Journal: The Journal of Biological Chemistry

Article Title: TIM-3 inhibition enhances breast tumor progression and metastasis: A paradoxical immune checkpoint response

doi: 10.1016/j.jbc.2025.111096

Figure Lengend Snippet: Treatment with anti-TIM3 monoclonal antibody increase tumor growth and weight. A , treatment schema. Six weeks old female Balb/c mice were inoculated with 4T1 (1.2 × 10 6 ) at the fourth abdominal mammary duct of the left flank (n = 3/group). Anti-TIM3 antibody (250 μg/injection) was given i.p. on day 18,21, 24 and day 28. B and C , in vivo tumor development at day 40. B , 4T1 induced control breast tumor ( C ) Breast tumor after anti-TIM3 administration. D , representative photograph of the visceral organs of Tumor mice and anti-TIM3 treated mice collected on day 40 after tumor inoculation. E , tumor length and width were measured on day 8, 16, 21, 24, 32 and until day 40 and tumor volume were determined. Bar plot represents mean ± SD. ∗∗ p < 0.01 (Unpaired t test with Holm-Šídák method). F , tumor weight was measured on day 40, the day of resection of each group of mice. Data represent mean ± SD. ∗∗∗ p < 0.001 (Unpaired two tailed t test).

Article Snippet: Murine triple-negative breast cancer (TNBC) cell line 4T1 was purchased from ATCC (ATCC, cat. #CRL-2539, RRID: CVCL_0125) and cultured in RPMI-1640 (Himedia, cat. #AL162S) supplemented with 10% fetal bovine serum (Himedia, cat. #RM112) and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO 2 and humidification.

Techniques: Injection, In Vivo, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: TIM-3 inhibition enhances breast tumor progression and metastasis: A paradoxical immune checkpoint response

doi: 10.1016/j.jbc.2025.111096

Figure Lengend Snippet: Histopathological and immunohistochemical evaluation of TIM-3 into 4T1 allograft breast tumors and detection of liver metastases following anti-TIM-3 monoclonal antibody treatment. A and B , TIM-3 immunohistochemical detection mostly observed into tumor infiltrating immune cells (indicated by red arrow ) in primary breast tumor tissue into both control tumor mice and anti-TIM3-treated mice, respectively, at × 400 magnification with a scale bar 20 μm. C , TIM-3 expression scoring into tumor (Control) and anti-TIM-3 treated group was determined semi-quantitatively by counting the percentage of TIM-3-positive cells and staining intensity of five × 400 magnification fields selected randomly and represented in the bar graph as mean ± SD, from three mice per group, ∗∗∗ p < 0.001 (Unpaired two-tailed t-test). D-G , primary breast tumor tissue section stained with H&E, ( D and E ) breast tumor tissue histology of control tumor mice and anti-TIM3 treated mice (at × 100 magnification respectively, with scale bar 100 μm), and ( F and G ) breast tumor tissue histology of control tumor mice and anti-TIM3 treated mice respectively (at × 400 magnification respectively, with scale bar 100 μm) resected after day 40. H – L , histological analysis of liver metastasis of tumor-bearing mice and treated group showed ( H ) tumor foci (indicated with a black circle ) of control tumor mice, and ( I ) anti-TIM3 mAb-treated tumor mice showed several tumor foci (indicated with several black circles ). J , the number of tumor metastatic foci into liver tissue section was determined by counting fifty high power field under × 400 magnification of each mouse of an experimental group (n = 3). Data represent mean ± SD. ∗ p < 0.05 (Unpaired two-tailed t -test). Infiltration of inflammatory cells into liver was observed and demarcated by ( K ) blue arrow indicated the moderate (not too severe) infiltration into control tumor-bearing mice, while ( L ) the green arrow indicated the severe inflammatory cell infiltration into the liver of anti- TIM-3 treated group. All data represent mean ± SD; n = 3.

Article Snippet: Murine triple-negative breast cancer (TNBC) cell line 4T1 was purchased from ATCC (ATCC, cat. #CRL-2539, RRID: CVCL_0125) and cultured in RPMI-1640 (Himedia, cat. #AL162S) supplemented with 10% fetal bovine serum (Himedia, cat. #RM112) and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO 2 and humidification.

