Self-triggered thermoelectric nanoheterojunction for cancer catalytic and immunotherapy

The exogenous excitation requirement and electron-hole recombination are the key elements limiting the application of catalytic therapies. Here a tumor microenvironment (TME)-specific self-triggered thermoelectric nanoheterojunction (Bi0.5Sb1.5Te3/CaO2 nanosheets, BST/CaO2 NSs) with self-built-in electric field facilitated charge separation is fabricated. Upon exposure to TME, the CaO2 coating undergoes rapid hydrolysis, releasing Ca2+, H2O2, and heat. The resulting temperature difference on the BST NSs initiates a thermoelectric effect, driving reactive oxygen species production. H2O2 not only serves as a substrate supplement for ROS generation but also dysregulates Ca2+ channels, preventing Ca2+ efflux. This further exacerbates calcium overload-mediated therapy. Additionally, Ca2+ promotes DC maturation and tumor antigen presentation, facilitating immunotherapy. It is worth noting that the CaO2 NP coating hydrolyzes very slowly in normal cells, releasing Ca2+ and O2 without causing any adverse effects. Tumor-specific self-triggered thermoelectric nanoheterojunction combined catalytic therapy, ion interference therapy, and immunotherapy exhibit excellent antitumor performance in female mice.

treatment, 3 doses of the nanosystems were injected but no information was shown regarding blood levels of calcium.
Results 5.The characterization of the nanosystem is comprehensive and well-executed, reflecting the authors' outstanding expertise in the field of nanomedicine and cancer theranostics. 6.For in vivo fluorescent imaging, Cy5.5 is known to have a strong background in the stomach and GI tract, making it a less ideal fluorophore for biodistribution analysis. 7.Also, FL/gram tissue is not very convincing for biodistribution studies, ICP/MS or other more accurate quantification methods are recommended. 8.According to Fig. 5C, liver, kidney, and lung share most of the injected nanosystems. Although toxicity analysis did not reveal any significant adverse effects, the nanosystem's metabolic pathway, biological fate, and comprehensive toxicity profile still stand as the rule of thumb before utilizing it in cancer treatment. Such analysis is considered a standard practice in the development of safe and effective drug, including nanotherapeutics.
Discussion and Figures 9.Reading the manuscript, the idea of ROS surge reminds the reviewer of radiotherapy, which basically employs targeted radiation to induce ROS generation in the tumor. As radiotherapy would encounter hypoxia and tumor resistance among other issues, do the authors foresee any potential resistant from the current method? More demonstration in this regard would interest many clinicians. 10.In Fig.1, what do the gray ovals on cell membrane mean? 11.The manuscript contains many not-so-frequently-used expressions, it would benefit from a thorough review for grammatical errors and typos.
Reviewer #3: Remarks to the Author: This paper describes an interesting thermal triggered nano-catalyst system that generate ROS in acidic TME. Both in vitro and in vivo data support multifunctional effects of the BST/CaO2 NSs in generating Ca2+ ion surge, heat, and ROS. However, some critical control experiments are missing. Background information and literature on prior work, especially prior work on CaO2, is completely omitted. BST and CaO2 nano-systems have been separately reported before. Control experiments needed for critical evaluation of the reported work: a. Synthesis and characterization of CaO2 NPs. b. Cell viability and cell death at acidic pH. c. Temperature change (ΔT) in culture media for CaO2, BST, and BST/CaO2. d. Intracellular concentration of Ca2+ ions.
Several minor points need to be addressed. 1. Figure 2: what are the values for scale bars? 2. Evidence of heat generated when BST/CaO2 NSs are exposed to aqueous media at different pHs. 3. Figure 5: what were injected dose for each nanoparticle? What were the concentrations of Ca2+ in tumor and lymph nodes?
Reviewer #1 (Remarks to the Author): The study by Yuan et al. is relevant and interesting to the broad readership of the journal. The study is well organized. However, I have doubts regarding the innovation and there are some major revisions that need to be addressed and in vivo data that need to be provided prior to reconsider the study: Response: Thank you for the reviewer's positive comments. We have followed the reviewer's comments and performed additional experiments to address the points raised by the reviewer. Please see point-by-point responses below.
For the innovation of our strategy reported in this manuscript, although both BST NSs and CaO2 NPs have been reported separately for tumor therapy, the intelligent combination of BST NSs and CaO2 NPs and their synergy-derived tumor-specific selftriggered thermoelectric therapy is what makes this paper innovative. The exogenous excitation requirement and electron-hole pair recombination are the key elements limiting the application of catalytic therapies. Tumor-specific self-triggered thermoelectric catalysis based on BST/CaO2 heterojunction combined catalytic therapy, ion interference therapy, and immunotherapy is first reported.

Central innovation:
Tumor microenvironment (TME)-specific and self-triggered thermoelectric therapy without any external stimulation is innovative and has great potential in tumor therapy.

Collaborative innovation:
(1) Upon exposure to the acidic TME, the CaO2 NP coating hydrolyzed rapidly and released Ca 2+ , H2O2, and heat.
(2) Heat: The heat induced a temperature difference on BST NSs, triggering the thermoelectric effect, which pyro-generates negative and positive charges for chemical oxidation-reduction reactions and reactive oxygen species (ROS) generation. The voltage inside the thermoelectric material (BST NSs)-induced self-built-in electric field can retard electron-hole recombination, ensuring the corresponding catalytic activity and high ROS production.

Biosafety:
In a normal physiological environment, the different and mild hydrolysis pathways of CaO2 NPs, producing Ca 2+ and O2 slowly and without heat, cannot trigger the thermoelectric catalysis of BST NSs, guaranteeing high biosafety to normal organs and tissues.
The innovation of our reported TME-specific and self-triggered thermoelectric therapy was reframed and emphasized in the Abstract, Introduction, and Discussion sections.
1) The text contains many grammar issues with errors. Please revise the language and fluency of the whole text. Response: Thank you for the reviewer's comments. We have revised the WHOLE manuscript carefully and tried to avoid any grammar or syntax errors. In addition, we have asked colleagues who are skilled in writing scientific papers in English to check the English. We believe that the language is much improved.
2) In Figure 5b, the authors need to show the in vivo fluorescence images for all animals or at least 3 animals. The same was true for the organs. Not just 1 animal or the organs from 1 animal. Please revise this. Response: Thank you for this valuable comment. The in vivo fluorescence images of 3 mice are shown in Figure 6. In addition, PA and CT imaging were carried out for more accurate characterization of the distribution of nanomaterials in vivo. The relevant statements were added in our revised manuscript.

