Repurposing Conventional Magnetic Functional Agents: A Novel Strategy for Long-Acting, Safe, Magnetically Mediated Precision Oncology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well written and properly designed. However, several issues must be addressed to make it appropriate for publication. 

1. Lines 57-65: the results are usually not discussed in the Introduction section. It would be better to transfer this part of the text to the Conclusion.
2. Line 108: you may have meant tumor vasculature?
3. Methods section: you must describe the magnetic field exposure in detail: what was the duration and intensity of exposure to which device was used? Also, the method of immunofluorescent staining must be described in detail, including which antibodies were used (clone number  manufacturer, etc).
4. Lines 178-179: you cannot claim that combination therapy is more effective, than monotherapy groups, since MVD was reduced in all the treated groups without statistical significance between them. The same refers to the lines 274-281.
5. Line 209: are you sure about CD3+ cells?

Author Response

Response to Reviewer

Original general comments: The manuscript is well written and properly designed. However, several issues must be addressed to make it appropriate for publication.

Author reply: Thank you very much for your positive comments and kind suggestion. The authors have made improvements in this revised version, according to your kind suggestion. We consider that the manuscript meets the standard of the journal now.
 

Q1) Lines 57-65: the results are usually not discussed in the Introduction section. It would be better to transfer this part of the text to the Conclusion.

Author reply: Thank you very much for your comments. The detailed results and discussion have been transferred in the Conclusion section, and the Introduction section has been accordingly modified in the “revised manuscript”.

Q2) Line 108: you may have meant tumor vasculature?

Author reply: Thank you very much for your comments. Line 108 indeed referred to the tumor vasculature system. We have made the corresponding modification in Line 113 of section: 2.6 Mechanism Study in the “revised manuscript”.

 

Q3) Methods section: you must describe the magnetic field exposure in detail: what was the duration and intensity of exposure to which device was used? Also, the method of immunofluorescent staining must be described in detail, including which antibodies were used (clone number manufacturer, etc).

Author reply: Thank you very much for your comments. The magnetic field exposure duration was described in Line 104 in the manuscript: “once daily for 4 hours from day 1 to day 9 post-grouping”. The description of the intensity and the device has been added in Line 104-105 in the “revised manuscript”: “A home-made MF generation device was used (CN120919537A, alternating MF, 20 Hz, 50 mT).”

The specific method for immunofluorescence staining and the antibodies used have been added to the “revised manuscript” in section 2.6 Mechanism Study, Lines 124-138:

“Paraffin sections derived from glioma-bearing mouse models were deparaffinized and the antigen retrieval was performed in EDTA buffer (pH 9.0). Nonspecific binding was blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. Sections were then incubated overnight at 4 oC with primary antibodies against CD3 (mouse-derived, GB12014, Servicebio, 1:2000), CD4 (rabbit-derived, GB15064, Servicebio, 1:2000), and CD8 (rabbit-derived, GB15068, Servicebio, 1:2000). Then sections were incubated with HRP-conjugated goat anti-mouse IgG (for mouse-derived CD3+ antibody, GB23301, Servicebio 1:500) or goat anti-rabbit IgG (for rabbit-derived CD4+/CD8 antibodies, GB23303, Servicebio, 1:500) for 50 min at room temperature. The corresponding TSA was added to incubate in the dark for 10 min: 488 nm for CD3, 555 nm for CD4, and 647 nm for CD8. Nuclei were counterstained with DAPI for 10 min. Fluorescence signals were visualized and acquired using a laser scanning confocal microscope (LSCM). Antibodies used were purchased from Servicebio (Wuhan, China) and Jackson (Shanghai, China).”

 

Q4) Lines 178-179: you cannot claim that combination therapy is more effective, than monotherapy groups, since MVD was reduced in all the treated groups without statistical significance between them. The same refers to the lines 274-281.

