Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: Identification and characterization of eccDNA in rat skin induced by ionizing radiation. A) Flowchart for eccDNA purification and sequencing ( n = 4 for each group). B) Quantification of unique eccDNA. C) Distribution of unique eccDNA lengths. D) Overlap of eccDNA across sample groups. E) Genomic origins of eccDNA. F) Identification of three eccDNAs using PCR and Sanger sequencing, with circle 17:44148731‐48208624 (4059.8 Kb) present in all samples (CE: crude eccDNA, EE: exonuclease‐treated eccDNA). G) Flowchart of semiquantitative analysis of PCR by gel electrophoresis on the eccDNA gene. H) PCR detected five genes on circle 17:44148731‐48208624 . I) Flowchart for eccDNA pre‐treatment of rats with radiation‐induced skin injury ( n = 3 for each group). J) Vps41 protein expression in rat skin 3 days post eccDNA transfection. K) Skin damage photos at 8, 40, and 65 days post‐irradiation in eccDNA‐pre‐treated rats (4 µg injection; scale bar: 1 cm). L) Radiation damage scores and affected areas in eccDNA pre‐treated rats. M,N) Immunofluorescence and analysis of inflammatory factors (IL‐6, IL‐10, TNF‐α) in irradiated skin of eccDNA‐pre‐treated rats (scale bar: 100 µm). O) Inflammatory cytokine array detection in eccDNA‐treated WS1 cells ( n = 5 per group). P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons, one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons, and limma's empirical Bayes moderated t‐statistics for high‐throughput expression data. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Purification, Sequencing, Nucleic Acid Electrophoresis, Expressing, Transfection, Irradiation, Injection, Immunofluorescence, MANN-WHITNEY, High Throughput Screening Assay