Methods section:
Fluorescence imaging and biodistribution study. Cy5.5-labeled BST/CaO2 NSs were injected intravenously into CT26 tumor-bearing mice. The fluorescence of the whole body of mice was recorded by a Maestro2 in vivo imaging system. Twenty-four hours postinjection, the mice were sacrificed, and the tumors and major organs were collected and imaged. The ImageJ analysis system was applied to measure the fluorescence intensity of Cy5.5-labeled BST/CaO2 NSs in major organs and tumors. Then, the intensity values were normalized using the weight (grams) of each organ and tumor.

PA imaging in vivo.
To test the PA imaging of BST/CaO2 NSs, the PA signal was detected by the MSOT inVision PA imaging system (inVision 256-TF, iThera Medical). In detail, tumorbearing mice were intravenously injected with BST/CaO2 NSs before imaging. After 12 and 24 hours, tumor-bearing mice were imaged by a small animal MSOT inVision PA imaging system. CT imaging in vivo. The in vivo CT imaging was carried out by a small mouse X-ray CT (Gamma Medica-Ideas). Imaging parameters were as follows: field of view, 80 mm by 80 mm; slice thickness, 154 m; effective pixel size, 50 m; tube voltage, 80 kV; tube current, 270 A. The CT images were analyzed using amira 4.1.2. In detail, tumor-bearing mice were intravenously injected with BST/CaO2 NSs before imaging. After 12 and 24 hours, tumor-bearing mice were imaged by small animal X-ray CT. The mouse whole-body 360° scan lasted approximately 20 min under isophane anesthesia.

Results section:
In vivo imaging and biodistribution of BST/CaO2 NSs. To evaluate the in vivo therapeutic performance of the BST/CaO2 NS-based selftriggered thermoelectric system, CT26 xenograft tumor models were established in BALB/c mice. To investigate the biodistribution of the Cy5.5-labeled BST/CaO2 NSs, they were intravenously injected into CT26 xenograft tumor models prior to evaluating their antitumor effect. The biodistribution of the BST/CaO2 NSs was observed at 4, 12, and 24 hours postinjection using in vivo imaging, and it was found that there was an effective and continuous accumulation of the nanoscale particles at the tumor site (Fig.  6a). This was further confirmed by semiquantitative analysis of BST/CaO2 NSs in the major organs (including the heart, liver, spleen, lung, and kidney) and tumors 24 hours after intravenous injection. As shown in Fig. 6b, a bright fluorescence signal was present in the dissected tumor, which was in agreement with the in vivo imaging results. Supplementary Fig. 13 shows the semiquantitative analysis of BST/CaO2 NSs in the major organs and tumor 24 h after intravenous injection, which was in agreement with the in vivo imaging results, further demonstrating the EPR effect-induced accumulation of nanoscale BST/CaO2 NSs at the tumor site. To more accurately characterize the distribution of the BST/CaO2 NSs in vivo, photoacoustic (PA) imaging and computerized tomography (CT) were used to conduct real-time monitoring. Because of the excellent photothermal conversion performance of the BST NSs, they served as a PA indicator for in vivo photoacoustic imaging. Real-time PA images of the tumorbearing mice were recorded after intravenous injection with BST/CaO2 NSs. The findings suggest that the BST/CaO2 NS-based self-triggered thermoelectric system has great potential for use as a synergistic antitumor therapy in vivo due to its effective accumulation at the tumor site. As shown in Fig. 6c, BST/CaO2 NSs accumulated in the tumor site well over time. Furthermore, it should be noted that the BST/CaO2 NSs also exhibit potential as CT imaging agents due to the high X-ray attenuation coefficient of Bi. In fact, as demonstrated in Fig. 6d and 6e, there is a positive correlation between the concentration of BST/CaO2 NSs and the Hounsfield unit (HU) value, indicating their ability to serve as effective contrast agents for CT imaging. To evaluate their in vivo CT imaging potential, BST/CaO2 NSs were intravenously injected into CT26 tumor-bearing mice and analyzed using coronal CT imaging. The results, displayed in Fig. 6f, showed enhanced contrast within the tumor area, suggesting the potential for BST/CaO2 NSs to serve as efficient CT imaging agents for cancer diagnosis. Moreover, to further investigate the biodistribution of BST/CaO2 NSs in vivo, ICP/MS analysis was utilized, as depicted in Supplementary Fig. 14. The results indicated a significant accumulation of NSs within the major organs and tumors over a period of 30 days, highlighting their effectiveness in targeting tumors. Importantly, Supplementary Fig. 14 also illustrates that the accumulated BST/CaO2 NSs within normal organs and tissues were gradually excreted by the body over time, indicating their biocompatibility and potential for clinical translation.  3) Regarding Figure 5f, photographs of CT26 xenograft tumor-bearing mice under different treatments, one can barely see anything with these type of images. The authors already have the measures of tumor volume in Figs. 5d and 5e. All these images can go to supporting information. Response: Thank you for your comments. As suggested by the reviewer, Figure 5f has been removed. In addition, an orthotopic colorectal cancer animal model was established by injecting CT26-luc cells (2 × 10 6 ) into the cecal wall of mice to further evaluate the antitumor effect of the BST/CaO2 NS-based self-triggered thermoelectric strategy.

Methods section:
Measurement of antitumor effects. Subcutaneous xenograft colorectal cancer animal model: The CT26 tumor-bearing mice were randomly divided into four treatment groups, and the tumors reached approximately 80 mm 3 with five mice each as follows: PBS, BST NSs, CaO2 NPs, and BST/CaO2 NSs. The BST NSs and BST/CaO2 NSs were injected intravenously at a dose of 5 mg/kg. Because the loading capacity of CaO2 NPs on BST/CaO2 NSs was 10 wt%, the CaO2 NPs were injected intravenously at a dose of 0.5 mg/kg. The body weight and tumor size of each mouse in the different groups were measured and recorded by a caliper and digital scale every 2 days during the treatment. The tumor volumes were calculated according to the following formula: tumor volume = (length × width 2 )/2. Orthotopic colorectal cancer animal model: Half a month after orthotopic tumor cell inoculation, the mice received intraperitoneal injection of d-luciferin (150 mg kg -1 ) to check the bioluminescence intensity of the tumor. Mice with a bioluminescence intensity of ~1 × 10 6 photons (p) s -1 cm -2 sr -1 were used for in vivo anticancer experiments. Then, the mice were randomly divided into four groups (n = 5) and received rectal perfusion of PBS, BST NSs (5 mg/kg), CaO2 NPs (0.5 mg/kg), and BST/CaO2 NSs (5 mg/kg) three times a week. Furthermore, the mice in each group received intraperitoneal injection of d-luciferin (150 mg kg -1 ) after three weeks of treatment. The mice were anesthetized and imaged under a small animal imaging system to monitor tumor bioluminescence.

