Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Local RNA in situ hybridization. ( A ) Schematic overview of a microfluidic implementation of chromogenic RNA in situ hybridization. The initial preparation of the tissue section including paraffin removal (1), heat (2) and protease (3) pretreatment, as well as fixation was performed globally. The primary probe was then hybridized locally to the RNA of interest (4). In this case the detection of HER2 is shown with internal negative ( dapB ) and positive ( ActB ) controls. Finally, the amplification of the signal using preamplifier and amplifier strands (5), the binding of enzyme (6), and the enzymatic reactions (7) were performed globally on the whole slide. ( B ) Fluorescence images of RNA-ISH experiments detecting the gene expression of HER2 in cell pellet sections of the breast cancer cell lines MCF7 (left), SKBR3 (center) and BT474 (right). Probes directed against bacterial dapB and ActB were used as negative and positive controls, respectively. Scale bar: 100 μm.

Article Snippet: FFPE cell blocks of the cell lines MCF7, BT474 and SKBR3 (AMS Biotechnology, Abingdon, UK) with HER2 expression levels of 1+, 2+ and 3+, respectively, were used as references.

Techniques: RNA In Situ Hybridization, Amplification, Binding Assay, Fluorescence, Expressing

Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Detection of HER2 expression status by RNA in situ hybridization using internal positive and negative controls. ( A ) Fluorescence images of the signals detected for HER2 , dapB , and ActB in a mammary carcinoma section. Scale bar: 100 μm. ( B ) Quantitative analysis of the fluorescence intensity of FISH signals detected for HER2 , dapB , and ActB integrated per sectioned cell for FFPE sections of the breast cancer cell lines MCF7, SKBR3, and BT474 (see Figure ) as well as the mammary carcinoma from (A). 100 ms excitation.

Article Snippet: FFPE cell blocks of the cell lines MCF7, BT474 and SKBR3 (AMS Biotechnology, Abingdon, UK) with HER2 expression levels of 1+, 2+ and 3+, respectively, were used as references.

Techniques: Expressing, RNA In Situ Hybridization, Fluorescence

Journal: Nucleic Acids Research

Article Title: Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

doi: 10.1093/nar/gkz1151

Figure Lengend Snippet: Single color multiplexed RNA-ISH for the detection of breast cancer biomarkers. ( A ) The primary probes for ER , PgR , and HER2 were delivered to spatially distinct regions of a mammary carcinoma section using a microfluidic chip (schematic in upper left corner). Fluorescence images of the signal detected for the breast cancer biomarkers ER , PgR and HER2 in a mammary carcinoma section. Scale bar: 100 μm. ( B ) Fluorescence intensity of FISH signals detected for ER , PgR and HER2 integrated per sectioned cell for FFPE sections of the breast cancer cell lines MCF7, SKBR3 and BT474 (see ) as well as the mammary carcinoma from (A). 500 ms excitation.

Article Snippet: FFPE cell blocks of the cell lines MCF7, BT474 and SKBR3 (AMS Biotechnology, Abingdon, UK) with HER2 expression levels of 1+, 2+ and 3+, respectively, were used as references.

Techniques: Fluorescence

Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia concentration is increased in cancer cell–conditioned medium and TIF. Ammonia concentration in the tumor-conditioned medium, collected after 48 hours of incubation, was measured using a Dimension Ammonia assay (Siemens; n = 3). Cells were cultured at different densities: lymphoma (0.5 × 10 6 , 1.0 × 10 6 , 1.5 × 10 6 /mL; A ), multiple myeloma (1 × 10 6 , 2 × 10 6 , 4 × 10 6 /mL; B ), and breast cancer cell lines (0.5 × 10 6 , 1 × 10 6 , 1.5 × 10 6 /mL; C ). In A–C , empty medium incubated for 48 hours (without the cells) at 4°C or 37°C is presented as a control. D, Schematic presentation of the TIF and SCF isolation from mice. TIF was collected from tumors not exceeding 1,500 mm 3 . SCF was isolated at the same time from the contralateral tight as a control tissue fluid. E–G, The concentration of ammonia in TIF and SCF isolated from Raji tumor–bearing NSG ( n = 5; E ), MM.1s tumor–bearing SCID mice ( n = 5; F ), and breast cancer–bearing mice (EMT6 in BALB/c mice, n = 5; E0771 in BALB/c mice, n = 5; 4T1 in BALB/c mice; n = 5; MCF7 in NSG mice, n = 4; MDA-MB-231 in NSG mice, n = 3; G ). P values were calculated using a paired t test. Data show individual values and means ± SEM. n values are the number of biological replicates in in vitro experiments or the number of mice used to obtain the data. D, Created with BioRender.com. Winiarska, M. (2025) https://BioRender.com/x56a982 .

Article Snippet: In the case of the experiments involving MCF7 cells, slow-release pellets containing 17β-estradiol (Innovative Research of America) were implanted subcutaneously 4 days before tumor cell inoculation.

Techniques: Concentration Assay, Incubation, Cell Culture, Control, Isolation, In Vitro

Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia inhibits natural cytotoxicity and ADCC of NK cells and CAR NK cells. A, The viability of NK cells incubated with different concentrations of NH 4 Cl for 4 hours was assessed using propidium iodide staining and flow cytometry ( n = 3). B, Natural cytotoxicity of NK cells against K562 cells in the presence of different concentrations of NH 4 Cl ( n = 3). K562 cells were stained with CFSE and incubated with NK cells in different concentrations of ammonia. C, RTX-dependent cell cytotoxicity of NK cells against Raji cells in the presence of different concentrations of NH 4 Cl ( n = 4). Raji cells were stained with CFSE and incubated with NK cells and 100 μg/mL RTX in different concentrations of ammonia. D, Daratumumab (Dara)-dependent cell cytotoxicity of NK cells against Daudi cells in the presence of different concentrations of NH 4 Cl ( n = 5). Daudi cells were stained with CFSE and incubated with NK cells and 1 μg/mL daratumumab in different concentrations of ammonia. In B–D , cytotoxicity was assessed after 4 hours using flow cytometry and plotted as the percentage of propidium iodide–positive CFSE-positive target tumor cells. E, Trastuzumab-dependent cell cytotoxicity of NK cells against MCF7 cells in the presence of different concentrations of NH 4 Cl ( n = 4). Cytotoxicity was assessed using RTCA for 12 hours. The right panel presents the normalized cell index at the 12-hour time point. F, CD19 CAR NK cell cytotoxicity against Raji cells in the presence of different concentrations of NH 4 Cl ( n = 3). Cytotoxicity was determined after 18 hours in a luciferase-based killing assay, with Raji cells stably expressing luciferase as target cells. G, PD-L1 CAR NK cell cytotoxicity against MDA-MB-231 cells in the presence of 5 mmol/L NH 4 Cl ( n = 3). Cytotoxicity was assessed using RTCA for 24 hours. P values were calculated using two-way ANOVA with Tukey post hoc test. Data show individual values and means ± SEM. n values are the numbers of biological replicates in in vitro experiments.

Article Snippet: In the case of the experiments involving MCF7 cells, slow-release pellets containing 17β-estradiol (Innovative Research of America) were implanted subcutaneously 4 days before tumor cell inoculation.

Techniques: Incubation, Staining, Flow Cytometry, Luciferase, Stable Transfection, Expressing, In Vitro

Journal: Frontiers in Oncology

Article Title: A tumor cell specific Zona Pellucida glycoprotein 3 RNA transcript encodes an intracellular cancer antigen

doi: 10.3389/fonc.2023.1233039

Figure Lengend Snippet: ZP3-Cancer is the dominant transcript in tumor cells and is differentially distributed among cancer types. (A) Expression of the seven annotated ZP3 transcripts in CCls (n=1339). Bars represent Tukey box plots (boxes are median+IQR). Median transcript expression levels (from left to right) are 2.47, 0.22, 0.0, 0.04, 0.42, 0.0 and 0.0 TPM. ZP3-Cancer expression is significantly higher compared to all other transcripts (Kruskal-Wallis test, p < 0.0001). NC = Non-protein coding. (B) Relative expression of ZP3-cancer and ZP3-Oocyte in the CCLs MCF7 and HeLa, and in healthy ovarian tissue containing follicles, as determined by qPCR. ZP3-Cancer expression is set to 1 for MCF7 and HeLa, ZP3-Oocyte expression is set to 1 for ovarian tissue. ‘ZP3-both’ served as a positive control and used primers designed to detect ZP3-Cancer and ZP3-Oocyte simultaneously. Bars represent mean+SD for the triplicate qPCR reactions. (C) Differential expression of ZP3-Cancer in the cancer types covered by the CCLs. Cancer types represented by less than 5 CCLs were not included. Bars represent Tukey box plots (boxes are median+IQR). Number of samples are indicated between brackets. The shaded area indicates CCLs with a ZP3-Cancer expression level below the overall median (2.54 TPM).

Article Snippet: Total RNA extracted from the breast cancer cell line MCF7 (R1255830-50) and the cervical cancer cell line HeLa (R1255811-50) was purchased from Amsbio (Abingdon, UK).

Techniques: Expressing, Positive Control