Journal: Nature Biomedical Engineering

Article Title: Ablation of prostaglandin E 2 signalling through dual receptor knockout in CAR T cells enhances therapeutic efficacy in solid tumours

doi: 10.1038/s41551-025-01610-6

Figure Lengend Snippet: ( a ) 5 ×10 6 CD45.1 x CD4 Cre Ptger2 − / − Ptger4 fl/fl OT-I T cells and 5 ×10 6 CD90.1 OT-I T cells were co-injected into D4M.3A-SIINFEKL (10 6 cells s. c.) bearing mice. On days 2, 5, 9 and 14 the abundance of T cell populations was analyzed using flow cytometry; gating strategy depicted. T cell populations in spleen ( b ) and blood ( c ) were analyzed (data shown as mean ± SEM of n = 5 mice per group). Statistical analysis was done with a two-way ANOVA. ( d ) 10 ×10 6 CD4 Cre Ptger2 − / − Ptger4 fl/fl anti-EpCAM-CAR T cells and 10 ×10 6 anti-EpCAM-CAR T cells were intravenously injected into Panc02-OVA-EpCAM (2 ×10 6 cells s. c.) bearing mice. Tumor growth and survival are depicted for n = 5 mice per group. ( e ) Schematic representation of Ep2 −/− Ep4 −/− OT-I T cells production workflow. OT-I T cells were submitted to genetic editing by inserting the RNP complex through electroporation. ( f ) OT-I T cells with Ep2 and/or Ep4 knockout were treated with 2000 ng/ml PGE 2 . CREB phosphorylation was measured by flow cytometry. Depicted is the gating strategy for flow cytometry analysis. ( g ) 10 7 OT-I splenocytes with Ep2 and/or Ep4 knockout were adoptively transferred (i. v.) into recipient mice bearing D4M.3A-SIINFEKL tumors (10 6 cells s. c.). Single tumor growth curves of n = 3 independent repetitions with n = 5 mice per group are shown. Illustrations in e created with BioRender.com .

Article Snippet: The following antibodies and staining reagents were used for flow cytometry or cell sorting: Fixable Viability Dye eFluor 780 (1:1,000, Invitrogen), APC anti-human CD3 (1:100, clone OKT3, BioLegend), Pacific Blue anti-mouse CD3 (1:100, clone 17A2, BioLegend), Pacific Blue anti-mouse CD4 (1:100, clone GK1.5, BioLegend), FITC anti-mouse CD4 (1:100, clone GK1.5, BioLegend), PE-Cy7 anti-human CD4 (1:100, clone OKT4, BioLegend), PerCP anti-human CD8 (1:100, clone HIT8a, BioLegend), Pacific Blue anti-mouse CD8a (1:100, clone 53-6.7, BioLegend), FITC anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), BV605 anti-human CD8 (1:100, clone SK1, BioLegend), BV785 anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), Alexa Fluor 647 mouse anti-CREB (pS133)/ATF-1 (pS63) (1:20, clone J151-21, BD Biosciences), BV650 anti-human CD69 (1:100, clone FN50, BioLegend), PE/Cy7 anti-human CD62L (1:100, clone DREG-56, BioLegend), BV510 anti-human CD44 (1:100, clone IM7, BioLegend), Alexa Fluor 700 anti-human PD-1 (1:100, clone EH12.2H7, BioLegend), BV510 anti-human TIM-3 (1:100, clone F38-2E2, BioLegend), APC anti-human LAG-3 (1:100, clone 7G2C65, BioLegend), PE-Cy7 anti-mouse IFNγ (1:100, clone XMG1.2, eBioscience), BV421 anti-human CD45 (1:100, clone 2D1, BioLegend), BV605 anti-human CD4 (1:100, clone UCHL1, BioLegend), Alexa Fluor 700 anti-mouse CD4 (1:100, clone GK1.5, BioLegend), BV711 anti-mouse CD45.1 (1:100, clone A20, BioLegend), APC anti-rat CD90/mouse CD90.1 (Thy-1.1) (1:100, clone OX-7, BioLegend), BV421 anti-human CD69 (1:100, clone F50, BioLegend), PerCP-Cy5.5 anti-human CD25 (1:100, clone BC96, BioLegend) and FITC anti-human/mouse/rat c-myc (1:100, clone SH1-26E7.1.3, Miltenyi).

Techniques: Injection, Flow Cytometry, Electroporation, Knock-Out, Phospho-proteomics

Journal: Nature Biomedical Engineering

Article Title: Ablation of prostaglandin E 2 signalling through dual receptor knockout in CAR T cells enhances therapeutic efficacy in solid tumours

doi: 10.1038/s41551-025-01610-6

Figure Lengend Snippet: a , 5 × 10 6 CD45.1 × CD4 Cre Ptger2 − / − Ptger4 fl/fl OT-I T cells and 5 × 10 6 CD90.1 OT-I T cells were co-injected into D4M.3A-SIINFEKL (10 6 cells s.c.) bearing mice. b , c , On days 2, 5, 9 and 14, the abundance of T cell populations in tumours ( b ) and lymph nodes ( c ) was determined by flow cytometry (data shown as mean ± s.e.m. of n = 5 mice per group). Statistical analysis was done with a two-way ANOVA. d , OT-I T cells with Ep2 and/or Ep4 knockout were treated with 2,000 ng ml −1 PGE 2 and cAMP levels were determined in a luciferase-based readout (data shown as mean ± s.d. of n = 3 independent experiments with 2 technical replicates each; statistical analysis was done with an ordinary one-way ANOVA). e , OT-I T cells with Ep2 and/or Ep4 knockout were treated with 1,600 ng ml −1 PGE 2 and CREB phosphorylation was measured by flow cytometry. Pooled data of n = 3 representative experiments (mean ± s.d.) with 2 technical replicates each and a representative change of the pCREB MFI are shown. Statistical analysis was done with a two-way ANOVA. Mice bearing D4M.3A-SIINFEKL tumours (10 6 cells s.c.) were treated with 10 7 adoptively transferred (i.v.) OT-I T cells with Ep2 and/or Ep4 knockout. f , g , Tumour growth ( f ) was monitored over time and survival ( g ) was determined. Pooled results of n = 3 independent repetitions with n = 5 mice per group are shown as mean ± s.e.m. Statistical analysis was done with a repeated measurements mixed-effects analysis with Dunnett’s multiple comparison correction ( f ) and log-rank (Mantel–Cox) test ( g ). Panel a created with BioRender.com .

