeneity. We appreciate the reviewer's valuable suggestion, which strengthens the translational perspective of our research.\n2. In the subcutaneous therapeutic release experiments (Fig. 2 J-K), fluorescently labeled CDA and TEVs were each mixed with FK F aqueous solution, and the mixtures were subsequently injected 12 subcutaneously into mice. Did the encapsulation of EVs occur before or after the injection? How long does the encapsulation process take?\nResponse: We thank the reviewer for raising this insightful question regarding the encapsulation timing and mechanism in our system. In our design, TEVs are encapsulated after subcutaneous injection rather than during the pre-injection mixing step.\nFK F exists as nanofibers in aqueous solution and undergoes rapid entanglement under physiological 12 conditions to form a hydrogel network (Fig. 2A, B). Zeta potential analysis indicates that both the CDA and TEVs can adhere to the surface of FK F nanofibers via electrostatic interactions prior to 12 injection (Fig. 2G). Upon administration, the FK F solution undergoes immediate in situ gelation in 12 the physiological environment. This rapidly forming hydrogel network effectively entraps the pre- adsorbed TEVs and CDA within its matrix.\nThe kinetics of this process were quantified by rheological measurements (Fig. 2C), which show a sharp increase in storage modulus (G′) within seconds under physiological conditions, confirming rapid hydrogel formation. Consistent with this, in vivo imaging of the subcutaneously injected material (Fig. 2J, K, fig. S5 and S6) shows effective retention and localization of CDA and TEVs at the injection site shortly after administration, verifying that encapsulation occurs primarily in situ.\nWe hope this detailed explanation clarifies the dynamic encapsulation mechanism and its kinetics for the reviewer.\n3. Does the granzyme B signal colocalize with CD8 T cells in the immunofluorescent images (Fig. 5E and fig. S10)?\nResponse: We appreciate the reviewer’s valuable suggestion regarding the spatial relationship between granzyme B and CD8+ T cells. To address this, we have performed additional immunofluorescence co-localization analyses. As shown in the updated supplementary figures (fig. S21), the merged images demonstrate that granzyme B signal is predominantly localized within CD8⁺ T cells at the tumor site.\nThese new data provide direct visual evidence that granzyme B expression is primarily associated with CD8⁺ T cells, reinforcing the conclusion that the observed cytotoxic activity is mediated by activated CD8⁺ T lymphocytes. The corresponding results have been updated on page 13 and in fig. S21 of the revised manuscript.\nAdded: Page 13\nFlow cytometry analysis of orthotopic tumor tissues revealed higher infiltration of CD8+ T cells and CD8+ IFN-γ+ T cells in the TCG/FK F and TCG/FK F + aPD-1 groups compared to the other groups 12 12 (fig. S19 and 20). Consistent with this, immunohistochemistry and immunofluorescence analyses revealed increased CD8⁺ T-cell infiltration and a higher abundance of granzyme B⁺ cytotoxic T cells, respectively, in tumors receiving TCG/FK F combined with aPD-1 (Fig. 5E and fig. S21). 12\nfig. S21. Granzyme B immunofluorescence of RM-1 orthotopic tumors in different treatment groups.\n4. Can the authors comment on how this platform can overcome the highly heterogeneous antigen profiles present in some patients?\nResponse: We thank the reviewer for raising this critical point regarding tumor antigen heterogeneity. Our TEV-based vaccine platform is designed to address this very challenge by utilizing vesicles derived from the patient’s own tumor, which intrinsically encapsulate the patient-specific antigenic landscape. Unlike conventional vaccines targeting a predefined, limited set of antigens, TEVs naturally present a broad repertoire of tumor antigens, including both known tumor-associated antigens and patient-specific neoantigens.\nConsequently, even in the setting of high intratumoral heterogeneity, an autologous TEV vaccine can deliver the unique and comprehensive antigenic profile of the individual’s tumor. This enables the immune system to mount a polyclonal T-cell response directed against multiple tumor subclones simultaneously. By achieving personalized therapy with broad antigenic coverage, this approach mitigates the risk of immune escape due to antigen loss or clonal variation. Thus, it represents a rational and clinically promising strategy for inducing robust, patient-specific anti-tumor immunity against heterogeneous tumors.