Polypeptide sono-adjuvant for ultrasound-activatable regulation of innate immunity and cancer vaccination therapy

Domain: physics
Published: 2025-12-16
Editorial outcome: minor_revision

Abstract
Spatiotemporally-controllable regulation of innate immunity in lymph node remains a critical challenge to implement effective cancer vaccination. Here we report an ultrasound-activatable strategy to precisely stimulate innate immunity activation in vivo. Mechanistically, ultrasound-triggered mechanical and oxidative forces synchronously activate innate immune pathways in antigen-presenting cells via calcium ion influx and mitochondria DNA release. We next design a polypeptide sono-adjuvant (SONA) library with adjustable physicochemical properties for lymph node-targeting delivery. The top-performed SONA specifically activates robust and durable innate immune responses in lymph node upon localized ultrasound stimulation. The combination of SONA-based vaccine with ultrasound stimulation induces about 3.0-fold higher antigen-specific T cell responses than conventional adjuvant-based vaccines. Moreover, in syngeneic mouse models of orthotopic breast and liver tumors, the combination of SONA-based neoantigen vaccine with ultrasound stimulation markedly boosts immune checkpoint blockade therapy to suppress tumor growth and distant metastasis. Collectively, the polypeptide sono-adjuvant offers great promise for precise regulation of innate immunity and cancer vaccine therapy.

Introduction
The innate immune system is the frontline of host defense against infection or tumor1–3. Antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, play crucial roles in bridging innate and adaptive immunity4–6. APCs can phagocytize and cross-present tumor-associated antigens to induce cytotoxic T lymphocytes (CTLs) response in a tumor-specific manner7,8. However, microenvironment within lymph nodes (LNs) often are ineffective to stimulate APCs maturation, migration, and cytokine secretion, thus deactivating the antigen cross-presentation ability of APCs9–11.

The activation of nucleotide-binding oligomerization domain (NOD)-like receptor pathways and cytosolic DNA-sensing pathways, such as NOD-like receptor protein 3 (NLRP3) inflammasome, stimulator of interferon genes (STING), and Toll-like receptor 9 (TLR9), can stimulate APCs maturation by upregulating type I interferon (IFN-I) and pro-inflammatory cytokine responses12–14. Accordingly, various types of adjuvants targeting the innate immune pathways have been discovered and evaluated in preclinical settings. However, small-molecule adjuvants have shown limited antitumor efficacy and dose-limiting toxicity in clinical trials, which could be associated with their unsatisfactory LNs-targeting efficacy15,16. Alternatively, various adjuvant-integrated nanoparticles have been developed to improve adjuvant delivery to LNs, and generated potent antitumor immunity in preclinical studies17–20. However, most of these strategies exhibited the “always on” profile to stimulate innate immunity in vivo, and could potentially induce immune-related adverse effects due to their non-specific distribution and immune stimulation on untargeted tissues, thus limiting their future clinical translation21–23. Therefore, spatiotemporally-controllable regulation of innate immunity in LNs is of high priority, but remains a critical challenge to implement safe and robust cancer vaccination therapy.

Ultrasound (US), as a clinically-accessible technology, can non-invasively deliver energy into deep tissues up to tens of centimeters. US-triggered mechanical and oxidative force have been exploited to locally activate the intratumoral adaptive T-cell immunity24–29. However, US-triggered activation of innate immunity in LNs has rarely been reported, and the underlying mechanisms of US stimulation on activating innate immune pathways in APCs remain largely unknown.

In this study, we demonstrated that US-triggered mechanical and oxidative forces could simultaneously activate multiple innate immune pathways (NLRP3, STING, and TLR9) in APCs by a US-dependent manner. We next engineered a class of polypeptide-based sonosensitive adjuvant (namely sono-adjuvant, SONA) to implement LNs-specific and APCs-targeting delivery through adjusting the physicochemical parameters (e.g., size, charge, surface ligand). The top-performed SONA specifically drained into LNs and effectively targeted APCs, while displaying marginal off-target distribution in normal organs. It was worth noting that SONA remained in an ‘immune-silent’ state in the absence of US stimulation, since the sonosensitizer itself was incapable of triggering mechanical and oxidative force to activate innate immune responses. In contrast, the combination of LNs-localized US stimulation could precisely trigger the innate immunostimulatory effect of SONA, and induced robust IFN-I and pro-inflammatory cytokine responses in vivo. Moreover, the combination of SONA-based vaccine with US stimulation (SONV+US) elicited antigen-specific T-cell immunity and promoted intratumoral CTLs infiltration, which ultimately displayed more potent antitumor efficacy than the conventional adjuvant-immunotherapies in different mouse models of orthotopic tumors.

Results
US-triggered mechanical and oxidative forces activated innate immune pathways in APCs

We selected the US stimulation frequency to 1 MHz with a 50% duty cycle, and altered the power density and duration of US stimulation to investigate the effect of different levels of mechanical and oxidative forces on activating innate immune pathways in APCs. We screened the power density of US stimulation to avoid unintended cellular damage caused by mechanical force. No obvious cytotoxicity was observed in DC2.4 cells and RAW264.7 cells at US power density below 0.2 W/cm2 (Supplementary Fig. 1). We next investigated the impact of US-triggered mechanical force on innate immune response of APCs (Fig. 1a). Flow cytometry analysis and confocal laser scanning microscope (CLSM) imaging data indicated that US stimulation affected the intracellular calcium ion (Ca2+) concentration of DCs as a function of power density. Specifically, 0.2 W/cm2 of US stimulation induced a 3.1-fold increase of intracellular Ca2+ concentration in DCs compared to PBS group (Fig. 1b–d). The mechanosensitive channel Piezo1 is generally considered to be activated by various types of mechanical stimuli and trigger Ca2+ influx to promote pro-inflammatory immune response30,31. The addition of Dooku-1, a Piezo1-specific antagonist, markedly abrogated Ca2+ influx induced by US stimulation (Fig. 1e), indicating the US-triggered activation of Piezo1 channel in DCs. Ca2+ influx can induce NLRP3 inflammasome assembly and lead to caspase-1-dependent secretion of pro-inflammatory cytokines (IL-1β, IL-18)32,33. Western blot and enzyme linked immunosorbent assay (ELISA) further validated that US-triggered mechanical force activated the NLRP3-Capase-1-IL-1β signaling in APCs (Fig. 1f, g). Specifically, 3 min of US stimulation (0.2 W/cm2, 1 MHz, 50% duty cycle) induced 3.9- and 4.3-fold increase of IL-1β release and activated caspase-1 expression in DCs compared to PBS group, respectively (Fig. 1h). Moreover, the addition of Dooku-1 significantly inhibited US-triggered active caspase-1 release in DCs (Fig. 1i), further confirming the critical role of Piezo1 in US-triggered innate immune activation.

On the basis of mechanical activation of innate immunity by US stimulation, we next investigated whether the combination of US-triggered oxidative force could amplify the innate immune activation of APCs. We thus synthesized a biodegradable polypeptide via ring-opening polymerization of tyrosine-N-carboxyanhydrides (NCA). A sonosensitive polypeptide was subsequently obtained by conjugating a clinically applied sonosensitizer, hematoporphyrin monomethyl ether (HMME), onto the end amino group of the polypeptide (Supplementary Figs. 2 and 3). The sonosensitive polypeptide could self-assemble into a sonosensitive nanoparticle (SONP) for precisely modulating oxidative forces in APCs (Fig. 1j). We optimized the US power density at 0.2 W/cm² and the HMME concentration at 1.0 μg/mL, and further screened the duration of US stimulation to avoid unintended cellular damage caused by the combination of mechanical and oxidative stress. The result showed that negligible cytotoxicity was observed in SONP+US-treated DC2.4 cells and RAW264.7 cells at US duration below 4 min (Supplementary Fig. 4). We next used the reactive oxygen species (ROS) indicator, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), to detect the generation level of oxidative force by SONP+US in DCs. CLSM images showed that only SONP incubation without US stimulation showed no ROS generation effect, while SONP+US could trigger the intracellular ROS generation in DCs as a function of US stimulation duration (Fig. 1k). Meanwhile, SONP+US was demonstrated to exert endoplasmic reticulum (ER) stress and trigger mitochondrial DNA release in DCs (Fig. 1m), and this effect could be specifically abrogated by N-Acetyl-L-cysteine (NAC)-mediated ROS scavenging (Fig. 1l).

ER stress-mediated mitochondrial DNA release was demonstrated to activate both TLR9 and STING pathways34,35. We therefore evaluated whether SONP+US activated these innate immune pathways in DCs. As expected, SONP+US (1 MHz, 50% duty cycle, 0.2 W/cm2, 3 min) obviously activated TLR9-myeloid differentiation primary response 88 (MyD88)-interferon regulatory factor 7 (IRF7) and STING-TANK-binding kinase-1 (TBK1)-interferon regulatory factor 3 (IRF3) axes, as well as their downstream canonical nuclear factor κB (NF-κB) pathway in DCs by a US-dependent manner (Fig. 1n). Moreover, the addition of TLR9 inhibitor (TLR9-i) and STING inhibitor (STING-i) effectively abrogated the activation of above innate immune pathways induced by SONP+US in DCs (Supplementary Fig. 5).

Next, we compared the effect of US-triggered mechanical and oxidative forces on inducing innate immune pathways activation in APCs. WB analysis showed that only US stimulation-mediated mechanical force moderately activated NLRP3 pathway in DC2.4 and RAW264.7 cells, while merely affecting STING and TLR9 pathways activation. In comparison, SONP+US could simultaneously trigger the mechanical and oxidative forces, and effectively activate STING, TLR9 and NLRP3 pathways in DC2.4 and RAW264.7 cells, as evident from the upregulation of indicated proteins expression, such as phosphorylated-TBK1 (p-TBK1), phosphorylated-IRF3 (p-IRF3) and phosphorylated-IRF7 (p-IRF7) (Supplementary Fig. 6). In addition, we evaluated the effect of mechanical and oxidative forces on stimulating DC maturation in vitro. The result showed that US-triggered mechanical force moderately enhanced DC maturation level (CD86+, 8.9%) compared with PBS group (CD86+, 5.7%). In comparison, the combination of mechanical and oxidative forces mediated by SONP+US significantly increased the frequency of CD86+ DCs (15.1%), showing a 2.6- and 1.7-fold higher than that in PBS and US groups, respectively (Supplementary Fig. 7).

Furthermore, we found that SONP+US obviously elicited pro-inflammatory cytokine (TNF-α, IL-6) secretion by DCs (Fig. 1o), and upregulated type-I interferon-related genes (ISG) expression in THP1-ISG-Blue and RAW264.7-ISG-Lucia cells (Fig. 1p). In contrast, SONP incubation without US stimulation negligibly affected DCs maturation and innate immune activation. We also examined whether SONP+US affected other immune cell types beyond APCs, such as T cells and B cells. The CD3+ T cells and CD19+ B cells were isolated from untreated-mouse LNs by using fluorescence-activated cell sorting (Supplementary Fig. 8). The isolated T cells and B cells were subsequently treated in vitro with US, SONP, SONP+US for 24 h. Western blot result showed that there were no significant changes in the expression levels of p-TBK1, p-IRF3, and NLRP3 proteins in either CD3+ T cells or CD19+ B cells following SONP+US treatment compared to PBS group (Supplementary Fig. 9). These findings suggested that SONP+US may not effectively activate innate immune pathways in T cells and B cells.

Collectively, these findings demonstrated that SONP+US offered a spatiotemporally-controllable approach to stimulate multiple innate immune pathways in APCs by combing US-triggered mechanical and oxidative forces.

Engineering polypeptide sono-adjuvant for US-activatable activation of innate immunity in LNs

It has been extensively demonstrated that the formulations to spatially confine adjuvant activity within the secondary lymphoid organs are crucial for enhancing cancer vaccine-mediated T-cell response36–39. To optimize LNs-specific and APCs-targeting delivery, we developed a set of polypeptide-based sonosensitive adjuvant (namely sono-adjuvant, SONA) with adjustable physicochemical properties, including particle size, surface charge, and ligand modification, as shown in the corresponding table in Fig. 2a. Inspired by the mannose receptor was highly expressed in APCs40–42, we introduced an active-targeting moiety, mannose, for modifying the sonosensitive polypeptide (SOP) to improve the APCs-targeting performance (Supplementary Figs. 10 and 11). Meanwhile, a series of charge-modified polypeptides (CMPn) were synthesized via ring-opening co-polymerization of lysine-NCA and tyrosine-NCA (Supplementary Figs. 12 and 13). Through the co-assembly of SOP with CMPn, we obtained SONA1-12 that displayed adjustable particle size ranging from 128.7 to 169.5 nm, surface charge ranging from −57.1 mV to +9.5 mV, and with or without mannose modification. SONA1-12 were subcutaneously injected into the tail base of C57BL/6 mice, and ex-vivo fluorescence imaging of major organs was conducted at 12 h post-injection. SONA1-12 all effectively accumulated in LNs with minimal distribution in heart, liver, spleen, lung and kidney (Fig. 2b). Semi-quantification of the fluorescence signal screened out the top-performed SONV4, with a particle size of 141.3 nm and surface charge of +8.7 mV, which exhibited the highest LNs distribution in vivo (Fig. 2c, d and Supplementary Fig. 14). In addition, compared with SONA3 that without mannose-modification, SONA4 displayed 2.1- and 2.3-fold higher internalization by DCs and macrophages in LNs, respectively (unless noted, all SONA mentioned below refers to SONA4) (Fig. 2e and Supplementary Fig. 15).

We investigated the influence of different US stimulation parameters (timepoint, power density, duration) on controlling the adjuvant activity of SONA in vivo. The biodistribution of SONA in LNs was evaluated by utilizing the fluorescence imaging capacity of porphyrin-based sonosensitizer. Upon subcutaneous injection at the tail base, SONA gradually accumulated into LNs and peaked at 6 h post-injection (Supplementary Fig. 16). We thus selected the 6 h post SONA injection as the experimental timepoint for LNs-localized US stimulation. We next altered the power density and duration of US stimulation to investigate the effect of different levels of mechanical and oxidative forces on innate immune activation in LNs. The LNs were locally stimulated with 5 min of indicated power density of US (0, 0.2, 0.5, 1.0, 1.5 W/cm2) at 6 h post SONA injection. The results revealed that only SONA injection induced negligible innate immunity in LNs. In contrast, the combination of LNs-localized US stimulation effectively activated innate immune responses in vivo, and 1.0 W/cm2 of US stimulation elicited the highest recruitment and maturation of DCs and macrophages in LNs (Fig. 2f, g and Supplementary Fig. 17). We subsequently optimized the duration of US stimulation to achieve the optimal LNs-specific innate immune activation in vivo. The LNs were locally stimulated with 1.0 W/cm2 of indicated duration of US (0, 1, 3, 5, 7 min) at 6 h post SONA injection. The results showed that the recruitment and maturation levels of DCs in LNs gradually increased with the increase of US stimulation duration, and it almost reached the peak value when combined with 5 min of US stimulation (Supplementary Fig. 18). Therefore, we screened out the timepoint (6 h post injection), power density (1.0 W/cm2) and duration (5 min) of LNs-localized US stimulation for activating innate immunity in vivo. Under these US stimulation parameters, we found the combination of SONA injection with US stimulation (namely SONA+US) could induce abundant and sustained IFN-β and IL-6 secretion in LNs for over 48 h (Fig. 2h), and these cytokine levels gradually declined toward baseline by 72 h post-treatment, indicating a transient but substantial local innate immune responses induced by SONV+US.

We next examined the innate and adaptive immune responses in tumor-draining lymph nodes (tdLNs) of 4T1 breast tumor-bearing mice (Fig. 2i). SONA treatment induced no effect on DCs maturation and T cell activation compared with PBS group. In marked contrast, the frequency of matured DCs (CD80+CD86+) in SONA+US group was about 16.1%, showing 2.0- and 1.8-fold higher than that in PBS group (8.1%) and SONA group (8.7%), respectively (Fig. 2j and Supplementary Fig. 19a). The frequency of activated CD8+ T cells (CD69+) in SONA+US group was about 15.1%, showing 2.2- and 2.4-fold higher than that in PBS group (6.74%) and SONA group (6.04%), respectively (Fig. 2l and Supplementary Fig. 19c). It was worth noting that IFN-β was demonstrated to directly inhibit Tregs proliferation and reprogram M2-type macrophages43–45. Therefore, a significant decrease in the frequency of immune-suppressive cells in tdLNs, including Tregs (FOXP3+) and M2-type macrophages (CD206+), could be observed after SONA+US treatment (Fig. 2k, m and Supplementary Fig. 19b, d). Western blot analysis revealed that SONA+US activated the STING, TLR9, and NLRP3 innate immune pathways, as demonstrated by the upregulation of indicated proteins expression in tdLNs (Fig. 2n, o). Immunofluorescence staining of tdLNs further validated that SONA+US significantly increased the proliferation level of T cells than SONA treatment (Fig. 2p). These results collectively demonstrated that SONA+US provided a spatiotemporally-controllable strategy to induce region-confined activation of antitumor immunity in LNs.

SONA+US-boosted cancer vaccination elicited robust antigen-specifc T cell immunity

Spatiotemporally-controllable orchestration of antigen cross-presentation with efficient innate immune stimulation in APCs remains a critical challenge for inducing safe and potent antigen-specific T-cell response46–48. Herein, we sought to explore the potential of SONA as a generalizable vaccine platform for US-activatable antigen cross-presentation and T cell activation (Fig. 3a). Through the electrostatic interaction, a model antigen ovalbumin (OVA) was loaded into SONA to prepare the sonosensitive vaccine (sono-vaccine, namely SONV). Dynamic light scattering (DLS) and transmission electron microscope (TEM) analysis revealed that SONV exhibited a uniform spherical morphology, with hydrodynamic diameter of 138.6 ± 11.1 nm and surface charge of +5.7 mV (Fig. 3b, c).

Lysosomal trapping of tumor antigen usually limits the cancer vaccination efficiency49,50, which prompts us to investigate the performance of SONV for facilitating antigen lysosomal escape and cytosolic release. We performed an acridine orange staining assay to investigate the influence of US-triggered mechanical and oxidative forces on lysosomal membrane permeability of DCs. The result showed that only US stimulation-mediated mechanical force was demonstrated to increase the permeability of lysosomal membrane through the cavitation effect, while only SONV incubation showed no obviously sosomal disruption effect (Supplementary Fig. 20). In comparison, the combination of mechanical and oxidative forces mediated by SONV+US (1 MHz, 50% duty cycle, 0.2 W/cm2, 3 min) could further increase the lysosomal membrane permeability of DCs. In addition, obviously disrupted lysosomal structures were observed in the Bio-TEM images of SONV+US-treated DCs (Supplementary Fig. 21).

We employed a FITC-labelled OVA (OVA-FITC) to monitor the internalization and intracellular distribution of tumor antigen. Flow cytometry analysis revealed that SONV-FITC displayed 2.6-fold higher cellular uptake efficacy in DCs than free OVA-FITC at 12 h post incubation (Supplementary Fig. 22). CLSM examination revealed that the OVA-FITC signal in SONV group was majorly entrapped inside the lysosomal vesicles. In contrast, SONV+US significantly induced antigen cytosolic release, with most of OVA-FITC signal detected in the cytosol while not in lysosome (Supplementary Fig. 23). Specifically, the Pearson’s R value (PR) of OVA-FITC signal (green) with lysosomal signal (red) in SONV and SONV+US groups were 0.25 and −0.12, respectively. These results collectively confirmed that SONV+US offered a US-activatable approach to promote antigen cytosolic delivery by combing the US-triggered mechanical and oxidative force.

We subsequently examined the ability of SONV+US to induce antigen cross-presentation and T cell activation in vitro. CpG as a TLR-9 agonist and MSA-2 as a STING agonist were selected as the control adjuvants. Only SONV incubation barely affected DCs maturation and T cell activation. In contrast, SONV+US efficiently induced DCs maturation and elicited 2.5 and 2.0-fold more IL-1β secretion compared to OVA+CpG and OVA+MSA-2 groups, respectively (Supplementary Fig. 24). Flow cytometry analysis revealed that SONV+US promoted 2.8 and 2.6-fold higher antigen presentation in DCs than OVA+CpG and OVA+MSA-2 groups, respectively (Supplementary Fig. 25a). CLSM examination further confirmed that SONV+US promoted antigen presentation on the surface of DCs (Supplementary Fig. 25b). In addition, SONV+US-pretreated DCs most efficiently activated OT-1 T cells, with 2.1 and 1.9-fold higher proliferation level than OVA + CpG and OVA+MSA-2 groups, respectively (Supplementary Fig. 26).

We further validated the significance of innate immune pathways activation for DCs maturation and antigen presentation induced by SONV+US. Treatment with STING-IN2, an inhibitor of STING, was able to decrease the SONV+US-induced BMDCs maturation and antigen presentation levels (Supplementary Fig. 27). Likewise, using the TLR9 inhibitor AT791 and the NLRP3 inflammasome assembly inhibitor NLRP3-IN-10, we demonstrated that SONV+US stimulated BMDCs maturation by activating both the TLR9 and NLRP3 pathways (Supplementary Fig. 28). Collectively, above data demonstrated that SONV+US provided a US-activatable approach to orchestrate antigen cross-presentation with efficient innate immune stimulation in DCs.

To investigate the performance of SONV for LNs-targeting antigen delivery in vivo, C57BL/6 mice were subcutaneously injected with free OVA-FITC or OVA-FITC-loaded SONV. Ex vivo fluorescence imaging showed minimal distribution of free OVA-FITC in LNs and major organs. In contrast, SONV-FITC effectively drained into LNs and showed approximately 2.5-fold higher fluorescence signal than free OVA-FITC at 6 h post injection (Fig. 3d). Immunofluorescence staining of LN observed obvious co-localization of antigen (OVA-FITC, green), sono-adjuvant (SONA, red) with CD11c+ DCs (Fig. 3e). Flow cytometry analysis further demonstrated that SONV-FITC 3.8- and 4.2-fold more efficiently delivered antigen to DCs and macrophages in LNs than free OVA-FITC, respectively (Supplementary Fig. 29). These results together confirmed the great targeting ability of SONV to deliver tumor antigen into APCs in LNs.

We next assessed the impact of different US stimulation parameters on the long-term T cell responses induced by SONV+US. We selected the US frequency of 1 MHz, the duty cycle of 50%, and altered the power density (0.5, 1.0, 1.5 W/cm²) and duration (0, 3, 5 min) of US stimulation. The dynamic activation of antigen-specific CD8+ T cells and memory CD8+ T cells in the peripheral blood of SONV+US-treated mice was examined by flow cytometry (Supplementary Figs. 30 and 31). The results revealed that only SONV treatment induced moderate activation of antigen-specific CD8+ T cell response, and the frequency of Tetramer+ CD8+ T cells was marginally increased compared to PBS group at day 28 (Supplementary Fig. 32). In contrast, the combination of SONV with LNs-localized US stimulation effectively enhanced antigen-specific CD8+ T cell activation. Specifically, it was observed that US stimulation under the conditions of 1.0 W/cm² for 5 min or 1.5 W/cm² for 3 min elicited robust and durable antigen-specific CD8+ T cell responses, reaching the highest levels among all tested conditions. We subsequently evaluated the activation of immune memory responses in the peripheral blood. The result showed that SONV+US (1.0 W/cm²+5 min) treatment induced high-quality and long-term T cell immune responses, showing the highest levels of central memory CD8+ T cell (CD44+CD62L+) and effector memory CD8+ T cell (CD44+CD62L−) responses among all tested conditions (Supplementary Fig. 33).

The performance of SONV+US (1.0 W/cm²+5 min) for inducing antigen-specific immune response in vivo was next examined. C57BL/6 mice were subcutaneously injected with SONV three times at a 7-day interval, followed with LNs-localized US stimulation at 6 h post per injection. We found that SONV+US treatment significantly triggered the expression of stimulatory markers (CD80 and CD86) in macrophages and DCs in the LNs (Fig. 3f, g and Supplementary Fig. 34a, b). The maturation degree of DCs (CD80+CD86+ in CD11c+ cells) in SONV+US group was 22.5%, showing 2.2- and 1.6-fold higher than that in OVA+CpG (10.1%) and OVA+MSA-2 (14.2%) groups, respectively. In addition, SONV+US treatment induced the highest level of antigen-specific peptide-MHC-I complex on the surface of DCs (8.2%, SIINFEKL-H2Kb+ in CD11c+ cells), with 2.5- and 1.9-fold higher than that in OVA+CpG and OVA+MSA-2 groups, respectively (Fig. 3h and Supplementary Fig. 34c). More importantly, the frequency of antigen-specific CD8+ T cells (SIINFEKL-H2Kb tetramer+ in CD8+ cells) in SONV+US group was about 10.1%, while that in OVA + CpG and OVA+MSA-2 groups were only about 2.5% and 3.4%, respectively (Fig. 3i and Supplementary Fig. 35a). Upon ex-vivo restimulation with the SIINFEKL peptide, the splenocytes from SONV+US group mice showed the highest IFN-γ+ CD8+ T cell and TNF-α+ CD8+ T cell responses among all the groups. Specifically, the frequency of IFN-γ+ in CD8+ T cells in SONV+US group was about 24.3%, showing 2.5- and 1.9-fold higher than that in OVA+CpG group (9.8%) and OVA+MSA-2 group (12.6%), respectively (Fig. 3k and Supplementary Fig. 35b, c). CTLs killing experiment confirmed that SONV+US elicited the highest antigen-specific T cell killing effect among all the groups, while no significant increase was observed after SONV treatment than PBS group (Fig. 3j and Supplementary Fig. 35d).

We next examined the transcriptomic features of LNs from SONV+US group mice to investigate the underlying mechanism of innate and adaptive immunity activation. 1329 differentially expressed genes (DEGs) were identified between SONV+US with PBS group (Fig. 3m), and some innate immune pathways-relevant genes (Tlr6, Tlr7, Tlr9, Cgas, Sting1, Ifnab, Nlrp3 and Il1b) were upregulated, while some immune tolerance-relevant genes (Tgfbr1, Tgfb3, Smad1 and Ly6g6e) were downregulated after SONV+US treatment (Fig. 3n, o). Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified that DEGs were enriched in multiple innate immune pathways, such as Toll-like receptor signaling pathway, cytosolic DNA-sensing pathway, NOD-like receptor signaling pathway and RIG-1-like receptor signaling pathway (Fig. 3p, q). These results confirmed that SONV+US provided a US-activatable strategy to coordinatively stimulate multiple innate immune pathways in APCs of LNs and amplify antigen-specific T-cell immunity (Fig. 3l).

We next evaluated and compared the immune-related side effects of SONV+US with OVA + CpG and OVA+MSA-2 by measuring the systemic levels of inflammatory cytokines (IL-6 and IL12p70). The results demonstrated that mice treated with OVA + CpG or OVA+MSA-2 exhibited pronounced systemic cytokine release in serum, with IL-6 concentrations reaching about 219.2 pg/mL (OVA+CpG), 126.0 pg/mL (OVA+MSA-2) and IL-12p70 concentrations reaching about 34.0 pg/mL (OVA + CpG), 26.2 pg/mL (OVA+MSA-2) at 1 day post each injection (Supplementary Fig. 36). These cytokine levels gradually declined but remained slightly above baseline by day 7 post each injection, indicating a transient but substantial systemic inflammatory response induced by OVA + CpG and OVA+MSA-2. In contrast, mice treated with the SONV+US showed only a modest increase in IL-6 (35.8 pg/mL) and IL-12p70 (10.1 pg/mL) secretion at 1 day post each injection. Importantly, inflammatory cytokine levels were found to decline toward baseline and remain at physiological levels by day 7 post injection. These results demonstrated that SONV+US reduced systemic immune activation and exhibited good long-term safety compared to conventional adjuvant-based vaccination strategies.

SONV+US reshaped intratumoral immune microenvironment to suppress tumor growth and metastasis

Given the promising effect of SONV+US to elicit antigen-specific T-cell immunity in vivo, we continued to evaluate the prophylactic efficacy of US-activatable vaccination strategy in the B16-OVA melanoma tumor model. C57BL/6 mice were subcutaneously injected with OVA+CpG, OVA+MSA-2 and SONV three times at 7-day intervals, followed with LNs-localized US stimulation of SONV+US group mice was conducted at 6 h post per injection. The mice were inoculated with B16-OVA cells at 7-day post final treatment, and tumor growth was subsequently monitored (Fig. 4a). SONV treatment showed little efficacy to prevent B16-OVA tumor growth and induced no obvious survival benefit compared to PBS group. In contrast, mice immunized with SONV+US maintained an evidently higher survival rate than OVA + CpG and OVA+MSA-2 groups, with 3/6 of mice had no detectable tumor mass up to 60 days (Fig. 4b and Supplementary Fig. 37a). To assess the induction of long-term immune memory effect, the cured mice in SONV+US group were rechallenged with B16-OVA cells, and the result demonstrated 100% of cured mice prevented secondary tumor growth (Fig. 4e, f and Supplementary Fig. 37b). Notably, the frequency of effector CD8+ T memory cells and central CD8+ T memory cells in SONV+US group mice were markedly increased compared to the naïve mice on day 100 (Fig. 4g).

To evaluate the potential of SONV+US to inhibit lung metastases of tumor cells, immunized mice were intravenously inoculated with B16-OVA cells, and the lung tissues were harvested to quantify the metastatic nodules at day 14 after tumor inoculation. SONV treatment induced negligible anti-metastases efficacy compared to PBS group, and OVA + CpG, OVA+MSA-2 afforded modest protection against tumor metastases. In contrast, SONV+US treatment effectively suppressed the formation of lung metastatic nodules, showing 4.0-fold higher anti-metastases efficacy than SONV treatment (Fig. 4d).

