human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human nk cell line nk92/product/ATCC
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human nk cell line nk92 - by Bioz Stars, 2024-02
    99/100 stars

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    1) Product Images from "A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets"

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.1089369

    Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Transduction, Flow Cytometry, Incubation

    The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Lysis

    Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.
    Figure Legend Snippet: Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Techniques Used: Cell Culture, Staining

    α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Techniques Used: Labeling, Cell Culture, Fluorescence, Microscopy, Incubation

    α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Techniques Used: In Vivo, Luciferase, Expressing, Imaging, Injection, Ex Vivo, Software

    Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Clone Assay, FACS, Expressing, Co-Culture Assay, Concentration Assay, Fluorescence, Activity Assay

    human il 2 dependent nk cell lines nk 92  (ATCC)


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    ATCC human il 2 dependent nk cell lines nk 92
    Human Il 2 Dependent Nk Cell Lines Nk 92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human il 2 dependent nk cell lines nk 92/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human il 2 dependent nk cell lines nk 92 - by Bioz Stars, 2024-02
    86/100 stars

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    human nk 92 cell line  (ATCC)


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    ATCC human nk 92 cell line
    Cell proliferation curve and density optimization. Three densities of NPC and NPC PDX target cells were automatically monitored every 1-h time interval over 7 days to identify a suitable seeding number and time point for addition of <t>NK-92</t> cells. Results were expressed as CI values. Microscopic images of the cells after 24 h post seeding for (A) HK1, (B) NPC43; after 72 h post seeding for (C) C666-1, (D) C17; 48 h post seeding for (E) B110 and (F) G517. Results were expressed as CI ± SEM values with experimental replicates n = 2 for NPC cell line and n = 3 for NPC PDX cells. 4× objective; scale bar 200 μm.
    Human Nk 92 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human nk 92 cell line/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human nk 92 cell line - by Bioz Stars, 2024-02
    86/100 stars

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    1) Product Images from "Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method"

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2023.e17480

    Cell proliferation curve and density optimization. Three densities of NPC and NPC PDX target cells were automatically monitored every 1-h time interval over 7 days to identify a suitable seeding number and time point for addition of NK-92 cells. Results were expressed as CI values. Microscopic images of the cells after 24 h post seeding for (A) HK1, (B) NPC43; after 72 h post seeding for (C) C666-1, (D) C17; 48 h post seeding for (E) B110 and (F) G517. Results were expressed as CI ± SEM values with experimental replicates n = 2 for NPC cell line and n = 3 for NPC PDX cells. 4× objective; scale bar 200 μm.
    Figure Legend Snippet: Cell proliferation curve and density optimization. Three densities of NPC and NPC PDX target cells were automatically monitored every 1-h time interval over 7 days to identify a suitable seeding number and time point for addition of NK-92 cells. Results were expressed as CI values. Microscopic images of the cells after 24 h post seeding for (A) HK1, (B) NPC43; after 72 h post seeding for (C) C666-1, (D) C17; 48 h post seeding for (E) B110 and (F) G517. Results were expressed as CI ± SEM values with experimental replicates n = 2 for NPC cell line and n = 3 for NPC PDX cells. 4× objective; scale bar 200 μm.

    Techniques Used:

    Optimization of NK-92 cells in co-culture medium. (A) The viability of effector NK-92 cells was measured by CTG assay and (B) the cells were observed for 72 h once the cell culture medium was changed into the co-culture medium (1:1 vol ratio of target cell medium and effector cell medium). 4× objective; scale bar 200 μm.
    Figure Legend Snippet: Optimization of NK-92 cells in co-culture medium. (A) The viability of effector NK-92 cells was measured by CTG assay and (B) the cells were observed for 72 h once the cell culture medium was changed into the co-culture medium (1:1 vol ratio of target cell medium and effector cell medium). 4× objective; scale bar 200 μm.

    Techniques Used: Co-Culture Assay, CTG Assay, Cell Culture

    Effect of co-culture of HK1 and NPC43 cells, respectively with NK-92 cells. Both HK1 and NPC43 cells were seeded in E-plate 96. NK-92 cells were added to the wells 24 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of HK1 and NPC43 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.
    Figure Legend Snippet: Effect of co-culture of HK1 and NPC43 cells, respectively with NK-92 cells. Both HK1 and NPC43 cells were seeded in E-plate 96. NK-92 cells were added to the wells 24 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of HK1 and NPC43 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Techniques Used: Co-Culture Assay

    Effect of co-culture of C666 – 1 and C17 cells, respectively with NK-92 cells. Both C666–1 and C17 cells were seeded in E-plate 96. NK-92 cells were added to the wells 72 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of C666–1 and C17 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.
    Figure Legend Snippet: Effect of co-culture of C666 – 1 and C17 cells, respectively with NK-92 cells. Both C666–1 and C17 cells were seeded in E-plate 96. NK-92 cells were added to the wells 72 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of C666–1 and C17 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Techniques Used: Co-Culture Assay

    Effect of co-culture of B110 and G517 cells, respectively with NK-92 cells. Both B110 and G517 cells were seeded in E-plate 96. NK-92 cells were added to the wells 48 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of B110 and G517 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.
    Figure Legend Snippet: Effect of co-culture of B110 and G517 cells, respectively with NK-92 cells. Both B110 and G517 cells were seeded in E-plate 96. NK-92 cells were added to the wells 48 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of B110 and G517 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Techniques Used: Co-Culture Assay

    Overall NK-92 cytotoxicity effect in NPC cell lines and PDXs. 50% killing time (KT50) of target cells was measured by the RTCA Pro software. Results were expressed as KT50 ± SEM values with experimental replicates n = 2 or 3. T:E ratio of 1:0 is unattainable. One-Way ANOVA result indicates significant difference between individual T:E ratio with control group (T:E 1:0) (*p < 0.05).
    Figure Legend Snippet: Overall NK-92 cytotoxicity effect in NPC cell lines and PDXs. 50% killing time (KT50) of target cells was measured by the RTCA Pro software. Results were expressed as KT50 ± SEM values with experimental replicates n = 2 or 3. T:E ratio of 1:0 is unattainable. One-Way ANOVA result indicates significant difference between individual T:E ratio with control group (T:E 1:0) (*p < 0.05).

