Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: Two-component vector system where Cas9 is integrated into the AAVS1 safe harbor locus to sustain stable expression. The second component is a lentiviral system containing a sgRNA targeting on and off-target sites downstream of an AcrIIA4 variant that is uniquely barcoded. B. Editing efficiency measured over time within cells expressing wild-type AcrIIA4 (circular points) versus those without wild-type AcrIIA4 (rectangular). C. Indel mutation profiles spanning the on- and off-target regions. Deletions are shown in blue and insertions are in orange. D. Experimental design in which AcrIIA4 variants were delivered to A549 cells expressing Cas9, which were cultured for 10 days prior to collection and sequencing of the AcrobaTx barcode. E. Structure of the AcrobaTx barcode and definitions of gene editing activity metrics derived from measuring editing outcomes within the on- and off-target sites. F. Editing precision of alanine scanning variants normalized to the wild-type control. D69A variant is highlighted as the variant with the highest precision within the alanine scan mutant group. G. Crystal structure of AcrIIA4 bound to the SpCas9-gRNA complex. The interaction between D69 and R1335, required for PAM recognition, is highlighted. H. Distribution of editing efficiency ( EE , left) and editing precision ( EP , right) across all AcrIIA4 variants. Variants that resulted in EP in the top 90 th percentile are highlighted in red. I. Deletion profiles of DNA repair outcomes from cells without AcrIIA4 (blue) versus cells that express a variant that promotes precise editing (red). J. Editing precision measured across AcrIIA4 variants at Day 10 versus at Day 7 (Pearson Correlation Coefficient r = 0.56).

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Plasmid Preparation, Expressing, Variant Assay, Mutagenesis, Cell Culture, Sequencing, Activity Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: A. Experimental design in which the top 2000 AcrIIA4 variants, rank-ordered by EP , were delivered to A549 and 293T cells for another round of screening. B. Distribution of EP across all sample groups in the second round of screening versus the first round, at left. C. Frequency of editing within the AcrobaTx barcode across seven lead candidate enAcr groups. Yellow rectangles indicate areas in which editing occurs frequently while violet depicts areas with low editing activity. D. Ratio of on- to off-target editing in cells expressing seven representative lead candidate enAcrs (purple) versus ratios observed from DNA repair outcomes within cells expressing previously described benchmark AcrIIA4 variants.

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Activity Assay, Expressing

Journal: Redox Biology

Article Title: Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer

doi: 10.1016/j.redox.2022.102395

Figure Lengend Snippet: (A) 2-ME significantly decreases A549 cell viability in a dose-dependent manner in the 2D culture model at concentrations less than or equal 100 μM. A549 cells were treated with serial dilutions of 2-ME at a concentration range from 100 μM to 0.43 μM for 24 h. Cell viability was determined by MTT assay. Presented values are the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells. (B) 2-ME reduces the cell viability of the A549 3D spheroidal models in a dose dependent manner with expenditures close to 200 μM. A549 cells were exposed to serial dilutions of 2-ME over a concentration range of 200 μM–0.78 μM for 48 h. Cell viability was determined by the WST-1 assay. Presented values are the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells. (C) IC50 values for the A549 cell line treated with 2-ME were >100 μM. These values were calculated by analyzing the relationship between concentrations and percentage (%) of inhibition using GraphPad Prism version 9.0 for Windows, GraphPad Software, CA, USA.

Article Snippet: Wild-type human lung epithelial carcinoma, A549 cell line as 3D cell culture spheroids were obtained by plating cells with an average density of 8000 cells/cm 2 in 96-well U-bottom and V-bottom Lipidure® cell culture plates (Amsbio, Abingdon, United Kingdom) coated with phosphorylcholine which is naturally found in cell membranes.

Techniques: Concentration Assay, MTT Assay, WST-1 Assay, Inhibition, Software

Journal: Redox Biology

Article Title: Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer

doi: 10.1016/j.redox.2022.102395

Figure Lengend Snippet: (A) A549 cells as well as cell medium alone were treated with 2-ME at 10 μM, 1 μM and 10 nM concentrations, respectively. 2-ME treated A549 cells generated higher total hydrogen peroxide levels as compared to untreated control A549 cells incubated with Nutrient Mixture F-12 Ham medium alone. The obtained result comparing ROS induction in the wells with no cells and in the wells with A549 cells, indicates an increased production of ROS by the cancer cells. Luminescence was determined with a Glo-Max® Luminometer from Promega (Mannheim, Germany). Statistical analysis was performed using the GraphPad TTEST function (GraphPad Prism 9 version 9.0.0.). For analysis, both the mean of average luminescence (RLU) and the standard deviation were calculated. (B) Time-dependent growth rate of 2-ME-treated A549 cells. A549 cells treated with 2-ME 10 μM concentration up to 24 h. The analysis of the obtained results evaluated by staining with propidium iodide (PI) was performed using the CytoSMART Analysis System (Lux 3, CytoSMART, Eindhoven, The Netherlands). Values are presented as the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells.

