Journal: Cell Death Discovery

Article Title: Ex.50.T aptamer impairs tumor–stroma cross-talk in breast cancer by targeting gremlin-1

doi: 10.1038/s41420-025-02363-6

Figure Lengend Snippet: A The binding ability of ex.50.T on parental cells of EVs used for exosome SELEX was analyzed by qPCR. Data are mean percentage ± SD over control (fibroadenoma) of two biological replicates. B qPCR analysis of ex.50.T binding on BC continuous cell lines; U87MG cells were used as control cell line. Data are mean percentage ± SD over control of two biological replicates. C Flow cytometry analysis of Alexa-680–ex.50.T binding on PBS-washed MCF7 and BT-549 cells (Total) and high-salt-treated MCF7 and BT-549 cells (Internalized). D Western blot of GREM1 (R&D Systems, # AF956) in MCF7 and BT-549 cell lines. β-actin was used as a loading control. E FACS analysis of ex.50.T in GREM1-overexpressing MCF7 cells (MCF7 24% vs. MCF7 GFP 36%). Data are mean percentage ± SD over control of two independent experiments in three replicates. Western blot analysis was used for overexpression control of GREM1 (Santa Cruz Biotechnology, # sc-515877); β-actin was used as loading control. F FACS analysis of ex.50.T in BT-549 cells after interference with GREM1. Data are mean percentage ± SD over control (BT-549) of all sh-GREM1 interfered points of two independent experiments in three replicates. Western blot analysis was used for down-modulation control of GREM1 (R&D Systems, # AF956); β-actin was used as loading control. G ELISA with GREM1 antibody (sc-515877) (right) and biotinylated ex.50.T ELONA (left) on total protein lysate of U87MG cells overexpressing GREM1. Data are mean percentage ± SD over control U87MG cells of three independent experiments in triplicates. Western blot analysis was used for overexpression control of GREM1 (R&D Systems, # AF956); β-actin was used as loading control. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For examination of immuno-gold-labeled EVs isolated from BT459, MCF7, and U87MG cell cultures, the mouse monoclonal antibody sc-515877 directed against gremlin-1 (Santa Cruz Biotechnology, Santa Cruz, CA) was used; the secondary antibody used was a Protein A gold-conjugate (10 nm) to revealed antigen staining.

Techniques: Binding Assay, Control, Flow Cytometry, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Cell Death Discovery

Article Title: Ex.50.T aptamer impairs tumor–stroma cross-talk in breast cancer by targeting gremlin-1

doi: 10.1038/s41420-025-02363-6

Figure Lengend Snippet: A Transmission electron micrographs of isolated EVs from BC and glioblastoma cell lines. Each dot is the binding of the primary antibody to GREM1. The GREM1 level appears highest in BT-549 EVs, followed by MCF7 EVs, and finally U87MG EVs. Scale bars = 100 nm. B Transmission electron micrographs of isolated EVs from BC and glioblastoma cell lines. Each dot is the binding of AuNPs functionalized ex.50.T aptamer. The GREM1 level appears higher in BT-549 EVs, compared to MCF7 EVs and U87MG EVs. Scale bars = 100 nm. C Representative images from confocal microscopy of NFs (NFs) from two patients, exposed to PKH26-labeled, BT-549-derived EVs, pre-incubated with either ex.50.T aptamer or CtrlApt. The image shows reduced EV internalization compared to CtrlApt, as evinced by the decreased merged signal (orange). PKH-26 alone was used as negative control (NT). All images were captured at the same settings, enabling direct comparison of staining patterns. NFs were stained using DAPI (blue) and ALEXA488-conjugated anti-β-actin antibody (green), respectively for nuclei and cytoskeleton detection. Magnification 63x. D Western blots on three different NFs for MMP1 and ITGb1 upon treatment of BT-549-derived-EVs, CtrlApt, and ex.50.T, compared to untreated NFs. β-actin was used as loading control. Right panel densitometric analysis of western blot quantification. Data are presented as mean value ± SD over control (NT) of the three biological replicates. E Representative images from confocal microscopy of FAPa and MCT4 (Green, FITC) immunostaining in NFs, exposed to BT-549-derived EVs, pre-incubated with either ex.50.T aptamer or control aptamer (CtrlApt). Nuclei were stained with DAPI (blu).

Article Snippet: For examination of immuno-gold-labeled EVs isolated from BT459, MCF7, and U87MG cell cultures, the mouse monoclonal antibody sc-515877 directed against gremlin-1 (Santa Cruz Biotechnology, Santa Cruz, CA) was used; the secondary antibody used was a Protein A gold-conjugate (10 nm) to revealed antigen staining.

Techniques: Transmission Assay, Isolation, Binding Assay, Confocal Microscopy, Labeling, Derivative Assay, Incubation, Negative Control, Comparison, Staining, Western Blot, Control, Immunostaining

Journal: Cells

Article Title: Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner

doi: 10.3390/cells13201711

Figure Lengend Snippet: MR1 expression in human cancer cell lines after pulsing with Ac-6-FP.

Article Snippet: HLA-E genes were knocked out in MCF7 cells by using the CRISPR-Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Fluorescence

Journal: Cells

Article Title: Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner

doi: 10.3390/cells13201711

Figure Lengend Snippet: HLA-E expression in human cancer cell lines after treatment with human IFN-γ.

Article Snippet: HLA-E genes were knocked out in MCF7 cells by using the CRISPR-Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Control, Fluorescence

Journal: Cells

Article Title: Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner

doi: 10.3390/cells13201711

Figure Lengend Snippet: Expression of CD1 molecules in human cancer cell lines after treatment with human IFN-γ.

Article Snippet: HLA-E genes were knocked out in MCF7 cells by using the CRISPR-Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Control, Fluorescence

Journal: Cells

Article Title: Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner

doi: 10.3390/cells13201711

Figure Lengend Snippet: The reactivity of TCR-transduced mouse splenic T cells and human PBMCs. ( A ) TCR-expressing mouse splenic T cells were cocultured with IFN-γ-stimulated MCF7 cells. The reactivity against the MCF7 cells was measured by mouse IFN-γ ELISA in triplicate. TCR-negative (TCR-ve) T cells were used as a negative control. (A single experiment is representative of three independent experiments). ( B ) Human PBMCs from healthy donor A were transduced with TCRs and cocultured with IFN-γ-stimulated MCF7 cells. IFN-γ-production in the supernatant was measured by ELISA in triplicate. TCR-negative (TCR-ve) T cells were used as a negative control. A single experiment is representative of 3 independent experiments. The mean and SD values from technical triplicate cultures are indicated. Unpaired, two-tailed t-tests were performed (** p <0.01 and *** p <0.001).

Article Snippet: HLA-E genes were knocked out in MCF7 cells by using the CRISPR-Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Negative Control, Transduction, Two Tailed Test

Journal: Cells

Article Title: Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner

doi: 10.3390/cells13201711

Figure Lengend Snippet: The reactivity of TCR10-59 to MCF7 WT and various KO cells. ( A , B ) TCR10-59 and TCR2-15 were transduced into human PBMCs from healthy individuals, and the cells were cocultured with IFN-γ-stimulated MCF7 cells. TNF-⍺ ( A ) and IFN-γ ( B ) production in the supernatant was measured by ELISA. TCR-negative (TCR-ve) T cells were used as a negative control. T cells stimulated with PMA + ionomycin were used as a positive control. (A single experiment is representative of three independent experiments). ( C ) TCR2-78- or TCR10-59-expressing BW-hCD8⍺β+ cells were cocultured with MR1-overexpressing MCF7WT cells or MCF7ΔMR1 cells, and mouse IL-2-secretion was analyzed by ELISA. The mean and SD values from technical triplicate cultures are indicated. ( D ) PBMCs were retrovirally transduced with conventional (TCR2-15) or unconventional (TCR10-59) TCR genes and cocultured with luciferase-expressing IFN-γ-stimulated MCF7WT, MCF7 WT+B*59:01, and MR1-deleted MCF7 cells at different effector-to-target (E:T) ratios. TCR-negative (TCR-ve) T cells were used as a negative control. The cytotoxic effects were observed by identifying the viable cells by luciferase activity. A single experiment is representative of three independent experiments with similar results. The mean and SD values from technical triplicate cultures are indicated. Unpaired, two-tailed t-tests were performed (** p < 0.01 and *** p < 0.001).

Article Snippet: HLA-E genes were knocked out in MCF7 cells by using the CRISPR-Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control, Expressing, Transduction, Luciferase, Activity Assay, Two Tailed Test

Journal: Cells

Article Title: Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner

doi: 10.3390/cells13201711

Figure Lengend Snippet: The breast cancer cell-specific reactivity of TCR10-59. ( A ) TCR 2-15- and TCR10-59-expressing PBMCs were cocultured with various IFN-γ-stimulated human cancer cell lines (MDA-MB-231 and ZR-75-1 breast cancer cells, Colo205 colon cancer cells, A549 lung cancer cells, K562 myeloid cells, and Karpas 299 lymphoid cells) for 24 h. TCR-untransduced PBMCs were used as a negative control. T cells stimulated with PMA + ionomycin were used as a positive control. ELISA was performed to measure IFN-γ secretion in the supernatants. The mean and SD values from technical triplicate cultures are indicated. ( B ) TCR-transduced human PBMCs from healthy individuals were cocultured with IFN-γ-stimulated MCF7 cells as well as normal breast cells (HMECs). IFN-γ production in the supernatant was measured by ELISA. TCR-negative (TCR-ve) T cells were used as a negative control. For the positive control, T cells stimulated with PMA and ionomycin (P/I) were used. (A single experiment is representative of 2 independent experiments). The mean and SD values from technical triplicate cultures are indicated. Unpaired, two-tailed t -tests were performed (** p < 0.01 and *** p < 0.001).

Article Snippet: HLA-E genes were knocked out in MCF7 cells by using the CRISPR-Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cells

Article Title: Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner

doi: 10.3390/cells13201711

Figure Lengend Snippet: The effects of known microbial MR1 antigens and dependency on K43 MR1-restricted TCRs. TCR2-78- and TCR10-59-expressing BW cells were cocultured with IFN-γ-stimulated MCF7 cells. For stimulation, 5-OP-RU ( A ), and for inhibition, Ac-6-FP ( B ) and anti-MR1 antibody ( C ) were added to the cultures at the indicated concentrations. The dose-dependent responses were assessed by analyzing the IL-2 production in the supernatant. The experiment was performed three times with similar results. ( D ) TCR2-78- and TCR10-59-expressing BW cells were cocultured with IFN-γ-stimulated MCF7WT, MCF7ΔMR1-MR1 K43A , and MCF7ΔMR1-MR1 ++ cells. 5-OP-RU (125 nM) and Ac-6-FP (40 μg mL −1 ) were added to the cultures for the stimulation and inhibition of TCR-expressing BW cells, respectively. The IL-2 production in the supernatant was measured as the assessment of responses. The mean and SD values from technical triplicate cultures are indicated. Unpaired, two-tailed t tests were performed (** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns—not significant).

Article Snippet: HLA-E genes were knocked out in MCF7 cells by using the CRISPR-Cas9 KO plasmid according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Inhibition, Two Tailed Test