Gas therapy potentiates aggregation-induced emission luminogen-based photoimmunotherapy of poorly immunogenic tumors through cGAS-STING pathway activation

The immunologically “cold” microenvironment of triple negative breast cancer results in resistance to current immunotherapy. Here, we reveal the immunoadjuvant property of gas therapy with cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway activation to augment aggregation-induced emission (AIE)-active luminogen (AIEgen)-based photoimmunotherapy. A virus-mimicking hollow mesoporous tetrasulfide-doped organosilica is developed for co-encapsulation of AIEgen and manganese carbonyl to fabricate gas nanoadjuvant. As tetra-sulfide bonds are responsive to intratumoral glutathione, the gas nanoadjuvant achieves tumor-specific drug release, promotes photodynamic therapy, and produces hydrogen sulfide (H2S). Upon near-infrared laser irradiation, the AIEgen-mediated phototherapy triggers the burst of carbon monoxide (CO)/Mn2+. Both H2S and CO can destroy mitochondrial integrity to induce leakage of mitochondrial DNA into the cytoplasm, serving as gas immunoadjuvants to activate cGAS-STING pathway. Meanwhile, Mn2+ can sensitize cGAS to augment STING-mediated type I interferon production. Consequently, the gas nanoadjuvant potentiates photoimmunotherapy of poorly immunogenic breast tumors in female mice.

1. The in vitro stability of MTHMS should be examined by dynamic light scattering (DLS). 2. The authors need to illustrate the preparation mechanism of tvHMS. For example, how did tetrasulfide bond participate in the formation of tvHMS? 3. For the fabrication of tvHMS, why did NaOH selectively etch the inner solid silica? The underlying mechanism for NaOH etching method to make the virus-like silica nanoparticles should be discussed in depth. 4. The excretion possibility should be discussed to guarantee the potential biocompatibility of nanocarrier. 5. The in-depth mechanism for higher cellular uptake efficiency of virus-like MTHMS than sphere-like MTHMS should be illustrated clearly. 6. There are many self-citations, the broader context for this work should be given. 7. The extensive studies have developed different kinds of nanomaterial-based combination of phototherapy and gas therapy. The authors need to further provide related discussions. 8. The authors need to pay attention to some details. The full name of compounds should be indicated in the first appearance, such as TEOS and CTAB. 9. For AIEgen-based photoimmunotherapy, some important ref. should be cited, such as J. Mater. Chem. C, 2020,8, 15622-15625;Inorganic Chemistry, 2022, doi:10.1021 Reviewer #3 (Remarks to the Author): with expertise in cancer immunology/therapy This is an interesting interdisciplinary manuscript dealing with the development of multifunctional phototheranostic nanoparticles with additional gas-mediated (H2S, CO) immunoadjuvant property. The method enables laser-induced intracellular/intratumoral gas release, which leads to release of mitochondrial DNA into the cytosol and thereby activation of the cGAS/STING/Type I IFN pathway. This leads to a potentially improved induction of tumor-specific CD8+ T cells through improved tumor antigen cross-presentation by dendritic cells. My criticism relates mainly to the question to what extent the observed antitumor effects actually depend on the immune system.
Major points 1. On p.11, line 230-234, the authors state: "the frequency of mature DCs induced by tumor cells with different treatments was evaluated as a consequence of cGAS/STING activation. … As expected, MTHMS+L treated cancer cells significantly boosted DC maturation (Fig. 3d,e), which was driven by the striking IFN-b release after cGAS-STING activation".
Fig. 3d, e suggest some upregulation of CD80/CD86 on BM-derived DCs, but these experiments do not show that these changes are due to IFNb release after cGAS-STING activation. Such a conclusion would require experiments with inhibitors of the cGAS-STING pathway and/or DCs from mutant mice with deficiencies in the cGAS/STING pathway.

2.
Do the antitumor effects observed (the slower growth of the orthotopic tumors, the reduced lung metastasis, the slower growth of the re-challenge tumors, the longer survival of the mice) following MTHMS+L in Fig. 4 depend on the immune system (on CD8 T cells)? This should be answered with T cell depletion experiments. What was the effect of MTHMS on tumor growth  and lung metastasis (Fig. 4h) without laser illumination? 3. p.13. The authors state that they did not find evidence for toxicity in normal organs. What about the region/tissue around the orthotopic tumor? Was there toxicity observed (e.g., edema due to necrosis induced in the tumor?). If so, could this affect the take rate of re-challenge tumors? 4. Fig.5h, p.15, line 337-340: the authors present an increase in effector memory CD62Llow CD44+ cells in the spleen from 16% in PBS-treated mice to 47.5% in MTHMS+L-treated mice, arguing that this result indicates durable immunological memory.
I have doubts whether such a great increase in the proportion of these cells in the spleen reflects tumor-specific memory. To draw conclusions as to tumor-specific memory or the induction of tumorspecific T cell immunity, tumor-specific T cells need to be measured (e.g., with MHC tetramers or peptide restimulation).