\n\nReviewer #3\nIn this manuscript, the authors presented a novel tumor-derived extracellular vesicle-based vaccine integrated into peptide-based supramolecular hydrogels. The vaccine demonstrates the ability to form in situ hydrogels that effectively recruit dendritic cells for subsequent T cell priming. Comprehensive evaluation across multiple tumor models revealed significant tumor growth inhibition, with mechanistic studies demonstrating CD8+ T cell-dependent anti-tumor immunity. This well-designed and executed study makes a substantial contribution to the development of personalized cancer vaccines, offering important insights into extracellular vesicle-based immunotherapy strategies.\nResponse: We thank the reviewer for his/her accurate evaluation of our manuscript and appreciate the positive and insightful comments.\n1. In Figure 3A, the authors claim that DCs were activated by TCG/FK F. The authors should provide 12 the DC activation data for TEV stimulation alone. The authors claimed that the vaccine induced “antigen-specific CD4+ (21.4%) and CD8+ (16.3%) T cells compare with the FK F (14.2% and 12.5%) 12 and CG/FK F (15.1% and 13.7%) treatment groups (Fig. 3, E and F)”. The data from Figure 3E and 12 F can not support this conclusion, as the elevated CD4/8 T cell population may be induced from non- specific expansion. The increase of CD4/8 population may be antigen-specific T cells, but the results in Figure E and F did not directly show antigen specific T cell population.\nResponse: We thank the reviewer for these constructive and insightful comments. To address the first point, we introduced a new treatment group in which TEVs were delivered in FK F hydrogel 12 (TG/FK F) without CDA and compared it with the tumor lysate (TL) group. As shown in the new 12 supplementary data (fig. S9), TEV treatment alone failed to markedly enhance the expression of co- stimulatory molecules CD80/CD86 on infiltrating DCs compared with TL vaccine. This observation aligns with the previously reported intrinsic suppressive effect of TEVs on DC maturation. Importantly, however, TEV vaccination significantly upregulated MHC class I expression on DCs compared to TL vaccination, suggesting an enhanced antigen-presentation capacity. These results collectively underscore the critical role of the CDA adjuvant in counteracting TEV-mediated suppression and achieving robust DC activation.\nRegarding the reviewer’s second point on antigen specificity, we conducted additional experiments using an OVA-expressing RM-1 tumor model to directly assess antigen-specific T-cell priming. Flow cytometry analysis confirmed that vaccination with TCG/FK F significantly increased the proportion 12 of OVA-specific (SIINFEKL–H-2Kᵇ tetramer⁺) CD8⁺ T cells in both draining lymph nodes and tumor tissues (Fig. 3J and S23). Furthermore, ex vivo restimulation of splenocytes from vaccinated mice with OVA peptide induced a marked increase in IFN-γ⁺ CD8⁺ T cells, demonstrating robust antigen-specific activation. These new data provide direct evidence that the vaccine-induced T-cell expansion observed in our original figures is antigen-specific. The corresponding results and descriptions have been incorporated into the revised Results section and supplementary figures.\nAdded: Page 8\nBy directly comparing the effects of TLs and TEVs on DC activation, we found that in the absence of CDA, TEVs did not markedly enhance the expression of co-stimulatory molecules CD80/CD86 on DCs relative to TLs, however, they induced a significantly higher level of MHC I expression (fig. S9). These data indicate that, although TEVs alone are insufficient to drive full DC maturation, they possess superior antigen-presenting potential compared with TLs.\nfig. S9. Effects of TL and TEV vaccines on DC stimulation. A, Representative flow cytometry plots of CD80+CD86+ DCs in different treatment groups: LG/FK F (FK F + TL + GM-CSF) and TG/FK F 12 12 12 (FK F + TEV + GM-CSF) (n = 5). B, Quantification of the percentage of CD80+CD86+ DCs. C, 12 Representative histograms of MHC class I mean fluorescence intensity (MFI) on DCs. D, Quantification of MHC class I MFI in DCs (n = 5). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001.