The therapeutic efficacy of SONV+US to eradicate established tumors was subsequently investigated in the B16-OVA melanoma model. C57BL/6 mice were inoculated with B16-OVA cells in the right flank and treated with indicated formulations 7 days later (Fig. 4h). SONV treatment negligibly affected tumor growth and mice survival compared with PBS group. OVA+CpG and OVA+MSA-2 only moderately inhibited tumor growth and all the mice succumbed to tumors within 30 days (Fig. 4i and Supplementary Fig. 38a). SONV+US treatment markedly inhibited tumor growth and prolonged the median survival of mice from 20 days (PBS group) to 36 days, with 1/6 of mice produced complete tumor regression over 60 days (Fig. 4j). The body weight of all mice changed negligibly when exposed to the indicated treatments (Supplementary Fig. 38b).

Mechanistic studies were next performed to investigate whether SONV+US treatment elicited antitumor immunity in tdLNs and tumor microenvironment (Supplementary Figs. 39 and 40). We found that SONV+US strongly promoted DCs maturation and macrophages activation in tdLNs (Supplementary Fig. 41). The maturation degree of DCs (CD80+ in CD11c+ cells) in SONV+US group was 28.8%, showing 1.8- and 1.5-fold higher than that in OVA+CpG (16.2%) and OVA+MSA-2 (19.2%) groups, respectively. Compared to the SONV treatment that elicited negligible antitumor immunity, SONV+US significantly enhanced tumor-infiltration of CD8+ T cells (Fig. 4k and Supplementary Fig. 42a), and increased the activation of CTLs. The frequency of IFN-γ+ CD8+ T cells (IFN-γ+ in CD8+ T cells) in SONV+US group was about 19.8%, with 2.5 and 1.7-fold higher than that in OVA+CpG (7.9%) and OVA+MSA-2 (11.6%) groups, respectively (Fig. 4l and Supplementary Fig. 42b). In addition, compared with PBS group, SONV+US treatment increased the intratumoral progenitor exhausted (PD1+TIM3−) CD8+ T cells infiltration, while decreased the tumor-infiltration of terminally exhausted (PD1+TIM3+) CD8+ T cells. The frequencies of progenitor exhausted CD8+ T cells (PD1+TIM3− in CD8+CD3+ cells) and terminally exhausted CD8+ T cells (PD1+TIM3+ in CD8+CD3+ cells) in SONV+US group were about 28.4% and 19.4%, showing 2.2-fold higher and 3.0-fold lower than that in PBS group, respectively (Supplementary Fig. 42c–e). These results indicated that SONV+US treatment not only enhanced intratumoral CD8+ T cell infiltration and activation, but also was promising to address intratumoral CD8+ T cell exhaustion.

Next, we measured the cytotoxic effect of tumor-infiltrating NK cells and the suppressive function of tumor-infiltrating Tregs in order to provide clearer insights about SONV+US-mediated antitumor immunity. The findings showed that SONV+US treatment increased the intratumor cytotoxic NK cell response (IFN-γ+ NK cells and GZMB+ NK cells) compared with PBS group. The frequencies of IFN-γ+ NK cells (IFN-γ+ in NK1.1+ cells) and GZMB+ NK cells (GZMB+ in NK1.1+ cells) in SONV+US group were about 12.5% and 11.6%, respectively, showing 2.1- and 2.0-fold higher than that in PBS group (IFN-γ+ NK, 5.96% and GZMB+ NK, 5.56%), respectively (Supplementary Fig. 43). In addition, SONV+US treatment not only decreased the frequency of tumor-infiltrating Tregs, but also inhibited the production of Tregs-derived suppressive cytokines (IL-4, IL-10). The frequencies of tumor-infiltrating IL10+ Tregs (12.6%, IL10+ in CD25+FOXP3+ cells) and IL4+ Tregs (10.6%, IL4+ in CD25+FOXP3+ cells) in SONV+US group displayed 3.1- and 3.2-fold lower than that in PBS group (IL10+ Tregs, 39.2% and IL4+ Tregs, 33.3%), respectively (Fig. 4m and Supplementary Fig. 44). Importantly, SONV+US also relived the immunosuppressive tumor microenvironment by reducing intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) (Fig. 4n and Supplementary Fig. 45a), and repolarizing tumor-supportive M2-type tumor-associated macrophages (TAMs) to tumor-suppressive M1-type TAMs (Fig. 4o and Supplementary Fig. 45b, c). ELISA result revealed that SONV+US significantly increased intratumoral secretion of TNF-α, while inhibited the typical immunosuppressive cytokine (TGF-β) secretion (Fig. 4p and Supplementary Fig. 46). The intratumoral TNF-α secretion levels in OVA+CpG, OVA+MSA-2, and SONV+US group was about 1189.6 pg/mL, 1996.3 pg/mL, and 2346.3 pg/mL, respectively. The intratumoral TGF-β secretion levels in OVA+CpG, OVA+MSA-2, and SONV+US group was about 2515.0 pg/mL, 1593.6 pg/mL, and 835.8 pg/mL, respectively. Immunofluorescence staining of tumor sections further confirmed the excellent performance of SONV+US to promote the intratumoral infiltration of CD8+ T cells (Fig. 4q). These results together suggested the potent prophylactic and therapeutic efficacy of the US-activatable vaccination strategy against tumor growth and metastases (Fig. 4r).

Multivalent activation of innate immunity was essential for the antitumor efficacy of SONV+US

We next investigated whether SONV+US-mediated antitumor efficacy were attributed to the US-activatable activation of innate immunity in vivo. A TLR9-specific antagonist, AT79151,52, was intraperitoneally injected to block the interaction of mtDNA with TLR9 (Fig. 5a). We found that TLR9 blockade not only significantly diminished the tumor-inhibitory effect of SONV+US, but also reduced the survival time of B16OVA tumor-bearing mice (Fig. 5b, c and Supplementary Fig. 47).

Further evaluation of antitumor immunity in tdLNs revealed that TLR9 blockade highly decreased co-stimulatory molecules (CD80, CD86) expression on macrophages and DCs that elicited by SONV+US treatment (Fig. 5d, f and Supplementary Fig. 48a, b). TLR9-i injection remarkably decreased the antigen presentation level on APCs and antigen-specific CD8+ T cell response in tdLNs induced by SONV+US treatment (Fig. 5e, g). Specifically, the frequency of antigen-specific CD8+ T cell in SONV+US group (6.9%) was 2.2-fold higher than that in TLR9-i+SONV+US group (3.1%) (Fig. 5h and Supplementary Fig. 48c–e). TLR9-i injection almost completely abolished SONV+US-mediated CTLs infiltration, and the frequency of tumor-infiltrating IFN-γ+ CD8T+ cell in SONV+US group (9.5%) was 4.5-fold higher than that in TLR9-i+SONV+US group (2.1%) (Fig. 5i, j and Supplementary Fig. 49a, b). Meanwhile, the ability of SONV+US to model immunosuppressive tumor microenvironment, including M2-type TAMs repolarization, decrease of Tregs and MDSCs, was markedly impaired by TLR9 blockade (Fig. 5k–m and Supplementary Fig. 49c–f).

Given the fact that IFN-I play essential roles in promoting antigen presentation and CTLs tumor-infiltration53–55, we next visualized antigen-presented DCs in tdLNs and tumor-infiltrating CD8+ T cells in wild-type or type I IFNs receptor (IFNAR)-KO mice after SONV+US treatment. Immunofluorescence staining results showed that IFNAR-knockout abrogated the antigen presentation of DCs in tdLNs and the recruitment of CD8+ T cells in tumors in SONV+US-treated mice (Fig. 5n, o). These data implied that activation of STING-IFN-I signaling is crucial for SONV+US-mediated adaptive antitumor immunity activation.

A monoclonal antibody against IL-1β (anti-IL-1β) was intraperitoneally injected to block the NLRP3-IL-1β signaling in vivo (Fig. 5p). We found that IL-1β neutralization not only markedly impaired the tumor-inhibitory effect of SONV+US, but also reduced the survival time of B16OVA tumor-bearing mice (Fig. 5q, r and Supplementary Fig. 50).

To identify whether activation of CD8+ T cell response was required for SONV+US-mediated antitumor efficacy, we used a monoclonal antibody against CD8 (anti-CD8) to deplete CD8+ T cells in vivo (Fig. 5s). We performed transient CD8⁺ T cell depletion by using monoclonal antibodies to preserve the overall immune architecture and better mimic a functional immune system under therapeutic pressure. This approach allowed to identify and specifically assess the role of CD8⁺ T cells without introducing broader developmental or compensatory alterations that often observed in genetically immunodeficient strains. Moreover, antibody-mediated depletion was a widely used, well-established method to interrogate T cell involvement in preclinical studies32,48. The result showed that the tumor-inhibitory effect of SONV+US was completely abolished by CD8+ T cell depletion, and resulted in largely reduced survival time of tumor-bearing mice, indicating the indispensable role of CD8+ T cells in SONV+US-mediated antitumor immunity (Fig. 5t, u and Supplementary Fig. 51). These results together demonstrated that the US-activatable multivalent innate immunity stimulation and subsequent CD8+ T cell activation is essential to generate SONV+US-based antitumor immunity.

Neoantigen-based SONV+US boosted ICB therapy in orthotopic tumor models

The identification of tumor-specific neoantigen has attracted great interest in developing personalized therapeutic cancer vaccines56,57. Although the synthetic neoantigen peptides elicit T-cell immunity in vitro, a major issue remaining is their overall low immunogenicity and poor LNs-specific distribution in vivo58. To demonstrate the therapeutic potential of SONA platform for neoantigen vaccine immunotherapy, a triple-negative breast cancer (TNBC) cell, 4T1-specific neoantigen peptide (SHRSCSHQTSAPSPKALAHNGTPRNAI) was synthesized and assembled with SONA for preparing neoantigen-based sonosensitive vaccine (Ne-SONV) (Fig. 6a). Ne-SONV displayed a uniform hydrodynamic diameter of 127.6 ± 24.1 nm and positive surface charge of +5.4 mV (Supplementary Fig. 52).

Compared to the admixture of neoantigen peptide with CpG adjuvant (Neop+CpG) that moderately inhibited the orthotopic 4T1 tumor growth and all the mice lost within 30 days, Ne-SONV+US markedly inhibited tumor growth and prolonged the median survival of mice from 26 days (PBS group) to 40 days (Fig. 6b and Supplementary Fig. 53). Notably, the combo of Ne-SONV+US with programmed death-ligand 1 (PD-L1) blockade (Ne-SONV+US+αPD-L1) further extended the mice survival and 3/6 of mice survived above 60 days (Fig. 6c, d). Lung metastasis is the major cause of death in patients with TNBC. The mice of PBS or Neop+CpG group were observed with severe lung metastasis in 30 days post primary tumor inoculation (Fig. 6e). In contrast, Ne-SONV+US and Ne-SONV+US+αPD-L1 treatment dramatically increased the CD4+ and CD8+ T cells infiltration into lung tissue, and effectively inhibited the lung metastasis formation (Fig. 6f, g).

We next investigated whether Ne-SONV+US elicited potent intratumoral antitumor immunity in the orthotopic 4T1 tumor model (Supplementary Fig. 54). According to the t-SNE plots, we could intuitively observe the changes in the tumor-infiltration and corresponding programmed cell death protein 1 (PD-1) expression levels of different immune cells after indicated treatments. Compared with Neop+CpG treatment, Ne-SONV+US significantly increased intratumoral immune cells infiltration, including DCs, TAMs, CD8+ T cells, CD4+ T cells, and NK cells (Fig. 6h, i). Meanwhile, Ne-SONV+US treatment downregulated the PD-1 expression on TAMs when compared with PBS group, while marginally influenced the PD-1 expression on intratumoral CD8+ or CD4+ T cells (Fig. 6j). We found that Ne-SONV+US treatment recruited 3.5- and 2.3-fold higher of IFN-γ+ CD8+ T cells (15.2%, IFN-γ+ in CD8+ T cells) and TNF-α+ CD8+ T cells (6.38%, TNF-α+ in CD8+ T cells) than Neop + CpG, respectively (Supplementary Fig. 55). Combination therapy with αPD-L1 further increased the proportion of tumor-infiltrating CTLs. The frequency of IFN-γ+ CD8+ T cells and TNF-α+ CD8+ T cells in Ne-SONV+US+αPD-L1 group was about 24.9% and 11.2%, showing 5.8- and 4.0-fold higher than that in Neop+CpG treatment, respectively. Ne-SONV+US and Ne-SONV+US+αPD-L1 treatment also reversed the immunosuppressive tumor microenvironment, as indicated by the significant decrease of Tregs, M2-type TAMs and increase of M1-type TAMs (Fig. 6k and Supplementary Fig. 56). These results together demonstrated that Ne-SONV+US was able to enhance the immunogenicity of neoantigen and elicit robust cytotoxic CD8+ T cell response in solid tumors.

To demonstrate the therapeutic genericity of Ne-SONV+US strategy in solid tumors, we further evaluated the performance of Ne-SONV+US for boosting ICB therapy in the orthotopic hepatocellular carcinoma (HCC) model. HCC remains one of the most diagnosed solid tumors with poor survival in the clinic59. ICB therapy have shown promising antitumor efficacy in partial HCC patients; however, the advanced HCC patients marginally benefit from ICB therapy due to limited tumor-infiltration of effector T cells60,61. Our previously identified HCC cell, Hepa1-6-specific neoantigen peptide (RWLYWQPTL), was synthesized and assembled with SONA for preparing Ne-SONV vaccine, which displayed a uniform hydrodynamic diameter of 130.6 ± 21.0 nm and positive surface charge of +3.7 mV (Fig. 7a).

Combination of Ne-SONV+US with αPD-L1 was examined in the orthotopic Hepa1-6-Luc liver tumor model. Bioluminescence imaging in vivo indicated that αPD-L1 moderately inhibited Hepa1-6-Luc tumor growth and 4/5 of mice lost within 40 days (Fig. 7b). In contrast, Ne-SONV+US markedly inhibited tumor growth and prolonged the mice survival, with 3/5 of mice survived above 60 days. Notably, the combo of Ne-SONV+US with αPD-L1 induced 100% survival of the orthotopic liver tumor-bearing mice in 60 days post tumor inoculation (Fig. 7c, d). Ex vivo imaging of the liver tissue further confirmed that Ne-SONV+US boosted αPD-L1 therapy to suppress orthotopic HCC development (Fig. 7e, f). The Ki67 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of tumor section indicated the obviously decreased tumor cell proliferation and increased cell apoptosis/necrosis after the combo of Ne-SONV+US with αPD-L1 treatment (Fig. 7g).

To assess the elicitation of long-term immune memory effect, the cured mice in Ne-SONV+US+αPD-L1 group were rechallenged with Hepa1-6-Luc cells at 60 days post the primary tumor inoculation (Fig. 7h). Bioluminescence imaging in vivo indicated that 3/4 of cured mice prevented secondary tumor growth over 90 days (Fig. 7i–k and Supplementary Fig. 57), which was mainly attributed to the induction of high-quality and long-lived effector CD8+ T memory cells and central CD8+ T memory cells (Fig. 7l).

Hematoxylin-eosin (H&E) staining of the mouse major organs displayed no inflammatory lesion or histopathological damage at 40 days post-final SONA+US treatment (Supplementary Fig. 58). Biochemical analysis of serum also indicated the favorable long-term safety of SONA+US treatment in vivo (Supplementary Fig. 59). These findings together confirmed that SONA+US treatment showed no obvious toxicity on non-target organs. Collectively, our study provided a US-activatable approach for achieving safe and effective cancer vaccination therapy.

Methods
Ethics statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (2024-09-YHJ-06). The study complied with the ethics committee’s limit on maximal tumor size (1500 mm3) and the requirement that animal body weight loss did not exceed 20%. Neither of these thresholds was exceeded at any point during the study.

Materials
HMME, MitoTracker Red CMXRos, Yoda-1, Dooku-1, Fluo-4AM, and DCFH-DA were purchased from MedChemExpress. The STING agonist MSA-2, NAC (SJ-MX0166), were purchased from Shandong Sparkjade Biotechnology Co., Ltd. DAPI, Lysotracker Red were purchased from Meilun Biotech CO., Ltd. OVA 257-263 peptide, FITC-labeled OVA 257–264 peptide, 4T1 tumor-specific neoantigen (SHRSCSHQTSAPSPKALAHNGTPRNAI) peptide, and Hepa1-6 tumor-specific neoantigen (RWLYWQPTL) peptide were purchased from Bank Peptide Biological Technology Co., Ltd. CpG-ODN1585 (SOC-A1585) was purchased from SynbioTech (Suzhou/NJ). The HRP-labeled goat-anti-rabbit IgG (H+L) (P8002) was purchased from New Cell & Molecular Biotech. The Cell Freezing Medium (S924JV) was purchased from Basal Media Co., Ltd. The pre-stained protein ladder (PM008) was purchased from Novoprotein. The D-Luciferin was purchased from Yeasen. Mouse GM-CSF (GMF-M5211) was purchased from ACROBiosystems. Mouse M-CSF (PCK043) was purchased from Procell. Mouse IL-4 protein (PRP2117) was purchased from Abbkine. ELISA kit for mouse TNF-α was purchased from Elabscience Biotechnology Co., Ltd. ELISA kit for mouse IL-6 was purchased from absin. Caspase 1 Activity Assay Kit was purchased from Abcam. ELISA kits for mouse IFN-γ, mouse IFN-β and mouse TGF-β were purchased from NeoBioscience Technology Co., Ltd. Glutamic-pyruvic Transaminase Assay Kit (BC1555) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Mouse Lymphocyte Separation Medium was purchased from Shenzhen Dakewe Bio-engineering Co., Ltd. L-Tyrosine (Tyr), 2-iodoxybenzoic acid, sodium dithionite (Na2S2O4), 1-Hydroxybenzotriazole, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDCI) and azodiisobutyronitrile were purchased from J&K chemical. Triphosgene was purchased from TCI. Methoxy PEG-NH2 (Mn=5000) was obtained from SINOPEG. N, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and ethyl ether were all offered by SinoPharm Chemical Reagent Co., Ltd.

Cell lines and animals
DC2.4 cells (cat. GNM51), RAW264.7 cells (cat. TCM13), B16-OVA cells (cat. TCM2), and 4T1 cells (cat. TCM32) were all purchased from the Cell Bank of Shanghai, Chinese Academy of Sciences. Hepa1-6-Luc cells were provided by Prof. XL Liu from Mengchao Hepatobiliary Hospital of Fujian Medical University. RAW264.7-Lucia ISG cells and THP1-Blue ISG cells were provided by Prof. MY Zheng from Shanghai Institute of Material Medica, Chinese Academy of Sciences. The construct expressing luciferase was introduced into Hepa1-6, and 4T1 cells by lentiviral transduction for bioluminescence imaging. The cell lines were authenticated by the supplier using STR analysis. All cell lines were tested negative for mycoplasma contamination. BMDCs and BMDMs were differentiated from bone marrow cells isolated from the femurs of 6-week-old C57BL/6 mice according to the established method. All the cells were maintained in DMEM medium containing 10% FBS (LONSERA, Suzhou Shuangru Biotechnology Co., Ltd.) and 1% penicillin/streptomycin, and maintained at 37°C in a humidified incubator with 5% CO2 supply.

BALB/c mice (cat. SM-003, 4–5 weeks, female) and C57BL/6 mice (cat. SM-001, 4–5 weeks, female) were purchased from Shanghai Model Organisms Center. Ifnar1-KO C57BL/6 mice (cat. C001268, female, 4–5 weeks) was purchased from Cyagen (Suzhou). All animal procedures were performed under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. According to the animal ethics of institute and animal welfare, animals were euthanized by carbon dioxide inhalation when their tumor volumes (V) exceeded 1500 mm3 in all the antitumor studies. V=(L×W×W)/2) (L, the longest axis, W, the shortest axis). The tumor size of mice was not exceeded 1500 mm3 in all the animal studies.

Synthesis of the Man-mPEG113-b-P(Tyr40)-HMME sonosensitive polypeptide
To synthesize the macroinitiator Man-mPEG113-NH2, NH2-mPEG5k-NH2 (100 mg, 0.02 mmol), α-D-mannopyranosylphenyl isothiocyanate (3.14 mg, 0.01 mmol), and DIEA (2.58 mg, 0.02 mmol) were reacted in anhydrous DMF for 24 h at room temperature. The product was obtained by dialyzing against ethanol and DI water. The structure was examined by 1H-NMR spectrum. Next, the Man-mPEG113-b-P(Tyr40) polypeptide was synthesized through ring-opening polymerization of L-tyrosine N-carboxyanhydride (TyrNCA) using macroinitiator Man-mPEG5k-NH2, as described in above statement. Finally, to synthesize sonosensitive Man-mPEG113-b-P(Tyr40)-HMME polypeptide, HMME (15.9 mg, 0.013 mmol), DMAP (8.0 mg, 0.33 mmol), EDCI (12.6 mg, 0.33 mmol), and DIEA (8.8 mg, 0.33 mmol) were dissolved in anhydrous DMF. After stirring for 1 h, the DMF solution of Man-mPEG113-b-P(Tyr40) (100.0 mg, 0.0082 mmol) was added dropwise into the mixture, and stirred continued for 24 h at room temperature. Then, the reaction was precipitated with cold diethyl ether to obtain Man-mPEG113-b-P(Tyr40)-HMME as black powder. All newly generated materials are available for reuse by contacting the corresponding author upon reasonable request.

Synthesis of the charge-modified polypeptides
The mPEG113-b-P(Lys40), mPEG113-b-P(Tyr10-Lys30), mPEG113-b-P(Tyr20-Lys20), mPEG113-b-P(Tyr30-Lys10), and mPEG113-b-P(Tyr40) polypeptides were synthesized through ring-opening co-polymerization of lysine N-carboxyanhydride (LysNCA) and tyrosine N-carboxyanhydride (TyrNCA) using mPEG113-NH2 as the macroinitiator. Typically, NH2-PEG5k (100 mg, 0.018 mmol) and indicated mass of Lys NCA and Tyr NCA were dissolved in anhydrous DMF under argon. The reaction was stirred for 3 days at room temperature and precipitated with cold diethyl ether to obtain indicated polypeptides as white powder. The mPEG113-b-P(TyrP10-Lys30), mPEG113-b-P(TyrP30-Lys10), and mPEG113-b-P(TyrP40) polypeptides were synthesized via phosphorylation of the phenolic hydroxyl groups of tyrosine. For instance. mPEG113-b-P(Tyr40) (100 mg, 0.0087 mmol) was suspended in anhydrous N-methylpyrrolidone under ice bath. Anhydrous triethylamine (200 μL) and phosphorus oxychloride (200 μL) were then slowly added. The mixture was stirred in the darkness overnight and saturated NaHCO3 solution was added. After stirring for another 3 h, the mixture was dialyzed against DI water and lyophilized to obtain the mPEG113-b-P(TyrP40) as yellow solid.

Preparation and characterization of the sono-adjuvant
The SONA library were prepared by a nanoprecipitation method. Typically, 1.0 mg of Man-mPEG113-b-P(Tyr40)-HMME sonosensitive polypeptide or mPEG113-b-P(Tyr40)-HMME sonosensitive polypeptide in 50 μL DMSO and 0.5 mg of indicated charge-modified polypeptides was drop by drop added into 1.0 mL deionized water under supersonic vibration (85 W, on 5 s, off 2 s, 3 min). The resultant nanoparticles were purified through ultrafiltration tube (Mw=100 kDa) and dispersed in deionized water for further use. The hydrodynamic diameter and zeta potential of nanoparticles were measured in pH 7.4 buffer solution by dynamic laser scattering (DLS).

US stimulation parameters
The US equipment was purchased from DJO France S.A.S. (Cat. number: DJO 2776, Frequency: 1 MHz, Power density: 0–1.5 W/cm2, Duty cycle: 50%, Transducer type: COLLIMATING, Effective radiating area: 4 cm2). We selected the US frequency of 1 MHz and the duty cycle of 50% based on the following considerations. First, the US frequency of 1 MHz has good penetration depth in biological tissues, which allows it to delivery energy into the LNs67. When combined with a 50% duty cycle, the energy is evenly distributed and can ensures sufficient energy transfer to achieve the treatment effect during the penetration process. Second, the combination of 1 MHz frequency and 50% duty cycle can avoid excessive damage to the tissue caused by continuous high-energy output, allowing to reduce adverse effects such as thermal effects on tissues68. Third, this combination was demonstrated to produce a moderate cavitation effect, and can avoid unnecessary damage to the surrounding tissues caused by overly intense cavitation69–71.

Western blot assay
The cell sort issues were harvested, and proteins were extracted using Intact Protein™ lysis kit (GenuIN, 415). Then the protein concentration was measured by the bicinchoninic acid protein assay kit according to the manufacturer’s instruction. Equal amounts of protein were mixed with an equal volume of 5× loading buffer and boiled at 100°C for 20 min. Gel electrophoresis and protein transformation were then performed. The target proteins were then probed with specific primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase. Immunoreactive proteins were visualized using a Tanon 5200 chemiluminescent imaging system (Tanon). Target protein expression was semi-quantified with ImageJ software.

Cytokine detection
For cytokine analysis in cell supernatant, the sample was collected at 24 h after US stimulation. For cytokine analysis in tumor tissue, the sample was collected and homogenized in cold PBS in the presence of digestive enzymes to form single-cell suspensions, and the supernatant after centrifugation was collected. The cell supernatant or intratumor cytokine levels of TNF-α, IL-6, IL-1β, IFN-β, and TGF-β were measured with ELISA kits according to the manufacturer’s instructions.

Analysis of antigen-specific immune response in vitro
BMDCs (2×105 cells per well) were incubated with various formulations with the concentration of HMME (1.0 μg/mL), OVA (10 μg/mL), MSA-2 (10 μM), and CpG (2.0 μg/mL). BMDCs in SONV+US group were stimulated with 0.2 W/cm2 of US (1 MHz, 50% duty cycle, 3 min) at 6 h post-incubation. The cells were continuously cultured for 24 h and then stained with corresponding antibodies to analyze the maturation and antigen presentation levels of BMDCs. For analyzing CD8+ OT-1 T cell proliferation in vitro, BMDCs were treated with the same formulations as described above, and then co-cultured with CFSE-stained OT-1 T cells for 72 h (T cell: BMDC = 10: 1). CD8+ T cell proliferation was then assessed by flow cytometry.

Preparation and characterization of sono-vaccine
SONA was used as the vaccine carrier, and protein antigen or neoantigen peptide was selected as the tumor antigen for preparing sonosensitive vaccine. Briefly, 100 μL of OVA protein solution (0.2 mg) was added into 1.0 mL of as-prepared SONA solution. The resultant mixture was kept stirring at room temperature for 60 min, and then purified using an ultrafiltration tube (Mw=100 kDa). The OVA loading efficiency and capacity in SONV was determined by a bicinchoninic acid protein assay kit. To prepare Ne-SONV, 100 μL of 4T1 tumor-specific neoantigen peptide (SHRSCSHQTSAPSPKALAHNGTPRNAI) or Hepa1-6 tumor-specific neoantigen peptide (RWLYWQPTL) (0.2 mg) was drop by drop added into 1.0 mL of as-prepared SONA solution. The resultant mixture was kept stirring at room temperature for 60 min, and then purified using an ultrafiltration tube (Mw=100 kDa). The particle size and zeta potential of resultant SONV, Ne-SONV were evaluated in pH 7.4 buffer solution by a Zeta-sizer Nano ZS90 (Malvern Instruments, England).

Lymph node-targeting and cellular uptake of SONV in vivo
C57BL/6 mice were subcutaneously injected at the tail base with free OVA-FITC or SONV-FITC at an identical OVA-FITC dose of 5.0 mg/kg. Fluorescence signal in LNs and major organs were examined 6 h or 12 h post-injection with an IVIS Spectrum Imaging System. To determine the cellular uptake of SONV-FITC by APCs in vivo, LNs were harvested and digested to obtain single-cell suspensions. The cells were washed with PBS three times and stained with corresponding antibodies and then analyzed by flow cytometry.

Vaccination and antigen-specific immune response in vivo
C57BL/6 mice were vaccinated with indicated formulations with the identical dose of HMME (1.0 mg/kg), OVA (1.0 mg/kg), MSA-2 (2.0 mg/kg), and CpG (2.0 mg/kg) three times at one week interval. The LNs of SONV+US group mice were stimulated with 1.0 W/cm2 US (1 MHz, 50% duty cycle, 5 min) at 6 h post per SONV injection. The LNs were harvested 3-day after the final vaccination and digested to obtain single cell suspensions. The cells were stained with corresponding antibodies to analyze the maturation and antigen presentation levels of DCs and macrophages. To evaluate antigen-specific CD8+ T cell response in vivo, the spleen of vaccinated mice was harvested, digested, and passed through 200-mesh nylon mesh filters to obtain single-cell suspensions. The splenocytes were then stained with corresponding antibodies to analyze the antigen-specific CD8+ T cell response using the tetramer staining assay. To evaluate the antigen-dependent cytotoxic effect of CD8+ T cells, 1×106 splenocytes were collected from the vaccinated mice and stimulated with 10 μg/mL SIINFEKL peptide for 24 h. The cells were then stained with corresponding antibodies to analyze the cytotoxic CD8+ T cell response.

In vivo cytotoxicity killing assay
We performed an in vivo cytotoxicity killing assay to detect the OVA-specific cytotoxic T cell response generated by SONV+US. Half of splenocytes from untreated mice were labeled with OVA peptide and high concentration of CFSE (5.0 μM, TargetMol, T6802), denoted as OVAp+ cells. Meanwhile, the other half of splenocytes were labeled with low concentration of CFSE (0.5 μM) and not labeled with OVA peptide, denoted as OVAp− cells. Then, the OVAp+ cells (5×106 per mouse) and OVAp− cells (5×106 per mouse) were equally mixed and injected intravenously into the SONV+US-treated mice. After 24 h, spleen from treated mice was collected and subjected to flow cytometry analysis. The numbers of CFSEhigh and CFSElow cells were determined and used to calculate the percentage of OVA-specific cell killing.
Percentage of specific lysis=[1 − non-transferred control ratio/experimental ratio]×100%.