Author reply: Thank you very much for your comments. We have modified the expressions more accurately in the “revised manuscript”, in section 3.1.2. Tumor Neovascularization, Lines 196-198: “The intratumoral CD31 positive cells percentage in the 25 % Fe₃O₄ NPs + MF combined treatment group was reduced the most dramatically in average”; and Line 199-204: “The average microvessel density (MVD) values for the standalone therapy groups were 58.1 after 25% after Fe3O4 NPs treatment alone and 68.7 after MF therapy alone, respectively (Figure 2c). It showed a significant decline in average compared to that in the control group (149.6). The average MVD showed the largest decrease to 41.4 in the 25 % Fe₃O₄ - enhanced MF therapy group, which was the only group showed a statistical difference with the control group.”

The same modification was added in the and 3.2.2. Tumor neovascularization, Lines 304-308: “Similar to the Fe₃O₄ NPs, only the MVD in the GA+MF combination therapy group (45.7) showed a statistical difference with the control group (149.6). The average MVD value after the GA treatment alone (85.7) and MF treatment alone (68.7) were also reduced in average compared to the control group, but in a lower level (Figure 5c).” 

 

Q5) Line 209: are you sure about CD3+ cells?

Author reply: Thank you very much for your comments. Yes, we are sure these are CD3+ cells. We have modified the Fig 3a and Fig 6a with the CD3+CD4+ and CD3+CD8+ co-stained images to better demonstrate the results. The corresponding description in Line 226-238 and in Line 320-333 has been modified.   

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

I read your manuscript and here are my comments:

1. Which is the novelty and added value of the paper with respect to existent literature?

2. The manuscript claims therapeutic enhancement via local MF amplification by Fe₃O₄/GA, but provides no direct MF evidence (mapping/simulation/gradient data or in vivo confirmation). The effects could also come from Fe₃O₄ hyperthermia/ROS, injection-related inflammation, or GA local toxicity. Please add MF validation.

3. Key magnetic field details are missing. The study relies on MF therapy, yet no essential parameters are reported (static vs alternating, strength, frequency, gradients, setup geometry/distance, temperature control, calibration). Only 4 h daily (days 1–9) is mentioned. The authors should add MF specifications.

4. The authors claim glioma precision oncology, but the study uses a subcutaneous CT-2A hindlimb model, which does not reflect the intracranial glioma environment or BBB-related translational relevance. Please try to justify this model selection.

5. Please clarify why the reduced CD8/CD4 ratio is explained as beneficial rather than a potentially adverse immune effect. Also, add evidence for hypoxia/starvation (HIF-1α, GLUT1, pimonidazole) and consider perfusion assessments (Doppler or contrast imaging).

6. The authors should check the concentrations and units. Fe₃O₄ is reported as a “25% dispersion” but it’s unclear whether this is w/v, v/v, or mg/mL. GA is given as 10 mg/kg in 50 µL, but the molarity and local injection concentration should also be reported.

7. Scale bars on microscopy images should be added.

8. Language and formatting need revision. There are frequent typos ( “alos” instead of “also”), inconsistent section numbering, and repeated text (duplicated “Changes in MVD…”). Please thoroughly edit for clarity and consistency.

Comments on the Quality of English Language

Language and formatting need revision. There are frequent typos ( “alos” instead of “also”), inconsistent section numbering, and repeated text (duplicated “Changes in MVD…”). Please thoroughly edit for clarity and consistency.

Author Response

Response to Reviewer

Q1) Which is the novelty and added value of the paper with respect to existent literature?

Author reply: Thank you very much for your comments. In this study, we pioneered the application of magnetic contrast agents in tumor therapy. These two magnetic contrast agents served as local amplifiers for magnetic fields, yielding favorable therapeutic outcomes. Simultaneously, under the influence of magnetic fields, the magnetic materials could precisely target tumor sites, thereby enabling accurate targeted treatment. They showed great potential for precision tumor theranostics.

 

Q2) The manuscript claims therapeutic enhancement via local MF amplification by Fe₃O₄/GA, but provides no direct MF evidence (mapping/simulation/gradient data or in vivo confirmation). The effects could also come from Fe₃O₄ hyperthermia/ROS, injection-related inflammation, or GA local toxicity. Please add MF validation.

Author reply: Thank you very much for your comments. To verify the synergistic therapy efficiency, we have set four groups for both Fe₃O₄ and GA enhanced MF therapy, i.e. control group without any therapies, MF treatment alone group, Fe₃O₄/GA treatment alone group and Fe₃O₄/GA + MF treatment group to demonstrate the impact of Fe₃O₄/GA on MF therapy. The results demonstrated that the enhanced glioma therapy efficiency via the application of Fe₃O₄/GA during MF treatment was attributed to the Fe₃O₄/GA enhanced MF effect, rather than MF and the magnetic materials themselves.

