Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Kyn induced Siglec‐15‐mediated immune escape. (A) The chromatin immunoprecipitation (ChIP)‐PCR assay was performed using an IgG or AhR antibody after treatment with 200 µmol/L Kyn for 1 h. Two primers targeting the promoter region of SIGLEC15 and CD274 mRNA were used for RT‐qPCR analysis. (B) 293T cells were co‐transfected with SIGLEC15 / CD274 promoter‐luciferase reporter PGL3 for 24 h and treated with the indicated concentration of Kyn for another 6 h, followed by an analysis of luciferase activity. (C) 293T cells were co‐transfected with SIGLEC15 / CD274 promoter‐luciferase reporter PGL3 for 24 h and treated with 200 µmol/L Kyn for the indicated time, followed by an analysis of luciferase activity. (D) 293T cells were co‐transfected with promoter‐luciferase reporter plasmids and siAhR /siScr for 24 h and treated with 200 µmol/L Kyn for 6 h. (E) SCCVII cells were transfected with lentivirus‐ SIGLEC15 , and transfection was confirmed using Western blotting analysis. (F) Vector or SIGLEC15 ‐overexpressing SCCVII cells were subcutaneously injected into C3H/He mice ( n = 5 per group). Tumor volumes were measured once every two days. (G) Tumor weights were measured after mice were euthanized. (H‐J) H&E (H), TUNEL (I), and Ki‐67 (J) staining analyses of tumor tissues in each group. (K‐P) Tumor‐infiltrating lymphocytes (TIL) harvested from xenograft tumors, and the percentages of CD3 + CD8 + (K), Ki‐67 + (L), PD‐1 + (M), IFN‐γ + (N), granzyme B + (O), and perforin + cells (P) were analyzed using flow cytometry. (Q) The percentages of CD8 + in the indicated tumors were analyzed by multiplex immunofluorescence staining. (R) The volume and Ki‐67 expression of tumor were compared in tongue orthotopic transplant models established by vector or SIGLEC15 ‐overexpressing SCCVII cells ( n = 6 mice per group). (S) PD‐1 + CD8 + T cells were compared in tongue orthotopic transplant models ( n = 6 mice per group), based on multiplex immunofluorescence staining. Three regions of interest (ROIs) in each tumor were analyzed and measured. Data are represented as mean ± SEM (A‐D, F‐G and I‐S) based on three independent experiments (A‐D). * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: Kyn, kynurenine; ChIP, chromatin immunoprecipitation; RT‐qPCR, real‐time quantitative polymerase chain reaction; AhR, aryl hydrocarbon receptor; siScr, siScramble; H&E, hematoxylin and eosin; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; PD‐1, programmed cell death protein 1; TIL, Tumor‐infiltrating lymphocyte; IFN‐γ, interferon‐gamma; ROI, region of interest; SEM, tandard error of the mean; ns, not significant.

Article Snippet: For TIL analysis, samples were incubated with membrane antibodies containing anti‐mouse CD3 allophycocyanin (APC)‐Cy7, anti‐mouse CD8‐FITC, anti‐mouse PD‐1 phycoerythrin (PE)‐Cy7, and incubated with transcription factor fixation/permeabilization working solution (92550‐00, BioGems) for 30 min at 4°C, and washed with permeabilization buffer (BioGems).

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Transfection, Luciferase, Concentration Assay, Activity Assay, Western Blot, Plasmid Preparation, Injection, TUNEL Assay, Staining, Flow Cytometry, Multiplex Assay, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, End Labeling

