Journal: Journal of Biomedical Science

Article Title: Unraveling Cathepsin S regulation in interleukin-7-mediated anti-tumor immunity reveals its targeting potential against oral cancer

doi: 10.1186/s12929-025-01154-6

Figure Lengend Snippet: Overexpression of CTSS in HNC tissue is inversely correlated with CD8 + T-cell infiltration. A & B The dot graphs show the mRNA expression levels of CTSS in the head and neck cancer (HNC) datasets from ( A ) TCGA and ( B ) GEO ( GSE6791 ). C The representative photos of the CTSS immunohistochemical (IHC) staining on the HNC tissue array. The dot graphs summarize the quantitative score of each sample by stratification. D & E The representative photos of IHC staining for ( D ) CTSS expression and ( E ) CD8 + T-cell infiltration of the in-house oral cancer (OC) samples were shown (N = 70). The score of each sample is plotted by different parameters on the right panels. F & G The correlation between the IHC staining score of CTSS and CD8 in the in-house OC samples was studied. The results are shown by dot graph plots for ( F ) the entire cohort (N = 70) and ( G ) samples with T1 and T2 Stage (N = 38). H The tumor volume of the subcutaneously-inoculated NHRI-HN1 cell is plotted. Mice were implanted with tumors carrying either sh Ctss or shControl, and were grouped by treatment with αCD8 or IgG antibody (N = 6 for each group). Arrowhead indicates days for antibody administration. I The representative photos of the IHC staining for CTSS and CD8 + T cells for each treatment group

Article Snippet: Antibodies were listed in the following: Polyclonal Armenian rat IgG2 isotype control (10 μg/mice) ( cat# BE0086; BioXcell, Lebanon, NH, USA), Anti-mouse/human IL-7 antibody (αIL-7) (10 μg/mice) ( cat# BE0048; BioXcell), Polyclonal Armenian hamster IgG (100 μg/mice) ( cat# BE0091; BioXcell), αPD-1 (100 μg/mice) ( cat# BE0033-2; BioXcell), Rat IgG1 isotype control, anti-horseradish peroxidase (200 μg/mice) ( cat# BP0088; BioXcell), Anti-mouse CD8b antibody (Lyt3.2) (αCD8) (200 μg/mice) ( cat# BE0223; BioXcell).

Techniques: Over Expression, Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Journal of Biomedical Science

Article Title: Unraveling Cathepsin S regulation in interleukin-7-mediated anti-tumor immunity reveals its targeting potential against oral cancer

doi: 10.1186/s12929-025-01154-6

Figure Lengend Snippet: CTSS inhibits the CD8 + T-cells infiltration and proliferation by downregulating IL-7. A The expression level of IL-7, IL-10, and MCP1 is plotted in the dot graph by each sample (N = 16 in each group). B The tumor volume of the subcutaneously-inoculated NHRI-HN1 cell is plotted. Mice were implanted with tumors carrying either sh Ctss or shControl, and were grouped by treatment with αIL-7 or IgG antibody (N = 10 for each group). Arrowhead indicates days for antibody administration. C The representative photos of the IHC staining for CTSS, IL-7, and CD8 + T-cells for each treatment group. D Tumor infiltrative leukocytes were isolated and analyzed by FACS, the percentage of CD8 + cells in the target quadrant is plotted in the dot graph by each sample. E The percentage of Ki67 + /CD8 + cells in the target quadrant is plotted in the dot graph by each sample. F The percentage of naïve CD8 + cells, central memory CD8 + cells, effector memory CD8 + cells, and tissue-resident CD8 + cells in the target quadrant is plotted in the dot graph by each sample. G & H Mouse CD8 + T-cells were treated with CM with or without the αIL-7. The CM was obtained from in vitro NHRI-HN1 or MOC-1 that were pretreated with siScramble or si Ctss , or that with co-incubation of the mouse IL-7 recombinant protein (mIL-7). ( G) The result of the WST proliferation test for CD8 + T-cells is shown in the dot-bar graph. ( H ) The indicated scale for CFSE cell proliferation is measured and plotted in the dot-bar graph