    Techniques Used: Software

    GFP-based microscopy. The GFP-transduced target cells (A) HK1, NPC43, (B) C666–1 and C17 were co-cultured with effector NK-92 cells. Images were acquired after 24 and 72 h of co-culture. 10× objective.
    Figure Legend Snippet: GFP-based microscopy. The GFP-transduced target cells (A) HK1, NPC43, (B) C666–1 and C17 were co-cultured with effector NK-92 cells. Images were acquired after 24 and 72 h of co-culture. 10× objective.

    Techniques Used: Microscopy, Cell Culture, Co-Culture Assay

    human nk cell leukemia derived nk 92 cell line  (ATCC)


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    ATCC human nk cell leukemia derived nk 92 cell line
    Human Nk Cell Leukemia Derived Nk 92 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human nk cell line nk 92  (ATCC)


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    ATCC human nk cell line nk 92
    L. indica leaf extract pretreated ovarian cancer cells showed enhanced sensitivity to cytolysis by activated <t>NK</t> <t>cells.</t> OVCAR-5 cells were pretreated overnight with or without 0.3 mg/mL ( A ) crude extract, ( B ) ethyl acetate fraction, ( C ) dichloromethane fraction, ( D ) water soluble fraction and ( E ) water insoluble fraction of L. indica . Subsequently cells were co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents untreated cells, while red line represents treated cells. *** p < 0.001; ns, not significant
    Human Nk Cell Line Nk 92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer"

    Article Title: Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

    Journal: BMC Complementary Medicine and Therapies

    doi: 10.1186/s12906-023-03904-1

    L. indica leaf extract pretreated ovarian cancer cells showed enhanced sensitivity to cytolysis by activated NK cells. OVCAR-5 cells were pretreated overnight with or without 0.3 mg/mL ( A ) crude extract, ( B ) ethyl acetate fraction, ( C ) dichloromethane fraction, ( D ) water soluble fraction and ( E ) water insoluble fraction of L. indica . Subsequently cells were co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents untreated cells, while red line represents treated cells. *** p < 0.001; ns, not significant
    Figure Legend Snippet: L. indica leaf extract pretreated ovarian cancer cells showed enhanced sensitivity to cytolysis by activated NK cells. OVCAR-5 cells were pretreated overnight with or without 0.3 mg/mL ( A ) crude extract, ( B ) ethyl acetate fraction, ( C ) dichloromethane fraction, ( D ) water soluble fraction and ( E ) water insoluble fraction of L. indica . Subsequently cells were co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents untreated cells, while red line represents treated cells. *** p < 0.001; ns, not significant

    Techniques Used: Cell Culture, Cytotoxicity Assay

    Methyl gallate pre-treatment increased susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. OVCAR-5 cells were pretreated overnight with or without ( A ) 0.03 mg/mL isolated gallic acid, ( B ) 0.1 mg/mL isolated methyl gallate, ( C ) 0.03 mg/mL gallic acid standard, ( D ) 0.1 mg/mL methyl gallate standard, ( E ) 20 µM oxaliplatin, and then co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents non-treated cells, while red line represents treated cells. ** p < 0.01; *** p < 0.001; ns, not significant
    Figure Legend Snippet: Methyl gallate pre-treatment increased susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. OVCAR-5 cells were pretreated overnight with or without ( A ) 0.03 mg/mL isolated gallic acid, ( B ) 0.1 mg/mL isolated methyl gallate, ( C ) 0.03 mg/mL gallic acid standard, ( D ) 0.1 mg/mL methyl gallate standard, ( E ) 20 µM oxaliplatin, and then co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents non-treated cells, while red line represents treated cells. ** p < 0.01; *** p < 0.001; ns, not significant

    Techniques Used: Isolation, Cell Culture, Cytotoxicity Assay

    Combination treatment of methyl gallate and oxaliplatin augmented susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. A OVCAR-5 cells were pretreated for 24 h with or without 20 μM oxaliplatin, 10 μM oxaliplatin, 0.1 mg/mL methyl gallate (MG) alone, or combination of 0.1 mg/mL MG and 10 μM oxaliplatin. B SK-OV-3 cells were pretreated for 48 h with or without 50 μM oxaliplatin, 25 μM oxaliplatin, 0.02 mg/mL methyl gallate (MG) alone, or combination of 0.02 mg/mL MG and 25 μM oxaliplatin. Cells were then co-cultured with NK-92 cells at the various E:T ratios. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant
    Figure Legend Snippet: Combination treatment of methyl gallate and oxaliplatin augmented susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. A OVCAR-5 cells were pretreated for 24 h with or without 20 μM oxaliplatin, 10 μM oxaliplatin, 0.1 mg/mL methyl gallate (MG) alone, or combination of 0.1 mg/mL MG and 10 μM oxaliplatin. B SK-OV-3 cells were pretreated for 48 h with or without 50 μM oxaliplatin, 25 μM oxaliplatin, 0.02 mg/mL methyl gallate (MG) alone, or combination of 0.02 mg/mL MG and 25 μM oxaliplatin. Cells were then co-cultured with NK-92 cells at the various E:T ratios. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant

    Techniques Used: Cell Culture

    Combination therapy of methyl gallate and NK-92 cells suppressed re-proliferation of ovarian cancer cells. A , B OVCAR-5 cells were pretreated for 24 h with 0.1 mg/mL methyl gallate (MG), and ( C , D ) SK-OV-3 cells were pre-treated for 48 h with 0.02 mg/mL methyl gallate (MG). Cells were then co-cultured in the presence or absence of NK-92 cells at E:T ratio of 1:4. Untreated cells co-cultured in the presence or absence of NK-92 cells served as control. Cells were counted every 3 days. Values presented are the average ± SD of three independent experiments performed in triplicates. *** p < 0.001
    Figure Legend Snippet: Combination therapy of methyl gallate and NK-92 cells suppressed re-proliferation of ovarian cancer cells. A , B OVCAR-5 cells were pretreated for 24 h with 0.1 mg/mL methyl gallate (MG), and ( C , D ) SK-OV-3 cells were pre-treated for 48 h with 0.02 mg/mL methyl gallate (MG). Cells were then co-cultured in the presence or absence of NK-92 cells at E:T ratio of 1:4. Untreated cells co-cultured in the presence or absence of NK-92 cells served as control. Cells were counted every 3 days. Values presented are the average ± SD of three independent experiments performed in triplicates. *** p < 0.001

    Techniques Used: Cell Culture

    Increased expression of stress ligands for NK cell receptors on ovarian cancer cells after treatment with ( A ) ethyl acetate fraction of L. indica , ( B ) methyl gallate, and ( C ) combination of methyl gallate and oxaliplatin. OVCAR-5 cells were treated for 48 h with or without ( A ) L. indica ethyl acetate fraction (EA, 0.3 mg/mL), ( B ) methyl gallate (MG, 0.1 mg/mL), or ( C ) combination of methyl gallate (MG, 0.1 mg/mL) and oxaliplatin (10 µM), and then phenotype analyzed by FACS for the indicated ligands of NK cells: (a) CD112, (b) CD115, (c) MIC-A/B, (d) ULBP-1, (e) ULBP-2, (f) ULBP-3, (g) DR4 (TRAIL-R1), and (h) DR5 (TRAIL-R2). The relative mean fluorescence intensities of each stress ligand were compared between untreated cells (blue bars) and treated cells (red bars), and results presented are mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; ns, not significant
    Figure Legend Snippet: Increased expression of stress ligands for NK cell receptors on ovarian cancer cells after treatment with ( A ) ethyl acetate fraction of L. indica , ( B ) methyl gallate, and ( C ) combination of methyl gallate and oxaliplatin. OVCAR-5 cells were treated for 48 h with or without ( A ) L. indica ethyl acetate fraction (EA, 0.3 mg/mL), ( B ) methyl gallate (MG, 0.1 mg/mL), or ( C ) combination of methyl gallate (MG, 0.1 mg/mL) and oxaliplatin (10 µM), and then phenotype analyzed by FACS for the indicated ligands of NK cells: (a) CD112, (b) CD115, (c) MIC-A/B, (d) ULBP-1, (e) ULBP-2, (f) ULBP-3, (g) DR4 (TRAIL-R1), and (h) DR5 (TRAIL-R2). The relative mean fluorescence intensities of each stress ligand were compared between untreated cells (blue bars) and treated cells (red bars), and results presented are mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; ns, not significant

    Techniques Used: Expressing, Fluorescence

    human nk cell line nk92  (ATCC)


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    ATCC human nk cell line nk92
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets"

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.1089369

    Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Transduction, Flow Cytometry, Incubation

    The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Expressing, Cell Culture, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Lysis

    Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.
    Figure Legend Snippet: Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Techniques Used: Cell Culture, Staining

    α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Techniques Used: Labeling, Cell Culture, Fluorescence, Microscopy, Incubation

    α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Techniques Used: In Vivo, Luciferase, Expressing, Imaging, Injection, Ex Vivo, Software

    Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Figure Legend Snippet: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Techniques Used: Clone Assay, FACS, Expressing, Co-Culture Assay, Concentration Assay, Fluorescence, Activity Assay

    nk92 egfp cd16 pta 8836 cell lines  (ATCC)


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    ATCC nk92 egfp cd16 pta 8836 cell lines
    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of <t>NK92-CD16-EGFP</t> cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Nk92 Egfp Cd16 Pta 8836 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy"

    Article Title: Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2021-004399

    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Figure Legend Snippet: Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.

