Journal: Advanced Science

Article Title: Targeting IL27RA Enhances Immunotherapy in Triple‐Negative Breast Cancer by Modulating Tumor Cells and the Tumor Microenvironment

doi: 10.1002/advs.202516703

Figure Lengend Snippet: IL27RA restricts antigen‐specific T cell cytotoxicity. (A) Schematic of the in vitro CD8⁺ T cell activation workflow. (B–E) Co‐culture assays of PY8119 or EO771 cells transduced with vector or Il27ra overexpression constructs and activated CD8⁺ T cells. LDH release after 48 h (B, D) and flow‐cytometry quantification of 7‐AAD⁺ tumor cells (C, E) are shown. Wells without CD8⁺ T cells served as controls; a.u., arbitrary units. Left: Representative dot plots; right: Quantification. (F–I) Co‐culture assays using EO771 or PY8119 cells transduced with sh‐NC or sh‐ Il27ra . LDH release (F, H) and 7‐AAD⁺ tumor‐cell frequencies (G, I) after 48 h are shown. (J) Schematic of the antigen‐specific OT‐I T cell cytotoxicity assay. (K–N) OT‐I mediated killing of EO771‐OVA cells transduced with sh‐NC or sh‐ Il27ra (K, L) or vector or Il27ra overexpression constructs (M, N). LDH release (K, M) and 7‐AAD⁺ tumor‐cell frequencies (L, N) after 48 h co‐culture are shown. Wells without OT‐I T cells served as controls. All data are presented as mean ± SD, n = 3. Statistical significance was determined using two‐tailed unpaired Student's t ‐tests. Ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The schematic diagram was created using https://BioRender.com .

Article Snippet: Human breast cancer cell lines MDA‐MB‐231, MDA‐MB‐468, and HCC1937 (purchased from Procell, Wuhan, China, in March 2022), the mouse breast cancer cell line EO771 (purchased from Procell, Wuhan, China, in March 2022), and human embryonic kidney cells HEK293T (purchased from Procell, Wuhan, China, in March 2022) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China).

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Transduction, Plasmid Preparation, Over Expression, Construct, Flow Cytometry, Cytotoxicity Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting IL27RA Enhances Immunotherapy in Triple‐Negative Breast Cancer by Modulating Tumor Cells and the Tumor Microenvironment

doi: 10.1002/advs.202516703

Figure Lengend Snippet: IL27RA suppresses MHC‐I expression in human and murine TNBC cells. (A) Top enriched pathways identified by combined GO/KEGG analysis of PY8119 cells overexpressing Il27ra compared with vector controls. (B) Heatmap showing the expression of antigen‐processing and presentation (APP)–related genes in Il27ra ‐overexpressing PY8119 cells (red) versus control cells (blue). (C) Immunoblot analysis of MHC‐I and GAPDH (loading control, 37 kDa) in MDA‐MB‐231 cells overexpressing IL27RA (vector as control, n = 3) and MDA‐MB‐468 cells with IL27RA knockdown (sh‐NC as control, n = 3). (D) Flow‐cytometry analysis of surface MHC‐I mean fluorescence intensity (MFI) in MDA‐MB‐231 cells overexpressing IL27RA (n = 3). Representative histograms (left) and quantification (right; fold change relative to control) are shown. (E) Flow‐cytometry analysis of surface MHC‐I MFI in MDA‐MB‐468 cells with IL27RA knockdown (sh‐NC as control, n = 3). Representative histograms (left) and quantification (right) are shown. (F) Immunoblot analysis of MHC‐I and GAPDH in PY8119 and EO771 cells overexpressing Il27ra (vector as control, n = 3) or with Il27ra knockdown (sh‐NC as control, n = 3). (G) Flow‐cytometry quantification of surface MHC‐I MFI in PY8119 cells overexpressing Il27ra (n = 3). Representative histograms (left) and summary data (right) are shown. (H) Flow‐cytometry quantification of surface MHC‐I MFI in EO771 cells with Il27ra knockdown (sh‐NC as control, n = 3). Representative histograms (left) and summary data (right) are shown. (I) Immunofluorescence staining of IL27RA (red), MHC‐I (green), and DAPI (blue) in MDA‐MB‐468 cells with IL27RA knockdown (sh‐NC as control). The merged image is shown on the right, with quantification of MHC‐I fluorescence intensity per cell (a.u., arbitrary units). A total of 20 cells per group were analyzed across four independent experiments (5 cells per experiment). (J) Representative immunofluorescence images showing MHC‐I expression in CK14⁺ tumor cells and quantification of MHC‐I fluorescence intensity in CK14⁺ cells. A total of 20 CK14⁺ cells per group were analyzed across four independent experiments (5 cells per experiment); a.u., arbitrary units. Data are presented as mean ± SD. Statistical significance was determined using two‐tailed unpaired Student's t‐ tests. ** p < 0.01, *** p < 0.001, **** p < 0.0001. The schematic diagram was created using https://BioRender.com .