Assessment of systemic immune side effects
To evaluate systemic immune activation associated with different adjuvant strategies, serum levels of IL-6 and IL-12p70 were measured following indicated treatments. Female C57BL/6 mice were randomly assigned into three groups: OVA+CpG (50 μg CpG), OVA+MSA-2 (50 μg MSA-2) and SONV+US (50 μg HMME). All formulations contained 50 μg OVA antigen and were subcutaneously injected at the tail base of mice on day 0, day 7, and day 14, respectively. For SONV+US group mice, the draining lymph nodes were stimulated with US (1 MHz frequency, 50% duty cycle, 1.0 W/cm² intensity, 5 min) at 6 h post each injection. Peripheral blood was collected via submandibular vein at 1 day and 7 day post each injection. Serum was isolated and stored at −80°C until analysis. Levels of IL-6 and IL-12p70 were quantified using indicated ELISA kits following the manufacturer’s protocol.

RNA-seq analysis
The LNs from PBS or SONV+US group mice were harvested to extract the total RNA according to the manufacturer’s protocol. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. according to the manufacturer’s instructions (Illumina, San Diego, CA). To identify DEGs between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. Differential expression analysis was performed using the DESeq2. DEGs with |log2FC|≥1 and adjusted p-value≤0.05 were considered to be significantly different expressed genes. In addition, functional-enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) was performed to identify which DEGs were significantly enriched in metabolic pathways at Bonferroni-corrected p-value≤0.05 compared with the transcriptome background. KEGG pathway analysis were carried out by Python scipy software. The Gene Set Enrichment Analysis (GSEA) did not use the MSigDB database. Instead, it used the annotation results from KEGG pathway analysis. The ranking algorithm used for GSEA was Signal2Noise. The protein-protein interaction network analysis on DEGs was conducted by using the STRING database (combined score≥0.4, http://string-db.org/).

Antitumor efficacy in multiple murine tumor models
To evaluate the prophylactic effect of SONV+US against tumor growth and metastasis, C57BL/6 mice were vaccinated with indicated formulations with the identical dose of HMME (1.0 mg/kg), OVA (1.0 mg/kg), MSA-2 (2.0 mg/kg), and CpG (2.0 mg/kg) three times at one week interval. The LNs of SONV+US group mice were stimulated with 1.0 W/cm2 US (1 MHz, 50% duty cycle, 5 min) at 6 h post per SONV injection. 1×106 B16-OVA cells were subcutaneously or intravenously injected into vaccinated mice on day 7 after the final vaccination. The body weight and tumor volume of group mice were monitored every two days. To evaluate the therapeutic effect of SONV+US against established tumor growth, 1×106 B16-OVA cells were subcutaneously injected into the right flank of C57BL/6 mice on day 0, and mice were randomly grouped on day 7 and treated with indicated formulations as described above three times at one week interval. The body weight and tumor volume of group mice were monitored every two days.

To evaluate the combinational antitumor effect of Ne-SONV+US with PD-L1 blockade therapy in orthotopic TNBC model, 1×106 4T1-Luc cells were injected into the breast pad of BALB/C mice on day 0, and mice were randomly grouped on day 7, and treated by subcutaneous injection of Neop+CpG or Ne-SONV with identical dose of HMME (1.0 mg/kg), neoantigen peptide (1.0 mg/kg), CpG (2.0 mg/kg), and intraperitoneal injection of αPDL1 (5.0 mg/kg). The LNs of Ne-SONV+US and Ne-SONV+US+αPDL1 group mice were stimulated with 1.0 W/cm2 US (1 MHz, 50% duty cycle, 5 min). The body weight, tumor volume of group mice were monitored every two days and the bioluminescence imaging of group mice were conducted every ten days.

To evaluate the combinational antitumor effect of Ne-SONV+US with PD-L1 blockade therapy in orthotopic HCC model, 5×106 Hepa1-6-Luc cells were injected into the liver of C57BL/6 mice on day 0, and mice were randomly grouped on day 7, and treated by Ne-SONV+US or αPDL1 as described above. The bioluminescence imaging of group mice were conducted every ten days.

mtDNA-TLR9 blocking assay
For the mtDNA-TLR9 blocking, 200 µg of AT-791 (TargetMol, USA) was administered to the C57BL/6 mice by intraperitoneal injection 1 day before SONV+US treatment and every third day thereafter for five times.

IL-1β blocking assay
For the IL-1β blocking, 100 µg of anti-IL-1β (BE0246) (BioXcell) was administered to the C57BL/6 mice by intraperitoneal injection 1 day before SONV+US treatment and every third day thereafter for five times.

CD8+ T cell blocking assay
For the CD8+ T cell blocking, 100 μg of anti-CD8α (53–6.7) (BioXcell) was administered to the C57BL/6 mice by intraperitoneal injection 1 day before SONV+US treatment and every third day thereafter for five times. The depletion of CD8+ T cells in spleen was confirmed by flow cytometry.

Analysis of tumor-infiltrating immune cells in vivo
B16-OVA, Hepa1-6 and 4T1 tumor-bearing mice model were established and treated as described above. Tumor tissues were collected 3-days post final treatment, and processed through mechanical disruption and digested by collagenase IV (0.2 mg/mL), hyaluronidase (0.2 mg/mL) and DNaseI (0.1 mg/mL) for 90 min at 37°C. The cells were then filtered through 200-mesh nylon filters and washed three times with PBS to obtain single-cell suspensions. The collected cells were stained with corresponding antibodies to analyze tumor-infiltrating CD4+ T cells, CD8+ T cells, NK cells, IFN-γ+CD8+ T cells, Gzmb+CD8+ T cells, IFN-γ+NK cells, Gzmb+NK cells, Tregs, IL-10+Tregs, IL-4+Tregs, MDSCs, M2-TAM, and M1-TAM.

