Journal: Frontiers in Immunology

Article Title: Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer

doi: 10.3389/fimmu.2017.02004

Figure Lengend Snippet: Metastasis-associated macrophage (MAM) precursor cells (MAMPCs) suppress cytotoxicity of CD8 + T cells through a reactive oxygen species (ROS)-mediated mechanism. (A,B) Effects of myeloid cells on the CD8 + T cell-induced tumor cell apoptosis. Splenic CD8 + T cells from normal C57BL/6 mice were cultured with anti-CD3/CD28 antibodies (effector; E) in the absence or presence of MAMPCs, MAMs, or resident macrophages (RMACs) from the metastatic lung of E0771-LG-injected mice (MAMPC/E, MAM/E, RMAC/E, respectively). The preincubated T cells were then isolated and cultured with E0771-LG cells expressing red fluorescent protein in the nuclei (target; T) at the indicated E:T ratio in the presence of green fluorogenic caspase-3 substrate. After 36 h, the number of apoptotic cancer cells indicated by red/green double positive nuclei was counted. (A) Representative images of cells cultured with the caspase-3 substrate for 36 h (E:T = 4:1). Scale bar; 50 µm, arrowhead; apoptotic cancer cell. (B) Number of apoptotic cancer cells cultured with preactivated CD8 + T cells ( n = 3, two independent experiments). Data are mean ± SEM, * P < 0.01 vs. E + T (4:1), # P < 0.01 vs. MAMPC/E + T. (C) Fold-change of genes encoding checkpoint T cell receptor ligands in MAMPCs and MAMs compared with classical monocytes (C-MOs) determined by microarray analyses in Figure (logFC > 1, FDR < 0.05). Data on expression values are presented as mean ± SEM. Note that the scale is exponential. (D) Mean fluorescence intensity of checkpoint T cell receptor ligands assessed by flow cytometry in C-MOs, MAMPCs, and MAMs ( n = 3, two independent experiments). Blood (for C-MOs) and lung digestion (for MAMPCs and MAMs) were prepared from E0771-LG-injected C57BL/6 mice at 14 days posttumor injection and stained with antibodies for indicated markers or isotype IgG. Data are mean ± SEM, * P < 0.01 vs. IM, # P < 0.01 vs. MAMPC. (E) Effects of checkpoint inhibitors on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and neutralizing antibodies for PD1 or CTLA4, or isotype IgG in the absence (effector; E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio in number of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with IgG in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. IgG. (F) Effects of inhibitors of nitric oxide or ROS production on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and L-NMMA, nor-NOHA, catalase and superoxide dismutase (SOD) (Cat/SOD), or vehicle (–) in the absence (E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with PBS in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. PBS.

Article Snippet: We intraperitoneally injected d -luciferin in PBS (GoldBio, 1.5 mg/100 μL/20 g mouse) into anesthetized E0771-LG:Fl tumor-bearing mice.

Techniques: Cell Culture, Injection, Isolation, Expressing, Microarray, Fluorescence, Flow Cytometry, Staining, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Transforming acidic coiled-coil-containing protein 3-mediated lipid metabolism reprogramming impairs CD8 + T-cell cytotoxicity in hepatocellular carcinoma

