Journal: Advanced Science

Article Title: Targeting the ATX‐LPA Axis Overcomes TKI Resistance and Immunosuppression in Renal Cell Carcinoma via Dual Inhibition of AKT/mTOR and TBK1/IRF3 Pathways

doi: 10.1002/advs.76352

Figure Lengend Snippet: ENPP2 drives immune evasion via the TBK1/IRF3/PD‐L1 axis and suppresses CD8 + T‐cell effector functions in renal cancer cells. (A) GSEA plots showing negative enrichment of interferon‐α and interferon‐γ response signatures following ENPP2 knockdown. (B) Representative flow cytometry histograms and quantification of median fluorescence intensity (MFI) for surface PD‐L1 expression in 786‐O and ACHN cells transduced with control shRNA (shNC) or shENPP2. (C) Immunoblot analysis of ENPP2, PD‐L1, total TBK1, phospho‐TBK1 (Ser172), total IRF3, and phospho‐IRF3 (Ser396) in 786‐O and ACHN cells following ENPP2 knockdown. (D) Immunoblot analysis of the TBK1/IRF3/PD‐L1 axis in ENPP2‐overexpressing 786‐O and ACHN cells treated with 10 µM GLPG1690, 10 µM GLPG1690 plus 20 µM LPA, or DMSO control for 48 h. (E) Immunoblot analysis of the specified cascade proteins in ENPP2‐knockout (sgENPP2) cells reconstituted with either wild‐type ENPP2 (oeENPP2) or the catalytically inactive T210A mutant. (F) Progenitor and terminal exhaustion scores of CD8+ T cells stratified by LPAR5 expression level. (G) UMAP visualization of T‐cell subclusters identified by single‐cell RNA‐seq (Tex, exhausted T cells; Teff, effector T cells; Tstr, stress‐response T cells; Trm, tissue‐resident memory T cells; Tcm, central memory T cells; Tpex, progenitor‐exhausted T cells; CXCL13+ T, CD8+CXCL13+ T cells). (H) Proportions of the indicated CD8+ T‐cell subpopulations in tumors with low versus high ENPP2 expression. (I) Feature plots showing co‐localization of LPAR5 and PDCD1 (PD‐1) expression in tumor‐infiltrating T cells. (J, K) LDH‐release cytotoxicity assays of primary human T cells co‐cultured with control (NC) or ENPP2‐knockout (sgENPP2) 786‐O cells across varying effector‐to‐target (E:T) ratios (J), and the corresponding rescue effect of exogenous LPA supplementation (K). (L, M) LDH‐release cytotoxicity assay (L) and representative crystal violet staining (M) of oeENPP2 RCC cells co‐cultured with T cells, demonstrating the restorative effects of pharmacological anti‐PD‐1 antibody treatment or genetic PD‐L1 knockdown (siPD‐L1) on T‐cell‐mediated killing. (N, O) Representative flow cytometry plots and quantification of Granzyme B (GZMB) and TNF‐α (N), as well as Perforin and IFN‐γ (O) in CD8 + T cells after co‐culture with control (NC) or ENPP2‐knockout (SG) 786‐O cells. (P, Q) In vivo antibody‐mediated CD4 + and CD8 + T‐cell depletion in an immunocompetent syngeneic RCC model. Tumor growth kinetics (P) and final endpoint tumor weights (Q) of the indicated depletion cohorts (αCD4, αCD8, or αCD4+αCD8) compared to the IgG control. Quantitative in vitro data are presented as mean ± SD from n = 3 independent experiments. In vivo data are presented as mean ± SD with n = 5 mice per group. Statistical significance was determined using an unpaired two‐tailed Student's t ‐test (B, F, N, O), one‐way ANOVA (L, Q), or two‐way ANOVA (J, K, P), followed by Tukey's post hoc test. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary human CD8+ T cells were purified using a CD8+ T‐cell Isolation Kit (Miltenyi Biotec, 130‐096‐495) following the manufacturer's instructions.

Techniques: Knockdown, Flow Cytometry, Fluorescence, Expressing, Transduction, Control, shRNA, Western Blot, Knock-Out, Mutagenesis, Single Cell, RNA Sequencing, Cell Culture, Cytotoxicity Assay, Staining, Co-Culture Assay, In Vivo, In Vitro, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting the ATX‐LPA Axis Overcomes TKI Resistance and Immunosuppression in Renal Cell Carcinoma via Dual Inhibition of AKT/mTOR and TBK1/IRF3 Pathways

doi: 10.1002/advs.76352

Figure Lengend Snippet: In vivo experiments demonstrate that targeting ENPP2 improves T cell function and enhances the efficacy of targeted‐immune combination therapy. (A–C) PDX subcutaneous tumor weights at the experimental endpoint (A), tumor volume changes recorded during the experiment (B), and representative tumor images (C) following treatment as outlined in the schematic. Immunotherapy refers to reinfusion of in vitro pre‐activated T cells and administration of αPD‐1 antibody. Combination therapy mimics clinical first‐line therapy, combining TKI with immune checkpoint inhibitor (ICI). (D, E) Representative flow cytometry histograms (D) and quantification of median fluorescence intensity (MFI) (E) for surface PD‐L1 expression on tumor cells dissociated from the excised xenografts. (F) Serum concentrations of TNF‐α and IFN‐γ in the indicated immune‐reconstituted murine cohorts, measured by ELISA. (G, H) Flow cytometric analysis and MFI quantification of the cytotoxic effector molecules Granzyme B (GZMB) (G) and Perforin (H) in tumor‐infiltrating CD8 + T cells. (I, J) Representative contour plots and quantitative analysis of the exhaustion markers PD‐1 (I) and LAG‐3 (J) on tumor‐infiltrating T cells. (K) Representative immunohistochemistry (IHC) images and quantification of the proliferation marker Ki‐67 in tumor sections from the specified treatment groups. Scale bars: 50 µm. All in vivo data are presented as mean ± SD with n = 5 independent biological replicates per group. Statistical significance was determined using one‐way ANOVA (A, E, F, G, H, I, J, K), or two‐way ANOVA (B), followed by Tukey's post hoc test. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary human CD8+ T cells were purified using a CD8+ T‐cell Isolation Kit (Miltenyi Biotec, 130‐096‐495) following the manufacturer's instructions.

Techniques: In Vivo, Cell Function Assay, In Vitro, Flow Cytometry, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Marker