Article Snippet: Wild-type human lung epithelial carcinoma, A549 cell line as 3D cell culture spheroids were obtained by plating cells with an average density of 8000 cells/cm 2 in 96-well U-bottom and V-bottom Lipidure® cell culture plates (Amsbio, Abingdon, United Kingdom) coated with phosphorylcholine which is naturally found in cell membranes.

Techniques: Generated, Incubation, Standard Deviation, Concentration Assay, Staining

Journal: Redox Biology

Article Title: Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer

doi: 10.1016/j.redox.2022.102395

Figure Lengend Snippet: (A). A549 cells were pretreated with V5 peptide at a concentration of 100 μM for 4 h, and then treated with 1 μM 2-ME for 24 h. Values are the mean ± SD of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells. (B) The cells were treated for 24 h with either 100 μM PalmB or 10 μM 2-ME, and a combination of both. Both PalmB and 2-ME alone, significantly decreased A549 cell viability. A synergistic effect on the viability decrease was observed for treatment with both PalmB and 2-ME used in combination. The cell viability was determined by MTT assay. Values are presented as the mean ± SE of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001 vs. control cells C. Serum levels of critical estrogen metabolites analyzed in lung cancer patients (LC) as compared with healthy control (C) are presented as median, as well as quartile 1 and 3, with the minimum and maximum values, in the form of a typical box and whiskers plot. Statistically significant differences are marked with an asterisk * and refer to p-value < 0.05. It is worth noting that 2-ME serum level is significantly lower in lung cancer patients as compared with the control group. Based on table No 2.

Article Snippet: Wild-type human lung epithelial carcinoma, A549 cell line as 3D cell culture spheroids were obtained by plating cells with an average density of 8000 cells/cm 2 in 96-well U-bottom and V-bottom Lipidure® cell culture plates (Amsbio, Abingdon, United Kingdom) coated with phosphorylcholine which is naturally found in cell membranes.

Techniques: Concentration Assay, MTT Assay

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: Quantitation and characterization of tumor-cell derived exosomes. NanoSight and ImageStream analyses of A549-derived exosomes determined size, concentration, and characterization. Human lung cancer cells A549, H358, and non-tumor cells HEK293 were cultured in exosome depleted media for 24 h. Exosomes were isolated from cultured supernatant by using differential centrifugation ( A ) Mean size and concentration of A549 cell-derived exosome determined by NanoSight analysis, mean size was recorded as 182.6 nm, 154.22 nm, and 142.7 nm for A549, H358 and HEK293 cells respectively. ( B ) NanoSight data analysis showing the small size of H358 and HEK293 exosomes compared to A549 cell-derived exosomes. ( C ) NanoSight data analysis showing significantly reduced concentration of exosomes produced from 160 mL of culture media collected of p53 null human lung cancer cells H358 compared to A549 and HEK293 cell exosomes. ( D ) Representative Figure of ImageStream analysis to characterize A549 derived exosomes by expression signals of CD63, CD9, TSG-101, CD81, and EpCAM. ( E ) Percentage of total expression of conventional exosomes markers expressed over A549, H358 and HEK293 cell-exosomes respectively * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Quantitation Assay, Derivative Assay, Concentration Assay, Cell Culture, Isolation, Centrifugation, Produced, Expressing

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: ImageStream and flow cytometry analyses quantify time-dependent internalization of tumor cell-derived exosomes by THP-1 cells. THP-1 cells were seeded and differentiated into M0 macrophages upon overnight stimulation with PMA (20–100 ng/mL). M0 macrophages were then co-cultured with PKH-26 stained A549-derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 to 72 h. Image Stream analysis showing Bright field, CD64 + , PKH-26 + , and composite images. ( A ) Time-dependent internalization of exosomes by CD64 + populations assessed by internalization of PKH-26 stained A549-derived exosomes. CD64 + population, without internalization indicates M0 phenotype. ( B ) MATLAB analysis of percentage of PKH-26 + and CD64 + PKH-26 + signals to show time-dependent internalization of exosomes. Fluorescent signals were collected from 300 cells for each time point. ( C ) Representative flow cytometry of exosome internalization analysis at 24 h and 72 h. Group comparisons of 24 h exosome-, 24 h exosome+, and 72h exosome+ were made. After co-culture, cells were prepared for flow cytometry and stained with CD64, CD11b, and PKH-26. The experiment was repeated twice, with three replicates per sample in each experiment. Exosome- sample was used as control ( D ) Percentage of internalization by THP-1 macrophages as CD11b + CD64 + PKH-26 + showing a significant increase of uptake in 72 h compared to 24 h * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Staining, Co-Culture Assay