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: eccDNA drives increased VPS41 expression. A) Diagram of Circle 17:44148731‐48208624 in rat skin. B) Validation of the PS enzyme for linear DNA removal. C) Standard curve for semiquantitative analysis of PCR by gel electrophoresis on the eccDNA gene. D) PCR analysis of RALA and VPS41 amplification in HaCaT cells post‐irradiation. E) Gel electrophoresis shows VPS41 copy number on gDNA remains unchanged after irradiation. F) Elevated mRNA and protein expression of VPS41 in HaCaT cells after irradiation. G) Vps41 protein expression in various tissues after 4 Gy (X‐ray) total body irradiation in rats. H) Vps41 expression (Log 2 Fold Change) from scRNA‐Seq data of irradiated rat skin ( n = 4 per group). I) Relative expression levels of Vps41 (Log 2 Fold Change) in different cell types in scRNA‐Seq data of irradiated rat skin. J) Immunohistochemical analysis of VPS41 expression in a patient with clinical radiation‐induced skin injury (scale bar: 100 µm). K) Significant upregulation of VPS41 mRNA levels after transfection of HaCaT cells‐derived eccDNA into WS1 cells. L) Increased VPS41 expression after HaCaT cells‐derived eccDNA transfection into HEK‐293T cells. M) PCR analysis suggests intact gene expression elements for VPS41 on eccDNA. N) Nuclear‐cytoplasmic separation and semiquantitative analysis of PCR by gel electrophoresis reveal VPS41 gene localization on eccDNA. O) Apoptosis rate of WS1 cells transfected with total eccDNA after irradiation. P) Apoptosis rate of WS1 cells transfected with purified eccDNA after irradiation. Q) UVB and paclitaxel treatment effects on VPS41 expression in skin cells (HaCaT and WS1). R) Semiquantitative analysis of PCR by gel electrophoresis of DNA damage inducers and inhibitors on eccDNA VPS41 amplification in HaCaT cells. Treatments include IR (6 Gy), UVB (20 mJ/cm 2 ), etoposide (2 µ m ), paclitaxel (20 n m ), and cisplatin (2 µ m ). P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Biomarker Discovery, Nucleic Acid Electrophoresis, Amplification, Irradiation, Immunohistochemical staining, Transfection, Derivative Assay, Gene Expression, Purification, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: VPS41 upregulation confers radioprotective effects at the cellular level. A) VPS41 protein localization after EGFP‐VPS41 plasmid transfection in HaCaT and WS1 cells (Hoechst: blue, EGFP: green, LysoTracker: red; scale bar: 20 µm). B) Western blot showing VPS41 plasmid overexpression efficiency in HaCaT and WS1 cells. C) ROS levels in HaCaT cells post‐irradiation after VPS41 plasmid transfection. D,E) Effect of VPS41 plasmid transfection on γH2AX levels in HaCaT cells after irradiation (scale bar: 20 µm). F) Cell viability in HaCaT and WS1 cells post‐irradiation with VPS41 plasmid transfection. G) LDH release in HaCaT and WS1 cells following irradiation and VPS41 plasmid transfection. H) Colony formation rate in HaCaT cells post‐irradiation with VPS41 plasmid. I) Reduced apoptosis in HaCaT and WS1 cells post‐irradiation after VPS41 plasmid transfection. J) Western blot showing shVPS41 knockdown efficiency in HaCaT and WS1 cells. K) Decreased irradiated cell viability after shVPS41 infection in HaCaT and WS1 cells. L) Increased LDH release in irradiated HaCaT and WS1 cells post‐shVPS41 infection. M) Elevated apoptosis rate in irradiated HaCaT and WS1 cells after shVPS41 infection. N) Reduced colony formation in irradiated HaCaT cells after shVPS41 infection. P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Plasmid Preparation, Transfection, Western Blot, Over Expression, Irradiation, Knockdown, Infection, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: Therapeutic effects of AAV‐Vps41 on radiation‐induced skin injuries in rats. A) Flowchart for skin radiation injury in rats pre‐treated with AAV‐Vps41 for 16 days ( n = 4 per group). B) Increased Vps41 protein expression in rat skin one month post AAV‐Vps41 infection. C) Photographs of skin radiation injury in AAV‐Vps41 pre‐treated rats on days 12, 44, and 72 post‐irradiation (scale bar: 1 cm). D) Radiation injury score statistics in AAV‐Vps41 pre‐treated rats. E) Analysis of the area of skin radiation injury in AAV‐Vps41‐treated rats. F) HE staining showing tissue resistance to ionizing radiation in AAV‐Vps41 pre‐treated rats (45 Gy for 72 days), scale bar 250 µm. G,H) Immunofluorescence detection and analysis of IL‐6, IL‐10, TNF‐α in irradiated skin of AAV‐Vps41 pre‐treated rats (scale bar: 100 µm). I) Schematic diagram of inflammatory factor chip detection in AAV‐Vps41 pre‐treated WS1 cells. J) GO classification of inflammatory factor chip results in AAV‐Vps41 pre‐treated WS1 cells. K) Heat Map of inflammatory factor chip results in AAV‐Vps41 pre‐treated WS1 cells. P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons, Fisher's exact test for GO enrichment analysis, and limma's empirical Bayes moderated t‐statistics for protein expression data. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Infection, Irradiation, Staining, Immunofluorescence, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: VPS41 negatively regulates KAI1 expression through the lysosomal pathway to confer resistance to apoptosis. A) Flowchart for screening VPS41 interaction proteins via differential protein analysis and mass spectrometry after VPS41 upregulation post‐irradiation. B) Volcano plot showing proteomic analysis (VPS41 vs Vector). C) Electron microscopy analysis reveals inhibited apoptosis progression in cells with upregulated VPS41 after irradiation (scale bar: 5 µm). D) Intersection of differential proteins identified four candidates: ISG15, KAI1, IFT20, and ATPAF1. E) Co‐localization of VPS41‐EGFP and KAI1‐BFP plasmids in WS1 and HaCaT cells assessed by confocal microscopy (scale bar: 20 µm). F) Immunoprecipitation confirms VPS41 binds KAI1. G) PNGase F treatment has minimal effect on VPS41‐KAI1 interaction. H) Western Blot shows upregulation of VPS41 decreases KAI1 expression, suppressing apoptosis, while VPS41 downregulation increases KAI1 expression and enhances apoptosis. I) VPS41 and KAI1 expression changes in HaCaT cells treated with CQ (20µ m ) or MG‐132 (20 µ m ) combined with X‐ray (10 Gy). J) Analysis of KAI1 decay rate after CHX (300 µ m ) treatment and X‐ray (10 Gy) in HaCaT cells. K) Effect of VPS41 knockdown and eccDNA transfection on apoptosis rates in irradiated cells with or without KAI1 overexpression. L) Apoptosis testing shows KAI1 reverses VPS41‐mediated radiation resistance. M) LDH measurement assesses the role of KAI1 in reversing VPS41‐mediated radiation resistance. P values were calculated using different statistical methods based on data type: unpaired two‐tailed t test for differential protein analysis and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified. [Correction added on 28 April 2025, after first online publication: figure 5 is updated in this version].

