Noninvasive ultrasound targeted modulation of calcium influx in splenic immunocytes potentiates antineoplastic immunity attenuating hepatocellular carcinoma proliferation
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eLife Assessment
This valuable study presents an innovative noninvasive immunotherapeutic strategy for hepatocellular carcinoma by combining ultrasound stimulation with calcium-loaded nanodroplets to activate splenic immune responses. The authors provide solid preclinical data, including single-cell transcriptomic analyses and evidence of tumor growth suppression, supported by a creative and well-executed methodology. Further validation of the calcium signaling mechanisms and assessment of long-term safety will strengthen the translational potential of this approach. The work will be of broad interest to researchers in oncology, immunotherapy, and biomedical engineering.
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Abstract
Abstract
The spleen, as the largest immune organ, plays a pivotal role in modulating immune responses, particularly in the context of carcinogenesis and tumor progression. Non-pharmacological manipulation, particularly splenic ultrasound stimulation (SUS), has demonstrated significant immunomodulatory efficacy in alleviating chronic inflammatory diseases, suggesting its potential to revitalize splenic immunocompetence suppressing tumor proliferation, yet remains underexplored. This study applied low-frequency pulsed focused ultrasound (FUS) noninvasively stimulating the spleen (FUS sti. spleen) to investigate the efficacy in enhancing antitumor immunity and suppressing hepatocellular carcinoma (HCC). The results showed that FUS sti. spleen significantly suppressed tumor proliferation, achieving a suppression rate of >70% for H22-HCC and >83% for Hepa1-6-HCC, along with significantly prolonged survival. Comprehensive flow cytometry, single-cell RNA sequencing (scRNA-seq) and cytokine analyses demonstrated that SUS profoundly reshaped the splenic and intratumoral immune landscape, specifically activating cytotoxic CD8+ T cells and NK cells while suppressing immunosuppressive cell populations. Mechanistically, FUS facilitated calcium influx in splenic immunocytes, activating multiple signaling pathways, such as TNF, NFκB, MAPK, HIF-1, and ErbB, thereby counteracting tumor-driven immunosuppressive polarization while potentiating robust immune activation that impedes malignant progression and neoplastic proliferation. Leveraging above insights, we developed spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STNDs@Ca²⁺), which, upon FUS stimulation, undergo cavitation-mediated controlled release of Ca²⁺, further amplifying immunocyte activation and tumor suppression, achieving a remarkable H22-HCC suppression rate of over 90%. This study highlights the therapeutic potential of ultrasound-mediated splenic immunomodulation, both as a standalone intervention and in synergy with STNDs@Ca²⁺, as a novel and noninvasive strategy for cancer immunotherapy.
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eLife Assessment
This valuable study presents an innovative noninvasive immunotherapeutic strategy for hepatocellular carcinoma by combining ultrasound stimulation with calcium-loaded nanodroplets to activate splenic immune responses. The authors provide solid preclinical data, including single-cell transcriptomic analyses and evidence of tumor growth suppression, supported by a creative and well-executed methodology. Further validation of the calcium signaling mechanisms and assessment of long-term safety will strengthen the translational potential of this approach. The work will be of broad interest to researchers in oncology, immunotherapy, and biomedical engineering.
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Reviewer #1 (Public review):
Summary:
This study presents a compelling strategy for ultrasound-mediated immunomodulation in HCC, supported by robust scRNA-Seq data. While the mechanistic depth and translational validation require further refinement, the work significantly advances the field of noninvasive cancer immunotherapy. Addressing the major concerns, particularly regarding calcium signaling specificity and STNDs@Ca²⁺ safety, will strengthen the manuscript's impact.
Strengths:
(1) Innovative Approach:
The integration of noninvasive ultrasound with calcium-targeted nanotechnology (STNDs@Ca²⁺) represents a significant advancement in cancer immunotherapy. The dual mechanism - direct immunomodulation via FUS and calcium delivery via nanoparticles - is both novel and promising.(2) Comprehensive Mechanistic Insights:
The use of …Reviewer #1 (Public review):
Summary:
This study presents a compelling strategy for ultrasound-mediated immunomodulation in HCC, supported by robust scRNA-Seq data. While the mechanistic depth and translational validation require further refinement, the work significantly advances the field of noninvasive cancer immunotherapy. Addressing the major concerns, particularly regarding calcium signaling specificity and STNDs@Ca²⁺ safety, will strengthen the manuscript's impact.