doi: 10.1038/s41392-025-02367-9

Figure Lengend Snippet: scRNA-seq illuminates the potential immunosuppressive role of TACC3. a ‒ d Representative images ( a ), growth curves ( b ), and weights ( c ) of a subcutaneous tumor model established with the human HCC cell line PLC/PRF/5 and nude mice. Parallel experiments were performed, and survival data were collected to obtain survival curves ( d ) (n = 6 per group). e ‒ h Representative images ( e ), growth curves ( f ), and weights ( g ) of a subcutaneous tumor model generated from the mouse HCC cell line Hepa1‒6 and nude mice. The survival curve ( h ) was drawn as described previously (n = 6 per group). i ‒ l Representative images ( i ), growth curves ( j ), and weights ( k ) of a subcutaneous tumor model generated from Hepa1‒6 cells and C57BL/6 mice. The survival curve ( l ) was drawn as described previously (n = 6 per group). m , n Representative images of the orthotopic tumor model ( m ) and the longest diameters ( n ) of the orthotopic tumors (n = 6 per group). o Overall uniform manifold approximation and projection (UMAP) visualization of scRNA-seq data from orthotopic tumor tissues, color-coded by annotated cell types. p UMAP plots showing different cell types in orthotopic tumor tissues of sgNC and sgTACC3 groups. q UMAP plot was created by subclustering the T/NK cells of the aforementioned scRNA-seq. CD8 Teff/Temra effector CD8 + T cells, CD4 Teff/Temra effector CD4 + T cells, Tcm central memory T cells, CD4 Tem effector memory CD4 + T cells, CD4 Treg CD4 + regulatory T cells, CD8 Tex exhausted CD8 + T cells, NK natural killer T cells, CD8 Tem effector memory CD8 + T cells, TN naive T cells, LQ low-quality cells. r Stacked column chart of the proportions of different T/NK cell subpopulations in the sgNC and sgTACC3 groups. s Bubble plot showing the relative TACC3 expression levels in nontumor, sgNC, and sgTACC3 tumor cells. Normal, nontumor cells. Data and error bars are presented as the means ± SDs. The data were analyzed via Student’s t test ( b , c , f , g , j , k , n ) and the log-rank (Mantel‒Cox) test ( d , h , l )

Article Snippet: For CD8 + T-cell depletion in the animal models, a mouse anti-CD8 mAb (100 μg/100 μL of PBS, BE0146, BioXcell, USA) and an isotype control antibody (100 μg/100 μL of PBS, BE0089, BioXcell, USA) were delivered through intraperitoneal injection to each group of C57BL/6 mice (n = 6 per group) on days 0, 4, 8, 12, and 16 to eliminate CD8 + T cells.

Techniques: Generated, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Transforming acidic coiled-coil-containing protein 3-mediated lipid metabolism reprogramming impairs CD8 + T-cell cytotoxicity in hepatocellular carcinoma

doi: 10.1038/s41392-025-02367-9

Figure Lengend Snippet: TACC3 promotes HCC progression by inhibiting the antitumor immunity of CD8 + T cells. a ‒ c Schematic diagrams of the CD8 + T-cell-mediated tumor killing assay ( a ), which was conducted by coculturing activated CD8 + T cells and wt HCC cells. Before cocultivation, activated CD8 + T cells were pretreated with supernatants from PLC/PRF/5 ( b ) or Huh7 cells ( c ) transfected with shTACC3 or ovTACC3. d , e Flow cytometry analysis was performed on CD45 + CD3 + cells from an orthotopic tumor model ( d ) to detect the infiltration of total CD8 + T cells, IFN-γ + CD8 + T cells, and PD-1 + CD8 + T cells ( e ) in sgNC or sgTACC3 HCC orthotopic tumor tissues (n = 6 per group). f , g Schematic diagram ( f ) and representative image ( g ) of the subcutaneous tumor model with anti-CD8a mAb administration. The anti-CD8a mAb was intraperitoneally injected every 4 days starting 6 days after the subcutaneous injection of the mouse HCC cell line Hepa1-6 (n = 6 per group). h , i Weights ( h ) and growth curves ( i ) of subcutaneous tumors as described in ( f , g ). j CD8a IHC analysis of our in-house cohort (n = 67); patients were divided into TACC3-high and TACC3-low groups according to the median TACC3 H score. Detailed information on the H scores is provided in the Supplementary Materials. k Violin plot of CD8a IHC analysis as described in ( j ). Scale bars, 50 μm. l , m mIHC analysis of TACC3, pancytokeratin, CD8a, GZMB, and PD-1 in an initial cohort of 6 HCC samples ( l ) and the expanded HCC tissue microarray cohort (n = 67) ( m ). For both analyses, patients were stratified into TACC3-high and TACC3-low groups on the basis of the median TACC3 mean fluorescence intensity (MFI) within the respective cohort. Scale bars, 50 μm. Data and error bars are presented as the means ± SDs. The data were analyzed by Student’s t test ( b , c , e , h , i , k )