Results section:
Additionally, an orthotopic colorectal cancer animal model was established by injecting CT26-luc cells (2 × 10 6 ) into the colorectal wall of mice to evaluate the antitumor effect of the BST/CaO2 NS-based self-triggered thermoelectric strategy. Once the bioluminescence intensity of the colon reached 1 × 10 6 photons (p) s -1 cm -2 sr -1 , the mice were randomly divided into four groups (n = 5) and received rectal perfusion of PBS, BST NSs, CaO2 NPs, or BST/CaO2 NSs three times a week. Three weeks later, the mice in each group received intraperitoneal injection of d-luciferin (150 mg kg -1 ) and were anesthetized and imaged under the small animal imaging system to monitor tumor bioluminescence. As shown in Fig. 7d, the representative bioluminescence images demonstrated that BST NSs alone did not delay tumor growth compared to the control group. However, the tumor bioluminescence change images and curves in Fig.  7e revealed that CaO2 NPs exhibited moderate in vivo anticancer effects during treatment. Encouragingly, BST/CaO2 NSs showed a marked inhibitory effect on tumor growth compared to saline, demonstrating the high antitumor efficiency of the BST/CaO2 NS-based self-triggered thermoelectric strategy in the orthotopic colorectal cancer animal model. Fig. 7. In vivo antitumor performance of the self-triggered thermoelectric system. a Experimental illustration of in vivo antitumor therapy. b, c Antitumor performance of different treatments, including control, BST, CaO2 and BST/CaO2, on a subcutaneous xenograft colorectal cancer animal model. Data are presented as the mean ± s.d. (n = 5 biologically independent mice). Statistical differences were analyzed by Student's twosided t test. d, e Antitumor performance of different treatments, including control, BST, CaO2 and BST/CaO2, on an orthotopic colorectal cancer animal model. Data are presented as the mean ± s.d. (n = 5 biologically independent mice). Statistical differences were analyzed by Student's two-sided t test. f The survival curves of tumorbearing mice under different treatments. g, h Flow cytometry analysis of the percentage of DC maturation and migration to lymph nodes (CD11c + CD80 + CD86 + ) under different treatments. Data are presented as the mean ± s.d. (n = 5 biologically independent mice). Statistical differences were analyzed by Student's two-sided t test. figure 6, the authors should quantify the level of damage for the tumors and each organ. This should be added in Figure 6. Response: The reviewer's comment is very constructive and useful. The level of damage to the tumors and each organ after treatment with BST/CaO2 NSs was quantified by ImageJ and added to our revised manuscript. Moreover, to quantitatively measure the apoptosis and DNA damage of organs and tumors in each treatment more accurately, we used flow cytometry to conduct detailed quantitative analysis of organs and tumors of mice in different treatment groups. The results and relevant statements have been added to our revised manuscript.

Results section:
To further investigate the underlying mechanism, tumor sections treated with BST/CaO2 NSs were analyzed using immunofluorescence (IF) staining of γ-H2AX and cleaved caspase-3 (C-CAS3) as markers for DNA double-strand breakage and cell apoptosis, respectively. As shown in Fig. 8a and Supplementary Fig. 21, high levels of irreparable DNA damage and cell apoptosis were observed in tumor sections treated with BST/CaO2 NSs due to the self-triggered thermoelectric catalysis and Ca 2+ -induced immunoregulation of the particles. Additionally, the self-triggered thermoelectric and immunotherapy of BST/CaO2 NSs was confirmed through TUNEL staining, which revealed a larger area of apoptosis in cancer cells after treatment with BST/CaO2 NSs (Fig. 8c). These findings demonstrate the efficient and synergistic effects of the selftriggered thermoelectric and immunotherapy of BST/CaO2 NSs. Furthermore, to obtain a more accurate quantitative measurement of apoptosis and DNA damage in organs and tumors in each treatment group, we used FCM to conduct detailed analysis. As depicted in Fig. 9 and Supplementary Figs. 22 and 23, no significant DNA damage or apoptosis was observed in normal organs (heart, liver, spleen, lung, kidney) across all treatment groups. However, BST/CaO2 NSs showed significant DNA damage and apoptosis in tumor tissues, confirming the tumor specificity and biosafety of the self-triggered thermoelectric strategy based on BST/CaO2 NSs.  Analysis of inflammatory factors in the heart, liver, lung, kidney, and spleen under different treatments. Data are presented as the mean ± s.d. (n = 5 biologically independent mice). Statistical differences were analyzed by Student's two-sided t test. c Blood hematology analysis of Balb/c mice under different treatments. Data are presented as the mean ± s.d. (n = 5 biologically independent mice). Statistical differences were analyzed by Student's two-sided t test. d Blood biochemical analysis of Balb/c mice under different treatments. Data are presented as the mean ± s.d. (n = 5 biologically independent mice). Statistical differences were analyzed by Student's two-sided t test.
Reviewer #2 (Remarks to the Author): In this research article, the authors investigated an interesting approach to induce a "ROS surge" by utilizing a pH-activated thermoelectric nanosystem, namely BST/CaO2 nanosheets (NS). The proposed mechanism involves the dissolution of the CaO2 surface layer in response to the acidic tumor microenvironment, leading to heat generation and subsequent activation of BST to generate ROS for temperature-driven cancer therapy. Furthermore, the released Ca 2+ ions in this process may trigger a series of tumorlimiting pathways, ultimately serving the purpose of self-triggered thermoelectric cancer/immunotherapy. Response: We very much appreciate the reviewer's thoughtful and helpful comments. During the past two months, we have performed a series of additional experiments to acquire more significant data. All these data have been added accordingly to the revised manuscript. Moreover, we have also made a series of modifications/corrections/additions to the manuscript. We hope that this revised version can now address all the concerns raised by the respected reviewer and satisfy the high publication standard in Nature Communications. Below, please also find our point-by-point responses.
Introduction: 1. When stating "photocatalysts have very limited access to light energy in vivo" and "most of photocatalysts with wide band gap can only respond to short wavelength light", please notice that at least 11 photoimmunotherapy clinical trials have been registered on clinicaltrials.gov, and most of them are using a NIR dye named IRDye700DX. Response: Thank you very much for this valuable comment. The inaccurate statement has been revised.

Introduction section:
Although photocatalytic therapy is capable of converting light energy into chemical energy, its practical application is limited by various factors. 10, 16-22 Therefore, alternative approaches such as piezocatalytic therapy have been explored to address these limitations and improve cancer treatment outcomes. In photocatalysis, light irradiation is necessary to initiate the process. However, this requirement adds complexity and limits its effectiveness since traditional photocatalysts have limited access to visible light energy in living organisms due to the barrier function of skin and other biological tissues. This scarcity of extrinsic light energy results in a limitation of the photocatalytic process. 17,22 To meet biomedical requirements, the fast recombination of photoexcited electron-hole pairs in both the surface and bulk phases of photocatalysts needs to be minimized. 11 This has been a significant challenge for photocatalytic therapy. However, piezocatalytic therapy based on the piezoelectric effect offers an alternative approach. By generating a piezoelectric potential, this therapy can drive charge separation or transfer and trigger redox reactions. [23][24][25][26][27] Essentially, this converts mechanical energy into chemical energy, but it does require the use of an additional external force, such as an ultrasonic generator.
2. The statement "High intensity or prolonged ultrasonic stimulation may cause mechanical and pathological damage to normal tissues or organs" reads a bit exaggerating and misleading. In fact, high-intensity focused ultrasound (HIFU) is a clinically verified method to treat cancer in many hospitals on a daily basis.

