Journal: Cell Death & Disease

Article Title: Targeting tumor intrinsic TAK1 engages TNF-α-driven cell death through distinct mechanisms and enhances cancer immunotherapy

doi: 10.1038/s41419-025-08013-0

Figure Lengend Snippet: A MC38 cells lacking Tak1 or B2m expression were pulsed with the MHC-I restricted chicken ovalbumin SIINFEKL peptide and subsequently cultured with increasing ratios of OT-I CD8 T cells, previously activated for 48 h by CD3 and CD28 stimulation. Tumor cell viability was assessed 24 h later by flow cytometry, using CD8 surface staining to discriminate T cells from tumor cells. B – D CD8 T cells isolated from naïve mouse spleens (Balb/c for CT-26 and EMT6, C57BL/6 for MC38) were activated by CD3 and CD28 stimulation in vitro and subsequently cultured with ( B ) MC38, ( C ) CT-26, or ( D ) EMT6 tumor cells lacking Tak1 expression. Viability was assessed using DRAQ7 uptake, imaging cells every 2 h for the indicated times. E – G Tak1- deficient MC38 ( E ) or CT-26 ( F ) cells were cultured in media derived from 48 h activated CD-8 T cells. Viability was assessed via DRAQ7 uptake. G Conditioned media from 48 h activated CD8 T cells was added to EMT6 cells deficient for Tak1 , Tnfr1 , or Tak1 and Tnfr1 and viability was assessed via DRAQ7 uptake. All panels represent the mean +/− SD from a single experiment, n = 2 independent experiments.

Article Snippet: For the CD8 depletion protocol, mice were injected with anti-mouse CD8 (BioXCell; BP0061, New Haven, CT) or IgG2a Isotype control (BioXCell; BP 00085) at 10 mg/kg i.p. at the dose schedule of Day -2, Day -1 prior to flank inoculation of cells, then again on Days 4, 8, and 11 post inoculation.

Techniques: Expressing, Cell Culture, Flow Cytometry, Staining, Isolation, In Vitro, Imaging, Derivative Assay

Journal: Cell Death & Disease

Article Title: Targeting tumor intrinsic TAK1 engages TNF-α-driven cell death through distinct mechanisms and enhances cancer immunotherapy

doi: 10.1038/s41419-025-08013-0

Figure Lengend Snippet: A Immunocompetent mice (C57BL/6, n = 15/group) were implanted with pooled clones from Tak1 -deficient or parental MC38 cells and tumor growth was monitored. B , C Immunocompetent mice (BALB/c, n = 8/group) were implanted with Tak1 -deficient CT-26 cells ( B ) and treated at the indicated days with an α-PD-1 antibody ( C , 10 mg/kg) and tumor growth was monitored. D Immunodeficient mice (NSG, n = 10/group) were implanted with Tak1 -deficient CT-26 cells and tumor growth was monitored. E Quantification of the percentage of complete tumor clearance from panels ( B – D ). F Balb/c mice implanted with Tak1 -deficient CT-26 tumors and exhibiting complete responses were rechallenged with CT-26 parental cells and tumor growth was monitored. Naïve mice challenged with parental CT-26 cells served as controls. G CT-26 parental and Tak1 -deficient tumor growth was monitored in Balb/c mice administered an α-CD8 depleting antibody for two sequential days prior to tumor engraftment and during tumor progression at the indicated days ( n = 10/group). H CT-26 parental and Tak1 -deficient tumor growth was assessed in Balb/c mice administered an α-TNF-α neutralizing antibody (15 mg/kg) at the indicated days ( n = 10/group). Tumor growth curves represent the mean tumor volume +/− SEM. Statistical differences between tumor volumes at the final measurement was determined using a two-way ANOVA with Tukey’s multiple correction where * p < 0.05; ** p < 0.01; and **** p < 0.0001 was considered significant.

Article Snippet: For the CD8 depletion protocol, mice were injected with anti-mouse CD8 (BioXCell; BP0061, New Haven, CT) or IgG2a Isotype control (BioXCell; BP 00085) at 10 mg/kg i.p. at the dose schedule of Day -2, Day -1 prior to flank inoculation of cells, then again on Days 4, 8, and 11 post inoculation.

Techniques: Clone Assay

Journal: Nature immunology

Article Title: Inhibition of ENT1 relieves intracellular adenosine-mediated T cell suppression in cancer.

doi: 10.1038/s41590-025-02153-3

Figure Lengend Snippet: Fig. 6 | Adenosine suppresses TIL function, which is restored by EOS301984. a, Proliferation of CD8+ T cells in dissociated tumor cell suspensions in the presence of ATP as a source of high adenosine concentrations and EOS301984 and anti-PD-1 as indicated. Representative example from n = 9 donors. An example gating strategy is provided in Supplementary Fig. 5. b–i, Summary data of EOS301984-mediated rescue of CD8+ (b) and CD4+ (c) T cell proliferation, TNF (d), IFNγ (e), granzyme A (f), granzyme B (g), perforin (h) and sFASL (i) production from dissociated tumor cell suspension cultures. Rescue refers to the degree to which adenosine-mediated suppression has been reversed. j, FACS analysis of TIM-3, PD-1 and TOX expression by PD-1− CD8+ TILs isolated from NSCLC tumor samples, stimulated with anti-CD3/CD28 microbeads for 72 h in the

Article Snippet: The following antibodies were used for Western blot: Anti-human ENT1 (SP120, Abcam, Ab182023, 1:1000) HRP anti-rabbit IgG (7074S, Cell signaling Technology, 7074S, 1:1000) Anti-B-actin (D6A8, Cell signaling Technology, 8457S, 1:1000) The following antibodies were used for in vivo experiments: InVivoPlus anti-mouse CD8α (53-6.7, BioXcell, BP004-1) Anti-PD-1 (OPDIVO® (nivolumab)), (Bristol Meyers Squibb) Validation SAHENTA was validated for the binding to human ENT1 in this publication: Robins, M. J., Peng, Y., Damaraju, V. L., Mowles, D., Barron, G., Tackaberry, T., Young, J. D. & Cass, C. E. Improved Syntheses of 5ʹ- S -(2-Aminoethyl)-6- N -(4-nitrobenzyl)-5ʹthioadenosine (SAENTA), Analogues, and Fluorescent Probe Conjugates: Analysis of Cell-Surface Human Equilibrative Nucleoside Transporter 1 (hENT1) Levels for Prediction of the Antitumor Efficacy of Gemcitabine.

Techniques: Suspension, Expressing, Isolation