Article Snippet: For CD8 + T-cell depletion in the animal models, a mouse anti-CD8 mAb (100 μg/100 μL of PBS, BE0146, BioXcell, USA) and an isotype control antibody (100 μg/100 μL of PBS, BE0089, BioXcell, USA) were delivered through intraperitoneal injection to each group of C57BL/6 mice (n = 6 per group) on days 0, 4, 8, 12, and 16 to eliminate CD8 + T cells.

Techniques: Transfection, Flow Cytometry, Injection, Microarray, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Transforming acidic coiled-coil-containing protein 3-mediated lipid metabolism reprogramming impairs CD8 + T-cell cytotoxicity in hepatocellular carcinoma

doi: 10.1038/s41392-025-02367-9

Figure Lengend Snippet: TACC3 reprograms PUFA metabolism to impair CD8 + T-cell antitumor immunity. a , b Heatmap ( a ) and volcano plot ( b ) of targeted metabolomics results showing the intracellular differentially abundant metabolites of PLC/PRF/5 cells (four shNC vs three shTACC3 PLC/PRF/5 cells as one sample in the shTACC3 group was excluded for quality control failure). c Bar chart showing the percentages of different types of differential intracellular metabolites. d Pie chart showing the percentages of intracellular differential PUFAs and non-PUFAs. PUFAs polyunsaturated fatty acids. e Fold-change of intracellular differential fatty acids via our intracellular targeted metabolomics data. f Intracellular DHA concentrations in PLC/PRF/5 and Huh7 cells detected via ELISA. g , h Heatmap ( g ) and volcano plot ( h ) of targeted metabolomics data showing the differentially abundant metabolites in CM supernatants from PLC/PRF/5 cells (shNC, n = 3; shTACC3, n = 3). i Bar chart showing the percentages of different types of differentially abundant metabolites in the supernatants. j Pie chart showing the percentages of differential PUFAs and non-PUFAs in the supernatant. k Fold-change of differential PUFAs detected by our supernatant-targeted metabolomics. l Venn diagram showing the 4 shared differential PUFAs between intracellular and supernatant-targeted metabolomics. DM differentially abundant metabolites, AA arachidonic acid, DGLA dihomogamma-linolenic acid, EPA eicosapentaenoic acid, DHA docosahexaenoic acid. m DHA concentrations in the supernatants of PLC/PRF/5 and Huh7 cells were measured via ELISA. n Schematic diagram of the CD8 + T-cell-mediated tumor killing assay (upper panel), which was conducted by coculturing activated CD8 + T cells and wt PLC/PRF/5 or Huh7 cells. Before cocultivation, activated CD8 + T cells were pretreated with 20 μmol DHA or NC. Representative images are shown in the bottom panel. CM conditioned medium; 0.1% v/v BSA-ethanol was used as a negative control (NC) for DHA treatment. o Schematic diagram of the CD8 + T-cell-mediated tumor killing assay (upper panel), which was conducted by coculturing activated CD8 + T cells and wt PLC/PRF/5 or Huh7 cells. Before cocultivation, activated CD8 + T cells were pretreated with 20 μmol DHA or NC plus CM supernatant from PLC/PRF/5 cells transfected with vector or ovTACC3. Representative images are shown in the bottom panel. CM conditioned medium; 0.1% v/v BSA-ethanol was used as a negative control (NC) for DHA treatment. p Representative image and statistical chart of the subcutaneous HCC model. Hepa1‒6 cells were transfected with or without ovTACC3. The mice were given 100 mg of DHA by daily gavage. n = 6 per group. q Flow cytometry analysis of the subcutaneous HCC model described above. The statistical charts of the total CD8 + T-cell percentage, IFN-γ + CD8 + T-cell percentage, and PD-1 + CD8 + T-cell percentage are shown in the right panel. n = 6 per group. The data and error bars represent the means ± SDs. The data were analyzed via Student’s t test ( e , f , k , m ‒ q )