Article Snippet: Human breast cancer cell lines MDA‐MB‐231, MDA‐MB‐468, and HCC1937 (purchased from Procell, Wuhan, China, in March 2022), the mouse breast cancer cell line EO771 (purchased from Procell, Wuhan, China, in March 2022), and human embryonic kidney cells HEK293T (purchased from Procell, Wuhan, China, in March 2022) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Knockdown, Flow Cytometry, Fluorescence, Immunofluorescence, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting IL27RA Enhances Immunotherapy in Triple‐Negative Breast Cancer by Modulating Tumor Cells and the Tumor Microenvironment

doi: 10.1002/advs.202516703

Figure Lengend Snippet: IL27RA downregulates MHC‐I in breast cancer cells through activation of the PI3K/AKT signaling pathway. (A, C) Immunoblot analysis of IL27RA, total AKT, p‐AKT (Thr308), p‐AKT (Ser473), and MHC‐I in MDA‐MB‐231 (A) and PY8119 (C) cells stably overexpressing IL27RA or Il27ra . GAPDH served as a loading control. (B and D) Immunoblot analysis of IL27RA, total AKT, p‐AKT (Thr308), p‐AKT (Ser473), and MHC‐I in MDA‐MB‐468 (B) and EO771 (D) cells with stable IL27RA or Il27ra knockdown (sh‐NC as control). (E–G) Immunoblot analysis of IL27RA, total AKT, p‐AKT (Thr308), p‐AKT (Ser473), and MHC‐I in MDA‐MB‐231 (E) and MDA‐MB‐468 (F) cells overexpressing IL27RA , and PY8119 (G) cells overexpressing Il27ra , cultured for 24 h with or without the AKT inhibitor MK‐2206. GAPDH served as a loading control. (H–K) Flow‐cytometry analysis of surface MHC‐I expression in PY8119 (H,I) and EO771 (J,K) cells following Il27ra overexpression or knockdown, with representative histograms (left) and quantification (right). (L,M) CFSE‐labeled EO771‐OVA cells (with or without Il27ra overexpression and with or without MK‐2206 treatment) were cocultured with activated OT‐I T cells for 24 h. Cytotoxicity was determined by quantifying the percentage of 7‐AAD⁺ cells within the CFSE⁺ population. Data are presented as mean ± SD, based on three independent biological replicates (n = 3). Statistical significance was determined using one‐way ANOVA with Tukey's multiple‐comparisons test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human breast cancer cell lines MDA‐MB‐231, MDA‐MB‐468, and HCC1937 (purchased from Procell, Wuhan, China, in March 2022), the mouse breast cancer cell line EO771 (purchased from Procell, Wuhan, China, in March 2022), and human embryonic kidney cells HEK293T (purchased from Procell, Wuhan, China, in March 2022) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China).

Techniques: Activation Assay, Western Blot, Stable Transfection, Control, Knockdown, Cell Culture, Flow Cytometry, Expressing, Over Expression, Labeling