Article Snippet: The following antibodies and staining reagents were used for flow cytometry or cell sorting: Fixable Viability Dye eFluor 780 (1:1,000, Invitrogen), APC anti-human CD3 (1:100, clone OKT3, BioLegend), Pacific Blue anti-mouse CD3 (1:100, clone 17A2, BioLegend), Pacific Blue anti-mouse CD4 (1:100, clone GK1.5, BioLegend), FITC anti-mouse CD4 (1:100, clone GK1.5, BioLegend), PE-Cy7 anti-human CD4 (1:100, clone OKT4, BioLegend), PerCP anti-human CD8 (1:100, clone HIT8a, BioLegend), Pacific Blue anti-mouse CD8a (1:100, clone 53-6.7, BioLegend), FITC anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), BV605 anti-human CD8 (1:100, clone SK1, BioLegend), BV785 anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), Alexa Fluor 647 mouse anti-CREB (pS133)/ATF-1 (pS63) (1:20, clone J151-21, BD Biosciences), BV650 anti-human CD69 (1:100, clone FN50, BioLegend), PE/Cy7 anti-human CD62L (1:100, clone DREG-56, BioLegend), BV510 anti-human CD44 (1:100, clone IM7, BioLegend), Alexa Fluor 700 anti-human PD-1 (1:100, clone EH12.2H7, BioLegend), BV510 anti-human TIM-3 (1:100, clone F38-2E2, BioLegend), APC anti-human LAG-3 (1:100, clone 7G2C65, BioLegend), PE-Cy7 anti-mouse IFNγ (1:100, clone XMG1.2, eBioscience), BV421 anti-human CD45 (1:100, clone 2D1, BioLegend), BV605 anti-human CD4 (1:100, clone UCHL1, BioLegend), Alexa Fluor 700 anti-mouse CD4 (1:100, clone GK1.5, BioLegend), BV711 anti-mouse CD45.1 (1:100, clone A20, BioLegend), APC anti-rat CD90/mouse CD90.1 (Thy-1.1) (1:100, clone OX-7, BioLegend), BV421 anti-human CD69 (1:100, clone F50, BioLegend), PerCP-Cy5.5 anti-human CD25 (1:100, clone BC96, BioLegend) and FITC anti-human/mouse/rat c-myc (1:100, clone SH1-26E7.1.3, Miltenyi).

Techniques: Injection, Flow Cytometry, Knock-Out, Luciferase, Phospho-proteomics, Comparison

Journal: International Journal of Molecular Medicine

Article Title: Deciphering the CAF-LCN2 axis: Key to overcoming anti-PD-L1 immunotherapy resistance in lung cancer

doi: 10.3892/ijmm.2026.5735

Figure Lengend Snippet: Influence of LCN2 on the growth of lung cancer in mice. (A) Simplified flowchart outlining the establishment of an anti-PD-L1 immunotherapy tolerance model in mice with lung cancer. (B) Western blot analysis of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice. (C) Immunohistochemical detection of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice (scale bar, 100 µm). (D) Establishment of a model for anti-PD-L1 immunotherapy in LCN2-silenced mice. (E) Evaluation of LCN2 mRNA expression in tumor tissues after LCN2 gene intervention in mice. (F) Assessment of LCN2 protein expression in tumor tissues following LCN2 gene intervention in mice. (G) Tumor size in LCN2 gene intervention mice (n=10 in each group). *P<0.05, **P<0.01 and ***P<0.001. Unpaired two-tailed Student's t-test was used for (B) and (C) and one-way ANOVA with Tukey's multiple comparisons test was used for (E) and (G). LCN2, lipocalin 2; PD-L1, programmed death-ligand 1; sh-, short hairpin; NC, negative control; COL1A1, collagen α-1(I) chain; THY1, Thy-1 membrane glycoprotein; i.p., intraperitoneal.

Article Snippet: The following primary antibodies were used: Rabbit anti-LCN2 (cat. no. ab125075; 1:100; Abcam), rabbit anti-COL1A1 (cat. no ab34710; 1:15; Abcam), rabbit anti-Thy-1 membrane glycoprotein (THY1) (cat. no 13801; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-E-cadherin (cat. no 3195; 1:400; Cell Signaling Technology, Inc.), rabbit anti-Vimentin (cat. no. 5741; 1:4,000; Cell Signaling Technology, Inc.), rabbit anti-IL-6 (cat. no. ab6672; 1:400; Abcam), rabbit anti-TNF-α (cat. no. ab307164; 1:1,000; Abcam), rabbit anti-Ki-67 (cat. no. ab16667; 1:200; Abcam) and rabbit anti-cleaved caspase-3 A (cat. no. 9661; 1:400; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Two Tailed Test, Negative Control, Membrane