\nAdded: Page 8\nFurthermore, using an OVA-expressing RM-1 tumor model (fig. S11), we assessed antigen-specific immunity induced by the TEV-based hydrogel vaccine. Flow cytometry revealed that vaccination significantly upregulated MHC class I on DCs both within the hydrogel and in draining lymph nodes (Fig. 3G, H and fig. S12). This was accompanied by a marked increase in OVA-specific DCs (Fig. 3I), confirming efficient antigen delivery and antigen-dependent DC activation. In parallel, we observed a substantial expansion of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes, and splenocytes from vaccinated mice exhibited increased frequencies of IFN-γ⁺ CD8⁺ T cells upon ex vivo OVA restimulation (Fig. 3J and K). Together, these data demonstrate that the hydrogel vaccine promotes robust antigen-specific DC activation and primes a functional antigen-specific CD8⁺ T-cell response.\nAdded: Page 13\nFurthermore, in the OVA-expressing RM-1 orthotopic model, flow cytometry demonstrated a significant increase in H-2Kᵇ–OVA tetramer⁺ CD8⁺ T cells within tumor tissues following TCG/FK F 12 vaccination (Fig. S23), indicating robust antigen-specific cytotoxic T-cell activation in the tumor microenvironment.\nfig. S11. A, Fluorescence microscopy image of OVA-expressing RM-1 cells (OVA fused with GFP), confirming stable expression of OVA. Scale bar, 100 μm. B, Western blot analysis of OVA protein expression in OVA-RM-1 cells and corresponding TEVs.\nFig. 3. TCG/FK F vaccine recruits and activates DCs in situ and promotes their lymph node 12 migration. A-C, Quantification using flow cytometry gating of DCs (A), CD80+CD86+ DCs (B) and CCR7+ DCs (C) within the hydrogel in mice (n = 5). D-F, Quantification using flow cytometry gating of (D) CD80+CD86+ DCs, (E) CD4+ T cells and (F) CD8+ T cells within the inguinal lymph nodes in mice (n = 5). G, MHC class I mean fluorescence intensity (MFI) in gel-infiltrating DCs (n = 5). H, MHC class I MFI in DCs from draining lymph nodes (n = 5). I, Percentage of OVA antigen-presenting DCs determined by flow cytometry (n = 5). J, Frequency of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in draining lymph nodes (n = 5). K, Percentage of IFN-γ-producing CD8⁺ T cells in splenocytes following ex vivo OVA peptide restimulation (n = 5). L, Schematic depicting the mechanism by which the TCG/FK F vaccine recruits and activates DCs in situ, thereby promoting the activation of tumor- 12 specific T cells within the lymph nodes. Data are mean ± SEM. Statistical significance in A-K was determined using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001.\nfig. S23. Quantification of SIINFEKL–H-2Kᵇ tetramer⁺ CD8⁺ T cells in orthotopic tumor in different treatment groups (n = 5). Data are mean ± SEM. Statistical significance was determined using one- way ANOVA. ***p < 0.001 and ****p< 0.0001.\n2. In Figure 4G and H, the authors showed the ELISPOT results for three mice in each group. The authors should provide the data for all the mice in supplementary information. The authors tested the synergistic anti-tumor effect with the immune checkpoint inhibitor aPD-1. Did the authors test whether the RM-1 cells express substantial PD-L1 or the vaccine boosted T cells infiltrated in tumors express high levels of PD-1?\nResponse: We thank the reviewer for these constructive comments and the opportunity to clarify the points raised. Regarding the ELISPOT assay in Fig. 4G and H, the three images shown for each group correspond to three independent biological replicates (rather than five), with each data point (well) representing splenocytes from an individual mouse. As the complete dataset for all individual replicates is already presented in the figure, no additional data were included in the Supplementary Information.\nWe fully agree with the reviewer’s second point on the mechanistic importance of the interplay between our vaccine and the PD-1/PD-L1 axis. As noted, sustained antigenic stimulation can drive CD8⁺ T cells toward an exhausted phenotype characterized by high PD-1 expression, which may limit their effector function15. To examine this in our model, we performed immunofluorescence staining on tumor tissues post-vaccination. The analysis revealed an increased frequency of CD8⁺PD- 1⁺ T cells within TCG/FK12F-treated tumors, consistent with robust vaccine-induced T-cell activation. Importantly, this upregulation was effectively reversed upon combination with anti-PD-1 therapy, providing a mechanistic basis for the observed synergistic antitumor effect. We are grateful to the reviewer for highlighting this aspect, which allowed us to strengthen the mechanistic link between vaccine-induced immunity and checkpoint blockade. These new data have been added to the revised Results and fig. S22.