Article Snippet: For CD8 + T-cell depletion in the animal models, a mouse anti-CD8 mAb (100 μg/100 μL of PBS, BE0146, BioXcell, USA) and an isotype control antibody (100 μg/100 μL of PBS, BE0089, BioXcell, USA) were delivered through intraperitoneal injection to each group of C57BL/6 mice (n = 6 per group) on days 0, 4, 8, 12, and 16 to eliminate CD8 + T cells.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Negative Control, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Transforming acidic coiled-coil-containing protein 3-mediated lipid metabolism reprogramming impairs CD8 + T-cell cytotoxicity in hepatocellular carcinoma

doi: 10.1038/s41392-025-02367-9

Figure Lengend Snippet: Key enzyme ACSL4 mediates TACC3-driven PUFA metabolic reprogramming. a Heatmap of RNA-seq data showing DEGs from 3 shNC and 3 shTACC3 PLC/PRF/5 cell lines. b Flow chart of n-3 PUFA metabolism. c ACSL4 IHC assay of a tissue microarray including 67 paired HCC tumor and peritumoral tissues from our center. Scale bars, 100 μm. d Correlation analysis between TACC3 and ACSL4 expression on the basis of the IHC staining intensity. Patients were divided into TACC3-high and TACC3-low groups according to the median TACC3 H score. Scale bars, 100 μm. e Statistical chart of the ACSL4 H score, which was determined via IHC of our tissue microarray. f Pearson’s correlation chart of the IHC intensity of TACC3 and ACSL4 in our tissue microarray. g , h The mRNA and protein levels of ACSL4 were detected after knockdown or overexpression of TACC3 via RT‒qPCR ( g ) and western blotting ( h ), respectively. i, k Intracellular DHA concentrations in PLC/PRF/5 ( i ) and Huh7 ( k ) cells treated with PRGL493 (50 μmol) were measured via ELISA. PRGL493 is a specific inhibitor of ACSL4. j, l DHA concentrations in the supernatants of PLC/PRF/5 ( j ) and Huh7 ( l ) cells treated with PRGL493 (50 μmol) were detected via ELISA. m, n Intracellular DHA concentrations of ovTACC3 PLC/PRF/5 ( m ) and ovTACC3 Huh7 ( n ) cells treated with PRGL493 (50 μmol) were measured via ELISA. o , p DHA concentrations in the supernatants of ovTACC3 PLC/PRF/5 ( o ) and ovTACC3 Huh7 ( p ) cells treated with PRGL493 (50 μmol) determined via ELISA. q Representative images of the CD8 + T-cell-mediated tumor killing assay, which was performed by coculturing activated CD8 + T cells and wt PLC/PRF/5 cells. Before cocultivation, activated CD8 + T cells were pretreated with CM from ovTACC3 PLC/PRF/5 cells treated with PRGL493 (50 μmol). The statistical chart is shown in the right panel. r mIHC analysis showing the colocalization of TACC3, ACSL4 and GZMB + CD8 + T cells or PD-1 + CD8 + T cells. Scale bars, 50 μm. Data and error bars are presented as the means ± SDs. The data were analyzed via the Wilcoxon signed-rank test ( e ), Pearson’s correlation analysis ( f ), and Student’s t test ( g , i ‒ q )

Article Snippet: For CD8 + T-cell depletion in the animal models, a mouse anti-CD8 mAb (100 μg/100 μL of PBS, BE0146, BioXcell, USA) and an isotype control antibody (100 μg/100 μL of PBS, BE0089, BioXcell, USA) were delivered through intraperitoneal injection to each group of C57BL/6 mice (n = 6 per group) on days 0, 4, 8, 12, and 16 to eliminate CD8 + T cells.