Statistics and reproducibility
For quantitative analysis, a minimum of three biological replicates were analyzed. Western blotting and immunofluorescent staining experiments were repeated three times independently with similar results. All data were presented as mean ± SD unless otherwise indicated. Statistical analysis was performed using GraphPad Prism software 8.2.1. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post-hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. The survival study was analyzed through log-rank (Mantel–Cox) test. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary: In this study, the authors combined ultrasound and polypeptide-based sono-sensitive adjuvant to selectively excite the innate immune responses in lymph nodes for cancer vaccine therapy. The study was performed both in vitro and in vivo with mice on various cancer models. Exciting results were claimed to have been acquired. But there are still some questions from the reviewers listed below.\nMajor comments on the manuscript:\n1. It is not clear to this reviewer what ‘an ultrasound-confined strategy’ is. How does ultrasound ‘confine’ the therapeutic interventions in this study? It is clear that the authors want to deliver ultrasound in a manner of accurate spatial and temporal control. But it still does not make sense to this reviewer how ‘confine’ is implemented.\n2. Throughout the study, ultrasound is typically applied 6 hours after the injection. Why 6 hours after the injection? Any justification?\n3. Although acronyms seem unavoidable in biological science articles, the large number of acronyms in this article greatly reduces the readability.\n4. Ultrasound plays an important role in this study. However, no specific information about the ultrasound equipment and related parameters were provided in this article. For example, when the authors say 1.0W/cm2 of ultrasound radiation was applied under certain conditions, how can we trust this quantity without knowing how ultrasound energy delivery was handled in the study?\n5. The authors claim to have spatiotemporally controlled the regulation of innate immunity using ultrasound (US). It would be helpful if the authors could elaborate on how ultrasound was utilized across different investigations, including the specific experimental procedures. Additionally, further clarification on the selection of various ultrasound parameters, such as frequency, duty cycle, and peak negative pressure, would enhance the readers’ understanding (Supplementary Information, Line 901, Line 929, Line 944).\n6. In Lines 600–603, the authors state that \"ultrasound-triggered mechanical and oxidative force allowed for spatiotemporal tailoring of innate immunity activation in LNs by adjusting the irradiation site, power, time interval, and duration of external ultrasound stimuli.\" However, the manuscript does not clearly explain how the mechanical and oxidative forces were controlled, particularly given that uncontrolled ultrasound irradiation could potentially lead to cellular damage. A discussion on the measures taken to minimize unintended cellular damage caused by mechanical and oxidative forces would strengthen the manuscript.\n7. Since mechanical effects and oxidative forces are two distinct phenomena triggered by ultrasound, it would be beneficial for the authors to clarify which of these plays a more dominant role in initiating immune pathways in antigen-presenting cells (APCs).\n8. Given that innate immune pathways in APCs are generally considered inflammatory, the authors may wish to provide additional mechanistic insights into how SONV + US remain unaffected by immune-suppressive cells.\n9. The manuscript would benefit from additional details on the experimental design, including batch sizes and statistical analyses. Providing this information in the supplementary section would improve the clarity and reproducibility of the study.\n10. Finally, improving the overall cohesiveness of the manuscript would enhance readability and comprehension.\nMinor comments on the manuscript:\n1. Line 46: Please provide a citation for the statement: \"Unfortunately, the microenvironment within the lymph nodes (LNs) is often ineffective in stimulating APC maturation, migration, and cytokine secretion, thus deactivating the antigen cross-presentation ability of APCs.\"\n2. Line 59: Please provide a citation to support the claim: \"However, most of these strategies exhibit an 'always on' profile to stimulate innate immunity in vivo and could potentially induce immune-related adverse effects due to their non-specific distribution and immune stimulation of untargeted tissues (e.g., liver, lung), thus limiting their future clinical translation.\"\n3. Line 90: Including an experimental figure illustrating how ultrasound (US) is applied would make this section more self-explanatory and improve clarity.\n4. The word “dramatically” appears multiple times throughout the manuscript (e.g., Line 91, Line 193, Line 246, Line 255, Line 369, Line 432, Line 434). Replacing it with a more precise term would improve the readability and scientific rigor of the text.\n5. Line 114: Please revise the sentence for improved clarity: \"CLSM images showed that SONP incubation plus ultrasound irradiation.\"\n6. Lines 115, 901, 929, 944: Please replace ‘MHZ’ with ‘MHz’ for consistency in unit notation.\n7. Line 166: The phrase “increasingly clear” could be replaced with a more specific description.\n8. Line 168: The sentence \"To optimize LN-confined distribution and APC-targeting internalization, we developed a set of sonosensitive adjuvants (namely sono-adjuvants, SONA) with adjustable physicochemical properties, including particle size, surface charge, and ligand modification.\" should reference the corresponding table in Figure 2 for clarity.\n9. Lines 186–188: Please provide either a citation or a brief explanation for selecting the reported range of power density. Additionally, specify the frequency and duty cycle used for ultrasound irradiation.\n10. Line 200: A more detailed explanation of why regulatory T cells (Tregs, FOXP3+CD25+CD4+CD3+) were downregulated by ultrasound irradiation would be beneficial.\n11. Figure 2(a): If this study is not a clinical study, the representation of a human lymph node may be misleading. Consider revising the figure to avoid potential misinterpretation.\n12. Lines 245–249: Please revise these sentences for improved clarity and readability.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This manuscript reports a new ultrasound-confined approach to regulate innate immunity and enhance cancer vaccination therapy. Chen et al. develops and characterizes a polypeptide-based sono-adjuvant (SONA) that activates multiple innate immune pathways (NLRP3, STING, and TLR9) in antigen-presenting cells (APCs) and demonstrates its efficacy in preclinical tumor models. This work could be of interest to the immunoengineering and cancer immunotherapy fields. Some comments are provided below:\nMajor issues:\n1.The manuscript is dense and difficult to follow. In some parts of the results section, the language is too vague and lacks quantitative evidence. For example, the sentence: ‘Flow cytometry analysis and confocal laser scanning microscope (CLSM) images indicated that ultrasound irradiation dramatically affects the intracellular Ca²⁺ concentration of DCs as a function of power density (Fig. 1b-d).’ How dramatic is the effect? The authors should provide specific data to support this claim.\n2.The authors analysed the transcriptomic features of LNs from SONV+US group mice. However, Figure 3m volcano plot doesn’t make sense. In the supplementary section, the authors stated that genes were selected using DESeq2 with log2FC ≥ 1 and FDR ≤ 0.05. But in the volcano plot, the p-value cutoff looks very close to 0. Since FDR ≤ 0.05 should correspond to a p-value at least around 0.01, the cutoff line should be closer to 2.5 on the y-axis. Please clarify this.\n3.The results section lacks a detailed description of the figures. In Figure 6h and 6i, it seems the authors performed some single-cell analysis, but I couldn't find any detailed explanation of this analysis. Figure 6i is also confusing, why do all the t-SNE plots look identical to Figure 6h? The four PD-1 expression panels represent four different methods, so there should be four separate single-cell datasets. It is hard to imagine that all four would produce identical t-SNE plots. Please clarify this.\n4.Please add details on the quantitative analysis procedures in supplementary. For example, in the GSEA analysis, which MSigDB dataset was used? Hallmark? Cancer genes? Please include all detailed analysis steps in the supplementary file\nMinor issues:\n1.The discussion is too optimistic and lacks a section on limitations. Addressing limitations makes the study more credible and realistic. How ultrasound penetration depth might vary in human tissues compared to mouse models?", "reviewer_4": "Chen et al. proposed a strategy for cancer vaccine immunotherapy using ultrasound-guided immune activation. The experimental design used a variety of mouse models to demonstrate the effectiveness of the strategy in different tumor settings. However, the manuscript should be enhanced before acceptance for publication.\n1. This study presented an ultrasound-guided immune activation approach, but did not adequately provide a detailed comparison with existing immunotherapeutic approaches, especially in terms of conventional immunotherapy such as immune adjuvants and immune checkpoint inhibitors. For a new immunotherapy platform, it is important to clearly demonstrate its advantages over existing approaches, especially in terms of improved efficacy and immune side effects.\n2. Although ultrasound therapy is a non-invasive method, the depth of penetration and tissue uptake of ultrasound may be suboptimal in the treatment of deep tumors, and different tissues may respond differently to ultrasound. How can ultrasound-guided immune activation protocols ensure the local precision of immune activation during treatment and the activation status of different immune cell populations be regulated?\n3. The manuscript mentioned the use of ultrasound and peptide adjuvants to activate immune pathways such as NLRP3, STING, and TLR9 to enhance the immune response, but the complexity of these immune pathways and their roles in different immune cells have not been fully considered.\n4. A variety of mouse models were used in the paper, including mammary tumor and hepatocellular carcinoma models, but there was fewer pairs in different immune contexts. For example, were immunodeficient mice (e.g., T-cell deficient mice) considered to validate the treatment? It is recommended that the validation of these models be increased to further illustrate the generalizability of the treatment in different immune settings.\n5. The immune response was detected mainly by methods such as flow cytometry and immunohistochemistry. However, the presentation of details in flow cytometry was insufficient. The significance of the treatment effect needs to be demonstrated more clearly in terms of indicators such as CD8+ T cell activation, maturation degree of DCs, and cytokine secretion level between different groups. In addition, the text mainly focuses on the response of CD8+ T cells and DCs, but ignored the role of other immune cells (e.g., Tregs, MDSCs, NK cells). It is recommended that the role of these cells be analyzed in detail, especially the inhibitory effect of Tregs and the effect of MDSCs on immune escape. Moreover, considering the complexity of immunotherapy, it is recommended to introduce a more fine-grained analysis of immune cell subpopulations (e.g., to distinguish between different T cell subpopulations by multiple-labelling flow cytometry).\n6. In mouse experiments, the article mentions that the treatment had a favorable biosafety profile, with no significant toxic effects found. However, the article did not perform long-term observations to verify the long-term safety of the strategy. Long-term follow-up is recommended in subsequent studies, especially to evaluate potential toxicity in non-target organs.\n7. Preliminary optimization of the parameters of ultrasound therapy was done in the paper, but it failed to further explore in depth the effects of different ultrasound parameters on the immune response. It is recommended to increase the study of the dose-response curve of ultrasound therapy, such as the change of effect under different ultrasound frequency and treatment time to find out the optimal therapeutic parameters.\n8. The effects of ultrasound stimulation were realized through the dual action of mechanical and oxidative forces, but did not elaborate on the specific mechanisms of interaction between the two. Experimental exploration of these mechanisms of action can be further increased, especially how ultrasound triggers intracellular calcium ion flow and ROS generation, whether it causes cellular damage and how to effectively circumvent these side effects.\n9. The article concluded with a briefer summary of the mechanisms of ultrasound-induced immune activation and therapeutic effects. It is suggested that the limitations and future directions of the study should be made more explicit in the conclusion section, especially how to overcome the limitations of short-term experimental observation and immune response monitoring that exist in the current experiments, and how to generalize the strategy across different cancer types.", "reviewer_5": "In this study, Chen et al reported an ultrasonic-activatable nano-adjuvant for vaccination therapy cancer. The polypeptide-based nano-adjuvant was screened by lymph node-targeting efficacy, DC activation profiles and so on. The outperformed nano-adjuvant was employed for lymph node-targeted delivery of protein antigen and neoantigen peptide. The antigen-loaded nano-vaccines highly efficiently elicited antitumor immune response for regressing orthotropic breast and liver tumors. Overall, the sono-sensitizer-conjugated polypeptide represents an elegant design of novel nano-adjuvants, which could provide a robust platform for ultrasonic-tunable regulation of innate immunity and boosting antitumor immune response. The data presented in the manuscript are comprehensive and convincing. I prefer to recommend publication upon addressing some specific concerns.\nMajor comments\n1. It has been reported that photodynamic therapy with laser irradiation could promote protein/siRNA escape from cytosol, the influence of US on cytosol release of the antigens should be discussed/investigated.\n2. The authors mentioned that oxidative stress could be crucial for innate immune response, the generation of oxidative stress should be validated.\n3. Ultrasonication is employed in this study to activate innate immune response, the advantages of US-activatable sono-adjuvant over the conventional adjuvants should be discussed.\n4. A more detailed explanation of US equipment and experimental settings for LNs-confined US irradiation should be provided.\n5. The synthesis of charge-modified polypeptide is quietly innovative, and a more detailed explanation of the synthetic methods should be provided.\n6. DLS-determined particle size and surface charge of neoantigen peptide-loaded nanovaccines should be provided.\n7. The necessity and advantages of spatiotemporally-controllable innate immunity activation in LNs should be more convincingly demonstrated\nSpecific comments\n1. Semi-quantification is required for Figure 2n and 2o.\n2. Fig 2f-g, statistical analysis should be performed.\n3. In figure 4m, n, the meaning of “in tumor (%)” is unclear. Please specify what percentage is being measured.\n4. In figure 5i, k, m, the meaning of “in tumor (%)” is unclear. Please specify what percentage is being measured.\n5. In figure 3j, the experimental method for analyzing OVA-specific cytotoxic effect in vivo is suggested to be presented in a more detailed manner.\n6. Supplementary figure 17, statistical analysis should be performed.\n7. The caption labeling for the statistical data in Supplementary figure 42 is missing.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to the reviewers\nWe sincerely appreciate the insightful comments and efforts of all the reviewers helping us to\nrevise the manuscript and improve our understanding of the polypeptide sono-adjuvant for\ncancer vaccine immunotherapy. To address the concerns of the reviewer, we conducted\nextended experimental studies and carefully revised the manuscript. Specifically,\n1. According to the comments of reviewers #1, 2, 4, 5, 6, we thoroughly screened the\nultrasound (US) procedures and parameters to perform lymph nodes (LNs)-specific US\nstimulation and maximize the innate immune activation efficacy in vivo. First, we evaluated\nthe biodistribution of sono-adjuvant (SONA) in LNs to find out the optimized US stimulation\ntime point, and the result showed that SONA gradually accumulated into LNs and peaked at\n6 h post subcutaneous injection. Therefore, we selected 6 h post SONA injection as the time\npoint for US stimulation. Next, we investigated the influence of different power density (0, 0.2,\n0.5, 1.0, 1.5 W/cm2) of US stimulation on the innate immune responses in vivo. Flow\ncytometry analysis revealed that 1.0 W/cm2 of US stimulation elicited the highest maturation\nratio of dendritic cells (DCs) in LNs. We subsequently investigated the influence of US\nstimulation duration (0, 1, 3, 5, 7 min) on innate immunity activation in vivo. The result showed\nthat the DCs maturation levels in LNs increased gradually upon US duration, which peaked\nwhen combined with 5 minutes of US stimulation. Therefore, we selected the optimal time\npoint (6 h post injection), power density (1.0 W/cm2) and duration (5 min) of US stimulation\nfor LNs-specific innate immune activation in vivo.\n2. According to the comments of Reviewers #1, 2, 4, 5, 6, we conducted additional\nexperimental studies to clarify the different immunogenic mechanism of mechanical and\noxidative forces elicited by US stimulation. Western blot assay showed that US-triggered\nmechanical force activated the NLRP3 pathway in DC2.4 cells and RAW264.7 cells in vitro,\nwhile merely activated the STING and TLR9 pathways. In contrast, the combination of\nmechanical and oxidative forces mediated by SONP+US effectively activated the STING,\nTLR9 and NLRP3 pathways in DC2.4 cells and RAW264.7 cells. It was worth noting that\nendoplasmic reticulum (ER) stress-mediated mtDNA release was demonstrated to activate\nboth TLR9 and STING pathways. Therefore, to further clarify the potential role of mechanical\nand oxidative forces to trigger ER stress and innate immune activation in APCs, we performed\na ROS scavenging assay to specifically eliminate SONP+US-mediated oxidative force while\nremaining the mechanical force. Western blot result showed that ROS scavenging by N-\nAcetyl-L-cysteine (NAC) almost completely abrogated SONP+US-induced ER stress in\nDC2.4 cells. Collectively, these findings indicated that US-triggered mechanical force was\nable to activate NLRP3 pathway, while the oxidative force was capable of activating both\nTLR9 and STING pathways through inducing ER stress and mtDNA release in APCs.\n3. According to the comments of reviewers #4, 5, 6, we provided a detailed validation of the\ntherapeutic benefits of our sono-adjuvant-based vaccination strategy over other conventional\nimmune reactive adjuvants, including typical STING agonist (MSA-2), TLR9 agonist (CpG)\nand immune checkpoint blockade antibody (anti-PDL1). These relevant experimental results\nwere presented in Fig. 3, Fig. 4, Fig. 6 and Fig. 7 in the revised manuscript. In addition, we\nprovided a detailed discussion about the critical advantages and current limitations of our\nsono-adjuvant strategy in the Discussion section, which could indicate the clinical translation\npotential and future research directions of the US-activatable adjuvant strategy.\n4. According to the comments of Reviewers #1, 2, 5, 6, we provided a detailed description\nabout the LNs-specific confined targeting to make sense to reviewer/readers. We engineered\na novel class of polypeptide-based sonosensitive adjuvant (SONA) to implement LNs-\ntargeting delivery through adjusting the physicochemical parameters (e.g., size, charge,\nsurface ligand). The result showed the top-performed SONA specifically drained into LNs and\neffectively targeted APCs, while displaying marginal off-target distribution in normal organs.\nThus, the rational design of polypeptide delivery platforms implemented the LNs-specific\ndistribution of sonosensitizer. More importantly, we found that SONA remained in an ‘immune-\nsilent’ state in the absence of US stimulation, while the combination of LNs-specific US\nstimulation could precisely trigger the innate immunostimulatory effect of SONA, and induced\nrobust innate immune responses in vivo. Therefore, we combined the LNs-targeting delivery\nstrategy and US-activatable strategy to ensure the LNs-specific confined activation of innate\nimmunity.\n5. According to the comments of Reviewer #3, we improved the detailing and completeness\nof transcriptomics analysis and interpretation in Methods section in the revised manuscript.\nTo identify DEGs between two different samples, the expression level of each transcript was\ncalculated according to the transcripts per million reads (TPM) method. Differential\nexpression analysis was performed using the DESeq2. DEGs with |log2FC| ≥ 1 and\nadjusted p-value ≤ 0.05 were considered to be significantly different expressed genes.\nFunctional-enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG,\nhttp://www.genome.jp/kegg/) was performed to identify which DEGs were significantly enriched in\nmetabolic pathways at Bonferroni-corrected p-value ≤ 0.05 compared with the transcriptome\nbackground. KEGG pathway analysis were carried out by Python scipy software. The Gene Set\nEnrichment Analysis (GSEA) did not use the MSigDB database. Instead, it used the annotation results\nfrom KEGG pathway analysis. The ranking algorithm used for GSEA was Signal2Noise. The protein-\nprotein interaction network analysis on DEGs was conducted by using the STRING database\n(combined score ≥ 0.4, http://string-db.org/).\n6. According to the comments of Reviewer #4, we re-established the B16-OVA tumor model and\nmeasured the suppressive function of tumor-infiltrating Tregs in order to provide clearer insights about\nSONV+US-mediated antitumor immunity. Our findings showed that SONV+US treatment not only\ndecreased the frequency of tumor-infiltrating Tregs, but also inhibited the production of Tregs-\nderived suppressive cytokines (IL-4, IL-10). The frequencies of tumor-infiltrating IL10+ Tregs\n(12.6%, IL10+ in CD25+FOXP3+ cells) and IL4+ Tregs (10.6%, IL4+ in CD25+FOXP3+ cells) in\nSONV+US group displayed 3.1- and 3.2-fold lower than that in PBS group (IL10+ Tregs, 39.2%\nand IL4+ Tregs, 33.3%), respectively.\n7. According to the comments of Reviewer #4, we re-established the B16-OVA tumor model\nand measured the cytotoxic effect of tumor-infiltrating NK cells to provide clearer insights\nabout SONV+US-mediated antitumor immunity. Our findings showed that SONV+US\ntreatment increased the intratumor cytotoxic NK cell response (IFN-γ+ NK cells and GZMB+\nNK cells) compared with PBS group. The frequencies of IFN-γ+ NK cells (IFN-γ+ in NK1.1+\ncells) and GZMB+ NK cells (GZMB+ in NK1.1+ cells) in SONV+US group were about 12.5%\nand 11.6%, respectively, showing 2.1- and 2.0-fold higher than that in PBS group (IFN-γ+ NK,\n5.96% and GZMB+ NK, 5.56%), respectively.\n8. According to the critical comments of Reviewer #4, we re-established the B16-OVA tumor\nmodel and introduced a more fine-grained analysis of tumor-infiltrating CD8+ T cell\nsubpopulations. Compared with PBS group, SONV+US treatment increased the intratumoral\nprogenitor exhausted (PD1+TIM3-) CD8+ T cells infiltration, while decreased the tumor-\ninfiltration of terminally exhausted (PD1+TIM3+) CD8+ T cells. The frequencies of progenitor\nexhausted CD8+ T cells (PD1+TIM3- in CD8+CD3+ cells) and terminally exhausted CD8+ T\ncells (PD1+TIM3+ in CD8+CD3+ cells) in SONV+US group were about 28.4% and 19.4%,\nshowing 2.2-fold higher and 3.0-fold lower than that in PBS group, respectively. These results\nindicated that SONV+US treatment not only enhanced intratumoral CD8+ T cell infiltration\nand activation, but also was promising to address intratumoral CD8+ T cell exhaustion.\n9. According to the comments of Reviewer #4, we performed additional experimental studies\nto verify the long-term safety of the sono-adjuvant strategy. C57BL/6 mice were treated with\nSONA+US (1 MHz, 50% duty cycle, 1.0 W/cm2, 5 min) three times at 3-days intervals, and\nthe major organs and serum of treated mice were collected at 40-days post final treatment.\nHematoxylin-eosin (H&E) staining of the major organs displayed no inflammatory lesion and\nhistopathological damage after SONA+US treatment. Biochemical analysis of serum also\nindicated the favorable long-term safety of SONA+US treatment in vivo. These findings\ntogether confirmed that SONA+US treatment showed no obvious toxicity on non-target\norgans.\n10. According to the critical comments of Reviewers #1, 2, 3, 4, we provided the specific\nquantitative evidence to support the claims about significant difference observed between\nexperimental groups. In addition, we added the critical reference citation for corresponding\nstatements to improve the scientific rigor of the text. Moreover, we provided a list of acronyms\nthat were used throughout the manuscript. For instance, the sonosensitive nanoparticle was\ndenoted as SONP. The sono-adjuvant was denoted as SONA. The sono-adjuvant-based\nvaccine was denoted as SONV. We were confident that these efforts could largely enhance\nthe text clarification and readability of the manuscript.\n11. According to the comments of Reviewers #1, 2, we clearly stated the batch sizes and\nstatistical analyses in all figure legends to improve the clarity and reproducibility of the results\nand conclusions.\nWe hope the revised manuscript could match the criteria of Nature Communications now.\nReferee’s comments are in black and our responses are in blue.\nReviewer #1 (Remarks to the Author):\nSummary: In this study, the authors combined ultrasound and polypeptide-based sono-\nsensitive adjuvant to selectively excite the innate immune responses in lymph nodes for\ncancer vaccine therapy. The study was performed both in vitro and in vivo with mice on\nvarious cancer models. Exciting results were claimed to have been acquired. But there are\nstill some questions from the reviewers listed below.\nMajor comments on the manuscript:\n1. It is not clear to this reviewer what ‘an ultrasound-confined strategy’ is. How does\nultrasound ‘confine’ the therapeutic interventions in this study? It is clear that the authors want\nto deliver ultrasound in a manner of accurate spatial and temporal control. But it still does not\nmake sense to this reviewer how ‘confine’ is implemented.\nResponse: We greatly appreciated the comments of the reviewer helping us to improve the\nreadability of our manuscript. We added the relevant discussion in Line 69-91 in the revised\nmanuscript as below:\n“In this study, we first demonstrated that US-triggered mechanical and oxidative force\nenabled to simultaneously activate multiple innate immune pathways (NLRP3, STING and\nTLR9) in APCs by a US-dependent manner. We subsequently engineered a novel class of\npolypeptide-based sonosensitive adjuvant (namely sono-adjuvant, SONA) to implement LNs-\ntargeting and APCs-targeting delivery through adjusting the physicochemical parameters\n(e.g., size, charge, surface ligand). We found the top-performed SONA specifically drained\ninto LNs and effectively targeted APCs, while displaying marginal off-target distribution in\nnormal organs (e.g., liver, lung, kidney). Therefore, the rational design of polypeptide delivery\nplatforms implemented the region-confined distribution of sonosensitizer in LNs. It was worth\nnoting that SONA remained in an ‘immune-silent’ state in the absence of US stimulation,\nsince the sonosensitizer itself was incapable of triggering mechanical and oxidative force to\nactivate innate immune pathways in APCs. In contrast, the combination of LNs-specific US\nstimulation could precisely trigger the innate immunostimulatory effect of SONA, and thus\ninduced robust type-I IFN and pro-inflammatory cytokine responses in vivo. Collectively, we\ncombined the LNs-targeting strategy and US-activatable strategy to ensure LNs-specific\nconfined activation of innate immunity.”\nMoreover, we revised the title of manuscript as ‘Polypeptide Sono-Adjuvant for\nUltrasound-Activatable Regulation of Innate Immunity and Cancer Vaccination Therapy’ to\nenable reviewers/readers to better understand how confined innate immunity activation was\nimplemented.\n2. Throughout the study, ultrasound is typically applied 6 hours after the injection. Why 6\nhours after the injection? Any justification?\nResponse: We greatly appreciated the comments of the reviewer. We evaluated the\nbiodistribution of SONA in LNs by utilizing the fluorescence imaging capacity of porphyrin-\nbased sonosensitizer. As shown in Supplementary Fig. 14, upon subcutaneous injection at\nthe tail base, SONA gradually accumulated into LNs and peaked at 6 h post injection.\nTherefore, we selected the 6 h post SONA injection as the experimental time point for LNs-\nspecific US stimulation.\nWe added the relevant discussion in Line 222-228 in the revised manuscript.\nSupplementary Fig. 14 (in this revised manuscript). a, Representative fluorescence images of mice at indicated\ntime points post SONA injection (n = 3 biologically independent mice). b, Representative fluorescence images and\nnormalized fluorescence signal of tissues collected at indicated time points post SONA injection (n = 3 biologically\nindependent mice). Statistical significance was calculated via one-way ANOVA with Tukey’s test and P values were\nindicated.\n3. Although acronyms seem unavoidable in biological science articles, the large number of\nacronyms in this article greatly reduces the readability.\nResponse: We greatly appreciated the comments of the reviewer reminding us to address\nthe largely used acronyms in our manuscript. To improve the readability of our manuscript,\nwe not only checked and avoided the unnecessary acronyms throughout the manuscript, but\nalso provided a list of the used acronyms in Supplementary Table S1 in the revised\nmanuscript.\nSupplementary Table S1 (in this revised manuscript). The list of used acronyms throughout the manuscript\nAcronyms Full name Acronyms Full name\nUS Ultrasound NF-κB Nuclear factor κB\nLNs Lymph nodes SOP Sonosensitive polypeptide\nAPCs Antigen-presenting cells CMP Charge-modified polypeptide\nICB Immune checkpoint blockade OVA Ovalbumin\nROS Reactive oxygen species DEGs Differentially expressed genes\nTregs Regulatory T cells\nNLRP3 NOD-like receptor protein 3\nMyeloid-derived suppressor\nSTING Stimulator of interferon genes MDSCs\ncells\nTLR9 Toll-like receptor 9 TAMs Tumor-associated macrophage\nIFN-I Type I interferon IFNAR Type I IFNs receptor\nCTLs Cytotoxic T lymphocytes PD-L1 Programmed death ligand 1\nSONP Sonosensitive nanoparticle HCC hepatocellular carcinoma\nSONA Sono-adjuvant TNBC Triple-negative breast cancer\nhematoporphyrin monomethyl\nSONV Sono-adjuvant-based vaccine HMME\nether\nNe-SONV- Neoantigen-based sono-vaccine ROP Ring-opening polymerization\nER Endoplasmic reticulum ISG Type-I interferon-related genes\nmtDNA mitochondrial DNA DLS Dynamic light scattering\nDCs Dendritic cells TEM Transmission Electron\nNAC N-Acetyl-L-cysteine tdLNs Tumor-draining lymph nodes\nH&E Hematoxylin and Eosin GSEA Gene set enrichment analysis\nTdLNs Tumor-draining lymph nodes IRF7 Interferon regulatory factor 7\nELISA Enzyme-linked immunosorbent TBK1 TANK-binding kinase-1\nMyeloid differentiation primary Confocal laser scanning\nMyD88 CLSM\nresponse 88 microscope\nWB Western blotting IRF3 Interferon regulatory factor 3\n4. Ultrasound plays an important role in this study. However, no specific information about\nthe ultrasound equipment and related parameters were provided in this article. For example,\nwhen the authors say 1.0 W/cm2 of ultrasound radiation was applied under certain conditions,\nhow can we trust this quantity without knowing how ultrasound energy delivery was handled\nin the study?\nResponse: We greatly appreciated the comments of the reviewer. We provided specific\ninformation about the US equipment and related parameters in Line 894-905 in the revised\nmanuscript as below:\n“The US equipment was purchased from DJO France S.A.S. (Cat. number: DJO 2776,\nFrequency: 1 MHz, Power density: 0-1.5 W/cm2, Duty cycle: 50%, Transducer type:\nCOLLIMATING, Effective radiating area: 4 cm2). We selected the US frequency of 1 MHz and\nthe duty cycle of 50% based on the following considerations. First, the US frequency of 1\nMHz has good penetration depth in biological tissues, which allows it to delivery energy into\nthe LNs66. When combined with a 50% duty cycle, the energy is evenly distributed and can\nensure sufficient energy transfer to achieve the treatment effect during the penetration\nprocess. Second, the combination of 1 MHz frequency and 50% duty cycle can avoid\nexcessive damage to the tissue caused by continuous high-energy output, allowing to reduce\nadverse effects such as thermal effects on tissues67. Third, this combination was\ndemonstrated to produce a moderate cavitation effect, and can avoid unnecessary damage\nto the surrounding tissues caused by overly intense cavitation68-70.”\nReference:\n66. Han Z, et al. Precise Cell Type Electrical Stimulation Therapy Via Force-electric Hydrogel Microspheres\nfor Cartilage Healing. Adv Mater 37, e2414555 (2025).\n67. Zhu K, Wang J, Wang Z, Chen Q, Song J, Chen X. Ultrasound-Activated Theranostic Materials and\nTheir Bioapplications. Angew Chem Int Ed Engl, e202422278 (2025).\n68. Moradi Kashkooli F, Jakhmola A, Hornsby TK, Tavakkoli JJ, Kolios MC. Ultrasound-mediated nano\ndrug delivery for treating cancer: Fundamental physics to future directions. J Control Release 355, 552-\n578 (2023).\n69. Paliwal S, Mitragotri S. Ultrasound-induced cavitation: applications in drug and gene delivery. Expert\nOpin Drug Deliv 3, 713-726 (2006).\n70. Yu CC, et al. A Conformable Ultrasound Patch for Cavitation-Enhanced Transdermal Cosmeceutical\nDelivery. Adv Mater 35, e2300066 (2023).\n5. The authors claim to have spatiotemporally controlled the regulation of innate immunity\nusing ultrasound (US). It would be helpful if the authors could elaborate on how ultrasound\nwas utilized across different investigations, including the specific experimental procedures.\nAdditionally, further clarification on the selection of various ultrasound parameters, such as\nfrequency, duty cycle, and peak negative pressure, would enhance the readers’\nunderstanding (Supplementary Information, Line 901, Line 929, Line 944).\nResponse: We sincerely thank the critical comments of the reviewer helping us to improve\nthe manuscript. We provided a detailed description of the experimental settings, including US\nprocedures and parameters, across different investigations in the revised manuscript as\nbelow:\n“In this study, we selected the US frequency of 1 MHz and the duty cycle of 50% based on\nthe following considerations. First, the US frequency of 1 MHz has good penetration depth in\nbiological tissues, which allows it to delivery energy into the LNs66. When combined with a\n50% duty cycle, the energy is evenly distributed and can ensure sufficient energy transfer to\nachieve the treatment effect during the penetration process. Second, the combination of 1\nMHz frequency and 50% duty cycle can avoid excessive damage to the tissue caused by\ncontinuous high-energy output, allowing to reduce adverse effects such as thermal effects on\ntissues67. Third, this combination was demonstrated to produce a moderate cavitation effect,\nand can avoid unnecessary damage to the surrounding tissues caused by overly intense\ncavitation68-70.”