\nReference:\n15. Blank, C. U. et al. Defining ‘T cell exhaustion’. Nat. Rev. Immunol. 19, 665–674 (2019).\nAdded: Page 13\nIn addition, immunofluorescence staining revealed elevated PD-1 expression on tumor-infiltrating CD8⁺ T cells following vaccination (fig. S22), suggesting that sustained immune activation induced a state of partial T-cell exhaustion. This finding provides a mechanistic rationale for the enhanced therapeutic benefit achieved upon combination with immune checkpoint blockade.\nfig. S22. Representative immunofluorescence images showing PD-1 expression in RM-1 orthotopic tumors from the different treatment groups.\n3. In Figure 5. The authors should provide the quantified data for tumor fluorescence values for each treatment mice group and indicate comparative analysis of the tumor inhibition efficacy.\nResponse: We thank the reviewer for the helpful suggestion to quantitatively evaluate tumor fluorescence. In response, we have performed this analysis and present the corresponding fluorescence intensity curves for all treatment groups in fig. S17. These curves depict the dynamic changes in tumor- associated fluorescence over the course of treatment. The quantitative data now provide a clearer comparison of therapeutic efficacy across the different groups. Statistical analyses evaluating the significance of tumor growth inhibition have been included. These new results have been incorporated into the revised Results and fig. S17.\nAdded: Page 13\nAs shown in Fig. 5B and fig. S17, treatment with the TCG/FK F vaccine not only significantly 12 inhibited tumor progression but also showed a further enhanced therapeutic effect when combined with aPD-1 therapy.\nfig. S17. In vivo fluorescence quantification curves for RM-1 orthotopic tumors. Data are represented as mean ± SEM (n = 5). Statistical significance was determined using one-way ANOVA. **p < 0.01 and ***p < 0.001.\n4. In Figure 4F and 5F, the authors report the elevated cytokines produced in tumor in TCG/FK F+aPD-1 group. Did the authors test the cytokine levels in serum? It will be a safety concern 12 if persistently elevated IL-6 was induced by this vaccine.\nResponse: We appreciate the reviewer’s concern regarding potential systemic inflammation and safety. To address this directly, we measured serum IL-6 levels by ELISA in mice from all treatment groups. The results show that serum IL-6 was not significantly elevated in mice receiving TCG/FK12F + aPD- 1 compared to the relevant control groups (fig. S8). This indicates that while the vaccine promotes cytokine production within the tumor microenvironment, it does not induce a systemic overproduction of the key pro-inflammatory cytokine IL-6. Together, these data support the favorable safety profile of our vaccine strategy, which achieves potent local immune activation without eliciting uncontrolled systemic inflammation. The corresponding results and interpretation have been added to the revised Results section and fig S8.\nAdded: Page 7\nSpecifically, serum IL-6 levels were comparable to those in healthy mice (fig. S8), indicating that localized TCG/FK F administration does not induce systemic inflammation and exhibits a favorable 12 safety profile.\nfig. S8. Serum IL-6 levels in mice from different treatment groups. Data are represented as mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA.\n5. In Figure 2B and C, PBS was used for the rheological tests. What if the PBS is replaced with serum or cell lysis?\nResponse: We appreciate the reviewer for this insightful question regarding the hydrogel's gelation behavior under physiologically relevant conditions. The addition of PBS to an aqueous FK F solution 12 induces the rapid formation of a self-supporting hydrogel. This is attributed to counterions from PBS, which screen electrostatic repulsion between the nanofibers, promoting their interaction, entanglement, and subsequent hydrogel formation.\nTo address the question directly, we performed additional rheological measurements using serum as the physiologically relevant medium. The results, now included in the revised fig. S3, demonstrate that robust gelation also occurs in serum, with a storage modulus (G′) and gelation kinetics comparable to those observed in PBS. To ensure experimental consistency, all rheological tests for this comparison were conducted under standardized concentration conditions. Consequently, the PBS data in the manuscript have been updated to reflect these unified parameters. These new findings have been incorporated into the revised Results section and fig. S3.