Journal: Genes & Cancer

Article Title: Novel high-grade serous epithelial ovarian cancer cell lines that reflect the molecular diversity of both the sporadic and hereditary disease

doi:

Figure Lengend Snippet: A) Immunohistochemical analysis of formalin-fixed paraffin-embedded solid tumors from patients 866, 2978, 3041, 3291, and 4485 with cytokeratin (CK7, CK8, CK18 and CK19) markers, WT1, PAX8, p53 and HER2. Brown color indicates positive staining, nuclei are counterstained with hematoxylin, and images are at 400x magnification. Topmost images are hematoxylin-eosin images of the corresponding tumor samples. B) Detection of CKs, WT1, PAX8, p53, HER2, E-cadherin and vimentin by Western blot of lysates of cell lines. Lower panel shows an agarose gel of TP53 PCR products from reversed-transcribed cell line mRNAs. β–actin was used as control in both procedures. Blk = Blank, RT = Reverse transcription

Article Snippet: WB and IHC analyses were performed using the following antibodies: p53 (D0-1, sc-126, Santa Cruz Biotechnology, Dallas, TX); HER2/ErbB2/Neu (C-18, sc-284, Santa Cruz Biotechnology, for WB; and OP15, Calbiochem®, Millipore (Canada) Ltd., Etobicoke, ON, for IHC); Cytokeratin (CK) 7 (Ab-2, MS-1352-P, Thermo Scientific, Waltham, MA); CK8 (Ab-4, MS-997-P, Thermo Scientific); CK18 (DC-10, sc-6259, Santa Cruz Biotechnology); CK19 (Ab-1, MS198-P, Thermo Scientific), WT1 (6F-H2, 05-753, Millipore (Canada) Ltd.), BRCA1 (OP92, MS110, Calbiochem®, Millipore (Canada) Ltd.), Vimentin (V9, sc-6260, Santa Cruz Biotechnology), E-cadherin (24E10, #3195, Cell Signaling Technology Inc., Danvers, MA), PAX8 (10336-1-AP, Proteintech, Chicago, IL) and beta-actin (AC-15, ab6276, Abcam Inc., Toronto, ON, Canada).

Techniques: Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Staining, Western Blot, Agarose Gel Electrophoresis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

doi: 10.1038/mtna.2013.54

Figure Lengend Snippet: Schematic of the different WT1-encoding vectors and quality control of the in vitro transcribed mRNA. ( a ) The T7 promoter, 5′-UTR, 3′-UTR, human β-globin UTR, poly(A)-tail (A64 or A120), the NLS, sig, and the lysosomal targeting sequence (DC-LAMP) are shown. All pGEM constructs have an overhang of nucleotides derived from the vector backbone, which remains as 3′ attachment to the poly(A)-tail after linearization with Spe I. The pST1 vector has a free-ending poly(A)-tail after linearization with Sap I . The original WT1 sequence is depicted in white and the optimized WT1 sequence in gray. ( b ) Electropherogram of the wild-type WT1 mRNA, representative of all mRNA samples, and ( c ) gel-like image of mRNA derived from the different WT1 constructs. NLS, nuclear localization sequence; sig, signal peptide; UTR, untranslated regions; WT1, Wilms' tumor 1.

Article Snippet: To analyze intracellular WT1 expression, cells were fixed and permeabilized using the BD Cytofix/Cytoperm plus kit and stained intracellularly with an anti-WT1 monoclonal antibody (clone 6F-H2; Dako Cytomation, Carpinteria, CA).

Techniques: In Vitro, Sequencing, Construct, Derivative Assay, Plasmid Preparation, Wilms Tumor Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