\nWe added the relevant discussion in Line 894-905 in the revised manuscript.\n“Since we fixed the US frequency and duty cycle as described above, we next altered the\npower density and duration of US stimulation to generate different levels of mechanical and\noxidative force, and then investigated their effect on innate immune activation in vitro and in\nvivo. For in vitro studies, we first screened the power density of US stimulation to avoid\nunintended cellular damage caused by mechanical force. The result showed that no obvious\ncytotoxicity was observed in DC2.4 cells and RAW264.7 cells at US power density below 0.2\nW/cm2 (Supplementary Fig. 1). We next selected the power density of US stimulation to be\n0.2 W/cm2, the HMME concentration to be 1.0 μg/mL, and screened the duration of US\nstimulation to avoid unintended cellular damage caused by the combination of mechanical\nand oxidative forces. The result showed that no obvious cytotoxicity was observed in\nSONP+US-treated DC2.4 cells and RAW264.7 cells at US duration below 4 minutes\n(Supplementary Fig. 4). Therefore, we screened out the power density (0.2 W/cm2) and\nduration (3 min) of US stimulation for innate immune activation in vitro.”\nWe added the relevant discussion in Line 98-101, 128-132 in the revised manuscript.\n“For in vivo studies, we first investigated the influence of different power density of US\nstimulation on inducing the innate immune responses in vivo. The LNs were locally stimulated\nwith 5 minutes of indicated power density of US (0, 0.2, 0.5, 1.0, 1.5 W/cm2) at 6 h post SONA\ninjection. Flow cytometry analysis revealed that only SONA treatment induced negligible\ninnate immunity in LNs. In contrast, the combination of LNs-localized US stimulation\neffectively activated the innate immune responses, and 1.0 W/cm2 of US stimulation elicited\nthe highest recruitment and maturation of DCs and macrophages in LNs (Fig. 2f, g and\nSupplementary Fig. 15). We subsequently investigated the influence of different duration of\nUS stimulation on innate immunity activation in vivo. The LNs were locally stimulated with 1.0\nW/cm2 of indicated duration of US (0, 1, 3, 5, 7 min) at 6 h post SONA injection. The results\nshowed that the recruitment and maturation levels of DCs in LNs gradually increased with\nthe increase of US duration, and it almost reached the peak value when combined with 5\nminutes of US stimulation (Supplementary Fig. 16). Therefore, we screened out the power\ndensity (1.0 W/cm2) and duration (5 min) of US stimulation for LNs-specific innate immune\nactivation in vivo.”\nWe added the relevant discussion in Line 230-243 in the revised manuscript.\nReference:\n66. Han Z, et al. Precise Cell Type Electrical Stimulation Therapy Via Force-electric Hydrogel Microspheres\nfor Cartilage Healing. Adv Mater 37, e2414555 (2025).\n67. Zhu K, Wang J, Wang Z, Chen Q, Song J, Chen X. Ultrasound-Activated Theranostic Materials and\nTheir Bioapplications. Angew Chem Int Ed Engl, e202422278 (2025).\n68. Moradi Kashkooli F, Jakhmola A, Hornsby TK, Tavakkoli JJ, Kolios MC. Ultrasound-mediated nano\ndrug delivery for treating cancer: Fundamental physics to future directions. J Control Release 355, 552-\n578 (2023).\n69. Paliwal S, Mitragotri S. Ultrasound-induced cavitation: applications in drug and gene delivery. Expert\nOpin Drug Deliv 3, 713-726 (2006).\n70. Yu CC, et al. A Conformable Ultrasound Patch for Cavitation-Enhanced Transdermal Cosmeceutical\nDelivery. Adv Mater 35, e2300066 (2023).\nSupplementary Fig. 1 (in this revised manuscript). The relative cell viability of DC2.4 and RAW264.7 cells after\ntreatment with different power density of US stimulation (n = 3 biologically independent replicates). Statistical\nsignificance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 4 (in this revised manuscript). The relative cell viability of DC2.4 and RAW264.7 cells after\nSONP+US treatment with different duration of US stimulation (n = 3 biologically independent replicates). Statistical\nsignificance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 15 (in this revised manuscript). a, The absolute number of CD11c+ DCs and CD11b+\nmacrophages in the LNs after SONA+US treatment with different power density of US stimulation (n = 3 biologically\nindependent mice). b, The CD80 or CD86 expression level on CD11c+ DCs after SONA+US treatment with different\npower density of US stimulation (n = 3 biologically independent mice). Statistical significance was calculated via\none-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 16 (in this revised manuscript). a, The absolute number of CD11c+ DCs and CD11b+\nmacrophages in the LNs after SONA+US treatment with different duration of US stimulation (n = 3 biologically\nindependent mice). b, The CD80 or CD86 expression level on CD11c+ DCs after SONA+US treatment with different\nduration of US stimulation (n = 3 biologically independent mice). Statistical significance was calculated via one-way\nANOVA with Tukey’s test and P values were indicated.\n6. In Lines 600–603, the authors state that \"ultrasound-triggered mechanical and oxidative\nforce allowed for spatiotemporal tailoring of innate immunity activation in LNs by adjusting\nthe irradiation site, power, time interval, and duration of external ultrasound stimuli.\" However,\nthe manuscript does not clearly explain how the mechanical and oxidative forces were\ncontrolled, particularly given that uncontrolled ultrasound irradiation could potentially lead to\ncellular damage. A discussion on the measures taken to minimize unintended cellular\ndamage caused by mechanical and oxidative forces would strengthen the manuscript.\nResponse: We greatly appreciated the comments of the reviewer reminding us to discuss\nthe minimization of unintended cellular damage caused by uncontrolled US stimulation. We\nadded the relevant discussion in Line 98-101, 128-132, 770-775 in the revised manuscript\nas below:\n“In this study, we selected the US frequency of 1 MHz and the duty cycle of 50%, since this\ncombination was demonstrated to not only exhibit high penetration depth in biological tissue,\nbut also avoid excessive damage to the tissues caused by continuous high-energy output66,67.\nIn addition, the combination of 1 MHz frequency and 50% duty cycle of US stimulation could\nproduce a moderate cavitation effect that not only facilitated the cytosolic delivery of payloads,\nbut also avoided unnecessary damage to the surrounding tissues caused by overly intense\ncavitation68-70. Meanwhile, for in vitro studies, we altered the power density and duration of\nUS stimulation to generate different levels of mechanical and oxidative force, and then\ninvestigated their cellular damage effect on APCs. The result showed that shorter than 4\nminutes of US stimulation at 0.2 W/cm2 exerted negligible cytotoxicity on DC2.4 cells and\nRAW264.7 cells (Supplementary Fig. 1 and 4). Therefore, we optimized the US parameters\nto minimize the unintended cellular damage on APCs in vitro.\nFor in vivo studies, the porphyrin-based sonosensitizer (HMME) integrated in the SONA\nplatform not only generated the US-activatable mechanical and oxidative forces, but also\nexhibited the fluorescence imaging capacity to precisely trace the distribution of SONA in\nLNs. In addition, the high-frequency ultrasound (HFUS) technique has been used to image\nand assess human LNs with potential diagnostic capacity65. Therefore, the fluorescence\nimaging and HFUS technique could be further combined to effectively visualize SONA-\ndraining LNs that located at the superficial internal, and thus guide us to perform LNs-specific\nUS stimulation and minimize the uncontrolled cellular damage on normal tissues.”\nReference:\n65. Najibi, A.J. et al. Durable lymph-node expansion is associated with the efficacy of therapeutic\nvaccination. Nat Biomed Eng 8, 1226-1242 (2024).\n66. Han Z, et al. Precise Cell Type Electrical Stimulation Therapy Via Force-electric Hydrogel Microspheres\nfor Cartilage Healing. Adv Mater 37, e2414555 (2025).\n67. Zhu K, Wang J, Wang Z, Chen Q, Song J, Chen X. Ultrasound-Activated Theranostic Materials and\nTheir Bioapplications. Angew Chem Int Ed Engl, e202422278 (2025).\n68. Moradi Kashkooli F, Jakhmola A, Hornsby TK, Tavakkoli JJ, Kolios MC. Ultrasound-mediated nano\ndrug delivery for treating cancer: Fundamental physics to future directions. J Control Release 355, 552-\n578 (2023).\n69. Paliwal S, Mitragotri S. Ultrasound-induced cavitation: applications in drug and gene delivery. Expert\nOpin Drug Deliv 3, 713-726 (2006).\n70. Yu CC, et al. A Conformable Ultrasound Patch for Cavitation-Enhanced Transdermal Cosmeceutical\nDelivery. Adv Mater 35, e2300066 (2023).\nSupplementary Fig. 1 (in this revised manuscript). The relative cell viability of DC2.4 and RAW264.7 cells after\ntreatment with different power density of US stimulation (n = 3 biologically independent replicates). Statistical\nsignificance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 4 (in this revised manuscript). The relative cell viability of DC2.4 and RAW264.7 cells after\nSONP+US treatment with different duration of US stimulation (n = 3 biologically independent replicates). Statistical\nsignificance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\n7. Since mechanical effects and oxidative forces are two distinct phenomena triggered by\nultrasound, it would be beneficial for the authors to clarify which of these plays a more\ndominant role in initiating immune pathways in antigen-presenting cells (APCs).\nResponse: We greatly appreciated the comments of the reviewer helping us to clarify the\ncontribution of mechanical and oxidative forces in initiating immune pathways in APCs. We\nadded the relevant discussion in Line 149-163 in the revised manuscript as below:\n“First, we compared the effect of mechanical and oxidative forces on inducing innate immune\npathways activation in DC2.4 cells and RAW264.7 cells by western blotting assay. The\nfindings showed that US-mediated mechanical force only moderately activated NLRP3\npathways in APCs, while merely affecting STING and TLR9 pathways activation. In contrast,\nSONP+US treatment could effectively activate STING, TLR9 and NLRP3 pathways in DC2.4\ncells and RAW264.7 cells, as evident from the significant upregulation of indicated proteins\nexpression, such as phosphorylated-TBK1 (p-TBK1), phosphorylated-IRF3 (p-IRF3) and\nphosphorylated-IRF7 (p-IRF7) (Supplementary Fig. 6). In addition, we compared the effect\nof mechanical and oxidative forces on triggering DCs maturation in vitro. The result showed\nthat US-mediated mechanical force only moderately enhanced DCs maturation level (CD86+,\n8.9%) compared with PBS group (CD86+, 5.7%). Only sonosensitive nanoparticle (SONP)\nincubation could not induce any mechanical and oxidative forces, and thus showed no\nobvious effect on DCs maturation. In contrast, the combination of mechanical and oxidative\nforces-mediated by SONP+US significantly increased the frequency of CD86+ DCs (15.1%),\nshowing a 2.6- and 1.7-fold higher than that in PBS and US groups, respectively\n(Supplementary Fig. 7). Collectively, these findings demonstrated that the oxidative force\ngenerated by SONP+US might play a more dominant role in initiating innate immune\npathways in APCs.”\nSupplementary Fig. 6 (in this revised manuscript). WB analysis of the activation of STING, TLR9 and NLRP3\npathways in DC2.4 cells (a) and RAW264.7 cells (b) after indicated treatments.\nSupplementary Fig. 7 (in this revised manuscript). Representative flow cytometric plots and the frequency of\nCD86+ DCs post indicated treatments (n = 3 biologically independent replicates). Statistical significance was\ncalculated via one-way ANOVA with Tukey’s test and P values were indicated.\n8. Given that innate immune pathways in APCs are generally considered inflammatory, the\nauthors may wish to provide additional mechanistic insights into how SONV + US remain\nunaffected by immune-suppressive cells.\nResponse: We greatly appreciated the comments of the reviewer reminding us to provide\nadditional mechanistic insights into how SONA+US remain unaffected by immune-\nsuppressive cells. We added the relevant discussion in Line 243-245, 253-257 in the revised\nmanuscript as below:\n“In this study, inspired by the mannose receptor was highly expressed in APCs40-42, we\nintroduced an active-targeting moiety, mannose, for surface modification of sono-adjuvant\n(SONA) to improve the APCs-targeting performance. Compared with SONA3 that without\nmannose-modification, SONA4 displayed 2.1- and 2.3-fold higher internalization by DCs and\nmacrophages in LNs, respectively (unless noted, all SONA mentioned below refers to SONA4)\n(Fig. 2e and Supplementary Fig. 13). Therefore, the combination of SONA with US\nstimulation could mainly affect the innate immune pathways in APCs and trigger abundant\nand sustained IFN-β secretion in LNs (Fig. 2h). It was worth noting that IFN-β was\ndemonstrated to directly inhibit Tregs proliferation and reprogram M2-type macrophages43-45.\nTherefore, a significant decrease in the frequency of immune-suppressive cells in the LNs,\nincluding Tregs (FOXP3+CD25+CD4+CD3+) and CD206+CD11b+ M2 macrophages, could be\nobserved after SONA+US treatment (Fig. 2k, m and Supplementary Fig. 17b, d).”\nReference:\n40. Singh A, et al. Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis. Proc\nNatl Acad Sci U S A 119, e2121098119 (2022).\n41. Li Y, et al. In situ tumor vaccine with optimized nanoadjuvants and lymph node targeting capacity to\ntreat ovarian cancer and metastases. Acta Pharm Sin B 14, 4102-4117 (2024).\n42. Liu Z, et al. Dual-Targeted Self-Adjuvant Heterocyclic Lipidoid@Polyester Hybrid Nanovaccines for\nBoosting Cancer Immunotherapy. ACS Nano 18, 15557-15575 (2024).\n43. Bacher N, et al. Interferon-alpha suppresses cAMP to disarm human regulatory T cells. Cancer Res\n73, 5647-5656 (2013).\n44. Yu R, Zhu B, Chen D. Type I interferon-mediated tumor immunity and its role in immunotherapy. Cell\nMol Life Sci 79, 191 (2022).\n45. Snell LM, McGaha TL, Brooks DG. Type I Interferon in Chronic Virus Infection and Cancer. Trends\nImmunol 38, 542-557 (2017).\nSupplementary Fig. 13 (in this revised manuscript). Representative flow cytometric plots and the frequency of\nSONA+CD11c+ DCs and SONA+CD11b+ macrophages in LNs at 6 h after SONA3 or SONA4 injection (n = 3\nbiologically independent mice). Statistical significance was calculated via unpaired two-tailed Student’s t-test and P\nvalues were indicated.\nFig. 2 (in this revised manuscript). h, The IFN-β production in LNs at different time points after SONA+US\ntreatment (n = 3 biologically independent mice). Statistical significance was calculated via unpaired two-tailed\nStudent’s t-test and P values were indicated.\nFig. 2 (in this revised manuscript). k, The ratio of M2-type (CD86−CD206+CD11b+) macrophages to M1-type\n(CD86+CD206−CD11b+) macrophages in tdLNs of indicated group mice (n = 5 biologically independent mice). m,\nThe frequency of Tregs (FOXP3+CD4+CD3+) in tdLNs of indicated group mice (n = 5 biologically independent mice).\nG1: PBS, G2: SONA, G3: SONA+US. Statistical significance was calculated via one-way ANOVA with Tukey’s\ntest and P values were indicated.\n9. The manuscript would benefit from additional details on the experimental design, including\nbatch sizes and statistical analyses. Providing this information in the supplementary section\nwould improve the clarity and reproducibility of the study.\nResponse: We greatly appreciated the comments of the reviewer helping us to improve the\nclarity and reproducibility of our study. We provided the corresponding batch sizes and\nstatistical analyses in all supplementary Figures in the revised manuscript. For instance, n = 3\nbiologically independent replicates or n = 5 biologically independent mice. Statistical\nsignificance was calculated via unpaired two-tailed Student’s t-test or one-way ANOVA with\nTukey’s test and P values were indicated.\n10. Finally, improving the overall cohesiveness of the manuscript would enhance readability\nand comprehension.\nResponse: We greatly appreciated the comments of the reviewer helping us to improve the\nreadability and comprehension of our manuscript. We checked and corrected unclear\nsentences throughout the manuscript, and provided the specific quantitative evidence to\nsupport the claims about significant difference observed between different groups. In addition,\nwe provided a critical discussion of the current limitations and future directions of our sono-\nadjuvant platform in the Discussion section. We were confident that these efforts would\nsignificantly improve the overall cohesiveness of the revised manuscript.\nMinor comments on the manuscript:\n1. Line 46: Please provide a citation for the statement: \"Unfortunately, the microenvironment\nwithin the lymph nodes (LNs) is often ineffective in stimulating APC maturation, migration,\nand cytokine secretion, thus deactivating the antigen cross-presentation ability of APCs.\"\nResponse: We provided corresponding citations for the statement in Line 47-49 in the\nrevised manuscript as below:\n“However, microenvironment within lymph nodes (LNs) often are ineffective to stimulate\nAPCs maturation, migration and cytokine secretion, thus deactivating the antigen cross-\npresentation ability of APCs9-11.”\nReference:\n9. Zhao T, et al. Vaccine adjuvants: mechanisms and platforms. Signal Transduct Target Ther 8, 283\n(2023).\n10. Su T, et al. Lymph node-targeting adjuvant/neoantigen-codelivering vaccines for combination\nglioblastoma radioimmunotherapy. Theranostics 13, 4304-4315 (2023).\n11. Wang Q, et al. Lymph node-targeting nanovaccines for cancer immunotherapy. J Control Release 351,\n102-122 (2022).\n2. Line 59: Please provide a citation to support the claim: \"However, most of these strategies\nexhibit an 'always on' profile to stimulate innate immunity in vivo and could potentially induce\nimmune-related adverse effects due to their non-specific distribution and immune stimulation\nof untargeted tissues (e.g., liver, lung), thus limiting their future clinical translation.\"\nResponse: We provided corresponding citations to support the claim in Line 60-63 in the\nrevised manuscript as below:\n“However, most of these strategies exhibited the ‘always on’ profile to stimulate innate\nimmunity in vivo, and could potentially induce immune-related adverse effects due to their\nnon-specific distribution and immune stimulation on untargeted tissues (e.g., liver, lung), thus\nlimiting their future clinical translation21-23.”\nReference:\n21. Ryu KA, Stutts L, Tom JK, Mancini RJ, Esser-Kahn AP. Stimulation of innate immune cells by light-\nactivated TLR7/8 agonists. J Am Chem Soc 136, 10823-10825 (2014).\n22. Ding C, et al. Photochemically controlled activation of STING by CAIX-targeting photocaged agonists\nto suppress tumor cell growth. Chem Sci 14, 5956-5964 (2023).\n23. Dou Y, et al. Optogenetic engineering of STING signaling allows remote immunomodulation to enhance\ncancer immunotherapy. Nat Commun 14, 5461 (2023).\n3. Line 90: Including an experimental figure illustrating how ultrasound (US) is applied would\nmake this section more self-explanatory and improve clarity.\nResponse: We provided an experimental figure in Supplementary Fig. 1a in the revised\nmanuscript for illustrating how US was applied to trigger mechanical and oxidative forces in\nvitro.\nSupplementary Fig. 1a. Experimental figure to illustrate the application of US stimulation in vitro.\n4. The word “dramatically” appears multiple times throughout the manuscript (e.g., Line 91,\nLine 193, Line 246, Line 255, Line 369, Line 432, Line 434). Replacing it with a more precise\nterm would improve the readability and scientific rigor of the text.\nResponse: We greatly appreciated the comments of the reviewer helping us to improve the\nreadability and scientific rigor of the text. We reduced the word “dramatically” throughout the\nmanuscript, and provided the specific quantitative evidence to support the claims about\nsignificant difference observed between different groups.\nFor instance, in Line 102-106 in the revised manuscript, the sentence was corrected as:\n“Flow cytometry analysis and confocal laser scanning microscope (CLSM) images indicated\nthat US stimulation affected the intracellular Ca²⁺ concentration of DCs as a function of power\ndensity. Specifically, US stimulation (0.2 W/cm2, 1 MHz, 50% duty cycle) induced 3.1-fold\nincrease of intracellular Ca²⁺ concentration in DCs compared to PBS group (Fig. 1b-d).”\nIn Line 243-245 in the revised manuscript, the sentence was corrected as: “Under these\nUS stimulation parameters, we found the combination of SONA injection with US stimulation\n(namely SONA+US) could induce abundant and sustained IFN-β and IL-6 secretion in LNs\nfor over 48 h (Fig. 2h).”\nIn Line 305-309 in the revised manuscript, the sentence was corrected as: “In\ncomparison, the combination of mechanical and oxidative forces mediated by SONV+US\n(1MHz, 50% duty cycle, 0.2 W/cm2, 3 min) could further increase the lysosomal membrane\npermeability of DCs. In addition, obviously disrupted lysosomal structures were observed in\nthe Bio-TEM images of SONV+US-treated DCs (Supplementary Fig. 19).”\nIn Line 314-318 in the revised manuscript, the sentence was corrected as: “In contrast,\nSONV+US significantly induced antigen cytosolic release, with most of OVA-FITC signal\ndetected in the cytosol while not in lysosome (Supplementary Fig. 21). Specifically, the\nPearson’s R value (PR) of OVA-FITC signal (green) with lysosomal signal (red) in SONV and\nSONV+US groups were 0.25 and -0.12, respectively.”\nIn Line 434-436 in the revised manuscript, the sentence was corrected as: “In contrast,\nSONV+US treatment effectively suppressed the formation of lung metastatic nodules,\nshowing 4.0-fold higher anti-metastases efficacy than SONV treatment (Fig. 4d).”\nIn Line 530-534 in the revised manuscript, the sentence was corrected as: “TLR9-i\ninjection remarkably decreased the antigen presentation level on APCs and antigen-specific\nCD8+ T cell response in tdLNs induced by SONV+US treatment. Specifically, the frequency\nof antigen-specific CD8+ T cell in SONV+US group (6.9%) was 2.2-fold higher than that in\nTLR9-i+SONV+US group (3.1%) (Fig. 5e, g and Supplementary Fig. 41c-e).”\nIn Line 534-537 in the revised manuscript, the sentence was corrected as: “TLR9-i\ninjection almost completely abolished SONV+US-mediated CTLs infiltration, and the\nfrequency of tumor-infiltrating IFN-γ+CD8 T+ cell in SONV+US group (9.5%) was 4.5-fold\nhigher than that in TLR9-i+SONV+US group (2.1%) (Fig. 5i, j and Supplementary Fig. 42a,\nb).”\n5. Line 114: Please revise the sentence for improved clarity: \"CLSM images showed that\nSONP incubation plus ultrasound irradiation.\"\nResponse: We revised the sentence in Line 132-137 in the revised manuscript as below:\n“We next used the reactive oxygen species (ROS) indicator, 2',7'-dichlorodihydrofluorescein\ndiacetate (DCFH-DA), to detect the generation level of oxidative force by SONP+US in DCs.\nCLSM images showed that only SONP incubation without US stimulation showed no ROS\ngeneration effect, while SONP+US could trigger the intracellular ROS generation of DCs as\na function of US stimulation duration (Fig. 1k).”\n6. Lines 115, 901, 929, 944: Please replace ‘MHZ’ with ‘MHz’ for consistency in unit notation.\nResponse: We replaced ‘MHZ’ with ‘MHz’ in Lines 115, 901, 929, 944 in the revised\nmanuscript for consistency in unit notation.\n7. Line 166: The phrase “increasingly clear” could be replaced with a more specific description.\nResponse: We revised the sentence in Line 200-202 in the revised manuscript as below:\n“It has been extensively demonstrated that the formulations to spatially confine adjuvant\nactivity within the secondary lymphoid organs are crucial for enhancing cancer vaccine-\nmediated T-cell response36-39.”\nReference:\n36. Lynn GM, et al. In vivo characterization of the physicochemical properties of polymer-linked TLR\nagonists that enhance vaccine immunogenicity. Nat Biotechnol 33, 1201-1210 (2015).\n37. Dane EL, Irvine DJ. Big thinking for adjuvants. Nat Biotechnol 33, 1146-1148 (2015).\n38. Ke X, et al. Physical and chemical profiles of nanoparticles for lymphatic targeting. Adv Drug Deliv Rev\n151-152, 72-93 (2019).\n39. Ding Y, Li Z, Jaklenec A, Hu Q. Vaccine delivery systems toward lymph nodes. Adv Drug Deliv Rev\n179, 113914 (2021).\n8. Line 168: The sentence \"To optimize LN-confined distribution and APC-targeting\ninternalization, we developed a set of sonosensitive adjuvants (namely sono-adjuvants,\nSONA) with adjustable physicochemical properties, including particle size, surface charge,\nand ligand modification.\" should reference the corresponding table in Figure 2 for clarity.\nResponse: We provided the corresponding reference in the sentence in Line 202-205 in the\nrevised manuscript as below:\n“To optimize LNs-specific and APCs-targeting delivery, we developed a set of polypeptide-\nbased sonosensitive adjuvant (namely sono-adjuvant, SONA) with adjustable\nphysicochemical properties, including particle size, surface charge, and ligand modification,\nas shown in the corresponding table in Fig. 2a.”\n9. Lines 186–188: Please provide either a citation or a brief explanation for selecting the\nreported range of power density. Additionally, specify the frequency and duty cycle used for\nultrasound irradiation.\nResponse: We provided corresponding citations and explanation for selecting the reported\npower density, frequency and duty cycle of US stimulation in Line 230-232, 896-903 in the\nrevised manuscript as below:\n“To investigate the effect of external US stimulation on controlling the adjuvant activity of\nSONA in vivo, the LNs were locally stimulated with 1 MHz, 50% duty cycle and indicated\npower density of US (0, 0.2, 0.5, 1.0, 1.5 W/cm2 ) at 6 h post SONA injection. The selected\nUS parameters not only ensured a good penetration depth in biological tissues, but also could\navoid excessive damage to the tissue caused by continuous high-energy output, allowing to\nreduce adverse effects such as thermal effects on tissues66,67.”\nReference:\n66. Han Z, et al. Precise Cell Type Electrical Stimulation Therapy Via Force-electric Hydrogel Microspheres\nfor Cartilage Healing. Adv Mater 37, e2414555 (2025).\n67. Zhu K, Wang J, Wang Z, Chen Q, Song J, Chen X. Ultrasound-Activated Theranostic Materials and\nTheir Bioapplications. Angew Chem Int Ed, e202422278 (2025).\n10. Line 200: A more detailed explanation of why regulatory T cells (Tregs,\nFOXP3+CD25+CD4+CD3+) were downregulated by ultrasound irradiation would be\nbeneficial.\nResponse: We provided a more detailed explanation of why regulatory T cells (Tregs,\nFOXP3+CD25+CD4+CD3+) were downregulated after SONA+US treatment in Line 243-245,\n253-257 in the revised manuscript as below:\n“Under these US stimulation parameters, we found the combination of SONA injection with\nUS stimulation (namely SONA+US) could induce abundant and sustained IFN-β and IL-6\nsecretion in LNs for over 48 h (Fig. 2h). It was worth noting that IFN-β was demonstrated to\ndirectly inhibit Tregs proliferation and reprogram M2-type macrophages43-45. Therefore, a\nsignificant decrease in the frequency of immune-suppressive cells in tdLNs, including Tregs\n(FOXP3+) and M2-type macrophages (CD206+), could be observed after SONA+US\ntreatment (Fig. 2m).”\nReference:\n43. Bacher N, et al. Interferon-alpha suppresses cAMP to disarm human regulatory T cells. Cancer Res\n73, 5647-5656 (2013).\n44. Yu R, Zhu B, Chen D. Type I interferon-mediated tumor immunity and its role in immunotherapy. Cell\nMol Life Sci 79, 191 (2022).\n45. Snell LM, McGaha TL, Brooks DG. Type I Interferon in Chronic Virus Infection and Cancer. Trends\nImmunol 38, 542-557 (2017).\nFig. 2 (in this revised manuscript). h, The IFN-β production in the LNs at different time points after SONA+US\ntreatment (n = 3 biologically independent mice). m, The frequency of Tregs (FOXP3+CD4+CD3+) in the tdLNs of\nindicated group mice (n = 5 biologically independent mice). G1: PBS, G2: SONA, G3: SONA+US. Statistical\nsignificance was calculated via unpaired two-tailed Student’s t-test (h) or one-way ANOVA with Tukey’s test (m)\nand P values were indicated.\n11. Figure 2(a): If this study is not a clinical study, the representation of a human lymph node\nmay be misleading. Consider revising the figure to avoid potential misinterpretation.\nResponse: We updated the representation of lymph node in Fig. 2a to not only avoid\npotential misinterpretation, but also indicate the potential of sono-adjuvant for clinical\ntranslation.\nFig. 2 (in this revised manuscript). a, Schematic illustration for developing SONA library with adjustable\nphysicochemical property and APCs-targeting ability to achieve US-activatable innate immunity activation in LNs.\n12. Lines 245–249: Please revise these sentences for improved clarity and readability.\nResponse: We revised these sentences in Line 302-309 in the revised manuscript as below:\n“The result showed that only US stimulation-mediated mechanical force was demonstrated\nto increase the permeability of lysosomal membrane through the cavitation effect, while only\nSONV incubation showed no obvious lysosomal disruption effect (Supplementary Fig. 18).\nIn comparison, the combination of mechanical and oxidative forces mediated by SONV+US\n(1MHz, 50% duty cycle, 0.2 W/cm2, 3 min) could further increase the lysosomal membrane\npermeability of DCs. In addition, obviously disrupted lysosomal structures were observed in\nthe Bio-TEM images of SONV+US-treated DCs (Supplementary Fig. 19).”\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We greatly appreciated the comments of the reviewer helping us to improve the\nquality of our manuscript.\nReviewer #3 (Remarks to the Author):\nThis manuscript reports a new ultrasound-confined approach to regulate innate immunity and\nenhance cancer vaccination therapy. Chen et al. develops and characterizes a polypeptide-\nbased sono-adjuvant (SONA) that activates multiple innate immune pathways (NLRP3,\nSTING, and TLR9) in antigen-presenting cells (APCs) and demonstrates its efficacy in\npreclinical tumor models. This work could be of interest to the immunoengineering and cancer\nimmunotherapy fields. Some comments are provided below:\nMajor issues:\n1. The manuscript is dense and difficult to follow. In some parts of the results section, the\nlanguage is too vague and lacks quantitative evidence. For example, the sentence: ‘Flow\ncytometry analysis and confocal laser scanning microscope (CLSM) images indicated that\nultrasound irradiation dramatically affects the intracellular Ca²⁺ concentration of DCs as a\nfunction of power density (Fig. 1b-d).’ How dramatic is the effect? The authors should provide\nspecific data to support this claim.\nResponse: We greatly appreciated the comments of the reviewer helping us to improve the\nreadability of the manuscript. We provided the quantitative evidence to support the claims\nabout dramatic difference observed between experimental groups throughout the manuscript.\nFor instance, in Line 102-106 in the revised manuscript, the sentence was corrected as:\n“Flow cytometry analysis and confocal laser scanning microscope (CLSM) images indicated\nthat US stimulation affected the intracellular Ca²⁺ concentration of DCs as a function of power\ndensity. Specifically, 0.2 W/cm2 of US stimulation (1 MHz, 50% duty cycle) induced 3.1-fold\nincrease of intracellular Ca²⁺ concentration in DCs compared to PBS group (Fig. 1b-d).”\nIn Line 112-117 in the revised manuscript, the sentence was corrected as: “Western blot\n(WB) and enzyme linked immunosorbent assay (ELISA) further validated that US-triggered\nmechanical force activated the NLRP3-Capase-1-IL-1β signaling in APCs (Fig. 1f, g).\nSpecifically, 3 minutes of US stimulation (0.2 W/cm2, 1 MHz, 50% duty cycle) induced 3.9-\nand 4.3-fold increase of IL-1β release and activated caspase-1 expression in DCs compared\nto PBS group, respectively (Fig. 1h).”\nIn Line 157-163 in the revised manuscript, the sentence was corrected as: “In addition,\nwe evaluated the effect of mechanical and oxidative forces on stimulating DCs maturation in\nvitro. The result showed that US-triggered mechanical force moderately enhanced DCs\nmaturation level (CD86+, 8.9%) compared with PBS group (CD86+, 5.7%). In comparison, the\ncombination of mechanical and oxidative forces mediated by SONP+US significantly\nincreased the frequency of CD86+ DCs (15.1%), showing a 2.6- and 1.7-fold higher than that\nin PBS and US groups, respectively (Supplementary Fig. 7).”\nIn Line 247-251 in the revised manuscript, the sentence was corrected as: “SONA\ntreatment induced no effect on DCs maturation and T cell activation compared with PBS\ngroup. In marked contrast, the frequency of matured DCs (CD80+CD86+) in SONA+US group\nwas about 16.1%, showing 2.0- and 1.8-fold higher than that in PBS group (8.1%) and SONA\ngroup (8.7%), respectively (Fig. 2j and Supplementary Fig. 17a).”\nIn Line 366-371 in the revised manuscript, the sentence was corrected as: “Upon ex-vivo\nrestimulation with the SIINFEKL peptide, the splenocytes from SONV+US group mice\nshowed the highest IFN-γ+CD8+ T cell and TNF-α+CD8+ T cell responses among all the\ngroups. Specifically, the frequency of IFN-γ+ in CD8+ T cells in SONV+US group was about\n24.3%, showing 2.5- and 1.9-fold higher than that in OVA+CpG group (9.8%) and OVA+MSA-\n2 group (12.6%), respectively (Fig. 3k and Supplementary Fig. 29b, c).”\nIn Line 484-489 in the revised manuscript, the sentence was corrected as: “ELISA result\nrevealed that SONV+US significantly increased intratumoral secretion of TNF-α, while\ninhibited the typical immunosuppressive cytokine (TGF-β) secretion (Fig. 4p and\nSupplementary Fig. 39). The intratumoral TNF-α secretion levels in OVA+CpG, OVA+MSA-\n2 and SONV+US group was about 1189.6 pg/mL, 1996.3 pg/mL and 2346.3 pg/mL,\nrespectively. The intratumoral TGF-β secretion levels in OVA+CpG, OVA+MSA-2 and\nSONV+US group was about 2515.0 pg/mL, 1593.6 pg/mL and 835.8 pg/mL,\nrespectively.”\n2. The authors analysed the transcriptomic features of LNs from SONV+US group mice.\nHowever, Figure 3m volcano plot doesn’t make sense. In the supplementary section, the\nauthors stated that genes were selected using DESeq2 with log2FC ≥ 1 and FDR ≤ 0.05.\nBut in the volcano plot, the p-value cutoff looks very close to 0. Since FDR ≤ 0.05 should\ncorrespond to a p-value at least around 0.01, the cutoff line should be closer to 2.5 on the y-\naxis. Please clarify this.\nResponse: We sincerely thank the critical comments of the reviewer for pointing out our\nincorrect presentation of the RNA-seq analysis and volcano plot. We corrected the statement\nabout differential expression analysis and updated the volcano plot in Fig. 3m in the revised\nmanuscript. Differential expression analysis was performed using the DESeq2. DEGs with\n|log2FC| ≥ 1 and adjusted p-value ≤ 0.05 were considered to be significantly different\nexpressed genes.\nWe added the relevant description in Line 980-983 in the revised manuscript.\nFig. 3 (in this revised manuscript). m, Volcano plot of DEGs in LNs after SONV+US versus PBS treatment.\n3. The results section lacks a detailed description of the figures. In Figure 6h and 6i, it seems\nthe authors performed some single-cell analysis, but I couldn't find any detailed explanation\nof this analysis. Figure 6i is also confusing, why do all the t-SNE plots look identical to Figure\n6h? The four PD-1 expression panels represent four different methods, so there should be\nfour separate single-cell datasets. It is hard to imagine that all four would produce identical t-\nSNE plots. Please clarify this.\nResponse: We greatly appreciated the comments of the reviewer reminding us to provide a\ndetailed description of the figures in the results section. Indeed, in Fig. 6h and 6i, we did not\nperform single-cell analysis to evaluate the antitumor immunity induced by Ne-SONV+US.