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Mass Spectrometry, Irradiation, Plasmid Preparation, Electron Microscopy, Confocal Microscopy, Immunoprecipitation, Western Blot, Knockdown, Transfection, Over Expression, Two Tailed Test

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: The interaction between VPS41 and KAI1 is critical for the radioprotection of VPS41. A) AlphaFold 3 prediction of structural domains for VPS41 and KAI1. B) IP experiments validate interaction domains between truncated VPS41 and KAI1 after transfection of various VPS41 truncation plasmids into HEK‐293T cells. C) IP experiments verify interaction domains between truncated KAI1 and VPS41 after transfection of KAI1 truncation plasmids into HEK‐293T cells. D) Co‐transfection of VPS41_WT‐EGFP and truncated variants with KAI1‐BFP in WS1 cells, followed by confocal microscopy to assess co‐localization (scale bar: 20 µm). E) The interaction between VPS41 and KAI1 remains unaffected by CQ treatment, which inhibits endosome and lysosome fusion. F) Apoptosis assays investigate the effects of truncated VPS41 on radiation‐induced apoptosis in HEK‐293T cells. G) Apoptosis assays assess the impact of different KAI1 truncation variants on radiation‐induced apoptosis in HEK‐293T cells. H) AlphaFold 3.0 predicts interaction sites of VPS41‐1‐286 and KAI1‐Δ111‐228. I) Schematic diagram of peptide array experiment. J) ECL imaging results of KAI‐Δ111‐228 peptide array. K) Peptide array and AlphaFold 3.0 analyze protein binding sites. L) Conservation of KAI1 binding peptide containing K263 among species. P values were calculated using one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Transfection, Cotransfection, Confocal Microscopy, Peptide Microarray, Imaging, Protein Binding, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: siRNA-Targeted Genes Affecting the A-T Parameter as Ranked by Z Score

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: Hierarchical clustering of high-content and high-throughput morphologic parameters with siRNA-targeted genes defines gene–morphology clusters. ( A ) Hierarchical clustering of both siRNA-targeted genes (side, vertical), at A-T parameter Z > 2 as the cut-off value, and associated cell morphologic parameters (n = 32, top horizontal) measured by In Cell Investigator suite, highlights 12 distinct morphologic clusters. ( B ) Z score bar chart of genes (A-T parameter Z ≤ 2) ordered by location within hierarchical clusters. ( C ) Zoomed screening images of cell shapes from selected siRNA-targeted genes ( KIF11 , GPS1 , RRM2 , PRC1 , CDC6 , CTGF , BTG4 , NOL1 , AXIN1 , MYO9B , UBE3A , PPP1R10 ) clusters. Control nontargeting siRNA (siNT) and GATA6 represent negative and positive screening controls, respectively. Green and red pseudocolorization represent α-tubulin and F-actin staining, respectively. Nc, nucleus.

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques: High Throughput Screening Assay, Control, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: Gene Membership of Cluster From Hierarchical Clustering in Figure 2

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: Changes in cellular morphology impacts cellular migration. ( A ) Changes in BE-HGD cell morphology as indicated by screening data were confirmed in independent experiments (n = 3) using an alternate siRNA format to that of the original screen (siOn-target plus). ( B ) Scratch-wound assays performed in confluent cells (n = 6) transfected with siRNAs targeting RRM2, ITGB8, GPS1, NOL1, MYO9B, and SPRY1. ( C ) Representative images of significantly affected wells. ( D ) Cell count and ( E ) cell viability after silencing of RRM2, ITGB8, GPS1, NOL1, MYO9B, and SPRY1. Negative, negative control growth media without fetal calf serum; Positive, positive control, epidermal growth factors in culture media. ∗ P < .01, ∗∗ P < .001, and ∗∗∗ P < .0001. NS, not significant in Student t testing.