Response:
We appreciate this helpful comment. The inaccurate statement has been deleted in our revised manuscript.
3. The proposed thermocatalytic therapy is interesting, but a quick search on PubMed showed that many two-dimensional nanosheets can elicit a similar effect. This leads to the question that why the authors chose Bi0.5Sb1.5Te3 with such a strong commitment. Is there any comparison between different types of nanosystems or different composition of BiSbTe nanosheets? Response: We thank the reviewer for this helpful comment. Although there are many thermoelectric materials with excellent properties, the thermoelectric materials in selftriggered thermoelectric therapy need to meet the following three conditions: first, a high Seebeck coefficient, high electrical conductivity, and low thermal conductivity are the basic conditions for an excellent thermoelectric material. Second, most prepared thermoelectric materials with extremely high thermoelectric conversion require very high temperature. However, in vivo applications require thermoelectric materials with high conversion efficiency at relatively low temperatures. Finally, a simple and economical synthesis strategy is also necessary. Based on the above conditions, we choose thermoelectric materials such as BixSb2-xTe3 as the research object. The effect of different compositions on the thermoelectric conversion efficiency of BixSb2-xTe3 has been reported (Nano Energy, 2016, 20, 144-155), and Bi0.5Sb1.5Te3 has the highest thermoelectric conversion efficiency at lower temperatures. In addition, the choice of thermoelectric materials is not truly the focus and innovation of this research. The concept of TME self-triggering thermoelectric therapy is what we want to share. The relevant statements have been added in our revised manuscript.

Introduction section:
To enable biomedical applications in vivo, it is essential to have thermoelectric materials with high conversion efficiency at low temperatures. BixSb2-xTe3 NSs exhibit a high Seebeck coefficient, high electrical conductivity, low thermal conductivity, and high thermoelectric conversion, making them suitable for self-triggered thermoelectric cancer therapy. 37,44 Bi0.5Sb1.5Te3 nanosheets (BST NSs) have been found to have the highest thermoelectric conversion efficiency at very low temperatures, making them ideal for biomedical applications in vivo. 44 Additionally, CaO2 nanoparticles serve as a reservoir of calcium ions (Ca 2+ ) and hydrogen peroxide (H2O2) and have shown promising results in calcium overload-mediated therapy. 45-47 However, CaO2 NPs also have the potential to act as an in vivo switch for self-triggering thermoelectric therapy due to their tumor microenvironment (low pH)-specific water liberation thermal effect, which has not yet been explored. 44.
4. The released Ca 2+ ions propose another concern. Calcium homeostasis is important in maintaining the stable functioning of a living organism. High levels of calcium in the blood, or hypercalcemia, are known to cause damage to the kidney, bone, brain, and digestive system. For treatment, 3 doses of the nanosystems were injected, but no information was shown regarding blood levels of calcium. Response: Thank you for this comment. In this research, we proposed a tumor microenvironment (TME)-responsive self-triggered thermoelectric therapy. When exposed to only the acidic TME, the CaO2 NP coating hydrolyzed rapidly and released Ca 2+ , H2O2, and heat. Ca 2+ released from the TME will induce ion interference therapy in tumor cells, which will be phagocytosed by DCs after tumor cell fragmentation and induce their maturation. The CaO2 NP coating hydrolyzed very slowly in normal cells to generate Ca 2+ and O2, in which the slowly released Ca 2+ was expelled from the cells via calcium channel proteins. Therefore, the relatively high Ca 2+ concentration only occurred in tumor cells and DCs. Moreover, the mice were given a very low dose of 0.5 mg/kg CaO2 NPs, in which approximately 10 g of CaO2 NPs was injected into each mouse. The concentrations of Ca 2+ in blood, tumor cells, and lymphocytes were detected and added to our revised manuscript. Moreover, the biosafety of BST/CaO2 NS-based cancer therapy has been comprehensively studied in our revised manuscript.

Results section:
Although three doses of BST/CaO2 NSs were injected into each mouse, the concentrations of both BST NSs and CaO2 NPs were very low, at 0.1 and 0.01 mg per mouse, respectively. The concentrations of Ca 2+ in blood exhibited no significant fluctuation due to the slow hydrolysis of CaO2 NPs under normal conditions ( Supplementary Fig. 15).
To further confirm the Ca 2+ -mediated ion interference therapy, the concentration of Ca 2+ in tumor cells was measured. The results demonstrated that each injection increased the Ca 2+ concentration in the subcutaneous graft tumor cells, which gradually returned to normal levels over time ( Supplementary Fig. 15).
To confirm the role of Ca 2+ in promoting the maturation of DC cells, we measured the concentration of Ca 2+ in lymphocytes at lymph nodes. As depicted in Supplementary  Fig. 15, the concentration of Ca 2+ in lymphocytes was positively correlated with the number of injections, indicating that an increase in Ca 2+ concentration could be detected in lymphocytes 12 hours after each injection. However, over time, the concentration of Ca 2+ in the lymphocytes gradually returned to normal levels. Results 5. The characterization of the nanosystem is comprehensive and well executed, reflecting the authors' outstanding expertise in the field of nanomedicine and cancer theranostics. Response: Thank you for this comment. We very much appreciate it.
6. For in vivo fluorescent imaging, Cy5.5 is known to have a strong background in the stomach and GI tract, making it a less ideal fluorophore for biodistribution analysis. Response: Thank you very much for this valuable comment. Although Cy5.5 is known to have a strong background in the stomach and GI tract, numerous studies have used Cy5.5 as a fluorescent agent for fluorescence imaging. In our revised manuscript, to more accurately characterize the distribution of BST/CaO2 NSs in vivo, photoacoustic (PA) imaging and computerized tomography (CT) were further used to conduct realtime monitoring of the distribution of BST/CaO2 NSs in vivo.

Methods section:
Fluorescence imaging and biodistribution study. Cy5.5-labeled BST/CaO2 NSs were injected intravenously into CT26 tumor-bearing mice. The fluorescence of the whole body of mice was recorded by a Maestro2 in vivo imaging system. Twenty-four hours postinjection, the mice were sacrificed, and the tumors and major organs were collected and imaged. The ImageJ analysis system was applied to measure the fluorescence intensity of Cy5.5-labeled BST/CaO2 NSs in major organs and tumors. Then, the intensity values were normalized using the weight (grams) of each organ and tumor.