doi: 10.1038/mtna.2013.54

Figure Lengend Snippet: Expression and presentation of WT1 protein after electroporation with mRNA derived from the different WT1-encoding vectors. ( a ) Intracellular staining of WT1 in K562 cells. K562 cells were electroporated with the indicated WT1 mRNA constructs and analyzed for WT1 expression by intracellular staining 4-, 24-, and 48-hour postelectroporation (open histograms). As a control for nonspecific WT1 immunoreactivity, parallel electroporations were performed with a control mRNA (pGEM/NEF-DC-LAMP), which yielded similar expression levels as the isotype controls (gray-filled histograms). This figure is representative of three independent experiments. ( b ) Comparison of WT1 ΔMFI (MFI of positive population substracted by the basal WT1 epxression) values of the total K562 population and the total immature DC population after electroporation with WT1-sh-DC-L-OPT mRNA encoded by the pGEM vector. Data are presented as mean ± SEM of three independent experiments ( **P = 0.0081). ( c ) Immunocytochemical detection of WT1 in TriMix-matured, WT1 mRNA-electroporated DCs. Immunocytochemistry for WT1 was performed on DCs fixated 24 hours after coelectroporation with WT1-encoding mRNA or control mRNA. Immunocytochemical staining patterns are shown from one representative experiment out of five (upper panel). Expression data were scored according to the staining intensity (middle panel) and according to the percentage of WT1-positive cells (lower panel; data are expressed as mean ± SEM of five independent experiments). ( d ) TriMix-DCs were coelectroporated with the indicated WT1 mRNA constructs or with the control mRNA. Twenty-four hours later, electroporated DCs were cocultured with the WT1-specific T-cell clone for 20 hours. IFN-γ release during this coculture was measured. Results are shown as mean ± SEM of three independent experiments ( *P < 0.05). ( e ) Human HLA-A2 + TriMix-DCs were loaded with the WT1 antigen either by electroporation with the pST1-derived WT1-sh-DC-L-OPT mRNA or by pulsing with the HLA-A2-restricted WT1 126–134 peptide. Negative controls included TriMix-DCs electroporated with the control mRNA specified above or pulsed with the HLA-A2-restricted Melan-A 26-35 peptide. Four, 24, or 48 hours after antigen loading, cells were used to stimulate a human HLA-A2-restricted WT1 126-134 -specific CD8 + T-cell clone. IFN-γ release was measured by ELISA after a 20-hour coculture. DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ MFI, mean fluorescence intensity; SEM, standard error of the mean; WT1, Wilms' tumor 1.

Article Snippet: To analyze intracellular WT1 expression, cells were fixed and permeabilized using the BD Cytofix/Cytoperm plus kit and stained intracellularly with an anti-WT1 monoclonal antibody (clone 6F-H2; Dako Cytomation, Carpinteria, CA).

Techniques: Expressing, Electroporation, Derivative Assay, Staining, Construct, Plasmid Preparation, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Fluorescence, Wilms Tumor Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

doi: 10.1038/mtna.2013.54

Figure Lengend Snippet: Functionality of the WT1 protein encoded by the optimized vector in an in vivo mouse model. ( a ) In vivo CTL assay after immunization with wild-type WT1 mRNA or mRNA encoded by the optimized vector. In vivo cytolysis of the target cells was assessed ex vivo by flow cytometry analysis of CFSE lo or CFSE hi cells in the spleen, injected and noninjected lymph nodes, and the peripheral blood. Data shown are from the blood and are representative for the other compartments. This figure is representative of three independent experiments. Data are presented as the percentage of specific lysis. ( b ) C57BL/6 mice ( n = 7) were inoculated with 5 × 10 5 C1498-WT1 cells s.c. 7 days later, and mice were immunized intranodally with control, wild-type WT1+TriMix, or optimized WT1+TriMix mRNA. Tumor volume was assessed (** P < 0.01; *** P < 0.001). WT1, Wilms' tumor 1.

Article Snippet: To analyze intracellular WT1 expression, cells were fixed and permeabilized using the BD Cytofix/Cytoperm plus kit and stained intracellularly with an anti-WT1 monoclonal antibody (clone 6F-H2; Dako Cytomation, Carpinteria, CA).

Techniques: Plasmid Preparation, In Vivo, CTL Assay, Ex Vivo, Flow Cytometry, Injection, Lysis, Wilms Tumor Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

doi: 10.1038/mtna.2013.54

Figure Lengend Snippet: Autotoxicity-related side effects after WT1 immunization. ( a ) WT1 expression in the kidney under physiological conditions. ( b ) No proteinuria was observed in mice immunized with the optimized WT1 mRNA, indicating no damage to the kidney ( n = 8 mice/group). ( c ) No pathological changes, such as lymphocyte infiltration or tissue destruction and repair, were observed in the kidney, lung, or liver (negative control) of WT1 mRNA-immunized mice. WT1, Wilms' tumor 1.

Article Snippet: To analyze intracellular WT1 expression, cells were fixed and permeabilized using the BD Cytofix/Cytoperm plus kit and stained intracellularly with an anti-WT1 monoclonal antibody (clone 6F-H2; Dako Cytomation, Carpinteria, CA).

Techniques: Expressing, Negative Control, Wilms Tumor Assay

Journal: International Journal of Molecular Sciences

Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

doi: 10.3390/ijms232012177

Figure Lengend Snippet: WT1 expression on EpCAM + cancer cells and detection of memory subsets of WT1-CTLs in MPE samples. ( A ) Expression of WT1 on EpCAM positive or negative cells sorted from whole cells in MPE 1st . PC, phase contrast; WT1 cells were stained with anti-WT1 antibody, followed by Alexa Fluor® Plus 488 secondary antibody; DNA, DAPI staining. The white bar indicates 100 μm. ( B ) The ratio of WT-CTLs is defined as WT1 tetramer + CD8 + T cells to MPE samples 1st or MPE 2nd (upper panel). The memory subsets of WT1-CTLs during disease progression (middle panel). CD62L + CD45RO + : central memory T cells (T CM ); CD62L + CD45RO − : naïve T cells (T N ); CD62L − CD45RO + : effector memory T cells (T EM ), CD62L − CD45RO − : terminal effector T cells (T TE ). The function of WT1-CTLs in MPE was evaluated by ELISpot assay for IFN-γ secretion under WT1 235 peptide stimulation (lower panel). The mean number of spots of WT1 235 peptide-specific was shown. The error bar indicated the mean and standard deviation. Unpaired t -test, * p < 0.05.

Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

Techniques: Expressing, Staining, Biomarker Discovery, Enzyme-linked Immunospot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

doi: 10.3390/ijms232012177

Figure Lengend Snippet: Detection of CD8 + T cell response to HLA-A*11:01 restricted SMAD4 P130L neoantigen in MPE. ( A ) WT1 tetramer-CD8 + T cells sorted from MPE 1st were expanded with HPL-IFN-DCs containing each predicted neoantigen peptide for SMAD4 P130L . For ELISpot assay to detect IFN-γ production after in vitro expansion, these cells were stimulated using each SMAD4 P130L peptide. Reactivity was shown in the box plot (per each well in 96 plates). DMSO was used as a negative control. A Wilcoxon signed-rank test was performed, * p < 0.05. ( B ) CD8 + T cells purified from in vitro expanded cells with HPL-IFN-DCs contained SMAD4-Neo1 peptides in A were stimulated by HEV0271 cells (HLA-A*11:01 homozygous) containing SMAD4-Neo1 (SVCVNLYH) or SMAD4-WT1 (SVCVNPYH). The reactivity to IFN-γ was evaluated by ELISpot assay. The error bar indicated the mean and standard deviation. Dunnett’s test, * p < 0.05.

Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

Techniques: Enzyme-linked Immunospot, In Vitro, Negative Control, Purification, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

doi: 10.3390/ijms232012177

Figure Lengend Snippet: Memory subsets of WT1 tetramer − CD8 + T cells and detection of SMAD4 P130L -specific CD8 + T cell expanded from MPE samples. ( A ) WT1 tetramer − CD8 + T cells in MPE 1st or MPE 2nd were stained with antibodies against CD62L and CD45RO for memory T cell subsets. ( B ) WT1 tetramer − CD8 + T cells were sorted from MPE 1st or MPE 2nd , expanding in vitro with HPL-IFN-DCs containing SMAD4-Neo1 peptide. These cells were stimulated with SMAD4-Neo1 or DMSO to detect IFN-γ-producing cells by ELISpot assay. The ratio of IFN-γ spots of SMAD4-Neo1 to control was shown in the box plot (per each well in 96 plates). Mann–Whitney U test, * p < 0.05.

Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

Techniques: Staining, In Vitro, Enzyme-linked Immunospot, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: A molecular atlas of the human postmenopausal fallopian tube and ovary from single-cell RNA and ATAC sequencing

doi: 10.1016/j.celrep.2022.111838

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse WT1 Clone 6F-H2 , Thermo Fisher Scientific , Cat#MA1–46028; RRID:AB_962464.

Techniques: Recombinant, Saline, RNAscope, Multiplex Assay, Diagnostic Assay, Software, Gene Expression, Microscopy, Real-time Polymerase Chain Reaction