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques: Migration, Transfection, Cell Counting, Negative Control, Positive Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: Low pH exposure, simulating GERD and tumor-associated environments, suppresses GPS1 and RRM2 expression in a temporal fashion. ( A ) Matrix heatmap of GPS1 and RRM2 expression in previously published gene expression microarray data of low pH-mediated signaling in SKGT4 cells. ( B ) Independent validation of GPS1 and RRM2 expression (n = 3) levels after continuous exposure to pH 6.5 in SKGT4 cells, ( C ) simulating a hypoxic tumor core with low pH over 4 hours or pulsatile exposure of BE-HGD cells simulating a reflux event (pH 4.5 for 10 min) followed by measurement over 8 hours. ∗ P < .01, ∗∗ P < .001, and ∗∗∗ P < .0001 in Mann–Whitney nonparametric testing. Rel., relative.

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques: Expressing, Gene Expression, Microarray, Biomarker Discovery, Reflux, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: GPS1 silencing results in consistent cell phenotype and β-catenin accumulation without affecting the cell cycle. Individual cell values from ( Ai ) morphologic A-T and ( Aii ) organelle inclusion/cell intensity parameters in siGPS1-transfected (n = 1059 individual cells) and nontargeting siRNA (n = 2818) wells from HCA data from an independent validation study. ( B ) Flow cytometric analysis of cell-cycle dynamics after silencing of GPS1 shows no changes from nontargeting control transfected cells. ( C and D ) Expression levels of GPS1, β-catenin, and actin (loading control) by Western blot after siRNA-mediated silencing of GPS1 in ( C ) BE-HGD CP-D cells and ( D ) SKGT4 EAC cells over 96 hours. ( E and F ) Densitometry of proteins normalized to actin loading control levels from 3 replicate experiments. ∗ P < .01, ∗∗ P < .001, and ∗∗∗ P < .0001 in Student t testing.

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques: Transfection, Biomarker Discovery, Control, Expressing, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: ( A and B ) Cortactin-positive pseudopodia formation is increased in GPS1-silenced BE-HGD cells. Confocal imaging of nucleus, β-tubulin, and F-actin in ( A ) control nontargeting or ( B ) GPS1-silenced cells. Expanded panels show zoomed composite image of all 4 panels with arrows highlighting nested tubulin, microtubule organizing center (MTOC) formation, nonfocal F-actin, and polymerized F-actin in pseudopodia extensions. ( C and D ) Confocal imaging of F-actin and cortactin immunostaining in ( C ) control nontargeting siRNA-transfected and ( D ) GPS1-silenced cells. Expanded panel shows zoomed composite image of all 4 panels from GPS1-silenced cells; arrow shows co-localization of F-actin with cortactin in pseudopodia-like protrusions. ( E ) Graphic representation (created with BioRender.com ) of nonmotile and motile cells where the MTOC is localized behind a leading-edge protrusion filled with polymerized F-actin and colocalized cortactin.

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques: Imaging, Control, Immunostaining, Transfection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

doi: 10.1016/j.jcmgh.2020.05.002

Figure Lengend Snippet: GPS1 suppresses cellular locomotive velocity and accumulated distances in BE-HGD and EAC cells. Individual live cell tracking data in BE-HGD CP-D cells from live imaging of ( A ) control nontargeting (siNT) and ( B ) GPS1-silenced (siGPS1) cells. ( C ) Velocity and ( D ) accumulated distances of BE-HGD CP-D cells from live imaging of control transfected (siNT) or cells after GPS1 silencing. Individual cell tracking data of SKGT4 EAC cells transfected with ( E ) control nontargeting (siNT) or ( F ) GPS1 targeting (siGPS1) siRNAs. ( G ) Velocity and ( H ) accumulated distances of SKGT4 EAC cells after GPS1 silencing or nontargeting transfection. ∗∗∗∗ P < .00001 in Student t testing.

Article Snippet: GPS1 (Hs00358804_m1), RRM1 (Hs01040698_m1), RRM2 (Hs00357247_g1), and RRM2B (Hs00968432_m1) primers also were used.