5.
In the abscopal model, the secondary tumor was implanted into the left mammary fat pad which is not so far away from the right fad pad.
Is it possible that reduced growth of the secondary tumor is affected by local inflammatory responses due to tissue destruction (e.g., edema following tissue necrosis)? Is the tumor growth affected by CD8+ T cells? This should be assessed by doing these experiments in mice depleted of CD8+ T cells by injecting depleting antibodies. 6. Fig. 6. In the abscopal experiments, the nanoparticles were injected one day after implantation of the secondary tumor. Is it possible that the slow growth of the secondary tumor is due to direct effects of the injected nanoparticles? If so, this would not be an abscopal effect. This should be ruled out by control experiments with MTHMS without laser. 7. p.11, lines 251-p.12, line 257: The time point of the apoptosis measurements seems not to be indicated. This should be done. In addition, a time course for the cell death should be provided.

8.
FMO, or isotope controls should be included for all FC examples, especially for the samples with no clear separation (e.g. Fig. 5e-g) to see how this populations were gated. Authors should provide a gating strategy to also show how other populations were gated (e.g. CD11c in Fig. 3d, and Fig. 5c).
Minor points 1. The manuscript contains many abbreviations, some of which seem to lack explanation. The readability could be improved by introducing each abbreviation where it comes up first. Examples: P.6, line 118: TSSI seems not to be defined/explained. Likewise, TEOS, CTAB (p.6); DCFH-DA (p.8); WSP-1 (p.9). Furthermore, it would be good to explain the full names of the different particle types at a certain place (e.g., a list of abbreviations or a suppl.  (Fig. 4), saying that on d10, 4T1 tumor cells were injected i.v. to mimic metastasis. 4T1 is spontaneously metastasizing to the lung. It would be good to explain in the main text why 4T1 tumor cells were additionally injected i.v.
In the main text, it should also be described that the treatment of the orthotopic tumor was done twice before the i.v. inoculation of the tumor cells.
Where and with how many tumor cells were the mice re-challenged? 3. p.16, line 360: the authors also measured IL-6 in the tumor lysates and argue that this demonstrates reversal of immunosuppression. However, IL-6 usually is associated with protumoral effects.

4.
Suppl. Methods line 236. The quantity of the samples loaded for SDS PAGE should be indicated.

5.
Suppl. Methods line 246: flow cytometry analysis of tumor single-cell suspensions. It should be indicated with which enzyme(s) the tumors were digested.
6. In Fig. 5d and Fig. 6e need to be indicated from which population % of CD8+ T cells is shown. In Fig. 5d, f, and Fig. 6e, the x-axis should be labeled.
Reviewer #4 (Remarks to the Author): with expertise in cancer nanotherapy In this manuscript, the authors propose the novel viewpoint of gas nanoadjuvant with robust cGAS-STING pathway activation to assist photoimmunotherapy of poorly immunogenic tumor. In this paper, the virus-like surface helps gas nanoadjuvant invade cancer cells more effectively through spike surface-assisted adhesion. And intratumoral overexpressed GSH could break the tetra-sulfide bond to release H2S. Following NIR laser irradiation, the AIEgen-based phototherapy in situ activated MnCO to generate Mn2+ and CO. Both H2S and CO induced the intracellular release of mtDNA, performing the immunoadjuvant property of engaging cGAS-STING pathway. Furthermore, Mn2+ sensitize cGAS to enhance STING-mediated type I IFN response in both tumor cells and DCs. This paper provides a new direction for breaking the bottleneck of poorly immunogenic tumor treatment. Overall, the idea of this paper is interesting and the conclusion is supported by data provided. The following concerns are suggested for improving the manuscript: Question 1: Photo images of bubble generation of CO gas upon NIR laser triggering in MTHMS with or without H2O2 should be added to visualize the phototherapy-facilitated CO generation. Question 2: In Supplementary Fig. 10a, the NIR-II fluorescence images at different time points after intravenous administration of free TSSI solution should be added as a control. Question 3: The IC50 values of different treatments in Fig. 3f should be listed in Supplementary and additional material as Supplementary Table. Question 4: In Supplementary Fig. 8, the variation between sphere-like MTHMS group and virus-like MTHMS group in flow cytometric quantification of cellular uptake should be assessed through Student's t-test. Question 5: In Supplementary Fig. 14, the legends should be noted in figure to point out the specific treatment of groups. Question 6: There are many methods to detect CO, authors used hemoglobin here. Authors should provide some references to support their choice in this aspect. Question 7: In the experimental section of in vitro ROS generation assessment, authors indicated that "the fluorescence was detected through PL instrument at 525 nm". Authors should add the excitation wavelength for the measurement. Response: We appreciate the reviewer's comments. As the reviewer suggested, we have conducted transmission electron microscopy (TEM) measurements to display more nanoparticles in the field of vision.

Comment 2. The intracellular GSH consumption by tvHMS should be evaluated by GSH probe such as ThiolTracker Violet.
Response: We appreciate the reviewer's comments. As suggested, we evaluated the intracellular GSH consumption by tvHMS using ThiolTracker Violet. These results were shown in the revised manuscript: "As seen in ThiolTracker Violet fluorescence images ( Supplementary Fig. 10), tvHMSincubated 4T1 cells showed attenuated green fluorescence due to GSH depletion."  Scale bar = 100 μm.

Comment 4. The intratumoural CO level should be determined using FL-CO-1 + PdCl2
fluorescence probe in mice after MTHMS+L treatment.
Response: We appreciate the reviewer's comments. As reviewer suggested, we determined the intratumoral CO level after MTHMS+L treatment using FL-CO-1 fluorescence probe. These results were discussed in the revised manuscript: "We then investigated the intratumoral CO level with FL-CO-1 fluorescence probe. As seen in Supplementary  Response: We appreciate the reviewer's comments. As reviewer suggested, we have adjusted the Response: We appreciate the reviewer's comments. We have carefully checked the description of Response: We appreciate the reviewer's comments. As suggested, we have increased the font size for better readability. Response: We appreciate the reviewer's comments. As suggested, we have added the experimental details in the figure captions. The details about the ELISA analysis have been supplemented in the revised manuscript: "The standard or sample was added to each well and incubated for 90 min at 37 ℃, then the wells were washed for five times. Next, biotin-labeled detection antibody was added and incubated for 60 min at 37 ℃, then the wells were washed five times. Subsequently, streptavidinconjugated HRP was added and incubated for 30 min at 37 ℃, then the wells were washed five times.
Finally, stop solution was added and monitored at 450 nm immediately." Moreover, "NIR-II fluorescence imaging was performed on NIR-OPTICS Series III 900/1700 (China) with the long pass (LP) filter of 1000 nm, and PSViewer software was employed to analyze the data. Photoacoustic imaging was recorded using the VisualSonics Vevo-2100 imaging system (Canada), and VevoLAZR software (Fujifilm, VisualSonics) was employed to analyze the data." Comment 10. The expression needs further check. I advise authors to standardize the use of technical term for avoiding misunderstanding to readers. For instance, whether "mean hydrodynamic diameter" and "size" mean the same meaning.
Response: We appreciate the reviewer's comments. We have carefully checked the expression and standardized the use of technical terms to avoiding misunderstanding. "Mean hydrodynamic diameter" and "size" have the same meaning, we have revised "size" to "mean hydrodynamic diameter". This study is innovative, the hypothesis is validated both in vitro and in vivo, and the conclusion is well supported by the experimental data. The relevant discussions and perspectives are profound and significant. The following aspects should be addressed to further promote the work.

The in vitro stability of MTHMS should be examined by dynamic light scattering (DLS).
Response: We appreciate the reviewer's comments. As suggested, we examined the in vitro 2. The authors need to illustrate the preparation mechanism of tvHMS. For example, how did tetrasulfide bond participate in the formation of tvHMS?
Response: We appreciate the reviewer's comments. It was reported that owing to the chemical homology between tetraethyl orthosilicate (TEOS) and bis(triethoxysilyl propyl) disulfide (BTESPD) ("chemical homology" principle, Adv Mater. 2015, 27, 215-222), BTESPD can be used to cohydrolyze and co-condensate with TEOS to form an organic-inorganic hybrid silica nanosystem on a molecular level (Biomaterials. 2018, 161, 292-305). Moreover, TEOS as a frequently-used silicon source could be co-hydrolyzed and co-condensated into the "functional bond"-doped (e.g.,  To improve the readability, The preparation mechanism of tvHMS has been supplemented in the revised manuscript: "The tvHMS was fabricated via post-co-condensation of TESPT with TEOS (v/v, 1: 3) through the chemical homology principle on the solid silica surface." 3. For the fabrication of tvHMS, why did NaOH selectively etch the inner solid silica? The underlying mechanism for NaOH etching method to make the virus-like silica nanoparticles should be discussed in depth.
Response: We appreciate the reviewer's comments. We suspect that the formation of surface morphology was likely due to the dosage and etching time of NaOH. In addition, the concentration and purity (> 99%, Sigma-Aldrich) of CTAB should not be ignored.
"We listed the underlying mechanism of the conversion of core-shell structure to the virusmimicking hollow mesoporous structure by NaOH etching: (1) NaOH rapidly etched the solid silica to produce dissolved silicate species, the core was removed to yield the hollow structure; (2) NaOH slowly etched a part of mesoporous silica to produce dissolved silicate species, leaving behind the disordered, pot-holed surface layer while generating the spike structure." The above detailed mechanism has been added to the revised manuscript.
4. The excretion possibility should be discussed to guarantee the potential biocompatibility of nanocarrier.
Response: We appreciate the reviewer's comments. The hollow mesoporous silica nanoparticles have attracted the interest of the scientific community due to their potential to be applied in the nanomedicine field (Biomaterials. 2010, 31, 5564-5574;Angewandte Chemie. 2016, 128(5): 1931-1935. The main advantages of hollow mesoporous silica nanoparticles arise from their simple, scalable, and cost-effective fabrication as well as their non-toxic matrix structure, large pore volume and surface area that is prone to be functionalized. A critical factor that affects nanocarrier application in biomedical applications is their biocompatibility. As a promising candidate material for efficient drug delivery, the biodegradation and excretion of mesoporous silica were characterized in many studies (Small. 2010, 6, 1794-1805, Microporous and Mesoporous Materials. 2016.
Silica is an endogenous substance of the human body that is particularly abundant in supporting tissues (Nanomedicine. 2012, 7, 111-120 highlighting the great potential of silica nanoparticles in clinical applications.
As reviewer suggested, the excretion possibility was discussed in the revised manuscript: "Moreover, mesoporous silica has been widely applied in biomedicine and exhibited great biocompatibility. It was demonstrated that mesoporous silica is excreted through feces and urine. It was reported that urinary excretion could account for 15-45% of the injected mesoporous silica nanoparticles at 0.5 h post-administration (Small. 2011, 7, 271-280). The excellent biodegradability guarantees safety for clinical application."

The in-depth mechanism for higher cellular uptake efficiency of virus-like MTHMS than
sphere-like MTHMS should be illustrated clearly.
Response: We appreciate the reviewer's comments. The surface topography and structure of nanoparticles have been demonstrated to significantly influence the cellular uptake process (Nat Mater. 2009, 8, 543-557 (Nanoscale. 2020, 12, 14911-14918), which further supported our study. Therefore, we believe that the in-depth mechanism behind the higher cell uptake of virus-like MTHMS than sphere-like MTHMS is the virus-like nanoparticles exhibited much more contacting sites per unit area with cell membranes compared to sphere-like nanoparticles, which was beneficial to elevate the adhesion interaction to significantly enhance the cell entry efficiency.
Following the reviewer's suggestion, the related discussion and references have been added in the revised manuscript: "Compared to the smooth surface of sphere-like MTHMS, the rough surface endowed virus-like MTHMS with much more contacting chances and elevated adhesion interaction with cytomembrane to remarkably improve the cellular internalization efficacy." 6. There are many self-citations, the broader context for this work should be given.  As expected, MTHMS+L treated cancer cells significantly boosted DC maturation (Fig. 3d, e), which was driven by the striking IFN-b release after cGAS-STING activation".  . 2018, 559, 269-273;Nat Commun. 2017, 8, 750;MedChemComm. 2019MedChemComm. , 10, 1999MedChemComm. -2023. "As shown in Supplementary Fig. 12, the cGAS-STING pathway inhibition significantly significance was calculated through one-way ANOVA using a Tukey post-hoc test.
2. Do the antitumor effects observed (the slower growth of the orthotopic tumors, the reduced lung metastasis, the slower growth of the re-challenge tumors, the longer survival of the mice) following MTHMS+L in Fig. 4