\nInstead, we performed a multicolor flow cytometry analysis to investigate the tumor-infiltration\nand corresponding PD-1 expression levels of various immune cells, such as CD8+ T cells,\nCD4+ T cells, NK cells, TAMs, DCs, MDSCs and neutrophils. The flow cytometry antibodies\nincluding anti-CD45-FITC, anti-CD3-Percp-Cy5.5, anti-CD8-AF700, anti-CD4-BV510, anti-\nCD11b-BV605, anti-F4/80-PE, anti-CD49b-PE-Cy7, anti-CD11c-BV421, anti-Ly6C-APC,\nanti-Ly6G-APC-Cy7 and anti-PD1-PE/Dazzle594 were integrated into one panel, and the flow\ncytometry data was collected by BD Fortessa. Next, we used the software DeNovo FCS\nExpress 7 to produce t-distributed random neighbor embedded (t-SNE) plots of the multicolor\nflow cytometry data. The t-SNE plot presented in Fig. 6h was merged from four different\ngroups data according to the instructions of software, and this analysis method and similar\nresult have been reported in other previous studies1,2. The t-SNE plots presented in Fig. 6i\nwere separated from the merged t-SNE plot based on the corresponding groups, and the\ncolor intensity value reflected the PD-1 expression levels on immune cells. Therefore,\naccording to the t-SNE plots, we could more intuitively observe the changes in the tumor-\ninfiltration and corresponding PD-1 expression levels of different immune cells after indicated\ntreatments.\nWe added the relevant description in Line 613-619 in the revised manuscript.\nReference: (for reviewer)\n1. Chen Y, Chen X, Bao W, Liu G, Wei W, Ping Y. An oncolytic virus-T cell chimera for cancer\nimmunotherapy. Nat Biotechnol 42, 1876-1887 (2024).\n2. Pu Y, et al. Ultrasound-triggered and glycosylation inhibition-enhanced tumor piezocatalytic\nimmunotherapy. Nat Commun 15, 9023 (2024).\n4. Please add details on the quantitative analysis procedures in supplementary. For example,\nin the GSEA analysis, which MSigDB dataset was used? Hallmark? Cancer genes? Please\ninclude all detailed analysis steps in the supplementary file\nResponse: We greatly appreciated the comments of the reviewer reminding us to provide\ndetailed RNA-seq analysis steps in Line 976-991 in the revised manuscript as below:\n“RNA-seq analysis. The LNs from PBS or SONV+US group mice were harvested to extract\nthe total RNA according to the manufacturer’s protocol. RNA purification, reverse\ntranscription, library construction and sequencing were performed at Shanghai Majorbio Bio-\npharm Biotechnology Co., Ltd. according to the manufacturer’s instructions (Illumina, San\nDiego, CA). To identify DEGs between two different samples, the expression level of each\ntranscript was calculated according to the transcripts per million reads (TPM) method.\nDifferential expression analysis was performed using the DESeq2. DEGs with |log2FC| ≥ 1\nand adjusted p-value ≤ 0.05 were considered to be significantly different expressed genes.\nIn addition, functional-enrichment analysis of Kyoto Encyclopedia of Genes and Genomes\n(KEGG, http://www.genome.jp/kegg/) was performed to identify which DEGs were\nsignificantly enriched in metabolic pathways at Bonferroni-corrected p-value ≤ 0.05 compared\nwith the transcriptome background. KEGG pathway analysis were carried out by Python scipy\nsoftware. The Gene Set Enrichment Analysis (GSEA) did not use the MSigDB database.\nInstead, it used the annotation results from KEGG pathway analysis. The ranking algorithm\nused for GSEA was Signal2Noise. The protein-protein interaction network analysis on DEGs\nwas conducted by using the STRING database (combined score ≥ 0.4, http://string-db.org/).”\nMinor issues:\n1. The discussion is too optimistic and lacks a section on limitations. Addressing limitations\nmakes the study more credible and realistic. How ultrasound penetration depth might vary in\nhuman tissues compared to mouse models?\nResponse: We greatly appreciated the comments of the reviewer helping us to enhance the\ncredibility and reality of our study. We provided a critical discussion of the current limitations\nof our sono-adjuvant platform in Line 753-784 in the revised manuscript as below:\n“It was worth noting that although the remarkable advantages of US-activatable sono-\nadjuvant in cancer vaccine immunotherapy, additional efforts are required to address the\ncurrent limitations for future clinical translation. First, from the manufacturing aspect, a more\nprecise control of polypeptides self-assembly by automated microfluidic devices should be\nexplored to produce uniform sono-adjuvant and minimize batch-to-batch variation, which\ncould address the critical translational challenges from bench to bedside. Second, from the\ninnate immune activation aspect, although sono-adjuvant represented a multiply adjuvanted\nplatform to synchronously activate STING, TLR9 and NLRP3 pathways in APCs, the\nunderlying potential and mechanism of the US-activatable innate immune stimulation strategy\nfor addressing APCs dysfunction and exhaustion were not extensively investigated. Third,\nthe induction of long-term antitumor immune responses was highly essential for preventing\ntumor recurrence and distant metastasis. Therefore, additional tumor-rechallenge assay and\nmore extensive immune memory response monitoring should be conducted to overcome the\ncurrent limitations of short-term experimental observation in this study. Moreover, the current\nsono-adjuvant platform was merely examined in various types of mouse tumor models.\nAlthough US therapy exhibited high tissue penetration depth and might be promising for\nstimulating human LNs that located at the superficial internal, the tissue uptake of US\nstimulation may be variable in the treatment of patients compared with mouse models.\nTherefore, an extensive and personalized exploration of the influence of different US\nparameters on innate immune responses should be conducted when applied for clinical\nimmunotherapy.\nFurthermore, the high-frequency ultrasound imaging (HFUS) technique should be further\nexplored and combined to precisely visualize the draining LNs at the superficial internal, and\nthus can guide to perform LNs-specific US stimulation and innate immune activation. The\nprecise monitoring of activation status of different immune cell populations during US-guided\nimmune activation treatment was highly essential for achieving personalized cancer\nimmunotherapy. In future studies, we could combine the multi-color flow cytometry assay to\ncontinuously monitor the activation status of different immune cell populations in the\nperipheral blood, such as detecting the expression of T cell activation-related markers (e.g.,\nCD69, CCR7, CD44, PD1). Based on these results, we could adjust the treatment parameters,\nsuch as the frequency, duration and power density of US stimulation, to accordingly regulate\nthe activation of immune cell populations. We expected the sono-adjuvant platform could be\noptimized in various aspects to improve its therapeutic application scopes, and implement a\nsafe and effective cancer vaccination strategy.”\nReviewer #4 (Remarks to the Author):\nChen et al. proposed a strategy for cancer vaccine immunotherapy using ultrasound-guided\nimmune activation. The experimental design used a variety of mouse models to demonstrate\nthe effectiveness of the strategy in different tumor settings. However, the manuscript should\nbe enhanced before acceptance for publication.\n1. This study presented an ultrasound-guided immune activation approach, but did not\nadequately provide a detailed comparison with existing immunotherapeutic approaches,\nespecially in terms of conventional immunotherapy such as immune adjuvants and immune\ncheckpoint inhibitors. For a new immunotherapy platform, it is important to clearly\ndemonstrate its advantages over existing approaches, especially in terms of improved\nefficacy and immune side effects.\nResponse: We greatly appreciated the comments of the reviewer. To demonstrate the\nadvantages of our sono-adjuvant strategy over the conventional immunotherapies in terms\nof improved antitumor efficacy, we added the relevant discussion in the revised manuscript\nas below:\n“We first selected the conventional TLRs agonist (CpG) and the STING agonist (MSA-2) as\nthe control treatment groups, and compared their effect with SONV+US to enhance antigen-\nspecific immune responses in vivo. As shown in Fig. 3, we found SONV+US treatment\nsignificantly triggered the expression of stimulatory markers (CD80 and CD86) in\nmacrophages and DCs in the LNs (Fig. 3f, g and Supplementary Fig. 28a, b). The\nmaturation degree of DCs (CD80+CD86+ in CD11c+ cells) in SONV+US group was 22.5%,\nshowing 2.2- and 1.6-fold higher than that in OVA+CpG (10.1%) and OVA+MSA-2 (14.2%)\ngroups, respectively. In addition, SONV+US treatment induced the highest level of antigen-\nspecific peptide-MHC-I complex on the surface of DCs (8.2%, SIINFEKL-H2Kb+ in CD11c+ cells),\nwith 2.5- and 1.9-fold higher than that in OVA+CpG and OVA+MSA-2 groups,\nrespectively (Fig. 3h and Supplementary Fig. 28c). More importantly, the frequency of\nantigen-specific CD8+ T cells (SIINFEKL-H2Kb tetramer+ in CD8+ cells) in SONV+US group\nwas about 10.1%, while that in OVA+CpG and OVA+MSA-2 groups were only about 2.5%\nand 3.4%, respectively (Fig. 3i and Supplementary Fig. 29a).”\nWe added the relevant discussion in Line 355-366 in the revised manuscript.\nFig. 3 (in this revised manuscript). SONV+US robustly elicited antigen-specific T-cell response via US-triggered\ninnate immunity activation.\n“We also compared the antitumor efficacy of SONV+US with the conventional TLRs\nagonist or STING agonist-based vaccines in the B16-OVA tumor models. We found that\nOVA+CpG and OVA+MSA-2 both afforded moderate tumor prophylactic and therapeutic\nefficacy. In contrast, SONV+US markedly prevented the formation and lung metastases of\nB16-OVA tumor, with 3/6 of mice had no detectable tumor mass up to 60 days (Fig. 4b and\nSupplementary Fig. 30). In addition, SONV+US effectively inhibited the established B16-\nOVA tumor growth and prolonged the median survival of mice from 20 days (PBS group) to\n36 days (Fig. 4i and Supplementary Fig. 31).\nWe next compared the effect of SONV+US with OVA+CpG and OVA+MSA-2 to promote\nintratumoral antitumor immunity in the B16-OVA tumor model. We found that SONV+US\nstrongly promoted DCs maturation and macrophages activation in the tdLNs\n(Supplementary Fig. 34). The maturation degree of DCs (CD80+ in CD11c+ cells) in\nSONV+US group was 28.8%, showing 1.8- and 1.5-fold higher than that in OVA+CpG (16.2%)\nand OVA+MSA-2 (19.2%) groups, respectively. Compared to the SONV treatment that\nelicited negligible antitumor immunity, SONV+US significantly enhanced tumor infiltration of\nCD8+ T cells (Fig. 4k and Supplementary Fig. 35a), and increased the activation of CTLs.\nThe frequency of IFN-γ+CD8+ T cells (IFN-γ+ in CD8+ T cells) in SONV+US group was about\n19.8%, with 2.5 and 1.7-fold higher than that in OVA+CpG (7.9%) and OVA+MSA-2 (11.6%)\ngroups, respectively (Fig. 4l and Supplementary Fig. 35b). ELISA result revealed that\nSONV+US significantly increased intratumoral secretion of TNF-α, while inhibited the typical\nimmunosuppressive cytokine (TGF-β) secretion (Fig. 4p and Supplementary Fig. 39). The\nintratumoral TNF-α secretion levels in OVA+CpG, OVA+MSA-2 and SONV+US group was\nabout 1189.6 pg/mL, 1996.3 pg/mL and 2346.3 pg/mL, respectively. The intratumoral TGF-β\nsecretion levels in OVA+CpG, OVA+MSA-2 and SONV+US group was about 2515.0 pg/mL,\n1593.6 pg/mL and 835.8 pg/mL, respectively.”\nWe added the relevant discussion in Line 420-446, 449-466 in the revised manuscript.\nFig. 4 (in this revised manuscript). SONV+US preformed potent prophylactic and therapeutic effect to mitigate\ntumor burden.\n“We also compared the antitumor efficacy of neoantigen-based SONV+US (namely Ne-\nSONV+US) with the conventional TLRs agonist-based neoantigen vaccine in the orthotopic\n4T1-Luc tumor model. As shown in Fig. 6, compared to the admixture of neoantigen peptide\nwith CpG adjuvant (Neop+CpG) that moderately inhibited the orthotopic 4T1 tumor growth\nand all the mice lost within 30 days, Ne-SONV+US markedly inhibited tumor growth and\nprolonged the median survival of mice from 26 days (PBS group) to 40 days (Fig. 6b and\nSupplementary Fig. 46). Notably, the combo of Ne-SONV+US with programmed death-\nligand 1 (PD-L1) blockade (Ne-SONV+US+αPD-L1) further extended the mice survival and\n3/6 of mice survived above 60 days (Fig. 6c, d).\nWe next compared the effect of Ne-SONV+US with Neop+CpG to promote intratumoral\nantitumor immunity in the orthotopic 4T1-Luc tumor model. We found that Ne-SONV+US\ntreatment recruited 3.5- and 2.3-fold higher of IFN-γ+ CD8+ T cells (15.2%, IFN-γ+ in CD8+ T\ncells) and TNF-α+ CD8+ T cells (6.38%, TNF-α+ in CD8+ T cells) than Neop+CpG, respectively\n(Supplementary Fig. 48). Combination therapy with αPD-L1 further increased the proportion\nof tumor-infiltrating CTLs. The frequency of IFN-γ+CD8+ T cells and TNF-α+ CD8+ T cells in\nNe-SONV+US+αPD-L1 group was about 24.9% and 11.2%, showing 5.8- and 4.0-fold\nhigher than that in Neop+CpG treatment, respectively.”\nWe added the relevant discussion in Line 605-627 in the revised manuscript.\nFig. 6 (in this revised manuscript). Ne-SONV+US combined with αPD-L1 therapy to suppress orthotopic TNBC\ngrowth and lung metastasis.\n“Furthermore, we compared the antitumor efficacy of neoantigen-based SONV+US\n(namely Ne-SONV+US) with anti-PD-L1 antibody (αPDL1) in the orthotopic Hepa1-6-Luc\ntumor model. As shown in Fig. 7, bioluminescence imaging in vivo indicated that αPD-L1\nmoderately inhibited Hepa1-6-Luc tumor growth and 4/5 of mice lost within 40 days (Fig. 7b).\nIn contrast, Ne-SONV+US markedly inhibited tumor growth and prolonged the mice survival,\nwith 3/5 of mice survived above 60 days. Notably, the combo of Ne-SONV+US with αPD-L1\ninduced 100% survival of the orthotopic liver tumor-bearing mice in 60 days post tumor\ninoculation (Fig. 7c, d).”\nWe added the relevant discussion in Line 659-664 in the revised manuscript.\nFig. 7 (in this revised manuscript). Ne-SONV+US boosted αPD-L1 therapy to establish durable immune memory\neffect in orthotopic HCC model.\n2. Although ultrasound therapy is a non-invasive method, the depth of penetration and tissue\nuptake of ultrasound may be suboptimal in the treatment of deep tumors, and different tissues\nmay respond differently to ultrasound. How can ultrasound-guided immune activation\nprotocols ensure the local precision of immune activation during treatment and the activation\nstatus of different immune cell populations be regulated?\nResponse: We greatly appreciated the comments of the reviewer. We added the relevant\ndiscussion in Line 200-212, 767-784 in the revised manuscript as below:\n“It has been extensively demonstrated that the formulations to spatially confine adjuvant\nactivity to the secondary lymphoid organs, such as LNs, are crucial for enhancing cancer\nvaccine-mediated T-cell response36-39. In this study, to optimize LNs-specific and APCs-\ntargeting delivery, we developed a set of sonosensitive adjuvant (namely sono-adjuvant,\nSONA) with adjustable physio-chemical properties including particle size, surface charge and\nligand-modification. The top-performed SONA could effectively and specifically accumulate\ninto LNs and was internalized by DCs and macrophages, while exhibited minimal off-target\ndistribution in other organs. Therefore, we performed the localized US stimulation on the LNs\nthat located at superficial internal, rather than on the deep tumors in different mouse tumor\nmodels.\nIt was worth noting that the current sono-adjuvant platform was merely examined in\nvarious types of mouse tumor models. Although US therapy exhibited high tissue penetration\ndepth and might be promising for stimulating human LNs in the clinic, the tissue uptake of\nUS stimulation may be variable in the treatment of patients compared with mouse models.\nTherefore, an extensive and personalized exploration of the influence of different US\nparameters on innate immune responses should be conducted when applied for clinical\nimmunotherapy. Furthermore, the high-frequency ultrasound imaging (HFUS) technique\nshould be further explored and combined to effectively visualize the draining LNs at the\nsuperficial internal65, and thus can guide to perform LNs-specific US stimulation and precise\ninnate immune activation.\nThe precise monitoring of activation status of different immune cell populations during\nUS-guided immune activation treatment was highly essential for achieving personalized\ncancer immunotherapy. In future studies, we could combine the multi-color flow cytometry\nassay to continuously monitor the activation status of different immune cell populations in the\nperipheral blood, such as detecting the expression of T cell activation-related markers (e.g.,\nCD69, CCR7, CD44, PD1). Based on these results, we could adjust the treatment parameters,\nsuch as the frequency, duration and power density of US stimulation, to accordingly regulate\nthe activation of immune cell populations.”\nReference:\n36. Lynn GM, et al. In vivo characterization of the physicochemical properties of polymer-linked TLR\nagonists that enhance vaccine immunogenicity. Nat Biotechnol 33, 1201-1210 (2015).\n37. Dane EL, Irvine DJ. Big thinking for adjuvants. Nat Biotechnol 33, 1146-1148 (2015).\n38. Ke X, et al. Physical and chemical profiles of nanoparticles for lymphatic targeting. Adv Drug Deliv Rev\n151-152, 72-93 (2019).\n39. Ding Y, Li Z, Jaklenec A, Hu Q. Vaccine delivery systems toward lymph nodes. Adv Drug Deliv Rev\n179, 113914 (2021).\n65. Najibi, A.J. et al. Durable lymph-node expansion is associated with the efficacy of therapeutic\nvaccination. Nat Biomed Eng 8, 1226-1242 (2024).\n3. The manuscript mentioned the use of ultrasound and peptide adjuvants to activate immune\npathways such as NLRP3, STING, and TLR9 to enhance the immune response, but the\ncomplexity of these immune pathways and their roles in different immune cells have not been\nfully considered.\nResponse: We greatly appreciated the comments of the reviewer helping us to elaborate on\nthe specific mechanisms of action of activating innate immune pathways by the use of US\nstimulation and polypeptide adjuvant. We added the relevant discussion in Line 149-153,\n205-208 in the revised manuscript as below:\n“In this study, inspired by the mannose receptor was highly expressed in APCs40-42, we\nintroduced an active-targeting moiety, mannose, for surface modification of sono-adjuvant\n(SONA) to improve the APCs-targeting performance. Compared with SONA3 that without\nmannose-modification, SONA4 displayed 2.1- and 2.3-fold higher internalization by DCs and\nmacrophages in LNs, respectively (unless noted, all SONA mentioned below refers to SONA4)\n(Fig. 2e and Supplementary Fig. 13). Therefore, the combination of SONA with US\nstimulation could mainly affect the innate immune pathways in APCs, such as DCs and\nmacrophages, while avoiding off-target effect on other immune cells.\nWe next performed experimental exploration of the effect of US-triggered mechanical and\noxidative forces on inducing innate immune pathways activation in APCs in vitro. The western\nblotting assay showed that only US stimulation-mediated mechanical force moderately\nactivated NLRP3 pathway in DC2.4 cells and RAW264.7 cells, while merely affecting STING\nand TLR9 pathways activation. The activation of NLRP3 pathway by US stimulation could be\nattributed to the intracellular Ca2+ influx through the Piezo1 channel30-33. In comparison, the\ncombination of sonosensitive nanoparticle (SONP) with US stimulation (namely SONP+US)\ncould simultaneously trigger the mechanical and oxidative forces, and was demonstrated to\neffectively activate STING, TLR9 and NLRP3 pathways in DC2.4 cells and RAW264.7 cells,\nas evident from the upregulation of indicated proteins expression, such as phosphorylated-\nTBK1 (p-TBK1), phosphorylated-IRF3 (p-IRF3) and phosphorylated-IRF7 (p-IRF7)\n(Supplementary Fig. 6). The activation of STING and TLR9 pathways by SONP+US\ntreatment could be attributed to the oxidative force-mediated endoplasmic reticulum (ER)\nstress, since previous studies have demonstrated that ER stress-triggered mtDNA release\nwas capable of activating both TLR9 and STING pathways in APCs34,35.”\nReference:\n30. Atcha H, et al. Mechanically activated ion channel Piezo1 modulates macrophage polarization and\nstiffness sensing. Nat Commun 12, 3256 (2021).\n31. Lin YC, Guo YR, Miyagi A, Levring J, MacKinnon R, Scheuring S. Force-induced conformational\nchanges in PIEZO1. Nature 573, 230-234 (2019).\n32. Tan M, et al. Metal-ion-chelating phenylalanine nanostructures reverse immune dysfunction and\nsensitize breast tumour to immune checkpoint blockade. Nat Nanotechnol, 19, 1903-1913 (2024).\n33. Lee GS, et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and\ncAMP. Nature 492, 123-127 (2012).\n34. Hu M, et al. ATM inhibition enhances cancer immunotherapy by promoting mtDNA leakage and\ncGAS/STING activation. J Clin Invest 131, e139333 (2021).\n35. Jia M, et al. Redox homeostasis maintained by GPX4 facilitates STING activation. Nat Immunol 21,\n727-735 (2020).\n40. Singh A, et al. Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis. Proc\nNatl Acad Sci U S A 119, e2121098119 (2022).\n41. Li Y, et al. In situ tumor vaccine with optimized nanoadjuvants and lymph node targeting capacity to\ntreat ovarian cancer and metastases. Acta Pharm Sin B 14, 4102-4117 (2024).\n42. Liu Z, et al. Dual-Targeted Self-Adjuvant Heterocyclic Lipidoid@Polyester Hybrid Nanovaccines for\nBoosting Cancer Immunotherapy. ACS Nano 18, 15557-15575 (2024).\nSupplementary Fig. 13 (in this revised manuscript). Representative flow cytometric plots and the frequency of\nSONA+CD11c+ DCs and SONA+CD11b+ macrophages in the LNs at 6 h after SONA3 or SONA4 injection (n = 3\nbiologically independent mice). Statistical significance was calculated via unpaired two-tailed Student’s t-test and P\nvalues were indicated.\nSupplementary Fig. 6 (in this revised manuscript). WB analysis of the activation of STING, TLR9 and NLRP3\npathways in DC2.4 cells (a) and RAW264.7 cells (b) after indicated treatments.\n4. A variety of mouse models were used in the paper, including mammary tumor and\nhepatocellular carcinoma models, but there was fewer pairs in different immune contexts. For\nexample, were immunodeficient mice (e.g., T-cell deficient mice) considered to validate the\ntreatment? It is recommended that the validation of these models be increased to further\nillustrate the generalizability of the treatment in different immune settings.\nResponse: We sincerely thank the comments of the reviewer for recommending us to\nillustrate the generalizability of SONV+US in different immune settings. We added the\nrelevant discussion in Line 552-560 in the revised manuscript as below:\n“In this study, we investigated the antitumor efficacy of SONV+US in T cell-depleted mice by\nsystemic injection of the CD8+ T cell-blockade antibody, rather than directing using the T-cell\ndeficient mice. The result showed that CD8+ T cells blockade almost completely abrogated\nthe antitumor performance of SONV+US in the B16-OVA tumor model, indicating the\nindispensable role of CD8+ T cells in SONV+US-mediated antitumor immunity (Fig. 5o, p).”\nFig. 5 (in this revised manuscript). s, t, Average B16-OVA tumor growth curves and corresponding survival curves\nof mice after SONV+US treatment with or without anti-CD8+ T cell blockade (n = 6 biologically independent\nreplicates). Statistical significance was calculated via one-way ANOVA with Tukey’s test (s) or log-rank (Mantel-\nCox) test (t) and P values were indicated.\n5. The immune response was detected mainly by methods such as flow cytometry and\nimmunohistochemistry. However, the presentation of details in flow cytometry was insufficient.\nThe significance of the treatment effect needs to be demonstrated more clearly in terms of\nindicators such as CD8+ T cell activation, maturation degree of DCs, and cytokine secretion\nlevel between different groups. In addition, the text mainly focuses on the response of CD8+\nT cells and DCs, but ignored the role of other immune cells (e.g., Tregs, MDSCs, NK cells).\nIt is recommended that the role of these cells be analyzed in detail, especially the inhibitory\neffect of Tregs and the effect of MDSCs on immune escape. Moreover, given the complexity\nof immunotherapy, it is recommended to introduce a more fine-grained analysis of immune\ncell subpopulations (e.g., to distinguish between different T cell subpopulations by multiple-\nlabelling flow cytometry).\nResponse: We sincerely thank the comments of the reviewer for helping us to improve the\npresentation of flow cytometry results in the manuscript. Following the comments of the\nreviewer, we first provided a clearer demonstration of the significance of the treatment effect\nin terms of indicators such as CD8+ T cell activation, maturation degree of DCs, and cytokine\nsecretion level between different groups.\nFor instance, in Line 355-366 in the revised manuscript, the sentence was corrected as:\n“In contrast, SONV+US treatment significantly triggered the expression of stimulatory\nmarkers (CD80 and CD86) in macrophages and DCs in the LNs (Fig. 3f, g and\nSupplementary Fig. 28a, b). The maturation degree of DCs (CD80+CD86+ in CD11c+ cells)\nin SONV+US group was 22.5%, showing 2.2- and 1.6-fold higher than that in OVA+CpG\n(10.1%) and OVA+MSA-2 (14.2%) groups, respectively. In addition, SONV+US treatment\ninduced the highest level of antigen-specific peptide-MHC-I complex on the surface of DCs\n(8.2%, SIINFEKL-H2Kb+ in CD11c+ cells), with 2.5- and 1.9-fold higher than that in OVA+CpG\nand OVA+MSA-2 groups, respectively (Fig. 3h and Supplementary Fig. 28c). More\nimportantly, the frequency of antigen-specific CD8+ T cells (SIINFEKL-H2Kb tetramer+ in\nCD8+ cells) in SONV+US group was about 10.1%, while that in OVA+CpG and OVA+MSA-2\ngroups were only about 2.5% and 3.4%, respectively (Fig. 3i and Supplementary Fig. 29a).”\nIn Line 449-458 in the revised manuscript, the sentence was corrected as: “SONV+US\nstrongly promoted DCs maturation and macrophages activation in tdLNs (Supplementary\nFig. 34). The maturation degree of DCs (CD80+ in CD11c+ cells) in SONV+US group was\n28.8%, showing 1.8- and 1.5-fold higher than that in OVA+CpG (16.2%) and OVA+MSA-2\n(19.2%) groups, respectively. Compared to the SONV treatment that elicited negligible\nantitumor immunity, SONV+US significantly enhanced tumor infiltration of CD8+ T cells (Fig.\n4k and Supplementary Fig. 35a), and increased the activation of CTLs. The frequency of\nIFN-γ+CD8+ T cells (IFN-γ+ in CD8+ T cells) in SONV+US group was about 19.8%, with 2.5\nand 1.7-fold higher than that in OVA+CpG (7.9%) and OVA+MSA-2 (11.6%) groups,\nrespectively (Fig. 4l and Supplementary Fig. 35b).”\nIn Line 484-489 in the revised manuscript, the sentence was corrected as: “ELISA result\nrevealed that SONV+US significantly increased intratumoral secretion of TNF-α, while\ninhibited the typical immunosuppressive cytokine (TGF-β) secretion (Fig. 4p and\nSupplementary Fig. 39). The intratumoral TNF-α secretion levels in OVA+CpG, OVA+MSA-\n2 and SONV+US group was about 1189.6 pg/mL, 1996.3 pg/mL and 2346.3 pg/mL,\nrespectively. The intratumoral TGF-β secretion levels in OVA+CpG, OVA+MSA-2 and\nSONV+US group was about 2515.0 pg/mL, 1593.6 pg/mL and 835.8 pg/mL, respectively.”\nIn Line 622-627 in the revised manuscript, the sentence was corrected as: “Importantly,\nNe-SONV+US treatment recruited 3.5- and 2.3-fold higher of IFN-γ+ CD8+ T cells (15.2%,\nIFN-γ+ in CD8+ T cells) and TNF-α+ CD8+ T cells (6.38%, TNF-α+ in CD8+ T cells) than\nNeop+CpG, respectively (Supplementary Fig. 48). Combination therapy with αPD-L1 further\nincreased the proportion of tumor-infiltrating CTLs. The frequency of IFN-γ+CD8+ T cells and\nTNF-α+ CD8+ T cells in Ne-SONV+US+αPDL1 group was about 24.9% and 11.2%, showing\n5.8- and 4.0-fold higher than that in Neop+CpG treatment, respectively.”\nNext, we re-established the B16-OVA tumor model and measured the cytotoxic effect of\ntumor-infiltrating NK cells and the suppressive function of tumor-infiltrating Tregs in order to\nprovide clearer insights about SONV+US-mediated antitumor immunity. We added the\nrelevant discussion in Line 467-479 in the revised manuscript as below:\n“The findings showed that SONV+US treatment increased the intratumor cytotoxic NK\ncell response (IFN-γ+ NK cells and GZMB+ NK cells) compared with PBS group. The\nfrequencies of IFN-γ+ NK cells (IFN-γ+ in NK1.1+ cells) and GZMB+ NK cells (GZMB+ in\nNK1.1+ cells) in SONV+US group were about 12.5% and 11.6%, respectively, showing 2.1-\nand 2.0-fold higher than that in PBS group (IFN-γ+ NK, 5.96% and GZMB+ NK, 5.56%),\nrespectively (Supplementary Fig. 36). In addition, SONV+US treatment not only decreased\nthe frequency of tumor-infiltrating Tregs, but also inhibited the production of Tregs-derived\nsuppressive cytokines (IL-4, IL-10). The frequencies of tumor-infiltrating IL10+ Tregs (12.6%,\nIL10+ in CD25+FOXP3+ cells) and IL4+ Tregs (10.6%, IL4+ in CD25+FOXP3+ cells) in\nSONV+US group displayed 3.1- and 3.2-fold lower than that in PBS group (IL10+ Tregs, 39.2%\nand IL4+ Tregs, 33.3%), respectively (Fig. 4m and Supplementary Fig. 37). Moreover, we\nintroduced a more fine-grained analysis of tumor-infiltrating CD8+ T cell subpopulations.\nCompared with PBS group, SONV+US treatment increased the intratumoral progenitor\nexhausted (PD1+TIM3-) CD8+ T cells infiltration, while decreased the tumor-infiltration of\nterminally exhausted (PD1+TIM3+) CD8+ T cells. The frequencies of progenitor exhausted\nCD8+ T cells (PD1+TIM3- in CD8+CD3+ cells) and terminally exhausted CD8+ T cells\n(PD1+TIM3+ in CD8+CD3+ cells) in SONV+US group were about 28.4% and 19.4%, showing\n2.2-fold higher and 3.0-fold lower than that in PBS group, respectively (Supplementary Fig.\n35c-e). These results indicated that SONV+US treatment not only enhanced intratumoral\nCD8+ T cell infiltration and activation, but also was promising to address intratumoral CD8+ T\ncell exhaustion.”\nSupplementary Fig. 36 (in this revised manuscript). Representative flow cytometric plots and the frequency of\ntumor-infiltrating IFN-γ+ NK cells (IFN-γ+ in NK1.1+ cells) (a) and Gzmb+ NK cells (Gzmb+ in NK1.1+ cells) (b) in\nindicated groups (n = 5 biologically independent mice). Statistical significance was calculated via one-way ANOVA\nwith Tukey’s test and P values were indicated.\nSupplementary Fig. 37 (in this revised manuscript). Representative flow cytometric plots and the frequency of\ntumor-infiltrating IL10+ Tregs (IL10+ in CD25+FOXP3+CD4+ T cells) (a) and IL4+ Tregs (IL4+ in\nCD25+FOXP3+CD4+ T cells) (b) in indicated groups (n = 5 biologically independent mice). Statistical significance\nwas calculated via one-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 35 (in this revised manuscript). Representative flow cytometric plots (a) and the frequency\nof tumor-infiltrating terminally exhausted CD8+ T cells ((PD1+TIM3+ in CD8+ T cells) (b) and progenitor exhausted\nCD8+ T cells ((PD1+TIM3- in CD8+ T cells) (c) in indicated groups (n = 5 biologically independent mice). Statistical\nsignificance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\n6. In mouse experiments, the article mentions that the treatment had a favorable biosafety\nprofile, with no significant toxic effects found. However, the article did not perform long-term\nobservations to verify the long-term safety of the strategy. Long-term follow-up is\nrecommended in subsequent studies, especially to evaluate potential toxicity in non-target\norgans.\nResponse: We greatly appreciated the comments of the reviewer. To verify the long-term\nsafety of our sono-adjuvant strategy, C57BL/6 mice were treated with SONA+US (1 MHz, 50%\nduty cycle, 1.0 W/cm2, 5 min) three times at 3-days intervals, and the major organs and serum\nof treated mice were collected at 40-days post final treatment. Hematoxylin-eosin (H&E)\nstaining of the major organs displayed no inflammatory lesion and histopathological damage\nafter SONA+US treatment (Supplementary Fig. 51). Biochemical analysis of serum also\nindicated the favorable long-term safety of SONA+US treatment in vivo (Supplementary Fig.\n52). These findings together confirmed that SONA+US treatment showed no obvious toxicity\nin non-target organs.\nWe added above data and the relevant discussion in Line 676-682 of the revised\nmanuscript.\nSupplementary Fig. 51 (in this revised manuscript). H&E staining of the major organs harvested 40-days post\nfinal treatment in indicated groups (Scale bar = 100 μm).\nSupplementary Fig. 52 (in this revised manuscript). Biochemical analysis of serum from treated mice on day 40\npost final treatment (n = 4 biologically independent replicates). UA, uric acid; GLU; blood glucose; ALT, alanine\naminotransferase; AST, aspartate transaminase; BUN, blood urea nitrogen; CREA, creatinine. Statistical significance\nwas calculated via unpaired two-tailed Student’s t-test and P values were indicated.\n7. Preliminary optimization of the parameters of ultrasound therapy was done in the paper,\nbut it failed to further explore in depth the effects of different ultrasound parameters on the\nimmune response. It is recommended to increase the study of the dose-response curve of\nultrasound therapy, such as the change of effect under different ultrasound frequency and\ntreatment time to find out the optimal therapeutic parameters.\nResponse: We greatly appreciated the comments of the reviewer. We added the relevant\ndiscussion in Line 222-243 in the revised manuscript as below:\n“To optimize the experimental parameters for LNs-specific US stimulation and innate immune\nactivation in vivo, we first evaluated the biodistribution of sono-adjuvant (SONA) in LNs by\nutilizing the fluorescence imaging capacity of porphyrin-based sonosensitizer. As shown in\nSupplementary Fig. 14, upon subcutaneous injection at the tail base, SONA gradually\naccumulated into the LNs and peaked at 6 h post injection. Therefore, we selected the 6 h\npost SONA injection as the experimental time point for LNs-localized US stimulation.\nWe selected the US frequency of 1 MHz and the duty cycle of 50%, since this combination\nwas demonstrated to not only exhibit high penetration depth in biological tissue, but also\navoid excessive damage to the tissue caused by continuous high-energy output66,67. In\naddition, the combination of a 1 MHz frequency and a 50% duty cycle could produce a\nmoderate cavitation effect that not only facilitated the cytosolic delivery of payloads, but also\navoided unnecessary damage to the surrounding tissues caused by overly intense\ncavitation68-70.\nWe next altered the power density and duration of US stimulation to generate different\nlevels of mechanical and oxidative force, and then investigated their effect on innate immune\nactivation. We first investigated the influence of different power density of US stimulation on\ninducing the innate immune responses in vivo. The LNs were locally stimulated with 5 minutes\nof indicated power density of US (0, 0.2, 0.5, 1.0, 1.5 W/cm2 ) at 6 h post SONA injection.\nFlow cytometry analysis revealed that only SONA treatment induced negligible innate\nimmunity in LNs. In contrast, LNs-specific US stimulation effectively activated the innate\nimmune responses, and 1.0 W/cm2 of US stimulation elicited the highest recruitment and\nmaturation of DCs and macrophages in the LNs (Fig. 2f, g and Supplementary Fig. 15). We\nsubsequently investigated the influence of different duration of US stimulation on innate\nimmunity activation in vivo. The LNs were locally stimulated with 1.0 W/cm2 of indicated\nduration of US (0, 1, 3, 5, 7 min) at 6 h post SONA injection. The results showed that the\nrecruitment and maturation levels of DCs in the LNs gradually increased with the increase of\nUS duration, and it almost reached the peak value when combined with 5 minutes of US\nstimulation (Supplementary Fig. 16). Collectively, we screened out the time point (6 h post\ninjection), power density (1.0 W/cm2) and duration (5 min) of US stimulation for LNs-specific\ninnate immune activation in vivo.”