Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting HK3 in tumor-associated macrophages enhances antitumor immunity through augmenting antigen cross-presentation in cervical cancer

doi: 10.1136/jitc-2025-011948

Figure Lengend Snippet: HK3 in tumor-associated macrophages compromises CD8 + T cell-mediated antitumor immunity. ( A ) Schema of the co-injection model using FigDraw. TC-1 cells were co-implanted with either WT or Hk3 -KO BMDMs at a 1:1 ratio subcutaneously into C57BL/6 mice. Tumors were resected for measurement at day 14 after inoculation. ( B ) Tumor growth after tumor inoculation (n=5 per group). ( C ) Tumor weight at day 14 after inoculation (n=5 per group). ( D ) Representative immunohistochemical images and quantification of Ki67 expression in tumor tissues. Scale bar, 50 µm. ( E ) Representative TUNEL staining of tumor tissues and quantification of TUNEL-positive nuclei (blue: DAPI, green: TUNEL; scale bar: 50 µm). ( F ) Percentages of tumor-infiltrating CD4 + or CD8 + T cells out of living cells at day 14. ( G ) Percentages of IFN-γ + CD8 + T cells, TNF-α + CD8 + T cells, GzmB + CD8 + T cells at day 14. ( H ) The gMFI of IFN-γ, TNF-α and GzmB in CD8 + T cells at day 14. ( I ) Experimental design for CD8 + T-cell depletion. Anti-CD8 neutralizing antibody (100 µg/mouse) was administered intraperitoneally at days 1, 4, and 9. ( J ) Tumor growth following CD8 + T-cell depletion (n=5 per group). ( K ) Percentages of IFN-γ or TNF-α-producing OT-I CD8 + T cells co-cultured with the OVA-loaded BMDMs. All values are expressed as the mean±SEM. *p<0.05, **p<0.01, ***p<0.001 and not significant (ns) by Student’s t-test or one-way analysis of variance test. BMDMs, bone marrow-derived macrophages; gMFI, geometric mean fluorescence intensity; GzmB, granzyme B; HK3, hexokinase 3; HK3-KO, HK3 knockout; IFN, interferon; i.p., intraperitoneally; PBS, phosphate buffered saline; TNF, tumor necrosis factor; WT, wild-type.

Article Snippet: During co-culture, anti-IFN-γ neutralizing antibody (100 μg/mL, clone R4-6A2, A2105, Selleck) was added.

Techniques: Injection, Immunohistochemical staining, Expressing, TUNEL Assay, Staining, Cell Culture, Derivative Assay, Fluorescence, Knock-Out, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting HK3 in tumor-associated macrophages enhances antitumor immunity through augmenting antigen cross-presentation in cervical cancer

doi: 10.1136/jitc-2025-011948

Figure Lengend Snippet: HK3 inhibition enhances cross-presentation by attenuating lysosomal antigen degradation. ( A ) KEGG enrichment analysis of marker genes in HK3 high versus HK3 low macrophages (median cut-off) in the cervical cancer single-cell dataset. ( B ) Top 20 upregulated KEGG pathways in WT versus Hk3 -KO BMDMs. ( C ) Dot plots showing the expression of selected genes from major histocompatibility complex class I antigen processing signature in HK3 high and HK3 low macrophages. Dot size indicates the percentage of cells expressing each gene, and color intensity represents the relative gene expression level. ( D ) RT-qPCR showing the mRNA expression of Lamp1 and Lamp2 in WT and Hk3 -KO BMDMs. ( E ) The gMFI of DQ-OVA in BMDMs at indicated time points post-treatment. ( F ) The gMFI of DQ-OVA in BMDMs pretreated with or without CQ for 30 min. ( G ) The gMFI of H-2K b -SIINFEKL complexes in OVA-loaded BMDMs following 24-hour treatment with either CQ or MG132. ( H ) Experimental design for the co-culture of naïve CD8 T cells derived from OT-I mice with OVA-loaded and PFA-fixed BMDMs using FigDraw. ( I ) Percentages of CD25 + or CD69 + OT-I cells following 24-hour co-culture with the OVA-loaded and PFA-fixed BMDMs. ( J ) Representative plots and percentages of IFN-γ-producing OT-I T cells following 72-hour co-culture with the OVA-loaded and PFA-fixed BMDMs. ( K ) Quantification of Annexin V + TC-1-OVA cells following 6-hour co-culture with OT-I T cells that were pre-co-cultured with OVA-loaded and PFA-fixed BMDMs. All values are expressed as the mean±SEM. *p<0.05, **p<0.01, ***p<0.001 not significant (ns) by Student’s t-test or one-way analysis of variance test. BMDMs, bone marrow-derived macrophages; CQ, chloroquine; gMFI, geometric mean fluorescence intensity; HK3, hexokinase 3; HK3-KO, HK3 knockout; IFN, interferon; Mφ, macrophages; KEGG, Kyoto Encyclopedia of Genes and Genomes; mRNA, messenger RNA; OVA, ovalbumin; PFA, paraformaldehyde; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; WT, wild-type.

