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: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

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

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: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

Techniques: Cell Culture, Staining

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: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

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

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: A549-Red-Fluc cells (Perkin-Elmer, Waltham, MA, USA) were intravenously injected into 6-week-old male BALB/c-nu/nu mice.

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

Journal: Journal of Cancer

Article Title: In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC

doi: 10.7150/jca.46320

Figure Lengend Snippet: Effect of 18 on phosphorylation of HER2 and Akt. A549 lung cancer cell lines were incubated with 18 and after 36 h, cells were washed and lysed. Western blot was performed to determine p-HER2 and p-Akt as well as total HER2 and Akt. (Blots were cropped and presented). Quantification of signal intensities were done by ImageJ). Lapatinib was used as positive control. A) Western blot of HER2, p-HER2, Akt, p-Akt. Equal loading of GAPDH was used for comparison. Experiments were repeated three times. Quantification of B) p-HER2 and C) p-Akt using densitometry. Band intensity was represented with scaling from GAPDH band intensity. * p< 0.05, ** p< 0.01.

Article Snippet: Bioware® Brite Cell Line A549 Red-FLuc cells that had been engineered to express firefly luciferase (Perkin Elmer) were injected into the tail vein (4.5 × 10 6 cells in 200 μl PBS per mouse) of 6-week-old Foxn1 nu - nude mice.

Techniques: Phospho-proteomics, Incubation, Western Blot, Positive Control, Comparison

Journal: Journal of Cancer

Article Title: In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC

doi: 10.7150/jca.46320

Figure Lengend Snippet: Therapeutic effect of 18 in NSCLC mice model. NSCLC tumors were induced in mice using luciferase transfected A549 cells. Mice were imaged using an in vivo imaging instrument after injection of luciferin for tumor growth was monitored. Compound 18 was injected via tail vein, and lapatinib (via intraperitoneal injection) was used as a positive control. A) Representative images of tumor growth in mice. Image was processed using software, and ROIs were drawn on the luciferase intensity to quantify the intensity. B) Plot of log transformed total flux representing the tumor volume vs. time in days using box and whisker plot. The total flux (p/s) values were log transformed to meet assumptions of an ANOVA. Compound 18 inhibits the tumor growth in lungs significantly compared to control.

Article Snippet: Bioware® Brite Cell Line A549 Red-FLuc cells that had been engineered to express firefly luciferase (Perkin Elmer) were injected into the tail vein (4.5 × 10 6 cells in 200 μl PBS per mouse) of 6-week-old Foxn1 nu - nude mice.

Techniques: Luciferase, Transfection, In Vivo Imaging, Injection, Positive Control, Software, Transformation Assay, Whisker Assay, Control