\nRevised: Page 5\nWe then simulated the in vivo environment by adding counterions (PBS or serum) to the FK F aqueous 12 solution, and found that the originally flowing solution immediately transformed into a self-supporting hydrogel (Fig. 2B and fig. S3A). Rheological tests corroborated this observation, as the storage modulus (G') rapidly exceeded the loss modulus (G'') upon PBS or serum addition (Fig. 2C and fig. S3B), indicative of a phase transition and enhanced elastic properties.\nfig. S3. Gelation behavior of FK 12 F in serum. A, Photographic demonstration of the rapid solution-to- hydrogel transition of FK F upon addition of serum. B, Rheological measurement (storage modulus 12 G′ and loss modulus G″) of the formed FK F hydrogel in serum. 12\n6. The authors claimed that the therapeutic efficacy was TEV specific, which was distinct from conventional EV vaccines loaded with shared tumor antigens. Did the authors test whether the EVs from unrelated tumor cell lines, or components from EVs, could induce any anti-tumor immune responses or tumor inhibition?\nResponse: We thank the reviewer for raising this important point regarding vaccine specificity. To address this directly, we performed a key control experiment in which TEVs were isolated from a heterologous tumor cell line (B16 melanoma) to formulate a control vaccine (T CG/FK F). This B16 12 was administered to RM-1 tumor-bearing mice using the same protocol as the autologous TCG/FK F 12 vaccine. As shown in the new data (Fig. S14), the heterologous T CG/FK F vaccine showed no B16 12 significant tumor-inhibitory effect compared to the PBS control. This result confirms that the therapeutic efficacy of our platform is antigen-specific and dependent on autologous TEVs, rather than being a nonspecific immune response to generic extracellular vesicle components.\nThis finding underscores that the potent antitumor immunity elicited by our system originates from the tumor-specific antigen repertoire enriched within patient-derived TEVs. These new data have been incorporated into the revised Results section and fig. S14.\nAdded: Page 12\nFurthermore, to confirm that the therapeutic effect depends on TEV-specific antigens, we generated a control vaccine (T CG/FK F) using TEVs from heterologous B16 cells. As shown in fig. S14, this B16 12 control vaccine lacked significant efficacy, supporting the antigen-specific mechanism of action.\nfig. S14. Tumor growth curves following administration of autologous or heterologous TEV vaccines. Mice bearing RM-1 tumors were treated with PBS, TCG/FK F (autologous TEV vaccine), 12 or T CG/FK F (heterologous, B16-derived TEV vaccine). Data are mean ± SEM (n=5). Statistical B16 12 significance was determined using one-way ANOVA. ****p< 0.0001.\n7. Line 489: Splenocytes (1 × 105) were stimulated for 24 h with 1 g/well TEVs (from RM-1 cells). The concentration of TEVs should be re-checked.\nResponse: We thank the reviewer for catching this error and apologize for the oversight. The correct peptide concentration is 1 μg/well. We have corrected this in the Methods section of the revised manuscript.\nRevised: Page 23\nSplenocytes (1 × 105) were stimulated for 24 h with 1 μg/well TEVs (from RM-1 cells).\n8. The reference formats were not consistent and should be carefully re-edited.\nResponse: We sincerely thank the reviewer for highlighting the formatting inconsistencies in the references. In response, we have thoroughly revised the reference list in its entirety to strictly conform to the journal’s required format. We apologize for any inconvenience caused and are grateful for the reviewer’s careful attention to detail."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for providing extensive and informative answers to my comments. The additional experiments and newly added results convincingly support the ability of hydrophobic gels incorporating the vaccine components to overcome tumor-induced immune suppression characterizing the TME. The specificity to T cell responses to the vaccine was also adequately addressed. The methodology used to address the comments was sound and experiments were convincingly presented. The manuscript represents a solid addition to the field of TEV and their potential role as promising anti-cancer vaccines.", "reviewer_2": "The authors have adequately addressed my comments.", "reviewer_3": "The authors have addressed all my concerns."}, "author_response": ""}]

Decision letter