Article Snippet: During co-culture, anti-IFN-γ neutralizing antibody (100 μg/mL, clone R4-6A2, A2105, Selleck) was added.

Techniques: Inhibition, Marker, Expressing, Immunopeptidomics, Gene Expression, Quantitative RT-PCR, Co-Culture Assay, Derivative Assay, Cell Culture, Fluorescence, Knock-Out, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting HK3 in tumor-associated macrophages enhances antitumor immunity through augmenting antigen cross-presentation in cervical cancer

doi: 10.1136/jitc-2025-011948

Figure Lengend Snippet: HK3 induces excessive lysosomal activation through promoting TFEB nuclear translocation after binding to mTOR. ( A ) Representative confocal images of BMDMs. Hoechst 33342 (blue) and LysoTracker Red (red) were used for cell nuclei and lysosome labeling, repectively (left). Scale bars, 5 µm. Representative histograms and gMFI of LysoTracker in BMDMs (right). ( B ) Representative histograms and gMFI of LysoSensor Green DND-189 in BMDMs. ( C ) RT-qPCR showing the mRNA expression of Atp6v1h and Ctsl in WT and Hk3 -KO BMDMs. ( D ) Representative confocal images of BMDMs. DAPI (blue) and TFEB (red) were used for cell nuclei and TFEB labeling, repectively (left). Western blotting of cytoplasmic and nuclear TFEB in WT and Hk3 -KO BMDMs (right). ( E ) Co-immunoprecipitation for the interaction between Flag-tagged HK3 and mTOR in HK3-OE HEK293T cells.( F ) Representative histograms and gMFI of phospho-mTOR (Ser2448) in BMDMs. ( G ) Representative histograms and gMFI of LAMP1 in BMDMs following 24-hour treatment with either rapamycin (Rapa, 25 µM) or TFEB activator 1 (TA1, 2 µM). ( H ) The gMFI of H-2K b -SIINFEKL complexes in OVA-loaded BMDMs following 24-hour treatment with either Rapa or TA1. ( I ) Percentages of IFN-γ-producing OT-I T cells following 72-hour co-culture with the OVA-loaded and then paraformaldehyde-fixed BMDMs pretreated with Rapa, TA1 and 2-deoxy-D-glucose (2-DG, 1 mM). All values are expressed as the mean±SEM. *p<0.05, **p<0.01, ***p<0.001 and not significant (ns) by Student’s t-test or one-way analysis of variance test. BMDMs, bone marrow-derived macrophages; gMFI, geometric mean fluorescence intensity; HK3, hexokinase 3; HK3-KO, HK3 knockout; HK3-OE, HK3 overexpressed; IFN, interferon; IP, immunoprecipitation; mRNA, messenger RNA; mTOR, mechanistic target of rapamycin; OVA, ovalbumin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TFEB, transcription factor EB; WT, wild-type.

Article Snippet: During co-culture, anti-IFN-γ neutralizing antibody (100 μg/mL, clone R4-6A2, A2105, Selleck) was added.

Techniques: Activation Assay, Translocation Assay, Binding Assay, Labeling, Quantitative RT-PCR, Expressing, Western Blot, Immunoprecipitation, Co-Culture Assay, Derivative Assay, Fluorescence, Knock-Out, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting HK3 in tumor-associated macrophages enhances antitumor immunity through augmenting antigen cross-presentation in cervical cancer

doi: 10.1136/jitc-2025-011948

Figure Lengend Snippet: HK3 is modulated by the IFN-γ-STAT1 signaling axis. ( A ) Upregulated pathways in HK3 high versus HK3 low samples (median cut-off) in the TCGA-CESC dataset using GSVA with Molecular Signatures Database Hallmark gene sets. ( B ) Correlation between HK3 and IFNG in TCGA-CESC dataset. r, Spearman correlation coefficient; p, p values for Spearman correlation. ( C ) RT-qPCR showing the mRNA expression of Hk3 in BMDMs treated with IFN-γ (20 ng/mL), LPS (100 ng/mL) or IL-4 (20 ng/mL) for 24 hours. ( D ) Dose-dependent effects of IFN-γ on Hk3 mRNA expression in BMDMs. RT-qPCR analysis was performed after 24-hour treatment with indicated concentrations of IFN-γ (0, 20, 50, 100 ng/mL). ( E ) Representative histograms and gMFI of HK3 in BMDMs treated with indicated concentrations of IFN-γ for 24 hours. ( F ) Experimental design for IFN-γ blockade. Splenic CD8 + T cells were activated with PMA and ionomycin for 6 hours, followed by 24-hour co-culture with BMDMs in the presence of IFN-γ neutralizing antibody. ( G ) Representative histograms and gMFI of HK3 in BMDMs following 24-hour co-culture with CD8 + T cells in the presence of IFN-γ neutralizing antibody. ( H ) Representative histograms and gMFI of HK3 in BMDMs treated for 6 hours with IFN-γ (20 ng/mL), JAK1/2 inhibitors ruxolitinib (1 µM) or STAT1 inhibitors fludarabine (1 µM). All values are expressed as the mean±SEM. *p<0.05, **p<0.01, ***p<0.001 and not significant (ns) by Student’s t-test or one-way analysis of variance test. BMDMs, bone marrow-derived macrophages; FACs, Fluorescence-Activated Cell Sorting; gMFI, geometric mean fluorescence intensity; GSVA, Gene Set Variation Analysis; HK3, hexokinase 3; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; mRNA, messenger RNA; PMA, phorbol 12-myristate 13-acetate; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TCGA-CESC, The Cancer Genome Atlas – Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma.