PA imaging in vivo.
To test the PA imaging of BST/CaO2 NSs, the PA signal was detected by the MSOT inVision PA imaging system (inVision 256-TF, iThera Medical). In detail, tumorbearing mice were intravenously injected with BST/CaO2 NSs before imaging. After 12 and 24 hours, tumor-bearing mice were imaged by a small animal MSOT inVision PA imaging system. CT imaging in vivo. The in vivo CT imaging was carried out by a small mouse X-ray CT (Gamma Medica-Ideas). Imaging parameters were as follows: field of view, 80 mm by 80 mm; slice thickness, 154 m; effective pixel size, 50 m; tube voltage, 80 kV; tube current, 270 A. The CT images were analyzed using amira 4.1.2. In detail, tumor-bearing mice were intravenously injected with BST/CaO2 NSs before imaging. After 12 and 24 hours, tumor-bearing mice were imaged by small animal X-ray CT. The mouse whole-body 360° scan lasted approximately 20 min under isophane anesthesia.

Results section: In vivo imaging and biodistribution of BST/CaO2 NSs.
To evaluate the in vivo therapeutic performance of the BST/CaO2 NS-based selftriggered thermoelectric system, CT26 xenograft tumor models were established in BALB/c mice. To investigate the biodistribution of the Cy5.5-labeled BST/CaO2 NSs, they were intravenously injected into CT26 xenograft tumor models prior to evaluating their antitumor effect. The biodistribution of the BST/CaO2 NSs was observed at 4, 12, and 24 hours postinjection using in vivo imaging, and it was found that there was an effective and continuous accumulation of the nanoscale particles at the tumor site (Fig.  6a). This was further confirmed by semiquantitative analysis of BST/CaO2 NSs in the major organs (including the heart, liver, spleen, lung, and kidney) and tumors 24 hours after intravenous injection. As shown in Fig. 6b, a bright fluorescence signal was present in the dissected tumor, which was in agreement with the in vivo imaging results. Supplementary Fig. 13 shows the semiquantitative analysis of BST/CaO2 NSs in the major organs and tumor 24 h after intravenous injection, which was in agreement with the in vivo imaging results, further demonstrating the EPR effect-induced accumulation of nanoscale BST/CaO2 NSs at the tumor site. To more accurately characterize the distribution of the BST/CaO2 NSs in vivo, photoacoustic (PA) imaging and computerized tomography (CT) were used to conduct real-time monitoring. Because of the excellent photothermal conversion performance of the BST NSs, they served as a PA indicator for in vivo photoacoustic imaging. Real-time PA images of the tumorbearing mice were recorded after intravenous injection with BST/CaO2 NSs. The findings suggest that the BST/CaO2 NS-based self-triggered thermoelectric system has great potential for use as a synergistic antitumor therapy in vivo due to its effective accumulation at the tumor site. As shown in Fig. 6c, BST/CaO2 NSs accumulated in the tumor site well over time. Furthermore, it should be noted that the BST/CaO2 NSs also exhibit potential as CT imaging agents due to the high X-ray attenuation coefficient of Bi. In fact, as demonstrated in Fig. 6d and 6e, there is a positive correlation between the concentration of BST/CaO2 NSs and the Hounsfield unit (HU) value, indicating their ability to serve as effective contrast agents for CT imaging. To evaluate their in vivo CT imaging potential, BST/CaO2 NSs were intravenously injected into CT26 tumor-bearing mice and analyzed using coronal CT imaging. The results, displayed in Fig. 6f, showed enhanced contrast within the tumor area, suggesting the potential for BST/CaO2 NSs to serve as efficient CT imaging agents for cancer diagnosis. Moreover, to further investigate the biodistribution of BST/CaO2 NSs in vivo, ICP/MS analysis was utilized, as depicted in Supplementary Fig. 14. The results indicated a significant accumulation of NSs within the major organs and tumors over a period of 30 days, highlighting their effectiveness in targeting tumors. Importantly, Supplementary Fig. 14 also illustrates that the accumulated BST/CaO2 NSs within normal organs and tissues were gradually excreted by the body over time, indicating their biocompatibility and potential for clinical translation.  8. According to Fig. 5C, the liver, kidney, and lung share most of the injected nanosystems. Although toxicity analysis did not reveal any significant adverse effects, the nanosystem's metabolic pathway, biological fate, and comprehensive toxicity profile still stand as the rule of thumb before utilizing it in cancer treatment. Such analysis is considered a standard practice in the development of safe and effective drugs, including nanotherapeutics. Response: We thank the reviewer very much for these professional comments. The long-term biological fate and possible metabolic pathway of nanomaterials were detected by analyzing urinary excretion and fecal excretion by ICP/MS. The comprehensive toxicity profile of the BST/CaO2 NS-based self-triggered thermoelectric strategy was further quantitatively tested by evaluating the levels of inflammatory factors, DNA damage, and cell apoptosis in each important organ (heart, liver, spleen, lung, and kidney) using quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM). All these results exhibited negligible adverse effects, which have also been added to our revised manuscript.