Techniques: Immunohistochemical staining, Control, Expressing, Staining, Two Tailed Test

Journal: Oncogene

Article Title: TBKBP1 induces capecitabine resistance through negative regulation of type I interferon pathway in triple-negative breast cancer

doi: 10.1038/s41388-025-03598-4

Figure Lengend Snippet: A Schematic outline of Tbkbp1-knockdown in tumors induced by capecitabine: 4T1 mouse breast cancer cells stably expressing shNC or shTbkbp1 were subcutaneously injected into BALB/c mice. The mice were treated with capecitabine (25 mg/kg i.g.), or PBS every two days since Day 9. ( n = 8 mice/group). B Representative images of the tumors illustrating the effect of TBKBP1-depletion in the PBS and CAP groups. ( n = 8 mice/group). C Effects of Tbkbp1 knockdown on tumor growth in the PBS and CAP groups. The data were represented as the mean ± SEM. The two-way ANOVA was used to compare the data. D Tumor weights of the six groups. The data were represented as the mean ± SEM. Data were compared by using the Student’s t test. E Bar plots showing the percentages of CD3e + cells among CD45 + cells, CD4 + cells among CD3e + CD45 + cells, CD8 + cells among CD3e + CD45 + cells, interferon-γ (IFNγ)-positive cells among CD8 + T cells, CD86 + cells among CD45 + CD11b + F4/80 + cells, and CD206 + cells among CD45 + CD11b + F4/80 + cells. Data were compared by using the Student’s t test. F Representative IHC images of CD4, CD8, CD86, and CD206 expression in shNC and shTbkbp1 tumors in the PBS and capecitabine group. Scale bar, 20 μm. G Boxplots showing the percentages of CD4-, CD8-, CD86-, and CD206-positive cells. The data were represented as the mean ± SEM. Data were compared by using the Student’s t test.

Article Snippet: The human embryonic kidney cell line HEK293T, human TNBC cell lines Hs 578 T, MDA-MB-468, BT-549, SUM159, LM2-4175, HCC1806, and the mouse TNBC cell line 4T1 were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Stable Transfection, Expressing, Injection

Journal: Oncogene

Article Title: TBKBP1 induces capecitabine resistance through negative regulation of type I interferon pathway in triple-negative breast cancer

doi: 10.1038/s41388-025-03598-4

Figure Lengend Snippet: A Bar plots of GO and KEGG analysis results showing that Tbkbp1 knockdown in CAP-treated breast tumors upregulated several pathways and biological processes. B GSEA analysis revealed that Tbkbp1 knockdown promoted CAP-induced upregulation of genes involved in interferon-related pathways. C Heatmaps showing that Tbkbp1-depletion upregulated genes involved in IFN- α pathway (left) and IFN- γ pathway (right). D qRT-PCR results showing that knockdown of Tbkbp1 upregulated the mRNA levels of Tbk1, Irf3, and Ifngr1 in 4T1 cells (upper) and 4T07 cells (bottom) under 5-FU treatment. The data were represented as the mean ± SEM. Data were compared by using the Student’s t test. E qRT-PCR results showing that overexpression of Tbkbp1 downregulated the mRNA levels of Irf3 and Ifngr1 in AT3 cells (upper) and TS/A cells (bottom) under 5-FU treatment. The data were represented as the mean ± SEM. Data were compared by using the Student’s t-test. F Western blot analysis showing that Tbkbp1 knockdown increased the protein levels of Tbk1, Irf3, and Ifngr1 in 4T1 cells (left) and 4T07 cells (right) induced by 5-FU. Densitometric quantification of Western blots was performed using ImageJ software. G Western blot analysis revealed that Tbkbp1 overexpression decreased the protein levels of Tbk1, Irf3, and Ifngr1 in AT3 cells (left) and TS/A cells (right) induced by 5-FU. Densitometric quantification of Western blots was performed using ImageJ software.