Article Snippet: Antibodies were listed in the following: Polyclonal Armenian rat IgG2 isotype control (10 μg/mice) ( cat# BE0086; BioXcell, Lebanon, NH, USA), Anti-mouse/human IL-7 antibody (αIL-7) (10 μg/mice) ( cat# BE0048; BioXcell), Polyclonal Armenian hamster IgG (100 μg/mice) ( cat# BE0091; BioXcell), αPD-1 (100 μg/mice) ( cat# BE0033-2; BioXcell), Rat IgG1 isotype control, anti-horseradish peroxidase (200 μg/mice) ( cat# BP0088; BioXcell), Anti-mouse CD8b antibody (Lyt3.2) (αCD8) (200 μg/mice) ( cat# BE0223; BioXcell).

Techniques: Expressing, Immunohistochemistry, Isolation, In Vitro, Incubation, Recombinant

Journal: Journal for immunotherapy of cancer

Article Title: Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8 + T-cell antitumor activity via the non-canonical P53/STING pathway.

doi: 10.1136/jitc-2024-010252

Figure Lengend Snippet: Figure 2 Clofarabine enhances the recruitment and cytotoxicity of CD8+ T cells. (A) Diagram of CD8+ T cell isolation and CD8+ T cell-related experiments. (B) The chemotaxis experiment of CD8+ T cells with A549 and B16F10. Clo was removed after treating tumor cells for 24 hours, and the remaining cells were cultured in a complete medium for 24 hours. After collection, the corresponding proportion of supernatants were added to the lower chamber of the transwell plate according to the tumor cell counts. CD8+ T cells (1×10∧5) were placed in the upper chamber, and cells in the lower chamber were collected and counted 48 hours later. In the blank group, the lower chamber was a complete medium. n=3. (C) CD8+ T cell killing assay of A549 and B16F10. After pretreatment with Clo for 24 hours, 1×10∧4 tumor cells and the corresponding CD8+ T cells were cultured for 48 hours. After eliminating the suspended CD8+ T cells, the absorbance of the remaining cells was detected by CCK-8, and the killing ratio was calculated. n=4. (D–E) CD8+ T cell cytotoxicity detection of A549 and B16F10. Clo was removed after treating A549 for 24 hours, and A549 was cultured in a complete medium for 24 hours, and the supernatants were collected. CD8+ T cells were cultured with the corresponding proportion of supernatants according to tumor cell counts and collected after 48 hours. Conduct flow cytometry to detect the proportion of GZMB+CD8+CD3+CD45+ T cells. n=3. (F) Schematic diagram of the melanoma treatment. (G) Tumor growth curves of B16F10-bearing mice with the indicated treatments. n=5. (H–K) Flow cytometry was conducted to measure the proportion of GZMB+CD8+CD3+ T cells/IFNγ+CD8+CD3+ T cells/LAG3+CD8+CD3+ T cells/PD-1+TIM3+CD8+CD3+ T cells in the tumors after the indicated treatments, and the statistical analysis was displayed. n=5. The p value was obtained using the unpaired Student’s t-test (C, D, E), and by multiple comparisons in an ordinary one- way analysis of variance (B, G, H, I, J, K), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PBMC, peripheral blood mononuclear cell; RBC, red blood cell; Clo, clofarabine; FSC-A, forward scatter area; IgG2a, immunoglobulin G2a; i.p., intraperitoneal; i.g., oral gavage.

Article Snippet: To test whether CD8+ T cell inhibition in melanoma reverses the antitumor effects of Clo, B16F10- bearing C57BL/6 mice randomly received one of the following treatments: (1) vehicle+immunoglobulin G2 (IgG2) a (#BE0089, Bio X Cell, USA); (2) Anti- CD8α (A2102, Selleck, USA); (3) Clo+IgG2 a; (4) Clo+Anti- CD8α.