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: A549 cell-derived exosomes differentiate non-committed M0 macrophages to M2 phenotype. A549 human lung cancer cells were cultured in exosome depleted media for 24 h. Exosomes were isolated from cultured supernatant using differential centrifugation. Exosomes were stained with PKH-26 for overnight. THP-1 cells were seeded and transformed into M0 macrophages upon overnight stimulation with PMA (20–100 ng/mL). M0 macrophages were then co-cultured with PKH-26 stained, A549 derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 to 72 h. ( A ) Representative flow cytometry plot showing in-vitro induction of M2 phenotype (CD11b + CD64 + CD163 + CD206 + ) with A549-derived exosomes. ( B ) Left to right panel-Total percentage of CD163 + macrophages, showing significant increase in CD163 + macrophages that have internalized PKH + exosomes. Total percentage of CD206 + macrophages, showing significant induction in CD206 + macrophages that have internalized PKH + exosomes. Total percentage of M2 macrophages as CD11b + CD64 + CD163 + CD206 + showing significantly induced M2 phenotypes with exosome-positive sample. ( C ) THP-1 cells were seeded and transformed into M0 macrophages upon overnight stimulation with PMA (20 ng/mL). M0 macrophages were then co-cultured with A549 derived exosomes in 10:1 ratio (10 exosomes/cell) for 24 h M2 gene signature, CHI3L1 (Ym1), IL-10, RETNLB (Fizz1), and Arg1 were upregulated on 24 h of A549-derived exosomes treatment, assessed by qRT-PCR in the cells. ( D ) ImageStream analyses showing CD64 + , PKH-26 + , CD206 + CD163 + macrophages before and after internalization of PKH+ exosomes. Left panel shows composite images from several M0 macrophages without exosome internalization. The right panel shows composite images of M0 macrophages with exosome internalization that have polarized to become M2 (CD206 + CD163 + ) ( E ) Time-dependent increase of M2 polarization induced by A549-derived exosomes showing by percentage of CD163 + , CD64 + CD163 + and PKH-26 + CD163 + analyzed by using MATLAB analysis with the signals collected from 300 cells in each time points. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Derivative Assay, Cell Culture, Isolation, Centrifugation, Staining, Transformation Assay, Flow Cytometry, In Vitro, Quantitative RT-PCR

Journal: Cells

Article Title: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

doi: 10.3390/cells9051303

Figure Lengend Snippet: Alterations in cellular bioenergetics of macrophages co-cultured with Tumor-derived exosomes by extracellular flux analysis. ( A ) Cellular bioenergetic profiles and ( B ) the cellular bioenergetic parameters (Basal OCR, ATP-linked OCR, Proton Leak, Maximal OCR, Reserve Capacity and Non-Mitochondrial OCR) of M0, M1 and M2 macrophages as determined by sequential injection of oligomycin (Oligo), FCCP, antimycin A (AntiA). Mean values from 6–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M1 macrophages. ( C ) Comparison of the cellular bioenergetic profiles of untreated M0 macrophages and those co-cultured with normal cell (HEK293) or tumor cell (A549)–derived exosomes, and ( D ) quantitation of the bioenergetic parameters of C. Mean values from 6–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M0-co-culture with normal cells-derived exosomes. ( E ) Inhibition of nonmitochondrial OCR in nonpermeabilized macrophages using DPI. Mean values from 4–8 replicates with ± sem. # p < 0.005 compared to M0 macrophages; * p < 0.01 compared to M1 macrophages. ( F ) PMP-sensitive mitochondrial OCR in macrophages. Mean values from 3–8 replicates with ± sem. # p < 0.05 relative to M0 macrophages and * p < 0.05 relative to M0-co-culture with normal cells-derived exosomes.

Article Snippet: Exosome-depleted A549 cell culture media (160 mL) were centrifuged at 300× g for 10 min at 4 °C to separate the supernatant from debris.

Techniques: Cell Culture, Derivative Assay, Injection, Comparison, Quantitation Assay, Co-Culture Assay, Inhibition

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