\nReference:\n66. Han Z, et al. Precise Cell Type Electrical Stimulation Therapy Via Force-electric Hydrogel Microspheres\nfor Cartilage Healing. Adv Mater 37, e2414555 (2025).\n67. Zhu K, Wang J, Wang Z, Chen Q, Song J, Chen X. Ultrasound-Activated Theranostic Materials and\nTheir Bioapplications. Angew Chem Int Ed Engl, e202422278 (2025).\n68. Moradi Kashkooli F, Jakhmola A, Hornsby TK, Tavakkoli JJ, Kolios MC. Ultrasound-mediated nano\ndrug delivery for treating cancer: Fundamental physics to future directions. J Control Release 355, 552-\n578 (2023).\n69. Paliwal S, Mitragotri S. Ultrasound-induced cavitation: applications in drug and gene delivery. Expert\nOpin Drug Deliv 3, 713-726 (2006).\n70. Yu CC, et al. A Conformable Ultrasound Patch for Cavitation-Enhanced Transdermal Cosmeceutical\nDelivery. Adv Mater 35, e2300066 (2023).\nSupplementary Fig. 14 (in this revised manuscript). a, Representative fluorescence images of mice at indicated\ntime points post SONA injection (n = 3 biologically independent mice). b, Representative fluorescence images and\nnormalized fluorescence signal of tissues collected at indicated time points post SONA injection (n = 3 biologically\nindependent mice). Statistical significance was calculated via one-way ANOVA with Tukey’s test and P values were\nindicated.\nSupplementary Fig. 15 (in this revised manuscript). a, The absolute number of CD11c+ DCs and CD11b+\nmacrophages in the LNs after SONA+US treatment with different power density of US stimulation (n = 3 biologically\nindependent mice). b, The CD80 or CD86 expression level on CD11c+ DCs after SONA+US treatment with different\npower density of US stimulation (n = 3 biologically independent mice). Statistical significance was calculated via\none-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 16 (in this revised manuscript). a, The absolute number of CD11c+ DCs and CD11b+\nmacrophages in the LNs after SONA+US treatment with different duration of US stimulation (n = 3 biologically\nindependent mice). b, The CD80 or CD86 expression level on CD11c+ DCs after SONA+US treatment with different\nduration of US stimulation (n = 3 biologically independent mice). Statistical significance was calculated via one-way\nANOVA with Tukey’s test and P values were indicated.\n8. The effects of ultrasound stimulation were realized through the dual action of mechanical\nand oxidative forces, but did not elaborate on the specific mechanisms of interaction between\nthe two. Experimental exploration of these mechanisms of action can be further increased,\nespecially how ultrasound triggers intracellular calcium ion flow and ROS generation,\nwhether it causes cellular damage and how to effectively circumvent these side effects.\nResponse: We appreciated the comments of the reviewer helping us to elaborate on the\nspecific mechanisms of interaction between ultrasound-triggered mechanical and oxidative\nforces in activating innate immune pathways. We added the relevant discussion in Line 98-\n101, 106-112, 128-131, 149-163 in the revised manuscript as below:\n“First, we provided a detailed explanation about how the combination of polypeptide adjuvant\nwith US stimulation (namely SONP+US) could trigger intracellular calcium ion flow and ROS\ngeneration in APCs. The mechanosensitive channel Piezo1 is generally considered to be\nactivated by various types of mechanical stimuli and trigger Ca2+ influx to promote pro-\ninflammatory immune response30-33. Therefore, US-triggered mechanical force was effective\nto activate the Piezo1 channel, and promote intracellular calcium ion flow in APCs. The\nporphyrin-based sonosensitizer, hematoporphyrin monomethyl ether (HMME), was\nintegrated into SONP to generate sonodynamic effect under the US stimulation. It has been\nextensively demonstrated that US-activatable sonodynamic effect could precisely induce\nintracellular ROS generation in a spatiotemporal-controllable manner26-29.\nWe next performed experimental exploration of the mechanisms of action of US-triggered\nmechanical and oxidative forces for stimulating innate immune activation. We compared the\neffect of mechanical and oxidative forces on inducing innate immune pathways activation in\nDC2.4 cells and RAW264.7 cells by western blotting assay. The result showed that only US\nstimulation-mediated mechanical force moderately activated NLRP3 pathway in APCs, while\nmerely affecting STING and TLR9 pathways activation. The activation of NLRP3 pathway by\nUS stimulation could be attributed to the intracellular Ca2+ influx through the Piezo1 channel30-\n33. In comparison, the combination of mechanical and oxidative forces mediated by\nSONP+US could effectively activate STING, TLR9 and NLRP3 pathways in DC2.4 cells and\nRAW264.7 cells, as evident from the upregulation of indicated proteins expression, such as\nphosphorylated-TBK1 (p-TBK1), phosphorylated-IRF3 (p-IRF3) and phosphorylated-IRF7 (p-\nIRF7) (Supplementary Fig. 6). The activation of STING and TLR9 pathways by SONP+US\ntreatment could be attributed to the oxidative force-mediated endoplasmic reticulum (ER)\nstress, since previous studies have demonstrated that ER stress-triggered mtDNA release\nwas capable of activating both TLR9 and STING pathways in APCs34,35. Similar result was\nalso observed in the DCs maturation mediated by US-triggered mechanical and oxidative\nforces. Only US stimulation-mediated mechanical force moderately enhanced DCs\nmaturation level (CD86+, 8.9%) compared with PBS group (CD86+, 5.7%). In contrast, the\ncombination of mechanical and oxidative forces significantly increased the frequency of\nCD86+ DCs (15.1%), showing a 2.6- and 1.7-fold higher than that in PBS and US groups,\nrespectively (Supplementary Fig. 7).\nWe also provided a detailed discussion about how to effectively circumvent the\nunintended cellular damage caused by US stimulation. We selected and fixed the US\nfrequency was 1 MHz and the duty cycle was 50%, since this combination was demonstrated\nto not only exhibit high penetration depth in biological tissue, but also avoid excessive\ndamage to the tissues caused by continuous high-energy output66,67. In addition, the\ncombination of a 1 MHz frequency and a 50% duty cycle could produce a moderate cavitation\neffect that not only facilitated the cytosolic delivery of payloads, but also avoided unnecessary\ndamage to the surrounding tissues caused by overly intense cavitation68-70. Meanwhile, for in\nvitro studies, we screened the power density and duration of US stimulation to minimize the\nunintended cellular damage effect on DCs and macrophages. The result showed that shorter\nthan 4 minutes of US stimulation (0.2 W/cm2) exerted negligible cytotoxicity on DC2.4 cells\nand RAW264.7 cells (Supplementary Fig. 1 and 4). For in vivo studies, the porphyrin-based\nsonosensitizer exhibited the fluorescence imaging capacity to precisely trace the distribution\nof sono-adjuvant in THE LNs. In addition, the high-frequency ultrasound (HFUS) technique\nhas been used to image and assess the human LNs with potential diagnostic capacity65.\nTherefore, the fluorescence imaging and HFUS technique could be further combined to\neffectively visualize the sono-adjuvant-draining LNs, and thus guide us to perform LNs-\nspecific US stimulation and minimize the uncontrolled cellular damage on normal tissues.”\nReference:\n26. Liang S, Yao J, Liu D, Rao L, Chen X, Wang Z. Harnessing Nanomaterials for Cancer Sonodynamic\nImmunotherapy. Adv Mater 35, e2211130 (2023).\n27. Xu C, Huang J, Jiang Y, He S, Zhang C, Pu K. Nanoparticles with ultrasound-induced afterglow\nluminescence for tumour-specific theranostics. Nat Biomed Eng 7, 298-312 (2023).\n28. Zhou M, et al. Vitamin B2-Based Ferroptosis Promoter for Sono-Enhanced Nanocatalytic Therapy of\nTriple-Negative Breast Cancer. 33, 2303899 (2023).\n29. Wang W, et al. Endogenous Stimuli-Activatable Nanomedicine for Immune Theranostics for Cancer.\n31, 2100386 (2021).\n30. Atcha H, et al. Mechanically activated ion channel Piezo1 modulates macrophage polarization and\nstiffness sensing. Nat Commun 12, 3256 (2021).\n31. Lin YC, Guo YR, Miyagi A, Levring J, MacKinnon R, Scheuring S. Force-induced conformational\nchanges in PIEZO1. Nature 573, 230-234 (2019).\n32. Tan M, et al. Metal-ion-chelating phenylalanine nanostructures reverse immune dysfunction and\nsensitize breast tumour to immune checkpoint blockade. Nat Nanotechnol, 19, 1903-1913 (2024).\n33. Lee GS, et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and\ncAMP. Nature 492, 123-127 (2012).\n34. Hu M, et al. ATM inhibition enhances cancer immunotherapy by promoting mtDNA leakage and\ncGAS/STING activation. J Clin Invest 131, e139333 (2021).\n35. Jia M, et al. Redox homeostasis maintained by GPX4 facilitates STING activation. Nat Immunol 21,\n727-735 (2020).\n65.Najibi AJ, et al. Durable lymph-node expansion is associated with the efficacy of therapeutic vaccination.\nNat Biomed Eng 8, 1226-1242 (2024).\n66. Han Z, et al. Precise Cell Type Electrical Stimulation Therapy Via Force-electric Hydrogel Microspheres\nfor Cartilage Healing. Adv Mater 37, e2414555 (2025).\n67. Zhu K, Wang J, Wang Z, Chen Q, Song J, Chen X. Ultrasound-Activated Theranostic Materials and\nTheir Bioapplications. Angew Chem Int Ed Engl, e202422278 (2025).\n68. Moradi Kashkooli F, Jakhmola A, Hornsby TK, Tavakkoli JJ, Kolios MC. Ultrasound-mediated nano\ndrug delivery for treating cancer: Fundamental physics to future directions. J Control Release 355, 552-\n578 (2023).\n69. Paliwal S, Mitragotri S. Ultrasound-induced cavitation: applications in drug and gene delivery. Expert\nOpin Drug Deliv 3, 713-726 (2006).\n70. Yu CC, et al. A Conformable Ultrasound Patch for Cavitation-Enhanced Transdermal Cosmeceutical\nDelivery. Adv Mater 35, e2300066 (2023).\nSupplementary Fig. 6 (in this revised manuscript). WB analysis of the activation of STING, TLR9 and NLRP3\npathways in DC2.4 cells (a) and RAW264.7 cells (b) after indicated treatments.\nSupplementary Fig. 7 (in this revised manuscript). Representative flow cytometric plots and the frequency of\nCD86+ DCs post indicated treatments (n = 3 biologically independent replicates). Statistical significance was\ncalculated via one-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 1 (in this revised manuscript). The relative cell viability of DC2.4 and RAW264.7 cells after\ntreatment with different power density of US stimulation (n = 3 biologically independent replicates). Statistical\nsignificance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 4 (in this revised manuscript). The relative cell viability of DC2.4 and RAW264.7 cells after\nSONP+US treatment with different duration of US stimulation (n = 3 biologically independent replicates). Statistical\nsignificance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\n9. The article concluded with a briefer summary of the mechanisms of ultrasound-induced\nimmune activation and therapeutic effects. It is suggested that the limitations and future\ndirections of the study should be made more explicit in the conclusion section, especially how\nto overcome the limitations of short-term experimental observation and immune response\nmonitoring that exist in the current experiments, and how to generalize the strategy across\ndifferent cancer types.\nResponse: We greatly appreciated the comments of the reviewer helping us to enhance the\ncredibility and reality of our study. We provided a critical discussion of the current limitations\nof our sono-adjuvant platform in Line 753-784 in the revised manuscript as below:\n“It was worth noting that although the remarkable advantages of the ultrasound-confined\nsono-adjuvant in cancer vaccine immunotherapy, additional efforts are required to address\nthe current limitations for future clinical translation. First, from the manufacturing aspect, a\nmore precise control of polypeptides self-assembly by automated microfluidic devices should\nbe explored to produce uniform sono-adjuvant and minimize batch-to-batch variation, which\ncould address the critical translational challenges from bench to bedside. Second, from the\ninnate immune activation aspect, although sono-adjuvant represented a multiply adjuvanted\nplatform to synchronously activate STING, TLR9 and NLRP3 pathways in APCs, the\nunderlying potential and mechanism of the US-activatable innate immune stimulation strategy\nfor addressing APCs dysfunction and exhaustion were not extensively investigated. Third,\nthe induction of long-term antitumor immune responses was highly essential for preventing\ntumor recurrence and distant metastasis. Therefore, additional tumor-rechallenge assay and\nmore extensive immune memory response monitoring should be conducted to overcome the\ncurrent limitations of short-term experimental observation in this study. Moreover, the current\nsono-adjuvant platform was merely examined in various types of mouse tumor models.\nAlthough US therapy exhibited high tissue penetration depth and might be promising for\nstimulating human LNs that located at the superficial internal, the tissue uptake of US\nstimulation may be variable in the treatment of patients compared with mouse models.\nTherefore, an extensive and personalized exploration of the influence of different US\nparameters on innate immune responses should be conducted when applied for clinical\nimmunotherapy.\nFurthermore, the high-frequency ultrasound imaging (HFUS) technique should be further\nexplored and combined to precisely visualize human LNs at the superficial internal, and thus\ncan guide to perform LNs-specific US stimulation and innate immune activation. The precise\nmonitoring of activation status of different immune cell populations during US-guided immune\nactivation treatment was highly essential for achieving personalized cancer immunotherapy.\nIn future investigations, a multi-color flow cytometry assay will be employed to monitor the\nactivation status of different immune cell populations in the peripheral blood, such as\ndetecting the expression of T cell activation-related markers (e.g., CD69, CCR7, CD44, PD1).\nBased on these results, the treatment parameters, such as the frequency, duration and power\ndensity of US stimulation will be well tunned to accordingly regulate the activation of immune\ncell populations. We expected the sono-adjuvant platform could be optimized in various\naspects to improve its therapeutic application scopes, and implement a safe and effective\ncancer vaccination strategy.”\nReviewer #5 (Remarks to the Author):\nIn this study, Chen et al reported an ultrasonic-activatable nano-adjuvant for vaccination\ntherapy cancer. The polypeptide-based nano-adjuvant was screened by lymph node-\ntargeting efficacy, DC activation profiles and so on. The outperformed nano-adjuvant was\nemployed for lymph node-targeted delivery of protein antigen and neoantigen peptide. The\nantigen-loaded nano-vaccines highly efficiently elicited antitumor immune response for\nregressing orthotropic breast and liver tumors. Overall, the sono-sensitizer-conjugated\npolypeptide represents an elegant design of novel nano-adjuvants, which could provide a\nrobust platform for ultrasonic-tunable regulation of innate immunity and boosting antitumor\nimmune response. The data presented in the manuscript are comprehensive and convincing.\nI prefer to recommend publication upon addressing some specific concerns.\nMajor comments\n1. It has been reported that photodynamic therapy with laser irradiation could promote\nprotein/siRNA escape from cytosol, the influence of US on cytosol release of the antigens\nshould be discussed/investigated.\nResponse: We appreciated the comments of the reviewer reminding us to discuss the\ninfluence of US-triggered mechanical and oxidative force on cytosol release of payloads.\nWe added the relevant discussion in Line 300-309, Line 314-320 in the revised manuscript\nas below:\n“An acridine orange staining assay was next performed to investigate the influence of\nmechanical and oxidative forces on endosome membrane permeability of DCs. As shown in\nSupplementary Fig. 18, only US stimulation-mediated mechanical force was demonstrated\nto increase the permeability of endosomal membrane through the cavitation effect, while only\nSONV incubation that could not induce any mechanical and oxidative forces showed no\nobvious endosomal disruption effect. In contrast, the combination of mechanical and\noxidative force mediated by SONV+US could further increase endosome membrane\npermeability of DCs, and significantly induced antigen cytosolic release, with most of OVA-\nFITC signal detected in the cytosol while not in endo-lysosomes (Supplementary Fig. 21).\nSpecifically, the Pearson’s R value (PR) of OVA-FITC signal (green) with endosomal signal\n(red) in SONV and SONV+US groups were 0.25 and -0.12, respectively. These results\ncollectively confirmed that SONV+US offered a US-activatable approach to promote antigen\ncytosolic delivery by combing the US-triggered mechanical and oxidative force.”\nSupplementary Fig. 18 (in this revised manuscript). Representative flow cytometry plots and the frequency of\nACO-FITC+ DCs after indicated treatments (n = 3 biologically independent replicates). Statistical significance was\ncalculated via one-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 21 (in this revised manuscript). Representative CLSM images to show the SONV distribution\nin DCs. OVA was labelled with FITC (green), the nuclei and endosome were stained with DAPI (blue) and Lyso-\nTracker (red), respectively, scale bar = 10 μm (n = 3 biologically independent replicates).\n2. The authors mentioned that oxidative stress could be crucial for innate immune response,\nthe generation of oxidative stress should be validated.\nResponse: We greatly appreciated the comments of the reviewer reminding us to evaluate\nthe generation of oxidative stress in DCs. We added the relevant discussion in Line 132-137\nin the revised manuscript as below:\n“We next used the reactive oxygen species (ROS) indicator, 2',7'-dichlorodihydrofluorescein\ndiacetate (DCFH-DA), to detect the generation level of oxidative force by SONP+US in DCs.\nCLSM images showed that only SONP incubation without US stimulation showed no ROS\ngeneration effect, while SONP+US could trigger the intracellular ROS generation of DCs as\na function of US stimulation duration (Fig. 1k).”\nFig. 1k (in this revised manuscript). Representative CLSM images to show the intracellular ROS generation in\nDC2.4 cells after SONP+US treatment with different duration of US stimulation, scale bar = 10 μm (n = 3 biologically\nindependent replicates).\n3. Ultrasonication is employed in this study to activate innate immune response, the\nadvantages of US-activatable sono-adjuvant over the conventional adjuvants should be\ndiscussed.\nResponse: We sincerely thank the comments of the reviewer for helping us to claim the\ncritical advantages of our US-activatable sono-adjuvant over the conventional adjuvants. We\nadded the relevant discussion in Line 732-752 in the revised manuscript as below:\n“First, there was no bioactive adjuvant integrated into the sono-adjuvant platform, and thereby\nthe elicitation and duration of innate immunity could be spatiotemporally tailored by the\nexternal US stimulation. The US-activatable turning on/off of innate immunity ensured to\nmitigate the immune-related adverse effects on untargeted tissues. Second, conventional\nadjuvant systems usually activated single innate immune pathways, and the sustained\nexposure to mono innate immune stimuli was likely to induce APCs dysfunction62-64. In\ncontrast, the sono-adjuvant represented a multivalent platform to synchronously activate\nmultiple innate immune pathways. Third, the sono-adjuvant represented a highly\nbiodegradable and generalizable vaccine platform to co-deliver various tumor antigens for\npersonalized cancer immunotherapy. In addition, through serial optimizations of sono-\nadjuvant library via adjusting the size, surface charge and targeting ligand, the sono-adjuvant\nnot only preferentially targeted LNs, but also was selectively internalized by APCs rather than\nnon-immune cells or T cells. More importantly, a critical challenge in inducing robust T-cell\nresponse is the spatiotemporal orchestration of antigen cross-presentation with innate\nimmune stimulation in APCs of LNs46-48. Despite various adjuvant systems have been\ndeveloped to co-deliver tumor antigen and adjuvant into LNs, most of them were ineffective\nto simultaneously promote antigen cytosolic release and innate immune stimulation in\nAPCs17-19. Our spatiotemporally-controllable sono-adjuvant platform could utilize the US-\ntriggered mechanical and oxidative force to synchronously promote antigen cytosolic release\nand innate immune pathways activation in APCs, and ultimately induced robust antigen-\nspecific T cell responses in vivo.”\nReference:\n17. Yu J, et al. Polymeric STING Pro-agonists for Tumor-Specific Sonodynamic Immunotherapy. Angew\nChem Int Ed Engl 62, e202307272 (2023).\n18. Luo M, et al. A STING-activating nanovaccine for cancer immunotherapy. Nat Nanotechnol 12, 648-\n654 (2017).\n19. Lee D, et al. Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune\npathways in antigen-presenting cells. Nat Biomed Eng 8, 593-610 (2024).\n46. Zeng YC, et al. Fine tuning of CpG spatial distribution with DNA origami for improved cancer\nvaccination. Nat Nanotechnol, 19, 1055-1065 (2024).\n47. Ye J, Hou B, Saeed M, Xu Z, Yu H. Engineering Bioinspired Nanomedicines to Mitigate the Resistance\nto Cancer Immunotherapy. Accounts of Materials Research 3, 697-708 (2022).\n48. Liu X, et al. In-situ vaccination immunotherapy of colorectal cancer with STING agonist-integrated\nsupramolecular nanovectors. Nano Today 56, 102273 (2024).\n62. Macagno A, Napolitani G, Lanzavecchia A, Sallusto F. Duration, combination and timing: the signal\nintegration model of dendritic cell activation. Trends Immunol 28, 227-233 (2007).\n63. Kasturi SP, et al. Programming the magnitude and persistence of antibody responses with innate\nimmunity. Nature 470, 543-547 (2011).\n64. Jin SM, et al. A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances\ncancer immunotherapy and reduces immune cell exhaustion. Nat Nanotechnol 18, 390-402 (2023).\n4. A more detailed explanation of US equipment and experimental settings for LNs-confined\nUS irradiation should be provided.\nResponse: We added a more detailed explanation of the experimental settings including US\nprocedures and parameters in Line 222-243, 894-905 in the revised manuscript as below:\n“The US equipment was purchased from DJO France S.A.S. (Cat. number: DJO 2776,\nFrequency: 1 MHz, Power density: 0-1.5 W/cm2, Duty cycle: 50%, Transducer type:\nCOLLIMATING, Effective radiating area: 4 cm2). We selected the US frequency of 1 MHz and\nthe duty cycle of 50% based on the following considerations. First, the US frequency of 1\nMHz has good penetration depth in biological tissues, which allows it to delivery energy into\nthe LNs66. When combined with a 50% duty cycle, the energy is evenly distributed and can\nensure sufficient energy transfer to achieve the treatment effect during the penetration\nprocess. Second, the combination of 1 MHz frequency and 50% duty cycle can avoid\nexcessive damage to the tissue caused by continuous high-energy output, allowing to reduce\nadverse effects such as thermal effects on tissues67. Third, this combination was\ndemonstrated to produce a moderate cavitation effect, and can avoid unnecessary damage\nto the surrounding tissues caused by overly intense cavitation68-70.\nTo optimize the experimental parameters for LNs-specific US stimulation and innate\nimmune activation in vivo, we first evaluated the biodistribution of sono-adjuvant (SONA) in\nLNs by utilizing the fluorescence imaging capacity of porphyrin-based sonosensitizer. As\nshown in Supplementary Fig. 14, upon subcutaneous injection at the tail base, SONA\ngradually accumulated into the LNs and peaked at 6 h post injection. Therefore, we selected\nthe 6 h post SONA injection as the experimental time point for LNs-localized US stimulation.\nWe next altered the power density and duration of US stimulation to generate different levels\nof mechanical and oxidative force, and then investigated their effect on innate immune\nactivation. We first investigated the influence of different power density of US stimulation on\ninducing the innate immune responses in vivo. The LNs were locally stimulated with 5 minutes\nof indicated power density of US (0, 0.2, 0.5, 1.0, 1.5 W/cm2) at 6 h post SONA injection.\nFlow cytometry analysis revealed that only SONA treatment induced negligible innate\nimmunity in LNs. In contrast, LNs-specific US stimulation effectively activated the innate\nimmune responses, and 1.0 W/cm2 of US stimulation elicited the highest recruitment and\nmaturation of DCs and macrophages in the LNs (Fig. 2f, g and Supplementary Fig. 15). We\nsubsequently investigated the influence of different duration of US stimulation on innate\nimmunity activation in vivo. The LNs were locally stimulated with 1.0 W/cm2 of indicated\nduration of US (0, 1, 3, 5, 7 min) at 6 h post SONA injection. The results showed that the\nrecruitment and maturation levels of DCs in the LNs gradually increased with the increase of\nUS duration, and it almost reached the peak value when combined with 5 minutes of US\nstimulation (Supplementary Fig. 16). Collectively, we screened out the time point (6 h post\ninjection), power density (1.0 W/cm2) and duration (5 min) of US stimulation for LNs-specific\ninnate immune activation in vivo.”\nReference:\n66. Han Z, et al. Precise Cell Type Electrical Stimulation Therapy Via Force-electric Hydrogel Microspheres\nfor Cartilage Healing. Adv Mater 37, e2414555 (2025).\n67. Zhu K, Wang J, Wang Z, Chen Q, Song J, Chen X. Ultrasound-Activated Theranostic Materials and\nTheir Bioapplications. Angew Chem Int Ed Engl, e202422278 (2025).\n68. Moradi Kashkooli F, Jakhmola A, Hornsby TK, Tavakkoli JJ, Kolios MC. Ultrasound-mediated nano\ndrug delivery for treating cancer: Fundamental physics to future directions. J Control Release 355, 552-\n578 (2023).\n69. Paliwal S, Mitragotri S. Ultrasound-induced cavitation: applications in drug and gene delivery. Expert\nOpin Drug Deliv 3, 713-726 (2006).\n70. Yu CC, et al. A Conformable Ultrasound Patch for Cavitation-Enhanced Transdermal Cosmeceutical\nDelivery. Adv Mater 35, e2300066 (2023).\nSupplementary Fig. 14 (in this revised manuscript). a, Representative fluorescence images of mice at indicated\ntime points post SONA injection (n = 3 biologically independent mice). b, Representative fluorescence images and\nnormalized fluorescence signal of tissues collected at indicated time points post SONA injection (n = 3 biologically\nindependent mice). Statistical significance was calculated via one-way ANOVA with Tukey’s test and P values were\nindicated.\nSupplementary Fig. 15 (in this revised manuscript). a, The absolute number of CD11c+ DCs and CD11b+\nmacrophages in the LNs after SONA+US treatment with different power density of US stimulation (n = 3 biologically\nindependent mice). b, The CD80 or CD86 expression level on CD11c+ DCs after SONA+US treatment with different\npower density of US stimulation (n = 3 biologically independent mice). Statistical significance was calculated via\none-way ANOVA with Tukey’s test and P values were indicated.\nSupplementary Fig. 16 (in this revised manuscript). a, The absolute number of CD11c+ DCs and CD11b+\nmacrophages in the LNs after SONA+US treatment with different duration of US stimulation (n = 3 biologically\nindependent mice). b, The CD80 or CD86 expression level on CD11c+ DCs after SONA+US treatment with different\nduration of US stimulation (n = 3 biologically independent mice). Statistical significance was calculated via one-way\nANOVA with Tukey’s test and P values were indicated.\n5. The synthesis of charge-modified polypeptide is quietly innovative, and a more detailed\nexplanation of the synthetic methods should be provided.\nResponse: We provided a more detailed explanation of the synthetic methods involving the\ncharge-modified polypeptides in Line 871-885 in the revised manuscript as below:\n“The mPEG -b-P(Lys ), mPEG -b-P(Tyr -Lys ), mPEG -b-P(Tyr -Lys ), mPEG -\n113 40 113 10 30 113 20 20 113\nb-P(Tyr -Lys ) and mPEG -b-P(Tyr ) polypeptides were synthesized through ring-\n30 10 113 40\nopening co-polymerization of lysine N-carboxyanhydride (Lys NCA) and tyrosine N-\ncarboxyanhydride (Tyr NCA) using mPEG -NH as the macroinitiator. Typically, NH -PEG\n113 2 2 5k\n(100 mg, 0.018 mmol) and indicated mass of Lys NCA and Tyr NCA were dissolved in\nanhydrous DMF under argon. The reaction was stirred for 3 days at room temperature and\nprecipitated with cold diethyl ether to obtain indicated polypeptides as white powder. The\nmPEG -b-P(TyrP -Lys ), mPEG -b-P(TyrP -Lys ) and mPEG -b-P(TyrP )\n113 10 30 113 30 10 113 40\npolypeptides were synthesized via phosphorylation of the phenolic hydroxyl groups of\ntyrosine. For instance. mPEG -b-P(Tyr ) (100 mg, 0.0087 mmol) was suspended in\n113 40\nanhydrous N-methylpyrrolidone under ice bath. Anhydrous triethylamine (200 μL) and\nphosphorus oxychloride (200 μL) were then slowly added. The mixture was stirred in the\ndarkness overnight and saturated NaHCO solution was added. After stirring for another 3 h,\n3\nthe mixture was dialyzed against DI water and lyophilized to obtain the mPEG -b-P(TyrP )\n113 40\nas yellow solid.”\n6. DLS-determined particle size and surface charge of neoantigen peptide-loaded\nnanovaccines should be provided.\nResponse: We performed DLS analysis to determine the particle size and surface charge of\nneoantigen peptide-loaded nanovaccines in Supplementary Fig. 45 in the revised\nmanuscript.\n“The 4T1 tumor cell-specific neoantigen peptide-loaded Ne-SONV displayed a uniform\nhydrodynamic diameter of 127.6 ± 24.1 nm and positive surface charge of 5.4 mV. The\nHepa1-6 tumor cell-specific neoantigen peptide-loaded Ne-SONV displayed a uniform\nhydrodynamic diameter of 130.6 ± 21.0 nm and positive surface charge of 3.7 mV.”\nSupplementary Fig. 45 (in this revised manuscript). a, DLS analysis of 4T1-Ne-SONV and Hepa1-6-Ne-SONV\nin PBS (pH 7.4). b, DLS-determined particle size and zeta potential of 4T1-Ne-SONV and Hepa1-6-Ne-SONV (n = 3\nbiologically independent replicates ).\n7. The necessity and advantages of spatiotemporally-controllable innate immunity activation\nin LNs should be more convincingly demonstrated.\nResponse: We appreciated the comments of the reviewer reminding us to highlight the\nnecessity and advantages of the spatiotemporally-controllable innate immunity activation\nstrategy. We added the relevant discussion and cited new references in Line 731-752 in the\nrevised manuscript as below:\n“Innate immune stimulation in antigen-presenting cells (APCs) of lymph nodes (LNs) is crucial\nfor bridging innate and adaptive immunity. However, current adjuvant strategies exhibited the\n‘always on’ innate immune stimulation profile and potentially induced immune-related\nadverse effects due to their non-specific distribution and activation on untargeted tissues (e.g.,\nliver, lung). In addition, conventional adjuvant systems usually activated single innate immune\npathways, and the sustained exposure to mono innate immune stimuli was likely to induce\nAPCs dysfunction62-64. Hence, it is of high priority to achieve spatiotemporally-controllable\nactivation of different innate immune pathways in APCs by a LNs-specific manner.\nIn this study, we introduced a US-activatable sono-adjuvant that exhibited critical\nadvantages over the conventional adjuvants to implement safe and effective cancer vaccine\nimmunotherapy. For instance, there was no bioactive adjuvant integrated into the sono-\nadjuvant platform, and thereby the elicitation and duration of innate immunity could be\nspatiotemporally tailored by the external US stimuli. The US-activatable turning on/off of\ninnate immunity ensured to mitigate the immune-related adverse effects on untargeted\ntissues. It was worth noting that the sono-adjuvant represented a multivalent platform to\nsynchronously activate multiple innate immune pathways (STING, TLR9, NLRP3) under\nexternal US stimuli. More importantly, a critical challenge in inducing robust T-cell response\nis the spatiotemporal orchestration of antigen cross-presentation with innate immune\nstimulation in APCs of LNs. Although various adjuvant systems have been developed to co-\ndeliver tumor antigen and adjuvant into LNs, most of them were ineffective to simultaneously\npromote antigen cytosolic release and innate immune stimulation in APCs, thus limiting the\nantigen presentation and T cell activation. Our spatiotemporally-controllable sono-adjuvant\nplatform could utilize the US-triggered mechanical and oxidative force to synchronously\npromote antigen cytosolic release and innate immune pathways activation in APCs, and\nultimately induced robust antigen-specific T cell responses in vivo.”\nReference:\n62. Macagno A, Napolitani G, Lanzavecchia A, Sallusto F. Duration, combination and timing: the signal\nintegration model of dendritic cell activation. Trends Immunol 28, 227-233 (2007).\n63. Kasturi SP, et al. Programming the magnitude and persistence of antibody responses with innate\nimmunity. Nature 470, 543-547 (2011).\n64. Jin SM, et al. A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances\ncancer immunotherapy and reduces immune cell exhaustion. Nat Nanotechnol 18, 390-402 (2023).\nSpecific comments\n1. Semi-quantification is required for Figure 2n and 2o.\nResponse: We provided the semi-quantification results of Fig. 2n and 2o in the revised\nmanuscript.\nSupplementary Table S2 (in this revised manuscript). The list of semi-quantification results of western blot assay\nin Fig. 2n and 2o.\n2. Fig 2f-g, statistical analysis should be performed.\nResponse: We performed the statistical analysis of Fig. 2f, g and Supplementary Fig. 15\nin the revised manuscript.\nSupplementary Fig. 15 (in this revised manuscript). a, The absolute number of CD11c+ DCs and CD11b+\nmacrophages in the LNs after SONA+US treatment with different power density of US stimulation (n = 3 biologically\nindependent mice). b, The CD80 or CD86 expression level on CD11c+ DCs after SONA+US treatment with different\npower density of US stimulation (n = 3 biologically independent mice). Statistical significance was calculated via\none-way ANOVA with Tukey’s test and P values were indicated.\n3. In figure 4m, n, the meaning of “in tumor (%)” is unclear. Please specify what percentage\nis being measured.\nResponse: We claimed the percentage being measured for analyzing tumor-infiltrating Tregs\nand MDSCs in Fig. 4m and 4n in the revised manuscript.\nFig. 4 (in this revised manuscript). m, The frequency of tumor-infiltrating Tregs (CD25+FOXP3+CD4+CD3+CD45+) (n = 5 biologically independent mice). n, The frequency of tumor-infiltrating MDSCs (Ly6G+CD11b+CD45+) (n = 5 biologically independent mice). Statistical significance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\n4. In figure 5i, k, m, the meaning of “in tumor (%)” is unclear. Please specify what percentage\nis being measured.\nResponse: We claimed the percentage being measured for analyzing tumor-infiltrating CD8+\nT cells, Tregs and MDSCs in Fig. 5i, 5k and 5m in the revised manuscript.\nFig. 5 (in this revised manuscript). i, The frequency of tumor-infiltrating CD8+ T cells (CD8+CD3+CD45+) (n = 5 biologically independent mice). k, The frequency of tumor-infiltrating Tregs (CD25+FOXP3+CD4+CD3+CD45+) (n = 5 biologically independent mice). m, The frequency of tumor-infiltrating MDSCs (Ly6G+CD11b+CD45+) (n = 5 biologically independent mice). Statistical significance was calculated via one-way ANOVA with Tukey’s test and P values were indicated.