Cold Exposure Therapy Enhances Breast Cancer Treatment

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Cold exposure therapy (CE) is a low-cost, non-invasive treatment for malignant tumors. When the body is exposed to a cold environment, it activates the thermogenic metabolism of brown adipose tissue (BAT), quickly inducing tumor starvation and inhibiting tumor growth. The microenvironmental changes caused by CE can further complement conventional treatments (such as radiotherapy and chemotherapy), enhancing the effectiveness of traditional therapies. Currently, there are no research reports on CE combined treatment, thus researchers are urgently needed to explore the combined antitumor mechanisms of CE with traditional therapies (such as radiotherapy).On October 14, 2024, Professor Ning Zhipeng from Guangxi Medical University and Academician Chen Xiaoyuan from the National University of Singapore jointly communicated in the top international journalACS Nano (IF=15.8) with the title “Cold Exposure Therapy Sensitizes Nanodrug-mediated Radioimmunotherapy for Breast Cancer” published related research papers.

Cold Exposure Therapy Enhances Breast Cancer Treatment

This study constructed a liposome (SL) loaded with glutathione (GSH) reactive nitric oxide (NO) prodrug S-Nitroso-N-acetyl-DL-penicillamine (SNAP). The GSH in the tumor microenvironment (TME) promotes the release of NO from SL, and then, the superoxide anion (O_2•^–) generated by radiotherapy reacts with NO to produce the highly oxidative peroxynitrite (ONOO-), inducing tumor cell death. CE has a series of effects on tumor tissues, including the reduction of ATP levels and myeloid-derived suppressor cells (MDSCs), as well as the decrease of GSH levels, all of which help improve the efficacy of radiotherapy (RT). This work studies the effects of CE combined with NO gas therapy on tumor radiotherapy sensitization and promotes tumor radiotherapy-immune therapy, highlighting the great clinical application prospects of the new CE therapy.

Figure 1: Schematic of SL-mediated radioimmunotherapy for breast cancer

This work provides a detailed characterization of the successfully prepared SL nanoplatform with GSH-responsive NO release and cellular internalization capability. The results show that the prepared SL is uniform in size, successfully loaded with SNAP, releases NO in a GSH-responsive manner, and has good colloidal stability. In addition, after co-culturing DIO-labeled SL with 4T1 cells, the fluorescence of DIO in the cells gradually increased, with the most significant DiO fluorescence observed after 4 hours, indicating that SL can be internalized by cancer cells.

Figure 2: Preparation and characterization of SL

Subsequently, low-glucose (LG) culture of tumor cells was used to simulate CE treatment-induced glucose reduction in tumor tissues, and the evaluation of the antitumor effects of SL at the cellular level was explored. After SL treatment, GSH in the tumor area promotes the release of NO from SNAP. Subsequently, the O_2•^– generated by RT reacts with NO to generate the highly oxidative ONOO-, inducing cell death and inhibiting cell colony formation. Immunofluorescence staining of the DNA double-strand break marker γ-H2AX showed that the SL+RT+LG group had the highest number of γ-H2AX, indicating that SL+LG treatment caused the greatest RT sensitization. At the same time, the survival fraction of 4T1 cells in different groups under different radiation doses was studied, showing that LG, SL, and SL+LG all had radiosensitizing effects, with SL+LG having the strongest effect. LG treatment induced a decrease in ATP and GSH content in tumors, with SL+LG+RT treatment maximizing the consumption of GSH and ATP.

Figure 3: In vitro antitumor efficacy evaluation of SL

Next, this work systematically characterized the immune cell death (ICD) induced by SL combined with NO gas promoting RT. Immunofluorescence images and flow cytometry quantitative analysis showed that the SL+RT+LG group had the strongest CRT expression in treated cells, indicating that SL combined with RT and LG can upregulate CRT expression in 4T1 cells. Immunofluorescence imaging demonstrated the release of HMGB1 from tumor cells, while ELISA showed that the amount of HMGB1 released from the SL+RT+LG group was significantly higher than that of other groups. The above results indicate that SL+RT+LG can induce ICD, which is beneficial for tumor immunotherapy. Transwell experiments further proved that SL+RT+LG-induced ICD can enhance the release of immunogenic antigens, leading to the maturation and infiltration of dendritic cells (DCs), further enhancing the T cell-mediated antitumor immune potential.

Figure 4: In vitro induction of ICD

Further exploration of the anti-tumor effects of the SL+RT+CE combination treatment strategy on 4T1 tumor-bearing mice showed that the SL+RT+CE combination treatment strategy exhibited significant therapeutic effects in vivo. Subsequently, to confirm the impact of combination treatment on the cellular reduction system, the levels of two major reducing substances NADPH and GSH were evaluated. Under oxidative conditions, NADPH and GSH are converted to their corresponding oxidized forms NADP+ and GSSG, thus losing their reducing capacity. The NADP+/NADPH ratio in the SL+RT+CE group significantly increased, while the total glutathione concentration and GSH/GSSG ratio decreased, further increasing ROS generation. To further explore the antitumor immune mechanisms of SL+RT+CE, flow cytometry analysis was performed on mature DC cells, tumor-infiltrating regulatory T cells (Tregs), and CD8⁺ T cells. The results showed that SL+RT+CE treatment maximally activated antitumor immunity.

Figure 5: Evaluation of in vivo antitumor effects and immune activation study

This work innovatively studied the effects of CE combined with NO gas therapy on tumor radiotherapy sensitization and promotion of tumor radiotherapy-immune therapy. The results confirmed that mice treated with CE under radiotherapy and NO gas treatment showed better antitumor immune stimulation than mice treated under warm conditions, with reduced levels of MDSCs and Tregs at the tumor site. This study provides a solid theoretical foundation and scientific evidence for the combination of CE with RT in antitumor therapy.

Original link:

https://pubs.acs.org/doi/10.1021/acsnano.4c09021

Cold Exposure Therapy Enhances Breast Cancer Treatment

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Cold Exposure Therapy Enhances Breast Cancer Treatment

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