 

Q3) Key magnetic field details are missing. The study relies on MF therapy, yet no essential parameters are reported (static vs alternating, strength, frequency, gradients, setup geometry/distance, temperature control, calibration). Only 4 h daily (days 1–9) is mentioned. The authors should add MF specifications.

Author reply: Thank you very much for your comments. The magnetic field exposure duration was described in Line 104 in the manuscript: “once daily for 4 hours from day 1 to day 9 post-grouping”. The description of the intensity and the device has been added in Line 104-105 in the “revised manuscript”: “A home-made MF generation device was used (CN120919537A, alternating MF, 20 Hz, 50 mT).”

 

Q4) The authors claim glioma precision oncology, but the study uses a subcutaneous CT-2A hindlimb model, which does not reflect the intracranial glioma environment or BBB-related translational relevance. Please try to justify this model selection.

Author reply: Thank you very much for your comments. This article primarily assessed whether this novel strategy of magnetic field enhancement by repurposing conventional magnetic functional agents could effectively treat CT-2A glioma. The subcutaneous tumor model is the preferred model for early-stage research on gliomas, which has advantages such as the feasibility of the model establishment, the operability of the experiments, and the reproducibility of the results. So we established the subcutaneous CT-2A hindlimb model for the preliminary study, which laid the groundwork for future in situ glioma therapy. 

 

Q5) Please clarify why the reduced CD8/CD4 ratio is explained as beneficial rather than a potentially adverse immune effect. Also, add evidence for hypoxia/starvation (HIF-1α, GLUT1, pimonidazole) and consider perfusion assessments (Doppler or contrast imaging).

Author reply: Thank you very much for your comments. In this study, reduced immune infiltration was used to indirectly demonstrate decreased vasculature at tumor sites, consistent with the findings for CD31 and MVD.

 

Q6) The authors should check the concentrations and units. Fe₃O₄ is reported as a “25% dispersion” but it’s unclear whether this is w/v, v/v, or mg/mL. GA is given as 10 mg/kg in 50 µL, but the molarity and local injection concentration should also be reported.

Author reply: Thank you very much for your comments. The 25% dispersion has been verified as 25% (w/w) in Line 69 in the “revised manuscript”. And the injection concentrations have been added in Line 91-92 in the “revised manuscript”: “50 μ of Fe₃O₄ solutions at predetermined concentrations (5 mg/mL, 0.5 mg/mL, 0.05 mg/mL).” To avoid the confusion, we have also changed the concentrations in the Fig 1-3 and Fig S1, S2 with the actual injection concentration and the corresponding description has also been modified.

The injection concentration of GA was added in Line 101 in the “revised manuscript”: “10 mg/kg, 50 μL, 4 mg/mL.” 

 

Q7) Scale bars on microscopy images should be added.

Author reply: Thank you very much for your comments. The scale bars have been added in the microscopy images in the “revised manuscript”.

 

Q8) Language and formatting need revision. There are frequent typos ( “alos” instead of “also”), inconsistent section numbering, and repeated text (duplicated “Changes in MVD…”). Please thoroughly edit for clarity and consistency.

Author reply: Thank you very much for your comments. We have carefully red the manuscript and corrected these errors in the “revised manuscript”.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have met all my comments. The paper can be published in the present form.

Author Response

Original general comments: The authors have met all my comments. The paper can be published in the present form.

Author reply: Thank you very much for your positive comments and kind suggestion, which has helped improve the quality of the article.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

I checked the revise version and these are my comments:

1. Novelty is still not very clear: try to add some phrases to clearly justify this (not pioneered or precision targeting without evidence).

2. MF amplification has not been proven. The four-group comparison shows efficacy, but does not confirm field amplification or exclude hyperthermia, ROS, or GA toxicity.

3. CD8/CD4 interpretation is still unclear. Reduced infiltration does not prove decreased vasculature, and the benefit of a lower CD8/CD4 ratio is really not justified.