Techniques: Cell Tracking Assay, Imaging, Control, Transfection

Journal: Immunity

Article Title: Influenza vaccine format mediates distinct cellular and antibody responses in human immune organoids

doi: 10.1016/j.immuni.2023.06.019

Figure Lengend Snippet: Influenza vaccine modality influences antibody magnitude, specificity, and breadth (A) Heatmap of antibody-binding magnitude against influenza proteins on a high-throughput protein microarray. Culture supernatants are from day 14 organoids. Column dendrogram represents unbiased sample grouping based on similarity; top bar color represents the antigen stimulation. Rows represent individual proteins on the microarray and were manually arranged based on influenza strain origin and protein type. (B) Summary antibody data from the protein microarray by protein type and virus source. Data represent median values. Each point is an individual donor. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using a Kruskal-Wallis test followed by paired Mann-Whitney U tests to compare groups. p values shown are for comparisons against the unstimulated control unless otherwise indicated by lines. Boxplots show the median, with hinges indicating the first and third quartiles and whiskers indicating the highest and lowest value within 1.5 times the interquartile range of the hinges. (C) Detection of cross-reactive Ab production from IIV- vs. LAIV-stimulated tonsil organoids. Each donor is a row. The numbers of protein antigens from H1N1 strains that circulated prior to each donor’s birth are shown to the right of the plot. Abs produced from the organoids were classified as either present or absent in the culture supernatants. (D) Protein targets from Ab responses unique to LAIV-stimulated organoids. NA, neuraminidase; NS, nonstructural; NP, nucleoprotein. The number of strains uniquely targeted by Abs from LAIV stimulation are shown in doughnut centers. (E) Organoid Ab responses to nonseasonal influenza strain proteins on the microarray. Ab presence or absence was classified and plotted as in ( C ) . n = 12 donors for all analyses (1 experiment). See also Tables 1 , , and .

Article Snippet: Influenza A H1N1 (A/California/07/2009) Nucleoprotein / NP Protein , Sino Biologicals , cat # 40205-V08B.

Techniques: Binding Assay, High Throughput Screening Assay, Microarray, MANN-WHITNEY, Produced

Journal: Immunity

Article Title: Influenza vaccine format mediates distinct cellular and antibody responses in human immune organoids

doi: 10.1016/j.immuni.2023.06.019

Figure Lengend Snippet: IIV and LAIV elicit distinct BCR repertoires and transcriptional profiles (A) Kinetics of HA + B cells with a given transcriptional profile in tonsil organoids. Phenotypes were defined based on scRNA-seq and manual annotation of cluster identities. Activated, Act. 1, Act. 2, and GC-entry clusters. Memory, MemB, Act. MemB, and FCRL4 Pre-MemB clusters. GC, LZ and DZ-like clusters. PB, PB1 and PB2 clusters. (B) BCR isotype usage in HA + memory, GC, and PB B cells at different time points. (C) BCR SHM frequencies of HA + B cell subsets following organoid stimulation with IIV, LAIV, or H1N1. Unpaired Mann-Whitney U tests were used to compare groups; ∗p < 0.05 and ∗∗∗∗p < 0.0001. (D) V gene usage of HA + plasmablasts following stimulation with different influenza antigens on day 7. (E) Shannon index as a metric of BCR diversity for HA+ plasmablasts. Boxplots show the median, with hinges indicating the first and third quartiles and whiskers indicating the highest and lowest value within 1.5 times the interquartile range of the hinges. (F) Representative data (1 of 4 donors) of the number and size of HA + BCR clonal families. Each wedge represents a clonal family. n = 4 donors (1 experiment) for all data. See also Table 1 and Figure S3 .

Article Snippet: Influenza A H1N1 (A/California/07/2009) Nucleoprotein / NP Protein , Sino Biologicals , cat # 40205-V08B.

Techniques: MANN-WHITNEY

Journal: Immunity

Article Title: Influenza vaccine format mediates distinct cellular and antibody responses in human immune organoids

doi: 10.1016/j.immuni.2023.06.019

Figure Lengend Snippet:

Article Snippet: Influenza A H1N1 (A/California/07/2009) Nucleoprotein / NP Protein , Sino Biologicals , cat # 40205-V08B.