Techniques: RNA Sequencing, Microarray, Expressing, Immunohistochemistry, Knockdown, Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Transforming acidic coiled-coil-containing protein 3-mediated lipid metabolism reprogramming impairs CD8 + T-cell cytotoxicity in hepatocellular carcinoma

doi: 10.1038/s41392-025-02367-9

Figure Lengend Snippet: GalNAc–siTACC3 synergizes with PD-1 blockade to enhance antitumor immunity. a ‒ c The graphic design of the construction process of GalNAc–siTACC3. Sequence and chemical modifications of si-mTACC3 ( a ). Synthesis of GalNAc–siTACC3 via a reaction between GalNAc-L96 and si-mTACC3 ( b ). Identification of GalNAc–siTACC3 by mass spectrometry ( c ). 2’-OMe 2’-O-Methyl, 2’-F 2’-Fluoro, GNA glycol nucleic acid. mTACC3 mouse TACC3. d Schematic diagram of the animal models and drug administration, including the PD-1 mAb and GalNAc–siTACC3. s.c. subcutaneous injection. e , f Representative images of the subcutaneous ( e ) and orthotopic ( f ) tumor models. Scale bars for the orthotopic tumor, 1 cm. g ‒ j Statistical charts of weight ( g ), growth curves ( i ), and survival curves ( j ) of the subcutaneous tumor models and the longest diameter of the orthotopic tumors ( h ). k ‒ n Flow cytometry analysis of orthotopic tumors ( k ) and statistical plots of total CD8 + T cells ( l ), IFN-γ + CD8 + T cells ( m ), and PD-1 + CD8 + T cells ( n ). n = 6 per group. o, p IHC experiments of TACC3, ACSL4, CD8, GZMB, and PD-1 in orthotopic tumors ( o ) and a statistical analysis of infiltrating CD8 + T cells ( p ). Scale bars, 100 μm. Data and error bars are presented as the means ± SDs. The data were analyzed via Student’s t test ( g , h , i , l ‒ p ) and the log-rank (Mantel‒Cox) test ( j )

Article Snippet: For CD8 + T-cell depletion in the animal models, a mouse anti-CD8 mAb (100 μg/100 μL of PBS, BE0146, BioXcell, USA) and an isotype control antibody (100 μg/100 μL of PBS, BE0089, BioXcell, USA) were delivered through intraperitoneal injection to each group of C57BL/6 mice (n = 6 per group) on days 0, 4, 8, 12, and 16 to eliminate CD8 + T cells.

Techniques: Sequencing, Mass Spectrometry, Injection, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Transforming acidic coiled-coil-containing protein 3-mediated lipid metabolism reprogramming impairs CD8 + T-cell cytotoxicity in hepatocellular carcinoma

doi: 10.1038/s41392-025-02367-9

Figure Lengend Snippet: Schematic diagram depicting that TACC3-mediated PUFA metabolic reprogramming via the LARP1/PABPC1-ACSL4 axis reduces DHA levels in the TME, thereby suppressing CD8 + T-cell antitumor immunity. Therapeutic targeting of TACC3 may increase the efficacy of HCC immunotherapy. The schematic diagram was created using BioRender and included a publication license (Chen (2025) https://BioRender.com/dsxgsmu )

Article Snippet: For CD8 + T-cell depletion in the animal models, a mouse anti-CD8 mAb (100 μg/100 μL of PBS, BE0146, BioXcell, USA) and an isotype control antibody (100 μg/100 μL of PBS, BE0089, BioXcell, USA) were delivered through intraperitoneal injection to each group of C57BL/6 mice (n = 6 per group) on days 0, 4, 8, 12, and 16 to eliminate CD8 + T cells.

Techniques: