CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model
Abstract Background CD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However, its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here, we investigated the role of C...
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BMC
2025-08-01
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| Series: | Journal of Biomedical Science |
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| Online Access: | https://doi.org/10.1186/s12929-025-01165-3 |
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| author | Shih-Hsuan Chan Chin-Yu Lin Hsuan-Jung Tseng Lu-Hai Wang |
| author_facet | Shih-Hsuan Chan Chin-Yu Lin Hsuan-Jung Tseng Lu-Hai Wang |
| author_sort | Shih-Hsuan Chan |
| collection | DOAJ |
| description | Abstract Background CD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However, its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here, we investigated the role of CD24a, the murine CD24 gene, in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model. Methods Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations, including myeloid-derived suppressor cells (MDSCs), natural killer cells, T cells, and macrophages, within tumors, spleens, and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors. Results Knocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8+ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage‐mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity, effects that were partially reversed by re‐expression of CD24a. Moreover, in vivo depletion of macrophages and CD8+ T cells reverted the delayed tumor growth caused by CD24a knockout, underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore, in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1, CXCL5, and CXCL10, chemokines known to recruit gMDSCs, further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation. Conclusions Our findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state, thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC. |
| format | Article |
| id | doaj-art-e1633dc0f4ca4b1ead88fd073b844215 |
| institution | Kabale University |
| issn | 1423-0127 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Biomedical Science |
| spelling | doaj-art-e1633dc0f4ca4b1ead88fd073b8442152025-08-20T04:03:02ZengBMCJournal of Biomedical Science1423-01272025-08-0132111910.1186/s12929-025-01165-3CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer modelShih-Hsuan Chan0Chin-Yu Lin1Hsuan-Jung Tseng2Lu-Hai Wang3School of Chinese Medicine, College of Chinese Medicine, China Medical UniversityDepartment of Biomedical Sciences and Engineering, Tzu Chi UniversitySchool of Chinese Medicine, College of Chinese Medicine, China Medical UniversitySchool of Chinese Medicine, College of Chinese Medicine, China Medical UniversityAbstract Background CD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However, its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here, we investigated the role of CD24a, the murine CD24 gene, in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model. Methods Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations, including myeloid-derived suppressor cells (MDSCs), natural killer cells, T cells, and macrophages, within tumors, spleens, and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors. Results Knocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8+ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage‐mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity, effects that were partially reversed by re‐expression of CD24a. Moreover, in vivo depletion of macrophages and CD8+ T cells reverted the delayed tumor growth caused by CD24a knockout, underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore, in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1, CXCL5, and CXCL10, chemokines known to recruit gMDSCs, further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation. Conclusions Our findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state, thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC.https://doi.org/10.1186/s12929-025-01165-3CD24aCRISPR/Cas9 knockoutTriple-negative breast cancerTumor microenvironmentMacrophagesCD8+ T cells |
| spellingShingle | Shih-Hsuan Chan Chin-Yu Lin Hsuan-Jung Tseng Lu-Hai Wang CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model Journal of Biomedical Science CD24a CRISPR/Cas9 knockout Triple-negative breast cancer Tumor microenvironment Macrophages CD8+ T cells |
| title | CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model |
| title_full | CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model |
| title_fullStr | CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model |
| title_full_unstemmed | CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model |
| title_short | CD24a knockout results in an enhanced macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses in tumor microenvironment in a murine triple-negative breast cancer model |
| title_sort | cd24a knockout results in an enhanced macrophage and cd8⁺ t cell mediated anti tumor immune responses in tumor microenvironment in a murine triple negative breast cancer model |
| topic | CD24a CRISPR/Cas9 knockout Triple-negative breast cancer Tumor microenvironment Macrophages CD8+ T cells |
| url | https://doi.org/10.1186/s12929-025-01165-3 |
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