Techniques: Recombinant, Plasmid Preparation, Amplification, Software

Journal: Journal of Cancer

Article Title: LINC00852 Promotes Lung Adenocarcinoma Spinal Metastasis by Targeting S100A9

doi: 10.7150/jca.26897

Figure Lengend Snippet: LINC00852 and its target S100A9 are overexpressed in human lung adenocarcinoma spinal metastasis (SM). (A) Clinical samples were collected and analyzed by immunohistochemical staining with S100A9 antibody (1:200). Representative staining is shown (400×). (B) Results of western blotting and real-time PCR assays showing that S100A9 was overexpressed in SM; *P < 0.05, **P < 0.01. (C,D) Results of lncRNA target prediction and signal pathway prediction using high-throughput microarray screening and the Kyoto Encyclopedia of Genes and Genomes (KEGG), showing that LINC00852 expression and MAPK pathway were enriched in SM tissues. (E) Results of real-time PCR assay showing that LINC00852 is overexpressed in cancer tissue of SM; **P < 0.01. (F) Duration of survival in patients with high LINC00852 expression was shorter than that of patients with low LINC00852 expression; however, the difference was not statistically significant. (G,H) RNA pull-down verifying LINC00852 binding to S100A9.

Article Snippet: The main primary antibodies were S100A9 (1:1000) (Abcam, Cambridge, MA, USA), ERK1/2 MAPK (1:2000), p-ERK1/2 MAPK (1:2000), P38 MAPK (1:1000), p-P38 MAPK (1:1000), SAKP/JNK (1:1000), p-SAKP/JNK (1:1000) (CST, Danvers, MA, USA), and GAPDH (1:5000) (Beyotime, Shanghai, China).

Techniques: Immunohistochemical staining, Staining, Western Blot, Real-time Polymerase Chain Reaction, High Throughput Screening Assay, Microarray, Expressing, Binding Assay

Journal: Journal of Cancer

Article Title: LINC00852 Promotes Lung Adenocarcinoma Spinal Metastasis by Targeting S100A9

doi: 10.7150/jca.26897

Figure Lengend Snippet: S100A9 activates the MAPK pathway. (A,B) P38, JNK, ERK, and their phosphorylation expression levels in transfected A549 and SPC-A1 cells were determined by western blotting. (C,D) A549 and SPC-A1 cells were transfected with or without LV-S100A9 and pretreated without or with SB203580 (0.5 μM or 5 μM for 24 hours), respectively. Expression levels of p-P38 and P38 were determined by western blotting; *P < 0.05; **P < 0.01. (E,F) A549 and SPC-A1 cells were transfected with or without LV-S100A9 and pretreated without or with PD98059 (1 μM or 10 μM for 24 hours), respectively; expression levels of p-ERK1/2 and ERK1/2 were determined by western blotting; *P < 0.05.

Article Snippet: The main primary antibodies were S100A9 (1:1000) (Abcam, Cambridge, MA, USA), ERK1/2 MAPK (1:2000), p-ERK1/2 MAPK (1:2000), P38 MAPK (1:1000), p-P38 MAPK (1:1000), SAKP/JNK (1:1000), p-SAKP/JNK (1:1000) (CST, Danvers, MA, USA), and GAPDH (1:5000) (Beyotime, Shanghai, China).

Techniques: Phospho-proteomics, Expressing, Transfection, Western Blot

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Localization of clusterin in normal human lung. Clusterin was detected immunohistochemically by staining formalin-fixed, paraffin embedded 3 μm sections of human control lung tissue. Representative images of clusterin (clu, A-C, brown/red, nuclei - blue) and elastic fibers (( D ), grey/black) in tissue obtained from control lung (n = 3). Clusterin localizes to fibroblast-like cells ( A ), to small areas of bronchial epithelial cells ( B ) and to elastic fibers in blood vessels and alveolar walls ( C , D ) serial sections). Clusterin was not detectable in macrophages, alveolar epithelial cells ( A ) or endothelial cells ( C ). Different cell populations/structures are indicated by arrows: f - fibroblast-like cell, m - macrophage, e – alveolar epithelial cell, be - bronchial epithelial cell, en - endothelial cell, smc – smooth muscle cell, ef - elastic fibers. Scale bar represents 25 µm.