depend on the immune system (on CD8 T cells)? This should be answered with T cell depletion experiments.
What was the effect of MTHMS on tumor growth (Fig. 4b-d) and lung metastasis (Fig. 4h) without laser illumination?
Response: We appreciate the reviewer's comments. To investigate whether the observed antitumor effects depend on the immune system (on CD8 T cells), 100 μg anti-CD8a antibody (BioXCell, clone 2.43) was intraperitoneally injected into the mice every four days since day 0. "The results showed that CD8 + T cell depletion significantly impaired tumor suppression, led to severe lung metastasis, and shortened the survival time of mice treated with MTHMS+L ( Supplementary Fig. 25a-e, 26, 27), confirming the central role of the immune system in gas nanoadjuvant-assisted photoimmunotherapy." Meanwhile, the survived mice in MTHMS+L group were also rechallenged with 4T1 cells with/without CD8 + T cell depletion (intraperitoneal injection of 100 μg anti-CD8a antibody every four days since tumor rechallenge). "As shown in Supplementary Fig. 25f, the tumor volume of CD8 + T cell-depleted mice exhibited an evident increase and reached ~780 mm 3 on day 20, validating the effect of the immune system in resistance to tumor relapse." We also evaluated the effect of MTHMS on tumor growth and lung metastasis without laser illumination. The lack of laser irradiation resulted in the failure of PDT/PTT therapy and insufficient MnCO activation, which remarkably hindered the sequentially initiated stimulation of gas nanoadjuvant. "As shown in Supplementary Fig. 25a-e, 26, 27, MTHMS treatment displayed a slight Figure R4. The secretion of cytokines (IL-6, TNF-α, and IFN-γ) in serum. Data represent the mean ± s.d. Statistical significance was calculated through two-tailed student's t-test.

In the abscopal model, the secondary tumor was implanted into the left mammary fat pad
which is not so far away from the right fat pad.
Is it possible that reduced growth of the secondary tumor is affected by local inflammatory responses due to tissue destruction (e.g., edema following tissue necrosis)?

Is the tumor growth affected by CD8 + T cells? This should be assessed by doing these experiments in mice depleted of CD8 + T cells by injecting depleting antibodies.
Response: We appreciate the reviewer's comments. The region/tissue around the orthotopic tumor after MTHMS+L treatment was dissected and no evidence of edema, hyperplasia, degeneration, hemorrhage, fibrosis, or necrosis was observed (Fig. R2). Additionally, the comparison of proliferation and apoptosis of normal tissue and tumor tissue after MTHMS+L treatment indicated that the tumor tissue exhibited widespread apoptosis, whereas the normal tissue showed high proliferative activity with negligible apoptosis (Fig. R3). To explore whether the tumor growth was affected by CD8 + T cells, 100 μg anti-CD8a antibody (BioXCell, clone 2.43) was intraperitoneally injected into the mice every four days since day 7. "The results showed that CD8 + T cell depletion remarkably impaired the suppression of the secondary tumor after MTHMS+L treatment ( Supplementary Fig. 37), verifying the critical role of CD8 + T cells in the reduced growth of the secondary tumor."  As shown in Supplementary Fig. 15a, b, the apoptosis ratio and cell death were gradually increased with prolonged incubation time after laser irradiation. Furthermore, it would be good to explain the full names of the different particle types at a certain place (e.g., a list of abbreviations or a suppl. In the main text, it should also be described that the treatment of the orthotopic tumor was done twice before the i.v. inoculation of the tumor cells.
Where and with how many tumor cells were the mice re-challenged?
Response: We appreciate the reviewer's comments. As suggested, we explained in the main text why 4T1 tumor cells were additionally injected i.v. "The additional i.v. injection of 4T1 tumor cells into tumor-bearing mice to simulate hematogenous metastasis has been widely applied as an artificial whole-body spreading tumor model (Nat Commun. 2019, 10, 2025Nat Commun. 2016, 7, 13193;ACS Nano. 2019, 13, 5662-5673;Nano Today. 2020, 35, 100987). Compared with spontaneous lung metastasis, the whole-body metastasis model was more aggressive and challenging, which was suitable for specialized anti-metastasis evaluation." According to the reviewer's suggestion, we described in the main text that "the treatment of the orthotopic tumor was done twice before the i.v. inoculation of the tumor cells".
For tumor rechallenge study, 2 × 10 5 4T1 cells were injected into the left mammary fat pad of survived mice.
3. p.16, line 360: the authors also measured IL-6 in the tumor lysates and argue that this demonstrates reversal of immunosuppression. However, IL-6 usually is associated with protumoral effects.
Response: We appreciate the reviewer's comments. Many studies have revealed that the activation of cGAS-STING pathway would exhibit apparent induction of type I IFNs and some proinflammatory cytokines including IL-6 and TNF-α (Cell Res. 2020, 30, 966-979;Adv Mater. 2022, 34, 2105783;ACS Nano. 2020, 14, 3927-3940). Actually, IL-6 exerts the dual faces in the tumor microenvironment; the dark face that drives malignancy, and the fairer aspect that promotes antitumor adaptive immunity (Semin Immunol. 2014, 26, 38-47). Of the proinflammatory cytokines, accumulating evidence establishes IL-6 as a key player in the activation, proliferation, and survival of lymphocytes during active immune responses. IL-6 signaling can also mobilize the T cell immune response, shifting it from a suppressive to a responsive state that can effectively act against tumors. Finally, IL-6 plays an indispensable role in boosting T cell trafficking to lymph nodes and to tumor sites, where they can become activated and execute their cytotoxic effector functions, respectively. To avoid misleading, the manuscript was revised to "MTHMS+L treatment led to high expression of proinflammatory cytokines in serum including TNF-α, IFN-γ, and IL-6 ( Fig. 6h)."

