Fig 1: TAM Promotes CTSB‐mediated Malignancy in the GBM Subcutaneous and Orthotopic Mouse Models. A. The images of subcutaneous tumor tissues with or without injection of TAM. B. The tumor volume at different time points in subcutaneous tumor tissues with or without injection of TAM (n = 6, two‐tailed Student's t test). C. The tumor weight in subcutaneous tumor tissues with or without injection of TAM (n = 6, two‐tailed Student's t test). D. The HE staining images of subcutaneous tumor tissues with or without injection of TAM. E. The IHC staining images of Ki67 in subcutaneous tumor tissues with or without injection of TAM. F. The statistical results of the IHC staining assay for Ki67 (n = 6, two‐tailed Student's t test). G. Western blot shows the protein expression of IL‐6, STAT3, and CTSB in subcutaneous tumor tissues with or without injection of TAM. The statistical results of the western blot assay for IL‐6, STAT3, and CTSB (n = 6, two‐tailed Student's t test). H. In vivo bioluminescence images of orthotopic tumor tissues with or without injection of TAM. I. The statistical analysis of bioluminescent tracking plots. J. The HE staining images of orthotopic tumor tissues with or without injection of TAM. K. The IHC staining images of Ki67 in orthotopic tumor tissues with or without injection of TAM. L. The statistical results of the IHC staining assay for Ki67 (n = 6, two‐tailed Student's t test). M. Western blot shows the protein expression of IL‐6, STAT3, and CTSB in orthotopic tumor tissues with or without injection of TAM. N. The statistical results of the western blot assay for IL‐6, STAT3, and CTSB (n = 6, two‐tailed Student's t test).
Fig 2: CTSB Promotes the Migration and TAM‐type Polarization of Macrophages by Binding to S100A10. A. Flow cytometry shows the proportion of CD163+ macrophages after co‐culture with U251 and T98G cells transfected with siRNA‐NC and siRNA‐CTSB. B. The statistical results of the flow cytometry assay for CD163+ macrophages after co‐culture with U251 and T98G cells, respectively (n = 3, two‐tailed Student's t test). C. Transwell shows the migrated TAM after co‐culture with U251 and T98G cells transfected with siRNA‐NC and siRNA‐CTSB. D. The statistical results of the Transwell assay for the migrated TAM after co‐culture with U251 and T98G cells, respectively (n = 3, two‐tailed Student's t test). E. ELISA measures the protein level of CTSB in the supernatant of U251 and T98G cells transfected with siRNA‐NC and siRNA‐CTSB (n = 3, two‐tailed Student's t test). F. The PPI network related to CTSB. G. The correlation between CTSB and S100A10 in the TCGA‐IDHWT GBM cohort and the meta‐IDHWT GBM cohort. H. Co‐IP assay confirmed the interaction the direct interaction between CTSB and S100A10 in M0 macrophages using anti‐S100A10 antibody. Immunoblotting showed the presence of CTSB in the IP lane. I. Co‐IP assay to detect the direct binding domain of CTSB on S100A10 using His‐CTSB and FLAG‐S100A10 truncation fragments (1‐79) in 293T cells. J. Molecular docking pattern between CTSB (Blue) and S100A10 (Yellow). K. ELISA measures the protein level of IL‐6 in the supernatant of TAM after co‐culture with U251 and T98G cells transfected with siRNA‐NC and siRNA‐CTSB (n = 3, two‐tailed Student's t test). All data were presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.
Fig 3: STAT3 Transcriptionally Activates CTSB and Promotes Malignancy in GBM. A. ChIP‐qPCR shows the STAT3 binding affinity to the different CTSB promoter regions in U251 and T98G cells (n = 3, two‐tailed Student's t test). B. The binding site of STAT3 on CTSB. C. Dual‐luciferase reporter assay shows the regulation of STAT3 in CTSB promoter activity in U251 cells (n = 3, one‐way ANOVA test). D. Western blot shows the protein expression of CTSB in U251 and T98G cells after transfection with STAT3‐overexpression plasmid and siRNA‐CTSB. E. The statistical results of the western blot assay for CTSB (n = 3, one‐way ANOVA test). F. Flow cytometry shows the proportion of apoptotic U251 and T98G cells after transfection with STAT3‐overexpression plasmid and siRNA‐CTSB. G. The statistical results of the flow cytometry assay for apoptosis detection in U251 and T98G cells (n = 3, one‐way ANOVA test). H. Transwell shows the migrated U251 and T98G cells in four groups. I. The statistical results of the Transwell assay for the migrated U251 and T98G cells (n = 3, one‐way ANOVA test). All data were presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.
Fig 4: TAM Promotes CTSB‐mediated Malignancy A. Western blot shows the protein expression of CTSB in U251 and T98G cells transfected with siRNA‐NC and siRNA‐CTSB. B. The statistical results of the western blot assay for CTSB in U251 and T98G cells (n = 3, two‐tailed Student's t test). C. CCK8 shows the OD values of U251 and T98G cells transfected with siRNA‐NC and siRNA‐CTSB (n = 3, one‐way ANOVA test). D. Transwell shows the migrated U251 and T98G cells transfected with siRNA‐NC and siRNA‐CTSB. E. The statistical results of the Transwell assay for the migrated U251 and T98G cells (n = 3, one‐way ANOVA test). F. Western blot shows the protein expression of CTSB in U251 and T98G cells transfected with siRNA and co‐cultured with or without TAM. G. The statistical results of the western blot assay for CTSB (n = 3, one‐way ANOVA test). H. CCK8 shows the OD values of U251 and T98G cells transfected with siRNA and co‐cultured with or without TAM (n = 3, one‐way ANOVA test). I. Transwell shows the migrated U251 and T98G cells transfected with siRNA and co‐cultured with or without TAM. J. The statistical results of the Transwell assay for the migrated U251 and T98G cells (n = 3, one‐way ANOVA test). All data were presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.
Fig 5: The scRNA‐seq Analysis on Orthotopic Tumor Tissues. A. UMAP shows the integration of samples in the two groups (shRNA‐NC and shRNA‐Ctsb). B. Seven major microenvironment cell types were identified. C. M0/M1/M2 macrophages and cancer cells were further identified. D. The percentage of microenvironment cells in the two groups. E. Cell‐cell interaction network among microenvironment cells. F. Enrichment analysis based on the DEGs between shRNA‐NC and shRNA‐Ctsb tumor cells. G. A working model showing that TAMs secrete IL‐6 to activate STAT3 in GBM cells, which transcriptionally upregulates CTSB expression. Secreted CTSB binds S100A10 on macrophages, reinforcing their M2 polarization and reciprocal IL‐6 production, establishing a feedforward IL‐6/STAT3/CTSB/S100A10 axis. Functionally, this loop enhances GBM proliferation, invasion, and immunotherapy resistance. All data were presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.
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