Techniques: Cell Isolation, Chemotaxis Assay, Cell Culture, CCK-8 Assay, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8 + T-cell antitumor activity via the non-canonical P53/STING pathway.

doi: 10.1136/jitc-2024-010252

Figure Lengend Snippet: Figure 3 Clofarabine activates the P53-induced non-canonical STING/NF-κB pathway and induces apoptosis, pyroptosis, and immunogenic cell death in melanoma and lung cancer cells. (A) Western blotting revealed the protein expression of P53, p-P53, cGAS, STING, p-IkBα, BAX, Cleaved Caspase-3, Cleaved Caspase-6, and Cleaved PARP in lysates collected from A375 and A549 treated with Clo. (B) ELISA was used to analyze the concentration of cGAMP in the lysate of A375 and A549 treated with Clo for 48 hours. n=2. (C) Western blotting revealed the nuclear protein expression of NF-κB p50 and p65 in lysates collected from A375 and A549 cells treated with Clo. (D) Immunoprecipitation was conducted to detect the interaction between P53 and STING. (E) Western blotting revealed the protein expression of GSDME-FL and GSDME-N in lysates collected from A375 and A549 treated with Clo. (F) The images of A375 treated with Clo for 48 hours show PI uptake. The statistical analysis was displayed. n=3. (G) qRT-PCR measurement of CCL5, CXCL10, HLA-A, HLA-B, HLA-C, and BAX mRNA expression in A375 and A549 treated with Clo for 48 hours. n=3. (H) ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of A375 and A549 treated with Clo for 48 hours. n=2. The p value was obtained by multiple comparisons in an ordinary one-way analysis of variance (B, F, G, H), and the results were presented as the mean±SD. ***p<0.001, ****p<0.0001. cGAS, cyclic GMP-AMP synthase; Clo, clofarabine; IgG, immunoglobulin G; PI, propidium iodidemRNA, messenger RNA; qRT- PCR, quantitative reverse transcription-PCR; STING, stimulator of interferon genes.

Article Snippet: To test whether CD8+ T cell inhibition in melanoma reverses the antitumor effects of Clo, B16F10- bearing C57BL/6 mice randomly received one of the following treatments: (1) vehicle+immunoglobulin G2 (IgG2) a (#BE0089, Bio X Cell, USA); (2) Anti- CD8α (A2102, Selleck, USA); (3) Clo+IgG2 a; (4) Clo+Anti- CD8α.

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Immunoprecipitation, Quantitative RT-PCR, Reverse Transcription

Journal: Journal for immunotherapy of cancer

Article Title: Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8 + T-cell antitumor activity via the non-canonical P53/STING pathway.

doi: 10.1136/jitc-2024-010252

Figure Lengend Snippet: Figure 5 Clofarabine regulates tumor cell death and downstream MHC-I/CCL5/CXCL10/BAX expression through NF-κB. (A) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then the cell viability of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours was measured by CCK8 assay. n=5. (B) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then flow cytometry was conducted to determine the apoptosis of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. n=3. (C) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then western blotting revealed the protein expression of GSDME-FL and GSDME-N in lysates collected from A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. (D–E) Pretreated A375 and A549 with JSH- 23 10 µM for 24 hours, then ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. n=2. (F) ChIP assay detects the binding of NF-κB p65 to the CCL5/CXCL10/HLA-B/BAX promoter of A549 after Clo 1.5 µM treatment for 24 hours. n=3. (G) ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of B16F10 with or without Gsdme knockdown after Clo 1.5 µM treatment for 48 hours. n=2. The p value was obtained by multiple comparisons in an ordinary one-way analysis of variance (A, B, D, E, F, G), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ChIP, chromatin immunoprecipitation; Clo, clofarabine; IgG, immunoglobulin G; MHC, major histocompatibility complex; NC, negative control.

Article Snippet: To test whether CD8+ T cell inhibition in melanoma reverses the antitumor effects of Clo, B16F10- bearing C57BL/6 mice randomly received one of the following treatments: (1) vehicle+immunoglobulin G2 (IgG2) a (#BE0089, Bio X Cell, USA); (2) Anti- CD8α (A2102, Selleck, USA); (3) Clo+IgG2 a; (4) Clo+Anti- CD8α.

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Binding Assay, Knockdown, Chromatin Immunoprecipitation, Immunopeptidomics, Negative Control