    Techniques Used: Derivative Assay, In Vivo, In Vitro, Flow Cytometry, Isolation, Staining, ADCC Assay

    human nk cell line  (ATCC)


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    ATCC human nk cell line
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    human nk 92 cell line  (ATCC)


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    ATCC human nk 92 cell line
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    natural killer nk 92 lineages  (ATCC)


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    ATCC natural killer nk 92 lineages
    Natural Killer Nk 92 Lineages, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human nk cell line  (ATCC)


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    ATCC human nk cell line
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    ATCC human nk cell line nk92
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Nk Cell Line Nk92, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human il 2 dependent nk cell lines nk 92
    Engineering and establishment of <t>α-Cot-NK92</t> cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.
    Human Il 2 Dependent Nk Cell Lines Nk 92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk 92 cell line
    Cell proliferation curve and density optimization. Three densities of NPC and NPC PDX target cells were automatically monitored every 1-h time interval over 7 days to identify a suitable seeding number and time point for addition of <t>NK-92</t> cells. Results were expressed as CI values. Microscopic images of the cells after 24 h post seeding for (A) HK1, (B) NPC43; after 72 h post seeding for (C) C666-1, (D) C17; 48 h post seeding for (E) B110 and (F) G517. Results were expressed as CI ± SEM values with experimental replicates n = 2 for NPC cell line and n = 3 for NPC PDX cells. 4× objective; scale bar 200 μm.
    Human Nk 92 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk cell leukemia derived nk 92 cell line
    Cell proliferation curve and density optimization. Three densities of NPC and NPC PDX target cells were automatically monitored every 1-h time interval over 7 days to identify a suitable seeding number and time point for addition of <t>NK-92</t> cells. Results were expressed as CI values. Microscopic images of the cells after 24 h post seeding for (A) HK1, (B) NPC43; after 72 h post seeding for (C) C666-1, (D) C17; 48 h post seeding for (E) B110 and (F) G517. Results were expressed as CI ± SEM values with experimental replicates n = 2 for NPC cell line and n = 3 for NPC PDX cells. 4× objective; scale bar 200 μm.
    Human Nk Cell Leukemia Derived Nk 92 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk cell line nk 92
    L. indica leaf extract pretreated ovarian cancer cells showed enhanced sensitivity to cytolysis by activated <t>NK</t> <t>cells.</t> OVCAR-5 cells were pretreated overnight with or without 0.3 mg/mL ( A ) crude extract, ( B ) ethyl acetate fraction, ( C ) dichloromethane fraction, ( D ) water soluble fraction and ( E ) water insoluble fraction of L. indica . Subsequently cells were co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents untreated cells, while red line represents treated cells. *** p < 0.001; ns, not significant
    Human Nk Cell Line Nk 92, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC nk92 egfp cd16 pta 8836 cell lines
    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of <t>NK92-CD16-EGFP</t> cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Nk92 Egfp Cd16 Pta 8836 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human nk cell line
    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of <t>NK92-CD16-EGFP</t> cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Human Nk Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC natural killer nk 92 lineages
    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of <t>NK92-CD16-EGFP</t> cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.
    Natural Killer Nk 92 Lineages, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: Engineering and establishment of α-Cot-NK92 cells as a universal CAR system (A) The conventional and universal CAR-NK system. (B) Levels of surface expression of CAR in NK92 or α-Cot-NK92 cells with/without α-HER2-Cot. The α-Cot-NK92 cells were cultured with puromycin for 3 d after α-Cot-CAR lentiviral transduction. Subsequently, transgene expression was evaluated using flow cytometry analysis (FACS) with α-Myc-PE (upper) and α-human IgG-PE conjugated antibody (hIgG, lower) after incubation with/without α-HER2-Cot. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Expressing, Cell Culture, Transduction, Flow Cytometry, Incubation

    The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: The action of α-Cot-NK92 cells depends on the type of conjugator. (A) Expression levels of HER2 and EGFR in various tumor cell lines, including mammary gland adenocarcinoma (AU565), ovarian adenocarcinoma (SK-OV-3), and epidermoid carcinoma (A431), were confirmed through FACS. Histograms are representative of at least three independent experiments. (B) Cytotoxicity against HER2 + , EGFR + , or HER2 + and EGFR + tumor cells mediated by α-Cot-NK92 cells with or without α-HER2-Cot or ZEGFR-Cot. The α-Cot-NK92 cells were co-cultured with calcein-stained tumor cells with or without a conjugator for 4 h at E:T ratios of 5:1, 1:1, and 0.5:1. The intensity of fluorescence emitted from the lysed target cells was measured using a fluorescence plate reader (SpectraMax i3x). All cytotoxicity data are represented as the mean ± S.D. of triplicate experiments. The statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05 vs. vehicle; ns: not significant. (C, D) IFN-γ and TNF-α secretion and CD107a expression in α-Cot-NK92 cells cultured with or without HER2 + EGFR + AU565 (C) or HER2 − EGFR + A431 (D) cells and with/without the conjugator (α-HER2-Cot or ZEGFR-Cot). Secreted IFN-γ and TNF-α levels were measured via ELISA using the medium of α-Cot-NK92 cells co-cultured with target cells at a 1:1 E:T ratio for 12 h. All experiments were performed in triplicate wells for each condition and repeated at least two times. Average values are shown as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t -test. *** P < 0.001; ** P < 0.01; ns: not significant. CD107a expression in α-Cot-NK92 cells was evaluated through FACS analysis after co-culture with target cells at a 5:1 E:T ratio for 4 h. Each value represents the percentage of CD56 + CD107a + cells in flow cytometric density plots. (E) Expression level of HER2 and EGFR in pulmonary adenocarcinoma (A549) confirmed through FACS analysis. Histograms are representative of at least three independent experiments. (F) Kinetics of α-Cot-NK92 cell-mediated tumor cell lysis using the xCELLigence real time cell analysis system. NK92 or α-Cot-NK92 cells were co-cultured with unlabeled A549 cells with/without ZEGFR-Cot at a 5:1 E:T ratio and monitored over time. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Expressing, Cell Culture, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Lysis

    Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR + HER2 − MDA-MB-231 and EGFR − HER2 + MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001; ** P < 0.01; * P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Cell Culture, Staining

    α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: α-Cot-NK92 cells acts in a conjugator-dependent manner at the immune synapse (A) Images depicting the sequential steps in the interaction between α-Cot-NK92 and AU565 cells with or without conjugators (α-HER2-Cot and ZEGFR-Cot). The α-Cot-NK92 and AU565 (HER2 + EGFR + ) cells were labeled with LysoSensor Green and DDAO-SE, respectively, and co-cultured in a medium containing propidium iodide to assess the lytic dynamics of α-Cot-NK92 cells with or without conjugators. Images were acquired using a fluorescence microscope. The snapshots are provided in Supplementary Mov1. (B) Percentage of α-Cot-NK92 cells remaining at each step after 2, 4, or 6 h of co-incubation with AU565 cells with/without conjugators. (C) Duration of the process, and (D) ratio of α-Cot-NK92 cells that kill target cells for target-contacting cells. Data are shown as the mean (red line) ± S.D. Statistical significance of differences between groups was evaluated using paired Student’s t- test. *** P < 0.001. Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Labeling, Cell Culture, Fluorescence, Microscopy, Incubation

    α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: α-Cot-NK92 cells with a conjugator inhibit tumor growth in an in vivo lung cancer model. (A) The schedule of in vivo studies using luciferase-expressing A549-Red-Fluc cells in mouse metastasis model treated with α-Cot-NK92 cells with or without ZEGFR-Cot. (B) Representative in vivo bioluminescence imaging of each group treated with α-Cot-NK92 cells with or without ZEGFR-Cot and Taxol (10 mg/kg body weight) compared to levels in the untreated group (none) on day 57 after tumor cell injection. (C) The tumor burden of each group (n = 10) in in vivo lungs quantified as total flux (photon/s), which was monitored weekly for 57 d after the A549-Red-Fluc injection. (D, E) Representative BLI (D) and total quantitative flux (E) in ex vivo lungs of each group extracted on day 57 after in vivo BLI. *** P < 0.001 vs. none; * P < 0.05 vs. vehicle; Tukey’s multiple comparison test; n.s.: not significant. Bioluminescent images were acquired after an i.p. injection of D -luciferin (150 mg/kg body weight) and analyzed using the Living Image Software. Total flux data were plotted as mean ± SD. Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: In Vivo, Luciferase, Expressing, Imaging, Injection, Ex Vivo, Software

    Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Journal: Frontiers in Immunology

    Article Title: A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets

    doi: 10.3389/fimmu.2022.1089369

    Figure Lengend Snippet: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.

    Article Snippet: The human NK cell line NK92 (CRL-2407; ATCC) was maintained in the ATCC-recommended medium.

    Techniques: Clone Assay, FACS, Expressing, Co-Culture Assay, Concentration Assay, Fluorescence, Activity Assay

    Cell proliferation curve and density optimization. Three densities of NPC and NPC PDX target cells were automatically monitored every 1-h time interval over 7 days to identify a suitable seeding number and time point for addition of NK-92 cells. Results were expressed as CI values. Microscopic images of the cells after 24 h post seeding for (A) HK1, (B) NPC43; after 72 h post seeding for (C) C666-1, (D) C17; 48 h post seeding for (E) B110 and (F) G517. Results were expressed as CI ± SEM values with experimental replicates n = 2 for NPC cell line and n = 3 for NPC PDX cells. 4× objective; scale bar 200 μm.

    Journal: Heliyon

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    doi: 10.1016/j.heliyon.2023.e17480

    Figure Lengend Snippet: Cell proliferation curve and density optimization. Three densities of NPC and NPC PDX target cells were automatically monitored every 1-h time interval over 7 days to identify a suitable seeding number and time point for addition of NK-92 cells. Results were expressed as CI values. Microscopic images of the cells after 24 h post seeding for (A) HK1, (B) NPC43; after 72 h post seeding for (C) C666-1, (D) C17; 48 h post seeding for (E) B110 and (F) G517. Results were expressed as CI ± SEM values with experimental replicates n = 2 for NPC cell line and n = 3 for NPC PDX cells. 4× objective; scale bar 200 μm.

    Article Snippet: Human NK-92 cell line (#CRL-2407™), an interleukin-2 (IL-2)-dependent natural killer cell line derived from a patient with malignant non-Hodgkin’s lymphoma, was obtained from ATCC, USA.

    Techniques:

    Optimization of NK-92 cells in co-culture medium. (A) The viability of effector NK-92 cells was measured by CTG assay and (B) the cells were observed for 72 h once the cell culture medium was changed into the co-culture medium (1:1 vol ratio of target cell medium and effector cell medium). 4× objective; scale bar 200 μm.

    Journal: Heliyon

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    doi: 10.1016/j.heliyon.2023.e17480

    Figure Lengend Snippet: Optimization of NK-92 cells in co-culture medium. (A) The viability of effector NK-92 cells was measured by CTG assay and (B) the cells were observed for 72 h once the cell culture medium was changed into the co-culture medium (1:1 vol ratio of target cell medium and effector cell medium). 4× objective; scale bar 200 μm.

    Article Snippet: Human NK-92 cell line (#CRL-2407™), an interleukin-2 (IL-2)-dependent natural killer cell line derived from a patient with malignant non-Hodgkin’s lymphoma, was obtained from ATCC, USA.

    Techniques: Co-Culture Assay, CTG Assay, Cell Culture

    Effect of co-culture of HK1 and NPC43 cells, respectively with NK-92 cells. Both HK1 and NPC43 cells were seeded in E-plate 96. NK-92 cells were added to the wells 24 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of HK1 and NPC43 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Journal: Heliyon

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    doi: 10.1016/j.heliyon.2023.e17480

    Figure Lengend Snippet: Effect of co-culture of HK1 and NPC43 cells, respectively with NK-92 cells. Both HK1 and NPC43 cells were seeded in E-plate 96. NK-92 cells were added to the wells 24 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of HK1 and NPC43 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Article Snippet: Human NK-92 cell line (#CRL-2407™), an interleukin-2 (IL-2)-dependent natural killer cell line derived from a patient with malignant non-Hodgkin’s lymphoma, was obtained from ATCC, USA.

    Techniques: Co-Culture Assay

    Effect of co-culture of C666 – 1 and C17 cells, respectively with NK-92 cells. Both C666–1 and C17 cells were seeded in E-plate 96. NK-92 cells were added to the wells 72 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of C666–1 and C17 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Journal: Heliyon

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    doi: 10.1016/j.heliyon.2023.e17480

    Figure Lengend Snippet: Effect of co-culture of C666 – 1 and C17 cells, respectively with NK-92 cells. Both C666–1 and C17 cells were seeded in E-plate 96. NK-92 cells were added to the wells 72 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of C666–1 and C17 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Article Snippet: Human NK-92 cell line (#CRL-2407™), an interleukin-2 (IL-2)-dependent natural killer cell line derived from a patient with malignant non-Hodgkin’s lymphoma, was obtained from ATCC, USA.

    Techniques: Co-Culture Assay

    Effect of co-culture of B110 and G517 cells, respectively with NK-92 cells. Both B110 and G517 cells were seeded in E-plate 96. NK-92 cells were added to the wells 48 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of B110 and G517 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Journal: Heliyon

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    doi: 10.1016/j.heliyon.2023.e17480

    Figure Lengend Snippet: Effect of co-culture of B110 and G517 cells, respectively with NK-92 cells. Both B110 and G517 cells were seeded in E-plate 96. NK-92 cells were added to the wells 48 h later. The rate of proliferation was monitored in real-time using xCELLigence system. (A) Cell index plot of B110 and G517 target cells treated with NK-92 cell with different T:E ratios. (B) Individual CI value was normalized to the CI value of T:E 1:0 at the time point of NK-92 addition and (C) normalized CI plot converted to % cytolysis plot. Results were expressed as y ± SEM values with experimental replicates n = 2.

    Article Snippet: Human NK-92 cell line (#CRL-2407™), an interleukin-2 (IL-2)-dependent natural killer cell line derived from a patient with malignant non-Hodgkin’s lymphoma, was obtained from ATCC, USA.

    Techniques: Co-Culture Assay

    Overall NK-92 cytotoxicity effect in NPC cell lines and PDXs. 50% killing time (KT50) of target cells was measured by the RTCA Pro software. Results were expressed as KT50 ± SEM values with experimental replicates n = 2 or 3. T:E ratio of 1:0 is unattainable. One-Way ANOVA result indicates significant difference between individual T:E ratio with control group (T:E 1:0) (*p < 0.05).

    Journal: Heliyon

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    doi: 10.1016/j.heliyon.2023.e17480

    Figure Lengend Snippet: Overall NK-92 cytotoxicity effect in NPC cell lines and PDXs. 50% killing time (KT50) of target cells was measured by the RTCA Pro software. Results were expressed as KT50 ± SEM values with experimental replicates n = 2 or 3. T:E ratio of 1:0 is unattainable. One-Way ANOVA result indicates significant difference between individual T:E ratio with control group (T:E 1:0) (*p < 0.05).

    Article Snippet: Human NK-92 cell line (#CRL-2407™), an interleukin-2 (IL-2)-dependent natural killer cell line derived from a patient with malignant non-Hodgkin’s lymphoma, was obtained from ATCC, USA.

    Techniques: Software

    GFP-based microscopy. The GFP-transduced target cells (A) HK1, NPC43, (B) C666–1 and C17 were co-cultured with effector NK-92 cells. Images were acquired after 24 and 72 h of co-culture. 10× objective.

    Journal: Heliyon

    Article Title: Analysis of NK-92 cytotoxicity in nasopharyngeal carcinoma cell lines and patient-derived xenografts using impedance-based growth method

    doi: 10.1016/j.heliyon.2023.e17480

    Figure Lengend Snippet: GFP-based microscopy. The GFP-transduced target cells (A) HK1, NPC43, (B) C666–1 and C17 were co-cultured with effector NK-92 cells. Images were acquired after 24 and 72 h of co-culture. 10× objective.

    Article Snippet: Human NK-92 cell line (#CRL-2407™), an interleukin-2 (IL-2)-dependent natural killer cell line derived from a patient with malignant non-Hodgkin’s lymphoma, was obtained from ATCC, USA.