Author Response

Response to Reviewer

Q1) Novelty is still not very clear: try to add some phrases to clearly justify this (not pioneered or precision targeting without evidence).

Author reply: Thank you very much for your comments. The novelty of this research lies in distinguishing from the traditional reliance of magnetic field therapy on high frequency and high field strength. Instead, the non-thermal physical effects and biological regulatory effects of magnetic field with a ultra-low frequency of 20 Hz and medium field strength of 50 mT were used as the core therapeutic driving force. They were enhanced with the inherent physical and biological properties of gadopentetic acid (GA, a clinically approved MRI contrast agent) and Fe₃O₄ (a classic superparamagnetic nanomaterial) to achieve enhanced anti-tumor efficiency. Each of the two innovations had its own focus. On the one hand, GA broke through the traditional limitation of contrast agents that only provided imaging but no treatment function. On the other hand, Fe₃O₄ focused on non-thermal magnetic response, overturning its classic application in magnetic therapy as only a magnetic hyperthermia medium. In common, they both could generate a co-directional local additional field that amplified local MF strength via superposition, which was generated through the alignment of spontaneously magnetized dipoles in Fe₃O₄ and unpaired electrons in GA with the applied external MF.

    We have added the novelty in the Abstract, Introduction and Conclusion part in the revised manuscript.

 

Q2) MF amplification has not been proven. The four-group comparison shows efficacy, but does not confirm field amplification or exclude hyperthermia, ROS, or GA toxicity.

Author reply: Thank you very much for your comments. The generation of the magnetic hyperthermia effect depends on the energy dissipation of magnetic nanoparticles (such Fe₃O₄) under an alternating magnetic field, which mainly generates heat through the Néel and Brownian relaxation mechanisms. Normally, magnetic nanoparticles are activated at frequencies ranging from 100 to 500 kHz and magnetic field intensities of 10 to 40 kA/m to generate therapeutic heat. In this study, we used the magnetic field with a ultra-low frequency of 20 Hz and medium field strength of 50 mT. Even if the heat could be generated by Fe₃O₄ through hysteresis loss or relaxation mechanisms under this magnetic field, it was extremely weak and insufficient to trigger magnetic hyperthermia effect. GA did not have magnetic domain structure, thus had no value for magnetic hyperthermia treatment. So the magnetic hyperthermia could be excluded.

In this study, we also checked the therapy efficiency of GA alone. It failed to inhibit tumor growth, thus we excluded GA toxicity. Since we used the Fe₃O₄ nanoparticles with a relatively large particle size (100-300 nm), the ROS generation capacity may be relatively weak.

However, the effect of magnetic field and its synergistic effect with Fe₃O₄ and GA on the tumor were complex, especially in the complex tumor microenvironment, the mechanism of the therapy enhancement was complex as you suggested. MF amplification was just one of their synergistic effects. So we have changed the description of MF amplification to MF effect amplification in the “revised manuscript” to make it more rigorous.

 

Q3) CD8/CD4 interpretation is still unclear. Reduced infiltration does not prove decreased vasculature, and the benefit of a lower CD8/CD4 ratio is really not justified.

Author reply: Thank you very much for your comments. We showed the result of the lower CD8/CD4 ratio after the therapy to illustrate it was a result of the decreased vasculature. These two factors formed a chain reaction through vasculature reduction - microenvironment remodeling - immune suppression. In this study, we have seen that the reduction of tumor vasculature was a sign of effective treatment, which blocked the tumor's nutrient supply, while the decrease in T cells was a transient secondary effect caused by the reduction of tumor vasculature. To avoid the misunderstanding, we have changed the corresponding description in 3.1.3 and 3.2.3 sections in the “revised manuscript”. 

In the long run, it may lead to the absence of immune surveillance in tumor tissues, increasing the risk of tumor recurrence/metastasis. This phenomenon provided a research direction for combined tumor treatment. On the basis of anti-angiogenesis, the strategy of vascular normalization regulation and immune activation could be combined with it to achieve a balance of effective tumor inhibition, moderate vascular preservation and promotion of immune cells infiltration. We added this to the Conclusion part in the “revised manuscript”.

Author Response File: Author Response.docx