Strengths:
(1) Innovative Approach:
The integration of noninvasive ultrasound with calcium-targeted nanotechnology (STNDs@Ca²⁺) represents a significant advancement in cancer immunotherapy. The dual mechanism - direct immunomodulation via FUS and calcium delivery via nanoparticles - is both novel and promising.(2) Comprehensive Mechanistic Insights:
The use of scRNA-seq and flow cytometry provides a detailed map of immune cell dynamics, highlighting key pathways (TNF, NFκB, MAPK) and cellular transitions (e.g., MDSC suppression, CD8⁺/NK cell activation).(3) Robust Preclinical Validation:
The study validates findings in two distinct HCC models (H22 and Hepal-6), demonstrating consistent tumor suppression (>70-90%) and prolonged survival, which strengthens translational relevance.Weaknesses:
Major Issues:
(1) Mechanistic Specificity of Calcium Influx:
While the study attributes immunomodulation to ultrasound-induced calcium influx, the exact mechanism (e.g., involvement of mechanosensitive channels like Piezo1 or TRP families) remains underdiscussed. The qRT-PCR data shows no changes in TRP channels, but the upregulation of Piezo1 warrants deeper exploration.Suggestion: The authors should include experiments to inhibit Piezo1 or other calcium channels to confirm their role in FUS-mediated effects.
(2) STNDs@Ca²⁺ Biodistribution and Safety:
Although biodistribution data show splenic accumulation, potential off-target effects (e.g., liver/lung uptake) and long-term toxicity are not fully addressed. The serum biochemical analysis (Table 2) lacks critical markers like inflammatory cytokines or immune cell counts.Suggestion: The authors should provide longitudinal toxicity data (e.g., histopathology beyond 3 hours) and assess systemic immune activation/inflammation.
(3) Statistical and Technical Clarifications:
The statistical methods for multi-group comparisons (e.g., ANOVA vs. t-test) are inconsistently described. For instance, Figure 1 labels significance without specifying correction for multiple comparisons.Suggestion: the authors should clarify statistical methods in figure legends and the Methods section; apply Bonferroni or FDR correction where appropriate.
(4) Interpretation of scRNA-seq Data:
The clustering of MDSCs using surface markers (Itgam/Ly6c2/Ly6g) overlaps with conventional myeloid populations (Supplementary Figure 16), raising questions about subset specificity.Suggestion: The authors should validate MDSC identity using functional assays (e.g., T cell suppression) or additional markers (e.g., Arg1, iNOS).
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Reviewer #2 (Public review):
Summary:
Wang et al. studied the therapeutic potential of focused ultrasound noninvasively stimulating the spleen (FUS sti. spleen) to modulate the splenic immunity, with an aim to exert anti-tumor effect. They found that the treatment enhanced antitumor capability in CD8 T/ NK cells and reduced the immunosuppression, facilitating the inhibition of HCC growth in vivo.
Strengths:
They have utilized bulk RNA sequencing, single cell RNA sequencing, and flow cytometry to investigate the immune and tumor cell profiling in the mouse models upon FUS sti. spleen. Moreover, they highlighted the importance of combining FUS with spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STND@Ca2+), which facilitated the calcium influx into the murine spleen and further enhanced the therapeutic efficacy of FUS.
Reviewer #2 (Public review):
Summary:
Wang et al. studied the therapeutic potential of focused ultrasound noninvasively stimulating the spleen (FUS sti. spleen) to modulate the splenic immunity, with an aim to exert anti-tumor effect. They found that the treatment enhanced antitumor capability in CD8 T/ NK cells and reduced the immunosuppression, facilitating the inhibition of HCC growth in vivo.
Strengths:
They have utilized bulk RNA sequencing, single cell RNA sequencing, and flow cytometry to investigate the immune and tumor cell profiling in the mouse models upon FUS sti. spleen. Moreover, they highlighted the importance of combining FUS with spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STND@Ca2+), which facilitated the calcium influx into the murine spleen and further enhanced the therapeutic efficacy of FUS.
Weaknesses:
While the study is interesting and potentially clinically impactful, the mechanism of action of the therapy is not fully elucidated. It would benefit from more rigorous approaches. With the theoretical part strengthened, this paper would be of interest to cancer cell biologists and clinician scientists working on the oncology field.
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