Results section:
In vivo biosafety evaluation of BST/CaO2 NSs.
The in vivo toxicity of nanomedicine is a crucial factor for translation from bench to practical applications. Therefore, we meticulously investigated the toxicity of BST/CaO2 NSs through histology examination, hematology assay, and immune analysis. Although three doses of BST/CaO2 NSs were injected into each mouse, the concentrations of both BST NSs and CaO2 NPs were very low, at 0.1 and 0.01 mg per mouse, respectively. The concentrations of Ca 2+ in blood exhibited no significant fluctuation due to the slow hydrolysis of CaO2 NPs under normal conditions ( Supplementary Fig. 15). As shown in Fig. 6 and Supplementary Fig. 14, BST/CaO2 NSs were distributed to a certain extent in the liver, spleen, lung, and other important organs, with their distribution decreasing over time. We hypothesize that the BST/CaO2 NSs will be partially excreted through renal urination and intestinal defecation, which was confirmed by ICP/MS analysis of urinary and fecal excretion ( Supplementary Fig.  24). This alleviated the long-term retention of BST/CaO2 NSs in the body to some extent. The IF staining of major organs, such as the heart, liver, spleen, lung, and kidney, of BST/CaO2 NS-treated mice with γ-H2AX and C-CAS3 as markers of DNA doublestrand breaks and cell apoptosis indicated no significant apoptosis or DNA damage in these normal organs ( Fig. 8b and 8d). Moreover, TUNEL staining of normal organs of mice treated with BST/CaO2 NSs confirmed their biosafety as an antitumor strategy. Furthermore, FCM analysis revealed no detectable DNA damage and apoptosis in the heart, liver, spleen, lung, and kidney for each treatment group compared to obvious DNA damage and apoptosis in the tumors (Fig. 9). Overall, these results suggest the biosafety of BST/CaO2 NS-based antitumor strategies. Hematoxylin and eosin (H&E) staining of major organs after treatment with BST/CaO2 NSs was carried out to further confirm the biocompatibility and specific targeted antitumor mechanism of BST/CaO2 NS-based therapy. As depicted in Fig. 10a, although intravenously injected BST/CaO2 NSs partially accumulated in normal organs (mainly in liver, spleen, and lung), they caused almost no damage. Moreover, real-time quantitative PCR (RT-qPCR) was applied to detect the damage and inflammatory response of each major organ exposed to BST/CaO2 NSs. The results presented in Fig.  10b confirmed the good biocompatibility and biosafety of BST/CaO2 NS-based cancer therapy. We also performed hematological detection to investigate the systematic biosafety properties of BST/CaO2 NSs. The mean corpuscular hemoglobin concentration (MCHC), red blood cells (RBCs), hematocrit (HCT), white blood cells (WBCs), platelets (PLTs), mean corpuscular volume (MCV), hemoglobin (HGB), and mean corpuscular hemoglobin (MCH) were measured (Fig. 10c). There was no statistically significant difference observed in the BST/CaO2 NS-treated groups 7 and 14 days after i.v. injection compared to the control group. Furthermore, blood biochemical parameters, including γ-glutamyl transpeptidase (γ-GT), total protein (TP), C-reactive protein (CRP), creatine kinase (CK), lactate dehydrogenase (LDH), creatinine (Cr), blood urea nitrogen (BUN), alanine aspartate aminotransferase (AST), amylase (AMY), aminotransferase (ALT), and albumin (ALB), between the control mice and the mice injected with BST/CaO2 NSs for 1, 7, and 14 days were tested. As presented in Fig. 10d, there were nearly no observable differences between the BST/CaO2 NSs and control groups. Therefore, all of the aforementioned results demonstrate that our prepared BST/CaO2 NSs should be considered a relatively biosafe and biocompatible nanomedicine.

Fig. 8. In vivo immunofluorescence staining and tissue damage analysis. a, b
Immunofluorescence images of the tumors and major organs (heart, liver, spleen, lung, and kidney) obtained from mice injected with BST/CaO2 NSs. The nucleus was stained with DAPI (blue), the damaged DNA was stained with γ-H2AX foci (red), and the apoptotic cells were stained using the apoptosis marker C-CAS3 (green). Scale bars: 1000 mm for the first line and 100 mm for the second and third lines. c, d TUNEL staining of the tumors and major organs (heart, liver, spleen, lung, and kidney) obtained from mice injected with BST/CaO2 NSs. Scale bars: 1000 mm for the first line and 100 mm for the second and third lines.  Discussion and Figures 9. Reading the manuscript, the idea of ROS surge reminds the reviewer of radiotherapy, which basically employs targeted radiation to induce ROS generation in the tumor. As radiotherapy would encounter hypoxia and tumor resistance among other issues, do the authors foresee any potential resistant from the current method? More demonstration in this regard would interest many clinicians. Response: We thank the reviewer for this professional and helpful comment. The advantages and potential resistance of the current method have been analyzed and added to the Discussion section.

Discussion section: Discussion
Catalytic therapies are a promising approach to cancer treatment, utilizing nanocatalysts that are nontoxic or low toxic to convert intracellular O2 or H2O into ROS such as · O2 -, · OH, and H2O2. These ROS induce effective tumor-specific oxidative damage and apoptosis without causing significant toxicity to normal organs or tissues. 1,11,12 However, most catalytic therapies rely on exogenous excitation, which means they require specific external stimuli such as light or ultrasound to trigger catalytic reactions. Unfortunately, exogenous excitation catalytic therapy faces several challenges in clinical application. First, the penetration of light is limited, and high-intensity ultrasound may cause collateral mechanical damage. Second, the catalytic efficiency of these therapies is often low due to the fast recombination of excited holes and electrons. Last, the use of additional excitation equipment can add operational complexity and inconvenience to the treatment process. 11,51,52 Recently, a new type of catalytic therapy has been developed for cancer treatment that combines thermoelectric effects and redox reactions. 37, 38 This approach is different from photocatalysis and piezocatalysis because it uses temperature fluctuation to generate pyro-generated negative and positive charges, which can trigger chemical oxidation-reduction reactions. Thermoelectric catalysis involves the creation of a selfbuilt-in electric field inside a thermoelectric catalyst, which retards electron-hole recombination and allows for greater catalytic activity and higher ROS generation. 43 However, current thermoelectric catalysis is triggered by laser irradiation through photothermal conversion, which limits its penetration into biological tissue and reduces its catalytic efficiency. To address this issue, there is a need to develop self-triggered thermoelectric catalytic materials or systems that maintain the advantages of thermoelectric catalysis while avoiding its limitations. Such developments hold great promise for clinical applications.
In this study, we have presented a novel self-triggered thermoelectric nanoheterojunction for enhanced tumor catalytic therapy and coupling with immunotherapy. We synthesized a conventional and efficient thermoelectric biomaterial, BST NSs, and selected it as the thermoelectric catalyst. The innovation lies in the in situ coating of CaO2 NPs, which not only acted as a trigger in response to the TME but also activated the immune system and imported immunotherapy. We explain that the CaO2 NPs were hydrolyzed rapidly into Ca 2+ and H2O2 in the acidic TME, generating a large amount of heat. The thermoelectric effect of BST NSs was activated by heat, producing negative and positive charges for chemical oxidation-reduction reactions and ROS generation. The self-built-in electric field inside BST NSs guided the separation of electron-hole pairs and retarded electron-hole recombination. H2O2 provided substrate (O2) supplementation for thermoelectric catalysis and ROS production and regulated Ca 2+ channels while delaying Ca 2+ efflux. The main hydrolysate Ca 2+ could mediate ion interference therapy, breaking intracellular ionic homeostasis and increasing the osmotic pressure of tumor cells. Additionally, it could promote DC maturation and tumor antigen presentation, thus activating an immune response and mediating effective immunotherapy. Moreover, the CaO2 NP coating hydrolyzed slowly in normal cells to generate Ca 2+ and O2, where the slowly released Ca 2+ was expelled from the cells via calcium channel proteins. Without the trigger of temperature fluctuation, the BST NSs possessed excellent biosafety and biocompatibility in normal organs and tissues. Overall, this study provides an intelligent strategy for the synthesis of a tumor-specific self-triggered thermoelectric catalyst and provides new insights into an advanced strategy to enhance the application scope and efficiency of catalytic therapy. Tumor-specific self-triggered thermoelectric catalysis based on BST/CaO2 heterojunction combined catalytic therapy, ion interference therapy, and immunotherapy exhibited excellent antitumor and biosafety properties both in vitro and in vivo.
Although the self-triggered synergistic thermoelectric, ionic interference, and immunotherapy demonstrated in this study show promising advantages and potential applications, further research is needed before clinical use. For instance, more detailed and comprehensive analysis of material metabolic pathways and toxicological implications should be conducted. Additionally, the thermoelectric materials based on BST NSs used in this study degrade slowly in vivo, leading to accumulation in vital organs. Although no adverse reactions were detected during the short-term study, it is challenging to predict long-term residual toxicity in vivo. Therefore, developing new safe and efficient degradable thermoelectric materials coupled with tumor-specific switches, such as hypoxia-responsive, low pH-responsive, and high ROS-responsive materials, would be a crucial strategy to promote the clinical transformation of selftriggered thermoelectric therapy.
10. In Fig. 1, what do the gray ovals on cell membrane mean? Response: We apologize for this careless omission. The gray ovals on the cell membrane indicate calcium channel proteins, which have been added to our revised manuscript. 11. The manuscript contains many not-so-frequently used expressions, it would benefit from a thorough review for grammatical errors and typos. Response: Thank you for the comments. We have revised the WHOLE manuscript carefully and tried to avoid any grammar or syntax errors. In addition, we have asked colleagues who are skilled in writing scientific papers in English to check the English. We believe that the language is much improved.
Reviewer #3 (Remarks to the Author): This paper describes an interesting thermal triggered nanocatalyst system that generate ROS in acidic TME. Both in vitro and in vivo data support the multifunctional effects of BST/CaO2 NSs in generating Ca 2+ ion surges, heat, and ROS. However, some critical control experiments are missing. Background information and literature on prior work, especially prior work on CaO2, is completely omitted. BST and CaO2 nanosystems have been separately reported before. Response: We very much appreciate the reviewer's thoughtful and helpful comments. During the past two months, we have performed a series of additional experiments to acquire more significant data. All these data have been added accordingly to the revised manuscript. Moreover, we have also made a series of modifications/corrections/additions to the manuscript. We hope that this revised version can now address all the concerns raised by the respected reviewer and satisfy the high publication standard in Nature Communications. Below, please also find our point-by-point responses.
For the innovation of our strategy reported in this manuscript, although both BST NSs and CaO2 NPs have been reported separately for tumor therapy, the intelligent combination of BST NSs and CaO2 NPs and their synergy-derived tumor-specific selftriggered thermoelectric therapy is what makes this paper innovative. The exogenous excitation requirement and electron-hole pair recombination are the key elements limiting the application of catalytic therapies. Tumor-specific self-triggered thermoelectric catalysis based on BST/CaO2 heterojunction combined catalytic therapy, ion interference therapy, and immunotherapy is first reported.