    Techniques: Microscopy, Cell Culture, Co-Culture Assay

    L. indica leaf extract pretreated ovarian cancer cells showed enhanced sensitivity to cytolysis by activated NK cells. OVCAR-5 cells were pretreated overnight with or without 0.3 mg/mL ( A ) crude extract, ( B ) ethyl acetate fraction, ( C ) dichloromethane fraction, ( D ) water soluble fraction and ( E ) water insoluble fraction of L. indica . Subsequently cells were co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents untreated cells, while red line represents treated cells. *** p < 0.001; ns, not significant

    Journal: BMC Complementary Medicine and Therapies

    Article Title: Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

    doi: 10.1186/s12906-023-03904-1

    Figure Lengend Snippet: L. indica leaf extract pretreated ovarian cancer cells showed enhanced sensitivity to cytolysis by activated NK cells. OVCAR-5 cells were pretreated overnight with or without 0.3 mg/mL ( A ) crude extract, ( B ) ethyl acetate fraction, ( C ) dichloromethane fraction, ( D ) water soluble fraction and ( E ) water insoluble fraction of L. indica . Subsequently cells were co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents untreated cells, while red line represents treated cells. *** p < 0.001; ns, not significant

    Article Snippet: Human advanced ovarian cancer cell lines OVCAR-5 (NCI Frederick, USA) and SK-OV-3 (ATCC, HTB-77), human monocytic cell line U937 (ATCC, CRL-1593.2), human NK cell line NK-92 (ATCC, CRL-2407) were purchased.

    Techniques: Cell Culture, Cytotoxicity Assay

    Methyl gallate pre-treatment increased susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. OVCAR-5 cells were pretreated overnight with or without ( A ) 0.03 mg/mL isolated gallic acid, ( B ) 0.1 mg/mL isolated methyl gallate, ( C ) 0.03 mg/mL gallic acid standard, ( D ) 0.1 mg/mL methyl gallate standard, ( E ) 20 µM oxaliplatin, and then co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents non-treated cells, while red line represents treated cells. ** p < 0.01; *** p < 0.001; ns, not significant

    Journal: BMC Complementary Medicine and Therapies

    Article Title: Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

    doi: 10.1186/s12906-023-03904-1

    Figure Lengend Snippet: Methyl gallate pre-treatment increased susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. OVCAR-5 cells were pretreated overnight with or without ( A ) 0.03 mg/mL isolated gallic acid, ( B ) 0.1 mg/mL isolated methyl gallate, ( C ) 0.03 mg/mL gallic acid standard, ( D ) 0.1 mg/mL methyl gallate standard, ( E ) 20 µM oxaliplatin, and then co-cultured with activated NK cells at the various E:T ratios. Cytotoxicity assay was determined by measuring the viability of PKH-26 labelled target cancer cells on 96-well plate. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. Blue line represents non-treated cells, while red line represents treated cells. ** p < 0.01; *** p < 0.001; ns, not significant

    Article Snippet: Human advanced ovarian cancer cell lines OVCAR-5 (NCI Frederick, USA) and SK-OV-3 (ATCC, HTB-77), human monocytic cell line U937 (ATCC, CRL-1593.2), human NK cell line NK-92 (ATCC, CRL-2407) were purchased.

    Techniques: Isolation, Cell Culture, Cytotoxicity Assay

    Combination treatment of methyl gallate and oxaliplatin augmented susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. A OVCAR-5 cells were pretreated for 24 h with or without 20 μM oxaliplatin, 10 μM oxaliplatin, 0.1 mg/mL methyl gallate (MG) alone, or combination of 0.1 mg/mL MG and 10 μM oxaliplatin. B SK-OV-3 cells were pretreated for 48 h with or without 50 μM oxaliplatin, 25 μM oxaliplatin, 0.02 mg/mL methyl gallate (MG) alone, or combination of 0.02 mg/mL MG and 25 μM oxaliplatin. Cells were then co-cultured with NK-92 cells at the various E:T ratios. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant

    Journal: BMC Complementary Medicine and Therapies

    Article Title: Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

    doi: 10.1186/s12906-023-03904-1

    Figure Lengend Snippet: Combination treatment of methyl gallate and oxaliplatin augmented susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. A OVCAR-5 cells were pretreated for 24 h with or without 20 μM oxaliplatin, 10 μM oxaliplatin, 0.1 mg/mL methyl gallate (MG) alone, or combination of 0.1 mg/mL MG and 10 μM oxaliplatin. B SK-OV-3 cells were pretreated for 48 h with or without 50 μM oxaliplatin, 25 μM oxaliplatin, 0.02 mg/mL methyl gallate (MG) alone, or combination of 0.02 mg/mL MG and 25 μM oxaliplatin. Cells were then co-cultured with NK-92 cells at the various E:T ratios. Experiment was performed in triplicates. Results from one representative experiment of three are shown and presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant

    Article Snippet: Human advanced ovarian cancer cell lines OVCAR-5 (NCI Frederick, USA) and SK-OV-3 (ATCC, HTB-77), human monocytic cell line U937 (ATCC, CRL-1593.2), human NK cell line NK-92 (ATCC, CRL-2407) were purchased.

    Techniques: Cell Culture

    Combination therapy of methyl gallate and NK-92 cells suppressed re-proliferation of ovarian cancer cells. A , B OVCAR-5 cells were pretreated for 24 h with 0.1 mg/mL methyl gallate (MG), and ( C , D ) SK-OV-3 cells were pre-treated for 48 h with 0.02 mg/mL methyl gallate (MG). Cells were then co-cultured in the presence or absence of NK-92 cells at E:T ratio of 1:4. Untreated cells co-cultured in the presence or absence of NK-92 cells served as control. Cells were counted every 3 days. Values presented are the average ± SD of three independent experiments performed in triplicates. *** p < 0.001

    Journal: BMC Complementary Medicine and Therapies

    Article Title: Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

    doi: 10.1186/s12906-023-03904-1

    Figure Lengend Snippet: Combination therapy of methyl gallate and NK-92 cells suppressed re-proliferation of ovarian cancer cells. A , B OVCAR-5 cells were pretreated for 24 h with 0.1 mg/mL methyl gallate (MG), and ( C , D ) SK-OV-3 cells were pre-treated for 48 h with 0.02 mg/mL methyl gallate (MG). Cells were then co-cultured in the presence or absence of NK-92 cells at E:T ratio of 1:4. Untreated cells co-cultured in the presence or absence of NK-92 cells served as control. Cells were counted every 3 days. Values presented are the average ± SD of three independent experiments performed in triplicates. *** p < 0.001

    Article Snippet: Human advanced ovarian cancer cell lines OVCAR-5 (NCI Frederick, USA) and SK-OV-3 (ATCC, HTB-77), human monocytic cell line U937 (ATCC, CRL-1593.2), human NK cell line NK-92 (ATCC, CRL-2407) were purchased.

    Techniques: Cell Culture

    Increased expression of stress ligands for NK cell receptors on ovarian cancer cells after treatment with ( A ) ethyl acetate fraction of L. indica , ( B ) methyl gallate, and ( C ) combination of methyl gallate and oxaliplatin. OVCAR-5 cells were treated for 48 h with or without ( A ) L. indica ethyl acetate fraction (EA, 0.3 mg/mL), ( B ) methyl gallate (MG, 0.1 mg/mL), or ( C ) combination of methyl gallate (MG, 0.1 mg/mL) and oxaliplatin (10 µM), and then phenotype analyzed by FACS for the indicated ligands of NK cells: (a) CD112, (b) CD115, (c) MIC-A/B, (d) ULBP-1, (e) ULBP-2, (f) ULBP-3, (g) DR4 (TRAIL-R1), and (h) DR5 (TRAIL-R2). The relative mean fluorescence intensities of each stress ligand were compared between untreated cells (blue bars) and treated cells (red bars), and results presented are mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; ns, not significant

    Journal: BMC Complementary Medicine and Therapies

    Article Title: Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer

    doi: 10.1186/s12906-023-03904-1

    Figure Lengend Snippet: Increased expression of stress ligands for NK cell receptors on ovarian cancer cells after treatment with ( A ) ethyl acetate fraction of L. indica , ( B ) methyl gallate, and ( C ) combination of methyl gallate and oxaliplatin. OVCAR-5 cells were treated for 48 h with or without ( A ) L. indica ethyl acetate fraction (EA, 0.3 mg/mL), ( B ) methyl gallate (MG, 0.1 mg/mL), or ( C ) combination of methyl gallate (MG, 0.1 mg/mL) and oxaliplatin (10 µM), and then phenotype analyzed by FACS for the indicated ligands of NK cells: (a) CD112, (b) CD115, (c) MIC-A/B, (d) ULBP-1, (e) ULBP-2, (f) ULBP-3, (g) DR4 (TRAIL-R1), and (h) DR5 (TRAIL-R2). The relative mean fluorescence intensities of each stress ligand were compared between untreated cells (blue bars) and treated cells (red bars), and results presented are mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; ns, not significant

    Article Snippet: Human advanced ovarian cancer cell lines OVCAR-5 (NCI Frederick, USA) and SK-OV-3 (ATCC, HTB-77), human monocytic cell line U937 (ATCC, CRL-1593.2), human NK cell line NK-92 (ATCC, CRL-2407) were purchased.

    Techniques: Expressing, Fluorescence

    Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy

    doi: 10.1136/jitc-2021-004399

    Figure Lengend Snippet: Neuroblastoma-derived sEVs modulate NK cell maturation in vivo and NK cell-mediated ADCC in vitro. (A) Schematic of NK cell subpopulations. Created with BioRender.com. (B) Representative flow cytometry plots of splenic NK cell subpopulations isolated from 9464D-GD2 tumor-bearing mice receiving the indicated treatments as described in . (C) Quantification of the percentage of immature splenic NK cell (CD27 +CD11b-; left panel) and mature NK cell (CD27- CD11b+; right panel) subpopulations in 9464D-GD2 tumor-bearing mice treated as described in . Mean±SEM, n=7 per group. Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. (D–G) Human (IMR32) or murine (9464D-GD2) neuroblastoma cells treated as indicated in the presence or absence of NK92-CD16-EGFP cells (NK) and monitored for viability utilizing cell impermeant nucleic acid stain YOYO-3 with the IncuCyte S3 Live-Cell Analysis System. The cell-by-cell analysis module was used to quantify viable tumor cells (YOYO3- EGFP-). (D) Kinetic analysis of the IMR32 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=6. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (E) Percentage of viable IMR32 cells at 24 hours, n=6. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. **p<0.01; ***p<0.001. (F) Kinetic analysis of the 9464D-GD2 in vitro NK-cell-mediated ADCC assay. Mean±SEM, n=4. Viable cells calculated as percentage of viable cells in each treatment condition divided by viable cells in untreated control group. (G) Percentage of viable 9464D-GD2 cells at 24 hours, n=4. Mean±SD. One-way ANOVA with Tukey’s/Sidak’s post hoc tests. *p<0.05; **p<0.01; ***p<0.001. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; DN, double negative; DP, double positive; sEV, small extracellular vesicle.

    Article Snippet: Human IMR32 neuroblastoma (CCL-127), HEK 293T/17 (CRL-11268), and NK92-EGFP-CD16 (PTA-8836) cell lines were purchased from ATCC.

    Techniques: Derivative Assay, In Vivo, In Vitro, Flow Cytometry, Isolation, Staining, ADCC Assay