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Targeting AhR reversed Kyn‐Siglec‐15‐mediated immune escape in vivo. (A) Mice with SCCVII were treated with Kyn (100 mg/kg) or/and CH‐223191 (10 µmol/L) once every two days for three times ( n = 5 per group). Tumor volumes were measured once every two days. (B) Tumor weights were measured after mice euthanized on day 20 ( n = 5 per group). (C) Western blotting analysis of Siglec‐15 expression after Kyn stimulation in mice. (D‐F) H&E (D), TUNEL (E and F upper), and Ki‐67 (E and F lower) staining analyses of tumor tissues in each group. (G) Concentration of serum TNF‐α and IFN‐γ in mice were measured by ELISA. (H‐M) The percentage of CD3 + CD8 + (H), Ki‐67 + (I), PD‐1 + (J), IFN‐γ + (K), granzyme B + (L), and perforin + cells (M) in cytotoxic T lymphocytes (CTLs) isolated from the indicated tumors were analyzed. (N) Representative images of multiplex immunofluorescence staining of CD3 + (green) and CD8 + (red) are shown, and quantification analysis were performed in tumor tissues ( n = 10 fields of 5 mice per group). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: AhR, aryl hydrocarbon receptor; Kyn, kynurenine; H&E, hematoxylin and eosin; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; TNF‐α, tumor necrosis factor‐alpha; IFN‐γ, interferon‐gamma; ELISA, enzyme‐linked immunosorbent assay; PD‐1, programmed cell death protein 1; CTL, cytotoxic T lymphocyte; DAPI, 4',6‐diamidino‐2‐phenylindole; SEM, tandard error of the mean; ns, not significant.

Article Snippet: For TIL analysis, samples were incubated with membrane antibodies containing anti‐mouse CD3 allophycocyanin (APC)‐Cy7, anti‐mouse CD8‐FITC, anti‐mouse PD‐1 phycoerythrin (PE)‐Cy7, and incubated with transcription factor fixation/permeabilization working solution (92550‐00, BioGems) for 30 min at 4°C, and washed with permeabilization buffer (BioGems).

Techniques: In Vivo, Western Blot, Expressing, TUNEL Assay, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Multiplex Assay, Immunofluorescence, End Labeling

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Siglec‐15 specific siRNA delivered by NH 2 ‐MSN nanoparticles enhances immunotherapy efficacy in vivo. (A) Synthesis routes of NH 2 ‐MSN nanoparticles loaded with siRNA. (B) Macroscopy characterization of NH 2 ‐MSNs with siS15. (C) Transmission electron microscopy images of NH 2 ‐MSNs and NP‐siS15 with indicated ratios. (D‐E) The morphology and Zeta potential of NH 2 ‐MSNs and NP‐siS15. (F) Scanning microscopy analysis of the cellular uptake of NH 2 ‐MSNs with or without the indicated siS15‐Cy5 by SCCVII cells after in vitro treatment for 24 h. (G) Western blotting analysis confirming Siglec‐15 gene‐silencing effect by siRNA released from nanoparticles in SCCVII cells. (H) Schematic diagram of the treatment strategy in SCCVII mouse model. (I) Probability of survival analysis for each group was performed. (J) Analysis of TUNEL and Ki‐67 staining of tumor tissues in each group. (K‐L) The percentage of CD3 + CD8 + (K) and PD‐1 + cells in CTLs (L) of the indicated treatment groups. (M) Analysis of multiplex immunofluorescence staining of CD3 + (green) and CD8 + (red) were shown and quantification analysis were performed in tumor tissues ( n = 10 fields of five mice per group). (N) Schematic diagram of the anti‐PD‐L1 and NP‐siS15 combination treatment strategy in C3H/He subcutaneous tumorigenesis models. (O‐P) Tumor volume and weights were measured and analyzed in the indicated groups. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: siS15, Siglec‐15 small interfering RNA; DAPI, 4',6‐diamidino‐2‐phenylindole; AhR, aryl hydrocarbon receptor; NP‐siS15, NH 2 ‐MSN‐si SIGLEC15 ; DAPI, 4',6‐diamidino‐2‐phenylindole; siScr, siScramble; anti‐PD‐1, anti‐programmed cell death protein 1 antibody; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; CTL, cytotoxic T lymphocyte; anti‐PD‐L1, anti‐programmed death‐ligand 1 antibody; SEM, tandard error of the mean; ns, not significant.

Article Snippet: For TIL analysis, samples were incubated with membrane antibodies containing anti‐mouse CD3 allophycocyanin (APC)‐Cy7, anti‐mouse CD8‐FITC, anti‐mouse PD‐1 phycoerythrin (PE)‐Cy7, and incubated with transcription factor fixation/permeabilization working solution (92550‐00, BioGems) for 30 min at 4°C, and washed with permeabilization buffer (BioGems).

Techniques: In Vivo, Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Microscopy, In Vitro, Western Blot, TUNEL Assay, Staining, Multiplex Assay, Immunofluorescence, Small Interfering RNA, End Labeling