Central innovation:
Tumor microenvironment (TME)-specific and self-triggered thermoelectric therapy without any external stimulation is innovative and has great potential in tumor therapy.

Collaborative innovation:
(1) Upon exposure to the acidic TME, the CaO2 NP coating hydrolyzed rapidly and released Ca 2+ , H2O2, and heat.
(2) Heat: The heat induced a temperature difference on BST NSs, triggering the thermoelectric effect, which pyro-generates negative and positive charges for chemical oxidation-reduction reactions and reactive oxygen species (ROS) generation. The voltage inside the thermoelectric material (BST NSs)-induced self-built-in electric field can retard electron-hole recombination, ensuring the corresponding catalytic activity and high ROS production.
(4) Ca 2+ : Ca 2+ mediates calcium overload-mediated therapy, which could be aggravated by dysregulation of calcium channels by H2O2. Additionally, Ca 2+ promotes DC maturation and tumor antigen presentation, thus activating the immune response and enabling immunotherapy.

Biosafety:
In a normal physiological environment, the different and mild hydrolysis pathways of CaO2 NPs, producing Ca 2+ and O2 slowly and without heat, cannot trigger the thermoelectric catalysis of BST NSs, guaranteeing high biosafety to normal organs and tissues.
The innovation of our reported TME-specific and self-triggered thermoelectric therapy was reframed and emphasized in the Abstract, Introduction, and Discussion sections.
Control experiments needed for critical evaluation of the reported work: a. Synthesis and characterization of CaO2 NPs. Response: Thank you for the reviewer's comments. The synthesis and characterization of CaO2 NPs have been added to our revised manuscript.

Methods section:
Preparation of CaO2 NPs. CaCl2 (0.1 g) and PVP (0.35 g) were weighed into a round flask and dissolved in 15 mL of ethanol using an ultrasound device. While stirring, 1 mL of ammonia and 0.2 mL of H2O2 solution were slowly added to the mixture to obtain a light blue milky white solution. The resulting product was collected by centrifugation at 15,000 rpm, washed three times with ethanol, and finally redispersed in deionized water.

Results section:
CaO2 NPs were synthesized using CaCl2, ammonia, and H2O2 as substrates in an ethanol solution. Both SEM and TEM images of CaO2 NPs showed that the synthesized CaO2 NPs had a uniform morphology with an average size of 10 nm ( Fig. 2b and 2f). EDS mapping of CaO2 NPs also confirmed successful preparation (Fig. 2j,  Supplementary Fig. 2). Response: Thank you for the reviewer's comments. Acidic pH due to high-efficiency glycolysis is one of the most representative specificities of tumor cells. Hence, tumor cells have good viability at acidic pH. c. Temperature change (ΔT) in culture media for CaO2, BST, and BST/CaO2. Response: Thank you for the reviewer's comments. Temperature changes (ΔT) in culture media with different pH values (7.4 and 5.5) for CaO2, BST, and BST/CaO2 were detected and added to our revised manuscript.

Results section:
The temperature difference of the thermoelectric catalyst is an indispensable condition for triggering the thermoelectric effect. Therefore, in this study, CaO2 was assembled onto the surface of BST NSs in situ to create BST/CaO2 NSs. The trigger (CaO2) could only be activated by the low pH of the tumor microenvironment (TME), which triggered the thermoelectric effect. To test the theory of the low pH-specific selftriggered thermoelectric effect, the temperature change of BST NSs, CaO2 NPs, and BST/CaO2 NSs at different pH values was detected. It was observed that there was a rapid temperature rise when CaO2 NPs or BST/CaO2 NSs were placed in a low pH solution (pH 5.5), but there was no significant temperature fluctuation at neutral pH (pH 7.4) (Fig. 3e).   Fig. 3. Analysis of the catalytic performance of the self-triggered thermoelectric system. a Zeta potential of ligand-free BST, BST-PAA-Ca 2+ , and BST/CaO2 NSs. Data are presented as the mean ± s.d. (n = 3 independent experiments). b X-ray diffraction (XRD) patterns of BST NSs and BST/CaO2 NSs. c X-ray photoelectron spectroscopy (XPS) spectra of BST NSs, CaO2 NPs, and BST/CaO2 NSs. d High-resolution XPS spectra of BST/CaO2 NSs (Bi 4f, Sb 3d, Te 3d, Ca 2p and O 1 s). e The temperature change of BST NSs, CaO2 NPs, and BST/CaO2 NSs at different pH values. Degradation of DPBF by f BST and g BST/CaO2 NSs at pH 5.5. h Reaction mechanism of DPBF detection· O2 -. i Degradation of DPBF by different groups. Data are presented as the mean ± s.d. (n = 3 independent experiments).
d. Intracellular concentration of Ca 2+ ions. Response: Thank you for the comments. Intracellular concentrations of Ca 2+ ions before and after treatment with CaO2, BST, and BST/CaO2 have been tested and added to our revised manuscript.