\n5. In figure 3j, the experimental method for analyzing OVA-specific cytotoxic effect in vivo is\nsuggested to be presented in a more detailed manner.\nResponse: We provided a detailed description of the experimental method for analyzing\nOVA-specific cytotoxic effect in Line 965-974 in the revised manuscript as below:\n“We performed an in vivo cytotoxicity assay to detect the OVA-specific cytotoxic T cell\nresponse generated by SONV+US. Half of splenocytes from untreated mice were labeled\nwith OVA peptide and high concentration of CFSE (5.0 μM), denoted as OVAp+ cells.\nMeanwhile, the other half of splenocytes were labeled with low concentration of CFSE (0.5\nμM) and not labeled with OVA peptide, denoted as OVAp− cells. Then, the OVAp+ cells (5×106\ncells per mouse) and OVAp− cells (5×106 cells per mouse) were mixed and intravenously\ninjected into the SONV+US-treated mice. After 24 h, spleen from treated mice was collected\nand subjected to flow cytometry analysis. The numbers of CFSEhigh and CFSElow cells were\ndetermined and used to calculate the percentage of OVA-specific cell killing.”\nPercentage of specific lysis = [1 − non-transferred control ratio/experimental ratio] × 100%.\n6. Supplementary figure 17, statistical analysis should be performed.\nResponse: We performed the statistical analysis of Supplementary Fig. 20 in the revised\nmanuscript.\nSupplementary Fig. 20 (in this revised manuscript). Representative flow cytometric plots and the statistical data\nof cellular uptake of free OVA-FITC antigen and SONV-FITC in DC2.4 cells (n = 3 biologically independent\nreplicates). Statistical significance was calculated via unpaired two-tailed Student’s t-test and P values were indicated.\n7. The caption labeling for the statistical data in Supplementary figure 42 is missing.\nResponse: We added the corresponding labeling in Supplementary Fig. 49 in the revised\nmanuscript.\nSupplementary Fig. 49 (in this revised manuscript). a, Representative flow cytometric plots of tumor-infiltrating\nCD45+CD3+CD8+ T cells. b, Representative flow cytometric plots of CD45+CD3-CD49b+ NK cells in 4T1 tumors.\nc, Representative flow cytometric plots of matured DCs in 4T1 tumors. d, Representative flow cytometric plots of\nTregs in 4T1 tumors. e, Representative flow cytometric plots of M1-pheotype TAMs in 4T1 tumors. f, Representative\nflow cytometric plots of M2-pheotype TAMs in 4T1 tumors. g, The frequency of tumor-infiltrating CD45+CD3+CD8+\nT cells in the indicated groups (n = 5 biologically independent mice). h, The frequency of CD45+CD3-CD49b+ NK\ncells in 4T1 tumors in indicated groups (n = 5 biologically independent mice). i, The frequency of matured DCs in\n4T1 tumors in indicated groups (n = 5 biologically independent mice). j, The frequency of Tregs\n(FOXP3+CD25+CD4+CD3+CD45+) in 4T1 tumors in indicated groups (n = 5 biologically independent mice). k, The\nfrequency of M1-pheotype TAMs (CD86+F4/80+CD11b+CD45+) in 4T1 tumors in indicated groups (n = 5\nbiologically independent mice). l, The frequency of M2-pheotype TAMs (CD206+F4/80+CD11b+CD45+) in 4T1\ntumors in the indicated groups (n = 5 biologically independent mice). Statistical significance was calculated via one-\nway ANOVA with Tukey’s test and P values were indicated.\nReviewer #6 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We greatly appreciated the comments of the reviewer helping us to improve the\nquality of our manuscript.\nResponse to the reviewers\nWe sincerely appreciate the insightful comments and efforts of all the reviewers helping us to\nrevise the manuscript and improve our understanding of the polypeptide sono-adjuvant for\ncancer vaccine immunotherapy. To address the concerns of the reviewer, we conducted\nadditional experimental studies and carefully revised the manuscript. Specifically,\n1. According to the comments of Reviewer #4, we compared the immune-related side\neffects of our ultrasound (US)-activatable vaccination strategy (SONV+US) with that of two\nrepresentative traditional immune agonists-based vaccines, OVA+CpG and OVA+MSA-2, by\nmeasuring the systemic levels of inflammatory cytokines (IL-6 and IL12p70). The results\ndemonstrated that mice treated with OVA+CpG or OVA+MSA-2 exhibited pronounced\nsystemic cytokine release in serum, with IL-6 concentrations reaching ~219.2 pg/mL\n(OVA+CpG), ~126.0 pg/mL (OVA+MSA-2) and IL-12p70 concentrations reaching ~34.0\npg/mL (OVA+CpG), ~26.2 pg/mL (OVA+MSA-2) at 1 day post each injection. These cytokine\nlevels gradually declined but remained slightly above baseline by day 7 post each injection,\nindicating a transient but substantial systemic inflammatory response induced by OVA+CpG\nand OVA+MSA-2. In contrast, mice treated with the SONV+US showed only a modest\nincrease in IL-6 (~35.8 pg/mL) and IL-12p70 (~10.1 pg/mL) secretion at 1 day post each\ninjection. Importantly, inflammatory cytokine levels were found to decline toward baseline and\nremain at physiological levels by day 7 post injection. These results demonstrated that\nSONV+US reduced systemic immune activation and exhibited good long-term safety\ncompared to traditional immune agonist-based vaccination strategies.\n2. According to the comments of Reviewer #4, we performed additional experiments to\nexamine whether SONP+US affected other immune cell types beyond antigen-presenting\ncells (APCs). Specifically, CD3⁺ T cells and CD19⁺ B cells were isolated from untreated-\nmouse lymph nodes by using fluorescence-activated cell sorting (FACS). The isolated T cells\nand B cells were treated in vitro with US, SONP, SONP+US for 24 h, and the activation of\ninnate immune pathways, including STING and NLRP3, was subsequently analyzed by\nwestern blot. The results showed that SONP+US treatment marginally affected the\nexpression of phosphorylated-TBK1 (p-TBK1), phosphorylated-IRF3 (p-IRF3) and NLRP3\nproteins in either CD3⁺ T cells or CD19⁺ B cells compared to the PBS group. These findings\nsuggested that SONP+US may not effectively activate innate immune pathways in T cells\nand B cells under our experimental conditions. A plausible explanation was the limited uptake\nand intracellular delivery of SONP by these non-professional phagocytes, including T cells\nand B cells, which may result in insufficient intracellular reactive oxygen species (ROS)\ngeneration, and consequently impair the activation of downstream signaling cascades.\n3. According to the comments of Reviewer #4, we evaluated the dose-response curve of\nlong-term immune responses by assessing the dynamic activation of antigen-specific CD8+\nT cells and memory CD8+ T cells in the peripheral blood of SONV+US-treated mice. We\nselected the US frequency of 1 MHz, the duty cycle of 50%, and altered the power density\n(0.5, 1.0, 1.5 W/cm²) and duration (0, 3, 5 min) of US stimulation to assess the impact of\ndifferent US parameters on antigen-specific and long-term T cell responses. Flow cytometry\nanalysis revealed that only SONV treatment induced moderate activation of antigen-specific\nCD8+ T cell response, and the frequency of Tetramer+CD8+ T cells was marginally increased\ncompared to PBS group at day 28. In contrast, the combination of SONV with LNs-localized\nUS stimulation effectively enhanced the activation of antigen-specific CD8+ T cell response\nin the peripheral blood. Specifically, it was observed that US stimulation under the conditions\nof 1.0 W/cm² for 5 minutes or 1.5 W/cm² for 3 minutes elicited robust and durable antigen-\nspecific CD8+ T cell responses, reaching the highest levels among all tested conditions. For\ninstance, the frequency of Tetramer+CD8+ T cells in SONV+US (1.0 W/cm² + 5 min) group\nwere approximately 4.6-fold higher than SONV group at day 28, respectively. We\nsubsequently evaluated the activation of immune memory responses in the peripheral blood.\nThe result showed that SONV+US (1.0 W/cm² + 5 min) treatment induced high-quality and\nlong-term T cell immune responses, showing the highest levels of central memory CD8+ T\ncell (CD44+CD62L+) and effector memory CD8+ T cell (CD44+CD62L-) responses among all\ntested conditions. Collectively, we found that US stimulation under the condition of 1.0 W/cm²\nfor 5 minutes could not only effectively promote innate immune activation in LNs, but also\nensure potent and durable tumor antigen-specific T cell response activation, which serves as\na critical indicator of the final therapeutic outcome.\n4. According to the comments of Reviewer #4 and editor, we addressed our decision criteria\nto use an antibody-based immune-depletion approach in the revised manuscript. In our study,\nwe chosen to perform transient CD8⁺ T cell depletion by using monoclonal antibodies to\npreserve the overall immune architecture and better mimic a functional immune system under\ntherapeutic pressure. This approach allowed us to identify and specifically assess the role of\nCD8⁺ T cells without introducing broader developmental or compensatory alterations that\noften observed in genetically immunodeficient strains. Moreover, antibody-mediated\ndepletion was a widely used, well-established method to interrogate T cell involvement in\npreclinical studies, especially when rapid experimental turnaround and immunocompetent\ntumor microenvironment were desired. Nevertheless, we agreed that immunodeficient mouse\nmodels, which lack functional T cells entirely, could serve as a rigorous tool to evaluate the\ndependence of therapeutic efficacy of our vaccination strategy on adaptive immunity in future\nstudies.\n5. According to the comments of Reviewer #4, we included critical references to recent\nstudies on US-activatable immune adjuvant strategies, and provided a comparative\ndiscussion highlighting the consistencies and distinctions between our data and existing\nrelated research in the Discussion section of our revised manuscript.\n6. According to the comments of Reviewer #3, we corrected the y-axis label in Figure 3m\nto “-log10(Padjust)” for consistency and clarity in the revised manuscript.\nWe hope the revised manuscript could match the criteria of Nature Communications now.\nReferee’s comments are in black and our responses are in blue.\nReviewer #1 (Remarks to the Author):\nReviewer #1 (Remarks to the Author):\nThe authors have successfully addressed this reviewer's questions. No more questions.\nResponse: We are grateful to the reviewers for insightful comments that helped improve our\nstudy.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We are grateful to the reviewers for insightful comments that helped improve our\nstudy.\nReviewer #3 (Remarks to the Author):\nThank you to the authors for thoroughly addressing all my concerns raised in the previous\nround. The revised manuscript is much improved. I would like to point out that the y-axis label\nin Figure 3m should be corrected to “–log10(Padjust)” for consistency and clarity, as\nmentioned in the response letter but not yet updated in the manuscript.\nResponse: We corrected the y-axis label in Figure 3m to “-log10(Padjust)” in the revised\nmanuscript.\nFig. 3. SONV+US robustly elicited antigen-specific T-cell response via US-triggered innate immunity\nactivation.\nReviewer #4 (Remarks to the Author):\nThe authors have revised the manuscript to address the reviewers' comments. However,\nseveral aspects of this response require further improvement.\n1. The authors compared the differences between ultrasound-guided immune activation\nstrategy and traditional immune agonists in activating antigen-specific immune responses\nthrough specific experimental data. Despite providing data on antitumor efficacy, no\ncomparison of immune side effects was made, failing to fully address concerns regarding\nimmune side effects. Additionally, the data showed insufficient engagement with existing\nrelated research.\nResponse: We greatly appreciated the critical comments of the reviewer. To address the\nreviewer’s concerns regarding potential immune-related adverse effects, we conducted\nadditional in vivo studies to evaluate and compare the systemic immune toxicity of our\nultrasound (US)-activatable vaccination strategy (SONV+US) with that of two representative\ntraditional immune agonists-based vaccines (e.g., OVA+CpG and OVA+MSA-2). The\nimmune agonists CpG and MSA-2 have been widely studied but were also known to trigger\noff-target systemic immune toxicity when administered systemically or subcutaneously,\nprimarily due to their non-specific biodistribution and innate immune-stimulatory profile21,23.\n“Our results demonstrated that mice treated with OVA+CpG or OVA+MSA-2 exhibited\npronounced systemic cytokine release in serum, with IL-6 concentrations reaching ~219.2\npg/mL (OVA+CpG), ~126.0 pg/mL (OVA+MSA-2) and IL-12p70 concentrations reaching\n~34.0 pg/mL (OVA+CpG), ~26.2 pg/mL (OVA+MSA-2) at 1 day post each injection\n(Supplementary Fig. 36). These cytokine levels gradually declined but remained slightly\nabove baseline by day 7 post each injection, indicating a transient but substantial systemic\ninflammatory response induced by OVA+CpG and OVA+MSA-2. In contrast, mice treated\nwith the SONV+US showed only a modest increase in IL-6 (~35.8 pg/mL) and IL-12p70\n(~10.1 pg/mL) secretion at 1 day post each injection. Importantly, the inflammatory cytokine\nlevels declined toward baseline and maintained at physiological levels by day 7 post injection.\nThese results demonstrated that SONV+US substantially reduced systemic immune\nactivation compared to traditional immune agonist-based vaccination strategies. The critical\nsafety advantage of SONV+US strategy could be attributed to the following aspects: 1. SONV\nexhibited efficient lymph node-targeting delivery via nanoparticle-mediated transport and\nmannose-mediated antigen-presenting cells (APCs) targeting, which ensured SONV\npreferentially accumulated into draining lymph nodes, while minimizing off-target distribution\nin other organs. 2. The adjuvant effect of SONV was highly dependent on external US\nstimulation, since the excitation of sonosensitizer requires US-triggered acoustic energy.\nTherefore, SONV+US offered a spatiotemporally controllable approach to precisely confine\ninnate immune activation within targeted tissues by adjusting the location, duration, and\nintervals of US stimulation.”\nThe relevant discussion was added in Line 418-432 in the revised manuscript. We also\nincluded the experimental method by assessing the systemic immune side effects in Line\n1015-1025 in the revised manuscript as below.\n“Assessment of systemic immune side effects. To evaluate systemic immune\nactivation associated with different adjuvant strategies, serum levels of IL-6 and IL-12p70\nwere measured following indicated treatments. Female C57BL/6 mice (4-5 weeks old) were\nrandomly assigned into three groups: OVA+CpG (50 μg CpG), OVA+MSA-2 (50 μg MSA-2)\nand SONV+US (50 μg HMME). All formulations contained 50 μg OVA antigen and were\nsubcutaneously injected at the tail base of mice on day 0, day 7, and day 14, respectively.\nFor SONV+US group mice, the draining lymph nodes were stimulated with US (1 MHz\nfrequency, 50% duty cycle, 1.0 W/cm² intensity, 5 min) at 6 h post each injection. Peripheral\nblood was collected via submandibular vein at 1 day and 7 day post each injection. Serum\nwas isolated and stored at -80 °C until analysis. Levels of IL-6 and IL-12p70 were quantified\nusing indicated ELISA kits (NeoBioscience) following the manufacturer’s protocol.”\nTo highlight the previous advance in the fields of stimuli-activatable vaccination\nstrategies, we included references to recent studies on US-activatable immune adjuvant\nstrategies, and provided a comparative discussion highlighting the consistencies and\ndistinctions between our data and existing related research. The relevant discussion was\nadded in Line 784-805 in the revised manuscript as below.\n“Various types of US-activatable immune adjuvant strategies have been developed to control\nthe activation of immune agonists in a region-specific manner. For instance, a US-responsive\npolymeric pro-agonist system was reported to specifically activated immune response within\nthe tumor microenvironment. Upon US stimulation, the pro-agonist system released bioactive\nSTING agonist, triggered potent local immune responses while minimizing systemic toxicity17.\nIn addition, a semiconducting polymeric nanoagonist enabling US-triggered activation of\nimmune agonist was reported for precise cancer immunotherapy. In combination with US\nstimulation, the nanoagonist generated abundant singlet oxygen to induce immunogenic cell\ndeath and simultaneously release caged immune agonist via ROS-cleavable linker. This US-\ntriggered activation enhanced innate immune activation within tumor tissue, promoted T cell\ninfiltration and elicited robust antitumor immunity62.\nCompared to the previous studies of spatiotemporally-controllable immune agonists for\ncancer immunotherapy, the US-activatable sono-adjuvant reported herein exhibited distinct\nadvantages for clinical application. First, the sono-adjuvant is rational designed to avoid the\nuse of conventional immune adjuvants (e.g., TLR agonists or STING agonist) with always-on\nimmune stimulation property. Thereby the elicitation and duration of innate immunity could be\nspatiotemporally tailored by the external US stimulation. The US-activatable turning on/off of\ninnate immunity ensured to mitigate the immune-related adverse effects on untargeted\ntissues. Second, the previously reported adjuvants generally activate single innate immune\npathways, and the sustained exposure to mono innate immune stimuli is intend to induce\nAPCs dysfunction63-65. In contrast, our sono-adjuvant represented a multivalent platform to\nsynchronously activate multiple innate immune pathways. Third, distinct from other strategies\nthat aimed to precisely activate immune agonists within tumor tissues, our sono-adjuvant\nstrategy specifically leveraged US stimulation to modulate innate immune responses within\nlymph nodes, which directly synergized with antigen cross-presentation in APCs to boost\nantigen-specific T cell responses. Moreover, the sono-adjuvant represented a highly\nbiodegradable and generalizable vaccine platform, and could have broad implications to co-\ndeliver various tumor antigens for personalized cancer immunotherapy.”\nReference:\n17. Yu J, et al. Polymeric STING Pro-agonists for Tumor-Specific Sonodynamic Immunotherapy. Angew\nChem Int Ed 62, e202307272 (2023).\n21. Ryu KA, Stutts L, Tom JK, Mancini RJ, Esser-Kahn AP. Stimulation of innate immune cells by light-\nactivated TLR7/8 agonists. J Am Chem Soc 136, 10823-10825 (2014).\n23. Dou Y, et al. Optogenetic engineering of STING signaling allows remote immunomodulation to\nenhance cancer immunotherapy. Nat Commun 14, 5461 (2023).\n62. Jiang J, et al. Sono-Driven STING Activation using Semiconducting Polymeric Nanoagonists for\nPrecision Sono-Immunotherapy of Head and Neck Squamous Cell Carcinoma. Adv Mater 35,\ne2300854 (2023).\n63. Macagno A, Napolitani G, Lanzavecchia A, Sallusto F. Duration, combination and timing: the signal\nintegration model of dendritic cell activation. Trends Immunol 28, 227-233 (2007).\n64. Kasturi SP, et al. Programming the magnitude and persistence of antibody responses with innate\nimmunity. Nature 470, 543-547 (2011).\n65. Jin SM, et al. A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances\ncancer immunotherapy and reduces immune cell exhaustion. Nat Nanotechnol 18, 390-402 (2023).\nSupplementary Fig. 36 (in this revised manuscript). a, The IL-6 secretion level in serum of treated mice at\nindicated time points (n = 3 biologically independent mice). b, The IL-12p70 secretion level in serum of treated mice\nat indicated time points (n = 3 biologically independent mice). The black arrows indicated the administration date.\n2. The authors provided more specific research data and results through experiments\ninvolving mannose-modified adjuvants to improve targeting, as well as in vitro experiments\non the effects of ultrasound stimulation and combined sonosensitive nanoparticle stimulation\non the activation of different immune pathways. This somewhat compensates for the lack of\nmechanistic explanation in the original manuscript. Although additional content was provided,\nthe response primarily focuses on the role of immune pathways in APCs, such as DCs and\nmacrophages, with less emphasis on other immune cells.\nResponse: We greatly appreciated the critical comments of the reviewer. To address the\nreviewer’s concern, we performed additional experiments to examine whether SONP+US\naffected other immune cell types beyond APCs. Specifically, CD3⁺ T cells and CD19⁺ B cells\nwere isolated from untreated-mouse lymph nodes by using fluorescence-activated cell sorting\n(FACS). Briefly, single-cell suspensions were prepared from harvested lymph nodes, stained\nwith anti-CD3-PE-Cy7 and anti-CD19-PE antibodies, and sorted on a BD FACSAria III cell\nsorter (Supplementary Fig. 8). The isolated T cells and B cells were subsequently treated\nin vitro with US, SONP, SONP+US for 24 h. US stimulation was applied at 1 MHz frequency,\n50% duty cycle, and an intensity of 0.2 W/cm² for 3 minutes. Western blot assay displayed\nthat SONP+US treatment marginally affected the expression of phosphorylated-TBK1 (p-\nTBK1), phosphorylated-IRF3 (p-IRF3) and NLRP3 proteins in either CD3⁺ T cells or CD19⁺\nB cells compared to the PBS group (Supplementary Fig. 9). These findings suggested that\nSONP+US may not effectively activate innate immune pathways in T cells and B cells under\nour experimental conditions. A plausible explanation was the limited uptake and intracellular\ndelivery of SONP by these non-professional phagocytes, including T cells and B cells, which\nmay result in insufficient intracellular reactive oxygen species (ROS) generation, and\nconsequently impair the activation of downstream signaling cascades.\nThe relevant discussion was added in Line 171-179 in the revised manuscript.\nSupplementary Fig. 8. Representative flow cytometry gating strategy for sorting the CD3+ T cells and CD19+ B\ncells from single-cell suspension prepared from LNs.\nSupplementary Fig. 9 (in this revised manuscript). WB analysis of the activation of STING and NLRP3 pathways\nin CD3+ T cells (a) and CD19+ B cells (b) after indicated treatments.\n3. The authors demonstrated that SONV+US relies on T cells for its effect through CD8+ T\ncell blockade experiments, which partially address concerns about the applicability of the\nimmune environment. However, the blockade of T cell function using antibodies is a\ntemporary immune suppression, not a true “immunodeficient background”. Immunodeficient\nmice lack the ability for complete T cell development and can be used to verify whether the\ntherapy depends on adaptive immunity. The authors only describe the experimental results\nwithout discussing why antibody blockade was chosen instead of using genetically deficient\nmice, nor do they explain the advantage of this approach. It is recommended that the authors\ninclude more rigorous experiments using immune-deficient models to strengthen the\ncomprehensiveness and persuasiveness of their conclusions.\nResponse: We totally agreed with the reviewer that immunodeficient mouse models, which\nlack functional T cells entirely, could serve as a rigorous tool to evaluate the dependence of\ntherapeutic efficacy of SONV+US on adaptive immunity. We have now clarified this rationale\nin Line 600-607 in the revised manuscript, and appreciated the kind suggestion of the\nreviewer, which is crucial for us to optimize the study design with genetically defined models.\nIn this study, we depleted CD8⁺ T cell using monoclonal antibodies to preserve the overall\nimmune architecture and better mimic a functional immune system under therapeutic\npressure. This approach allowed us to identify and specifically assess the role of CD8⁺ T cells\nwithout introducing broader developmental or compensatory alterations that often observed\nin genetically immunodeficient strains. Moreover, antibody-mediated depletion was a widely\nused, well-established method to interrogate T cell involvement in preclinical studies32,48,\nespecially when rapid experimental turnaround and immunocompetent tumor\nmicroenvironment were desired. Nevertheless, we acknowledged that the antibody-mediated\nT cell blockade approach exhibited inherent limitations and did not fully replicate the\nconditions of congenital immunodeficiency.\nReference:\n32. Tan M, et al. Metal-ion-chelating phenylalanine nanostructures reverse immune dysfunction and\nsensitize breast tumour to immune checkpoint blockade. Nat Nanotechnol 19, 1903-1913 (2024).\n48. Liu X, et al. In-situ vaccination immunotherapy of colorectal cancer with STING agonist-integrated\nsupramolecular nanovectors. Nano Today 56, 102273 (2024).\n4. The safety assessment lacks immunogenicity testing, making it unclear whether the\ntreatment-induced immune response is “temporary activation” or “long-term memory”. This\nalso makes it difficult to verify whether the therapy can continue to exert antitumor effects\nafter cessation, and prevents the evaluation of potential chronic effects on immune organs\nfrom long-term immune activation.\nResponse: We greatly appreciated the critical comments of the reviewer. To address the\nreviewer’s concerns about lacking immunogenicity testing for safety assessment, we\nconducted additional in vivo studies to evaluate the biosafety of SONV+US strategy by\nmeasuring the secretion levels of inflammatory cytokines (IL-6 and IL-12p70) in the serum of\ntreated mice. C57BL/6 mice were vaccinated with OVA+CpG, OVA+MSA-2 or SONV+US on\nday 0, day 7, and day 14, respectively. Peripheral blood was collected via submandibular vein\nat 1-day and 7-day post each injection. Serum was isolated and the levels of IL-6 and IL-\n12p70 were quantified using indicated ELISA kits (NeoBioscience) following the\nmanufacturer’s protocol. The results demonstrated that mice treated with OVA+CpG or\nOVA+MSA-2 exhibited pronounced systemic cytokine release in serum, with IL-6\nconcentrations reaching ~219.2 pg/mL (OVA+CpG), ~126.0 pg/mL (OVA+MSA-2) and IL-\n12p70 concentrations reaching ~34.0 pg/mL (OVA+CpG), ~26.2 pg/mL (OVA+MSA-2) at 1\nday post each injection (Supplementary Fig. 36). These cytokine levels gradually declined\nbut remained slightly above baseline at 7-day post each injection, indicating the combination\nof OVA+CpG and OVA+MSA-2 both induced a transient but substantial systemic\ninflammatory response. In contrast, the SONV+US group showed a modest increase in IL-6\n(~35.8 pg/mL) and IL-12p70 (~10.1 pg/mL) secretion at 1-day post each injection. Importantly,\nthe inflammatory cytokines declined toward baseline and remained at physiological levels by\n7-day post injection. The immunogenicity testing result demonstrated good biosafety of\nSONV+US compared to traditional immune agonist-based vaccination strategies.\nIn addition to measuring the secretion levels of inflammatory cytokines in the serum of\nSONV+US-treated mice, we also examined the dynamic secretion levels of IL-6 and IFN-β\nwithin the lymph nodes (LNs), as the local innate immune activation in LNs was essential for\neliciting safe and effective adaptive T cell responses. The result showed that SONA+US\ninduced abundant and sustained IFN-β and IL-6 secretion in LNs for over 48 h (Fig. 2h), and\nthese cytokine levels gradually declined toward baseline by 72 h post treatment, indicating a\ntransient but substantial local innate immune responses induced by SONV+US.\nNext, we assessed the dynamic activation of tumor antigen-specific CD8+ T cell response\nin the peripheral blood of SONV+US-treated mice (1 MHz frequency, 50% duty cycle,\n1.0 W/cm² intensity, 5 min), in order to determine the durability of antitumor effect following\ntreatment cessation (Supplementary Fig. 30, 31). The result showed that the frequency of\nantigen-specific CD8+ T cells (SIINFEKL-H2Kb tetramer+ in CD8+CD3+ cells) was significantly\nelevated and remained at high levels for up to 28 days following the initial treatment\n(Supplementary Fig. 32). Furthermore, elevated levels of both central memory CD8+ T cells\n(CD44+CD62L+ in CD8+CD3+ cells) and effector memory CD8+ T cells (CD44+CD62L- in\nCD8+CD3+ cells) were observed on 28-days following the initial treatment (Supplementary\nFig. 33), indicating the establishment of durable antitumor immune memory effects.\nCollectively, these results demonstrated that SONV+US strategy could safely and effectively\nactivate innate immune responses within LNs, thereby directly synergizing with antigen cross-\npresentation to elicit durable and tumor-specific T cell responses for long-term control of\ntumor growth, while minimizing off-target immune toxicity in non-tumor tissues.\nThe relevant discussion was added in Line 254-258, 366-382, 418-432 of the revised\nmanuscript.\nSupplementary Fig. 36 (in this revised manuscript). a, The IL-6 secretion level in serum of treated mice at\nindicated time points (n = 3 biologically independent mice). b, The IL-12p70 secretion level in serum of treated mice\nat indicated time points (n = 3 biologically independent mice). The black arrows indicated the administration date.\nFig. 2 (in this revised manuscript). h, The IFN-β and IL-1β production in LNs at different time points after\nSONA+US treatment (n = 3 biologically independent mice).\nSupplementary Fig. 30 (in this revised manuscript). Representative flow cytometry gating strategy for analyzing\nthe activation of antigen-specific CD8+ T cells and memory CD8+ T cells in the peripheral blood.\nSupplementary Fig. 31(in this revised manuscript). Representative flow cytometric plots of antigen-specifc CD8+\nT cells and memory CD8+ T cells in the peripheral blood at day 28.\nSupplementary Fig. 32 (in this revised manuscript). The frequency of Tetramer+CD8+ T cells in the peripheral\nblood of treated mice at the indicated time points (n = 3 biologically independent mice). The black arrows indicated\nthe administration date.\nSupplementary Fig. 33 (in this revised manuscript). a, The frequency of effector memory CD8+ T cell\n(CD44+CD62L-) in the peripheral blood of treated mice at the indicated time points (n = 3 biologically independent\nmice). b, The frequency of central memory CD8+ T cell (CD44+CD62L+) in the peripheral blood of treated mice at\nthe indicated time points (n = 3 biologically independent mice). The black arrows indicated the administration date.\n5. The authors identified the optimal time point for ultrasound stimulation and selected the\nbest experimental parameters through power density gradient and duration gradient\nexperiments. However, only the immediate immune activation after ultrasound stimulation\nwas observed, and the impact of different parameters on long-term immune responses was\nnot assessed. The dose-response curve should not only focus on acute effects but also\ncorrelate with final efficacy indicators. Or is immediate immune activation considered the final\nefficacy?\nResponse: We greatly appreciated the critical comments of the reviewer. To address the\nreviewer’s concerns about evaluating the dose-response curve of long-term immune\nresponses, we conducted additional in vivo studies to assess the dynamic activation of\nantigen-specific CD8+ T cell response and memory CD8+ T cell response in the peripheral\nblood of SONV+US-treated mice (Supplementary Fig. 30&31). C57BL/6 mice were\nsubcutaneously injected with SONV (HMME: 1.0 mg/kg, OVA: 2.0 mg/kg), and LNs-localized\nUS stimulation was conducted at 6 h post each injection. We selected the US frequency of 1\nMHz, the duty cycle of 50%, and altered the power density (0.5, 1.0, 1.5 W/cm²) and duration\n(0, 3, 5 min) of US stimulation to assess the impact of different US parameters on antigen-\nspecific and long-term T cell responses. Flow cytometry analysis revealed that only SONV\ntreatment induced moderate activation of antigen-specific CD8+ T cell response, and the\nfrequency of Tetramer+CD8+ T cells was marginally increased compared to PBS group at day\n28 (Supplementary Fig. 32). In contrast, the combination of SONV with LNs-localized\nUS stimulation effectively enhanced the activation of antigen-specific CD8+ T cell response\nin the peripheral blood. Specifically, it was observed that US stimulation under the conditions\nof 1.0 W/cm² for 5 minutes or 1.5 W/cm² for 3 minutes elicited robust and durable antigen-\nspecific CD8+ T cell responses, reaching the highest levels among all tested conditions. For\ninstance, the frequency of Tetramer+CD8+ T cells in SONV+US (1.0 W/cm² + 5 min) group\nwere approximately 4.6-fold higher than SONV group at day 28, respectively. We\nsubsequently evaluated the activation of immune memory responses in the peripheral blood.\nThe result showed that SONV+US (1.0 W/cm² + 5 min) treatment induced high-quality and\nlong-term T cell immune responses, showing the highest levels of central memory CD8+ T\ncell (CD44+CD62L+) and effector memory CD8+ T cell (CD44+CD62L-) responses among all\ntested conditions (Supplementary Fig. 33). Collectively, we found that US stimulation under\nthe condition of 1.0 W/cm² for 5 minutes could not only effectively promote innate immune\nactivation in LNs, but also ensure potent and durable tumor antigen-specific T cell response\nactivation, which serves as a critical indicator of the final therapeutic outcome.\nThe relevant discussion was added in Line 366-382 in the revised manuscript.\nSupplementary Fig. 30 (in this revised manuscript). Representative flow cytometry gating strategy for analyzing\nthe activation of antigen-specific CD8+ T cells and memory CD8+ T cells in the peripheral blood.\nSupplementary Fig. 31 (in this revised manuscript). Representative flow cytometric plots of antigen-specific CD8+\nT cells and memory CD8+ T cells in the peripheral blood at day 28.\nSupplementary Fig. 32 (in this revised manuscript). The frequency of Tetramer+CD8+ T cells in the peripheral\nblood of treated mice at indicated time points (n = 3 biologically independent mice). The black arrows indicated the\nadministration date.\nSupplementary Fig. 33 (in this revised manuscript). a, The frequency of effector memory CD8+ T cell\n(CD44+CD62L-) in the peripheral blood of treated mice at indicated time points (n = 3 biologically independent mice).\nb, The frequency of central memory CD8+ T cell (CD44+CD62L+) in the peripheral blood of treated mice at indicated\ntime points (n = 3 biologically independent mice). The black arrows indicated the administration date.\nReviewer #5 (Remarks to the Author):\nSince the authors have answered the all of questions very carefully, there are no more further\ncorrections in this final version, it can be published without further modifications.\nResponse: We are grateful to the reviewers for insightful comments that helped improve our\nstudy.\nReviewer #6 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We are grateful to the reviewers for insightful comments that helped improve our\nstudy.\nResponse to the reviewers\nReviewer #3 (Remarks to the Author):\nI have no additional comments, and the manuscript is now clear and well-prepared for\npublication.\nResponse: We are grateful to the reviewers for insightful comments that helped improve our\nstudy.\nReviewer #4 (Remarks to the Author):\nThe authors have made considerable effort to revise the manuscript that well address the\nreviewers' comments. The revised manuscript is suitable to be accepted for publication at\ncurrent stage.\nResponse: We are grateful to the reviewers for insightful comments that helped improve our\nstudy."}, {"round": 2, "reviewer_comments": {"reviewer_3": "I have no additional comments, and the manuscript is now clear and well-prepared for publication.", "reviewer_4": "The authors have made considerable effort to revise the manuscript that well address the reviewers' comments. The revised manuscript is suitable to be accepted for publication at current stage."}, "author_response": ""}]

Decision letter