Article Snippet: The human embryonic kidney cell line HEK293T, human TNBC cell lines Hs 578 T, MDA-MB-468, BT-549, SUM159, LM2-4175, HCC1806, and the mouse TNBC cell line 4T1 were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Quantitative RT-PCR, Over Expression, Western Blot, Software

Journal: Oncogene

Article Title: TBKBP1 induces capecitabine resistance through negative regulation of type I interferon pathway in triple-negative breast cancer

doi: 10.1038/s41388-025-03598-4

Figure Lengend Snippet: A Bar plots of GSEA, GO, KEGG, and Reactome analysis results based on RNA-seq data of breast tumors from BALB/c mice treated with PBS, or CAP. B Heatmaps showing that CAP upregulated genes involved in DNA damage (upper) and cytosolic DNA-sensing pathway (bottom). C qRT-PCR results showing that 5-FU upregulated the mRNA levels of Cgas, and Sting in 4T1 cells (upper) or 4T07 cells (bottom) stably expressing shNC or shTbkbp1. The data were represented as the mean ± SEM. Data were compared by using the Student’s t test. D qRT-PCR results showing that 5-FU upregulated the mRNA levels of Cgas, and Sting in AT3 cells (left) or TS/A cells (right) stably expressing Vector or Flag-Tbkbp1. The data were represented as the mean ± SEM. Data were compared by using the Student’s t test. E Western blot analysis showing that 5-FU increased the protein levels of Cgas, and Sting in 4T1 cells (left) or 4T07 cells (right) stably expressing shNC or shTbkbp1. Densitometric quantification of Western blots was performed using ImageJ software. F Western blot analysis showing that 5-FU increased the levels of Cgas, and Sting protein in AT3 cells (left) or TS/A cells (right) stably expressing Vector or Flag-Tbkbp1. Densitometric quantification of Western blots was performed using ImageJ software.

Article Snippet: The human embryonic kidney cell line HEK293T, human TNBC cell lines Hs 578 T, MDA-MB-468, BT-549, SUM159, LM2-4175, HCC1806, and the mouse TNBC cell line 4T1 were obtained from American Type Culture Collection (ATCC).

Techniques: RNA Sequencing, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Software

Journal: Oncogene

Article Title: TBKBP1 induces capecitabine resistance through negative regulation of type I interferon pathway in triple-negative breast cancer

doi: 10.1038/s41388-025-03598-4

Figure Lengend Snippet: A Viability of AT3 cells stably overexpressing Tbkbp1 (AT3-Tbkbp1, left) and TS/A cells stably overexpressing Tbkbp1 (TS/A-Tbkbp1, right) treated with DMSO (negative control), 5-FU (4 μ M), 5-FU (4 μM) with MG132 (10 μ M), 5-FU (4 μM) with BafA1 (200 nM), or 5-FU (4 μM) with 3-MA (5 mM) for 18 h, respectively. The data were represented as the mean ± SEM. Data were compared by using the Student’s t test. B Western blot analysis showing the expression of Tbk1 in AT3-Tbkbp1 cells (left) and TS/A-Tbkbp1 cells (right) treated with DMSO, 5-FU, or 5-FU plus the indicated inhibitors. Densitometric quantification of Western blots was performed using ImageJ software. C AT3-Tbkbp1 cells (left) and TS/A-Tbkbp1 cells (right) were treated with or without 200 nM BafA1 in combination of 4 μM 5-FU for the indicated times, and then subjected to Western blot analysis with Tbk1 and p62 antibodies. Densitometric quantification of Western blots was performed using ImageJ software. D Western blot analysis showing the expression of Tbk1, Irf3, and Ifngr1 when Tbkbp1-knockdown 4T1 cells (left) and 4T07 cells (right) were co-transfected with siTbk1 under 5-FU treatment for 18 h. Densitometric quantification of Western blots was performed using ImageJ software.

Article Snippet: The human embryonic kidney cell line HEK293T, human TNBC cell lines Hs 578 T, MDA-MB-468, BT-549, SUM159, LM2-4175, HCC1806, and the mouse TNBC cell line 4T1 were obtained from American Type Culture Collection (ATCC).

Techniques: Stable Transfection, Negative Control, Western Blot, Expressing, Software, Knockdown, Transfection