Results section:
To validate the efficacy of calcium overload-mediated ion interference therapy, the intracellular concentrations of Ca 2+ were evaluated both qualitatively and quantitatively using confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) with a Fluo-4 AM probe. The results depicted in Supplementary Fig. 10a indicate a significant increase in green fluorescence after treatment with CaO2 NPs and BST/CaO2 NSs, indicating successful endocytosis of these particles by tumor cells and hydrolysis of CaO2 at low pH. Additionally, the quantitative data on intracellular Ca 2+ concentrations obtained via FCM analysis ( Supplementary Fig. 10b) confirmed the high level of endocytosis observed for CaO2 NPs and BST/CaO2 NSs and the low pH responsive hydrolysis of CaO2 in tumor cells. Several minor points need to be addressed. 1. Figure 2: what are the values for scale bars? Response: We apologize for this careless omission. The values for scale bars have been added in our revised manuscript.

Fig. 5.
In vitro antitumor performance of the self-triggered thermoelectric system. Cell viability of BST NSs, CaO2 NPs and BST/CaO2 NS-treated a CT26 cells and b TE1 cells by CCK8 assays. Data are presented as the mean ± s.d. (n = 5 biologically independent cells). Statistical differences were analyzed by Student's two-sided t test. Representative fluorescence images and quantification of intracellular ROS by c CLSM and d FCM. Scale bar = 100 m. e Representative confocal microscopy images of mitochondria-selective JC-1-stained CT26 cells after different treatments. Scale bar = 10 m. f Representative confocal microscopy images of γ-H2AX-stained CT26 cells after different treatments. Scale bar = 10 m. g Confocal imaging of CT26 cells stained with PI (red fluorescence) and Calcein-AM (green fluorescence) to distinguish dead cells and live cells after different treatments. Scale bar = 100 m. h FCM images of CT26 cells stained with PI (red fluorescence) and Annexin V-FITC (green fluorescence) to measure cell apoptosis after treatment under different conditions.
2. Evidence of heat generated when BST/CaO2 NSs are exposed to aqueous media at different pH values. Response: Thank you for the reviewer's comments. Temperature changes (ΔT) in culture media with different pH values (7.4 and 5.5) for CaO2, BST, and BST/CaO2 were detected and added to our revised manuscript.

Results section:
The temperature difference of the thermoelectric catalyst is an indispensable condition for triggering the thermoelectric effect. Therefore, in this study, CaO2 was assembled onto the surface of BST NSs in situ to create BST/CaO2 NSs. The trigger (CaO2) could only be activated by the low pH of the tumor microenvironment (TME), which triggered the thermoelectric effect. To test the theory of the low pH-specific selftriggered thermoelectric effect, the temperature change of BST NSs, CaO2 NPs, and BST/CaO2 NSs at different pH values was detected. It was observed that there was a rapid temperature rise when CaO2 NPs or BST/CaO2 NSs were placed in a low pH solution (pH 5.5), but there was no significant temperature fluctuation at neutral pH (pH 7.4) (Fig. 3e).   Fig. 3. Analysis of the catalytic performance of the self-triggered thermoelectric system. a Zeta potential of ligand-free BST, BST-PAA-Ca 2+ , and BST/CaO2 NSs. Data are presented as the mean ± s.d. (n = 3 independent experiments). b X-ray diffraction (XRD) patterns of BST NSs and BST/CaO2 NSs. c X-ray photoelectron spectroscopy (XPS) spectra of BST NSs, CaO2 NPs, and BST/CaO2 NSs. d High-resolution XPS spectra of BST/CaO2 NSs (Bi 4f, Sb 3d, Te 3d, Ca 2p and O 1 s). e The temperature change of BST NSs, CaO2 NPs, and BST/CaO2 NSs at different pH values. Degradation of DPBF by f BST and g BST/CaO2 NSs at pH 5.5. h Reaction mechanism of DPBF detection· O2 -. i Degradation of DPBF by different groups. Data are presented as the mean ± s.d. (n = 3 independent experiments).
3. Figure 5: what were injected dose for each nanoparticle? What were the concentrations of Ca 2+ in tumor and lymph nodes? Response: Thank you for the helpful comments from the reviewer. The injected dose of each treatment has been added to our Methods section. The concentrations of Ca 2+ in the tumor and lymph nodes were also detected and added to our revised manuscript.

Methods section:
Measurement of antitumor effects. Subcutaneous xenograft colorectal cancer animal model: The CT26 tumor-bearing mice were randomly divided into four treatment groups, and the tumors reached approximately 80 mm 3 with five mice each as follows: PBS, BST NSs, CaO2 NPs, and BST/CaO2 NSs. The BST NSs and BST/CaO2 NSs were injected intravenously at a dose of 5 mg/kg. Because the loading capacity of CaO2 NPs on BST/CaO2 NSs was 10 wt%, the CaO2 NPs were injected intravenously at a dose of 0.5 mg/kg. The body weight and tumor size of each mouse in the different groups were measured and recorded by a caliper and digital scale every 2 days during the treatment. The tumor volumes were calculated according to the following formula: tumor volume = (length × width 2 )/2.

Results section:
To further confirm the Ca 2+ -mediated ion interference therapy, the concentration of Ca 2+ in tumor cells was measured. The results demonstrated that each injection increased the Ca 2+ concentration in the subcutaneous graft tumor cells, which gradually returned to normal levels over time ( Supplementary Fig. 15).
To confirm the role of Ca 2+ in promoting the maturation of DC cells, we measured the concentration of Ca 2+ in lymphocytes at lymph nodes. As depicted in Supplementary  Fig. 15, the concentration of Ca 2+ in lymphocytes was positively correlated with the number of injections, indicating that an increase in Ca 2+ concentration could be detected in lymphocytes 12 hours after each injection. However, over time, the concentration of Ca 2+ in the lymphocytes gradually returned to normal levels.
Although three doses of BST/CaO2 NSs were injected into each mouse, the concentrations of both BST NSs and CaO2 NPs were very low, at 0.1 and 0.01 mg per mouse, respectively. The concentrations of Ca 2+ in blood exhibited no significant fluctuation due to the slow hydrolysis of CaO2 NPs under normal conditions ( Supplementary Fig. 15).