Article Snippet: During co-culture, anti-IFN-γ neutralizing antibody (100 μg/mL, clone R4-6A2, A2105, Selleck) was added.

Techniques: Quantitative RT-PCR, Expressing, Co-Culture Assay, Derivative Assay, Fluorescence, FACS, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Dynamic reciprocal interactions between activated T cells and tumor associated macrophages drive macrophage reprogramming and proinflammatory T cell migration within prostate tumor models

doi: 10.1038/s41598-024-75265-9

Figure Lengend Snippet: IFNG neutralization impacts T cell effect on MDMs. ( A ) Primary M0 MDMs were cultured stepwise in mono-culture, to co-culture with established LNCaP or 22Rv1 tumor cultures; T cells were added for the last 24 h with vehicle control (Ctrl), or IgG control (IgG Iso), or anti-IFNG neutralizing antibody (anti-IFNG). ( B ) IFNG protein concentration (pg/ml) in cell culture supernatants; n = 5; data expressed as mean ± SEM. ( C ) MDM mRNA expression in the six tri-culture cellular and treatment conditions shown as normalized relative quantity (NRQ) as related to house-keeping genes RPLP0 and POLR2A . One-way ANOVA nonparametric with Friedman post-hoc analysis comparing the mean rank of each column with the rank of every other column; n = 5; Data represent mean ± SEM. ( D , F ) Concentrations of secreted cytokines and chemokines in supernatant from the six tri-culture cellular and treatment conditions, expressed as pg/mL. One-way ANOVA nonparametric with Friedman post-hoc analysis comparing the mean rank of each column with the rank of every other column; n = 5; Data represent mean ± SEM. ( E ) T cell migration analysis measured by in-chip confocal microscopy imaging in individual microdevice wells. Data is expressed as each sample’s fold change in migration distance as relative to its corresponding tri-culture vehicle control condition quantified by NIS-Elements. One-way ANOVA nonparametric with Friedman post-hoc analysis comparing the mean ranks of vehicle control IgG control, and vehicle control to Anti-IFNG neutralizing antibody; n = 5; Data represent mean ± SEM.

Article Snippet: For migration experiments, T cells were seeded on Stacks hydrogel wells at a concentration of either 5 × 10 5 or 2.5 × 10 4 cells per ml and allowed to migrate for 24 h. For IFNG neutralization experiments, either 1 μg ml − 1 anti-IFNG neutralizing antibody (R&D Systems by bio-techne, Minneapolis, MN USA Cat# AF-285-NA, RRID: AB_354445) (1:1000), 1 μg ml − 1 IgG control R&D Systems by bio-techne, Minneapolis, MN USA Cat# AB-108-C, RRID: AB_354267) (1:1000), or 0.1% 1xPBS (1:1000) were added to a T cell resuspension media immediately before the addition of the T cells to their culture layer for the 24 h migration period.

Techniques: Neutralization, Cell Culture, Co-Culture Assay, Control, Protein Concentration, Expressing, Migration, Confocal Microscopy, Imaging

Journal: Scientific Reports

Article Title: Dynamic reciprocal interactions between activated T cells and tumor associated macrophages drive macrophage reprogramming and proinflammatory T cell migration within prostate tumor models

doi: 10.1038/s41598-024-75265-9

Figure Lengend Snippet: TAMs retain proinflammatory plasticity in the TME. Dynamic, reciprocal interaction between tumor-polarized macrophages and activated T cells allows paracrine reprogramming of TAM via IFNG secretion.

Article Snippet: For migration experiments, T cells were seeded on Stacks hydrogel wells at a concentration of either 5 × 10 5 or 2.5 × 10 4 cells per ml and allowed to migrate for 24 h. For IFNG neutralization experiments, either 1 μg ml − 1 anti-IFNG neutralizing antibody (R&D Systems by bio-techne, Minneapolis, MN USA Cat# AF-285-NA, RRID: AB_354445) (1:1000), 1 μg ml − 1 IgG control R&D Systems by bio-techne, Minneapolis, MN USA Cat# AB-108-C, RRID: AB_354267) (1:1000), or 0.1% 1xPBS (1:1000) were added to a T cell resuspension media immediately before the addition of the T cells to their culture layer for the 24 h migration period.

Techniques: