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