Article Snippet: Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 μg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 μg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT.

Techniques: Staining, Formalin-fixed Paraffin-Embedded, Control

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Localization of clusterin in IPF lung. Immunohistochemical staining for clusterin was performed on formalin-fixed, paraffin embedded 3 μm sections of IPF lung tissue (n = 3). Clusterin staining (clu, A, C-G, I brown/red, nuclei - blue) and staining for elastin (H, J, EvG, grey/black) in representative tissue sections. Clusterin is undetectable in αSMA positive myofibroblasts ( A,B ), forming and in cells overlying fibroblastic foci ( A,C ), compared to strong staining of fibroblast-like cells in morphologically normal non-fibrotic areas ( D ). Clusterin was observed sporadically in bronchial epithelial cells but more frequently than in controls ( E ). Similar to control lung, clusterin colocalized with elastin ( G,H ) and was undetectable in macrophages, smooth muscle and endothelial cells ( F,G ). Clusterin also colocalized with amorphous elastin aggregates in dense fibrotic regions ( I,J ). Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, be - bronchial epithelial cell, en – endothelial cell, smc – smooth muscle cell, ef- elastic fibers. Scale bar represents 25 µm ( A–J ).

Article Snippet: Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 μg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 μg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT.

Techniques: Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Control

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Clusterin gene expression and protein levels are decreased in fibrotic compared with control lung fibroblasts. Fibroblasts isolated from human control and fibrotic lung were grown in monolayer culture and clusterin mRNA and protein levels were detected via microarray, proteome profiler and immunofluoresecence analysis. ( A ) Microarray analysis of mRNA shows decreased clusterin gene expression in fibroblasts derived from fibrotic lungs (open circles; n = 5 IPF and 7 SSc) compared with controls (closed circles; n = 6). Proteome profiler array analysis ( B , clusterin - CLU) and immunofluorescence staining of control and fibrotic lung fibroblasts ( C,D ) confirms low clusterin protein expression in fibrotic compared with control fibroblasts in vitr o. ( E ) Semi-quantitative analysis of clusterin staining ( C,D ), clusterin signal (pixel intensity) normalized to cell numbers per visual field (n = 6). Data is representative of three individual experiments. * P < 0.05, **** P < 0.0001; Scale bar in D represents 10 µm.

Article Snippet: Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 μg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 μg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT.

Techniques: Gene Expression, Control, Isolation, Microarray, Derivative Assay, Immunofluorescence, Staining, Expressing

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: TGF-β 1 associates with areas of decreased clusterin expression in fibrotic lung and down-regulates fibroblast clusterin mRNA and protein expression in vitro . Serial sections prepared from IPF lung (n = 3) were stained immunohistochemically to localize TGF-β 1 staining (( A ), red/brown, nuclei blue) and clusterin (( B ), red/brown, nuclei blue) in fibroblastic foci. ( A,B ) Representative images of immunohistochemical staining suggest that TGF-β 1 localizes to ECM, fibroblasts and macrophages, whilst staining for clusterin is weak or undetectable. ( C ) In vitro analysis of clusterin mRNA levels in TGF-β 1 stimulated (40 pM) human lung fibroblasts was performed via qRT-PCR and shows a time-dependent down-regulation of clusterin expression that was maximal at 24–48 h (n = 3) in response to TGF-β 1 . Clusterin protein levels were also down-regulated in response to TGF-β 1 (40 pM) compared to control at 24 h and 48 h as demonstrated by western blotting at 24 h ( D ) and immunofluorescent staining at 48 h (E, red, nuclei - blue). ( F ) Semi-quantitative analysis of fluorescent signal of panel E: clusterin signal (pixel intensity) was normalized to cell numbers per visual field and compared to control (n = 6). Full-length western blots are presented in Supplementary Figure . Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, ecm – extracellular matrix. * P < 0.05, ** P < 0.01; scale bar 100 µm (A) and 10 µm ( E ).

Article Snippet: Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 μg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 μg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT.