Suppl. Methods line 236. The quantity of the samples loaded for SDS PAGE should be indicated.
Response: We appreciate the reviewer's comments. As reviewer suggested, we indicated that equal amount of proteins (20 μg) were loaded for SDS PAGE. Response: We appreciate the reviewer's comments. As reviewer suggested, we indicated that the enzymes used to digest tumors were collagenase IV, hyaluronidase, and deoxyribonuclease I.
6. In Fig. 5d and Fig. 6e need to be indicated from which population % of CD8+ T cells is shown.
In Fig. 5d, f, and Fig. 6e, the x-axis should be labeled.
Response: We appreciate the reviewer's comments. In Fig. 5d and Fig. 6e, we indicated that the population % of CD8 + in CD3 + T cells is shown. And the x-axis was labeled in Fig. 5d, f, and Fig. 6e.

Reviewer #4:
In this manuscript, the authors propose the novel viewpoint of gas nanoadjuvant with robust cGAS-STING pathway activation to assist photoimmunotherapy of poorly immunogenic tumor.
In this paper, the virus-like surface helps gas Response: We appreciate the reviewer's comments. To confirm the CO generation, the phototherapy-facilitated CO burst from MTHMS upon NIR laser irradiation with or without H2O2 was directly visualized by a digital camera. As shown in Fig. R5, a certain number of bubbles from H2O2-incubated MTHMS were observed, which was consistent with the previous report that H2O2 could trigger MnCO to release CO gas. Moreover, a significantly higher number of bubbles were achieved in the MTHMS solution with both NIR laser and H2O2 treatment, due to PDT-triggered ROS generation enhanced oxidant sensitivity of MnCO and the PTT-induced temperature elevation breakdown of the Mn-CO coordination bond to further release extra CO gas. Figure R5. Photo images of bubble generation of CO gas upon NIR laser irradiation in MTHMS with or without H2O2.
Question 2: In Supplementary Fig. 10a, the NIR-II fluorescence images at different time points after intravenous administration of free TSSI solution should be added as a control.
Response: We appreciate the reviewer's comments. According to the reviewer's suggestion, we added the NIR-II fluorescence images at different time points after intravenous administration of free TSSI solution as a control in Supplementary Fig. 16.   Supplementary Fig. 16. NIR-II fluorescence images of tumor-bearing mice at different time points after intravenous administration of free TSSI solution and MTHMS.  Response: We appreciate the reviewer's comments. As suggested, we assessed the variation between sphere-like MTHMS group and virus-like MTHMS group in flow cytometric quantification of cellular uptake through Student's t-test ( Supplementary Fig. 9).
Supplementary Fig. 9. Flow cytometric quantification of 4T1 cells incubated with sphere-like MTHMS and virus-like MTHMS for 1 h and 4 h. Data represent the mean ± s.d. Statistical significance was calculated through two-tailed student's t-test.
Question 5: In Supplementary Fig. 14, the legends should be noted in figure to point out the specific treatment of groups.
Response: We agree with the reviewer's comments. We added the legends in Supplementary Fig.