Techniques: Expressing, In Vitro, Staining, Immunohistochemical staining, Quantitative RT-PCR, Control, Western Blot

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Effect of clusterin deficiency on TGF-β 1 -induced myofibroblast differentiation and collagen deposition. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untransfected (black bars). αSMA mRNA was assessed via qRT-PCR ( A ) and clusterin and αSMA protein levels detected by western blotting ( B,C quantification). 48 h following TGF-β 1 stimulation (40 pM) αSMA mRNA and protein were increased. Basal and increased levels of αSMA mRNA and protein, however, did not vary between clusterin deficient, mock-transduced and control fibroblasts. Collagen mRNA ( D ) and deposition ( E , F quantification) assessed by qRT-PCR and immunofluorescence staining were significantly increased in response to TGF-β 1 . Although, basal and TGF-β 1 induced changes in collagen levels varied between clusterin deficient, mock and control fibroblasts, the overall fold-increase in collagen mRNA and deposition levels did not significantly change between clusterin deficient fibroblasts and control/mock fibroblasts. Data generated in A-F is representative of two individual experiments with fibroblasts derived from 2 donors. Full-length western blots are presented in Supplementary Figure . Scale bar in E represents 10 µm. ** P < 0.01, *** P < 0.001 compared with untreated controls respectively.

Article Snippet: Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 μg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 μg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT.

Techniques: Transduction, shRNA, Quantitative RT-PCR, Western Blot, Control, Immunofluorescence, Staining, Generated, Derivative Assay

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Effect of clusterin deficiency on lung fibroblast proliferation.Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untreated (black bars). Proliferation in shRNA-mediated clusterin deficient fibroblasts ( A ) or fibrotic lung fibroblasts ( B ) compared with controls was assessed in response to the indicated stimuli for 48 h or 72 h for FBS by counting DAPI-positive nuclei in a high-throughput immunofluorescence assay. Cell numbers were normalized to cell counts of controls (0.4% FBS in DMEM) and expressed as percent change in proliferation relative to control (n = 6). Data representatives of two individual experiments with fibroblasts derived from 1 donor per group. Significances compared with controls are marked with (#) symbol and significances between controls vs. shCLU or non-fibrotic vs. fibrotic lung fibroblasts are indicated with (*). */# P < 0.05, **/## P < 0.01, ***/ ### P < 0.001, ****/ #### P < 0.0001.

Article Snippet: Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 μg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 μg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT.

Techniques: Transduction, shRNA, High Throughput Screening Assay, Immunofluorescence, Control, Derivative Assay

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Effect of clusterin deficiency on apoptosis. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open circles) and mock shRNA vectors (grey circles) or remained untransfected (black circles). Lung fibroblasts were seeded and treated with FasL (3 nM – 6 nM) and/or exogenous clusterin (CLU, 125 nM) for 19 h or remained untreated. Apoptotic cells (Annexin V+ and Annexin V+/ DAPI+ cells) were assessed via FACS analysis post staining of apoptotic cells with annexin V – Alexa647 and DAPI (mean ± SEM, n = 5). ( A ) shRNA-induced clusterin deficiency sensitized fibroblasts to basal and FasL-induced apoptosis, and this could be overcome by addition of exogenous clusterin ( B ). ( C ) Basal apoptosis in representative control (9.37 ± 0.63%) and fibrotic (11.6 ± 0.69%) lung fibroblast isolates was not significantly different. Fibrotic lung fibroblasts were more resistant to FasL-induced apoptosis compared with controls ( C ) and exogenous clusterin tends to reduce basal and FasL-induced apoptotic levels further ( D ). Data representative of at least two individual experiments with fibroblasts derived from 1 donor per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 μg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 μg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT.

Techniques: Transduction, shRNA, Staining, Control, Derivative Assay

Journal: Biomedical Optics Express

Article Title: Multiplexed COVID-19 antibody quantification from human sera using label-free nanoplasmonic biosensors

doi: 10.1364/BOE.454919

Figure Lengend Snippet: Imaging-based nanoplasmonic biosensor for multiplexed anti-SARS-CoV-2 antibody detection. (a) Schematic of a hyperspectral wide-field optical imaging platform showing a microarray-functionalized plasmonic Au nanohole array (Au-NHA) biosensor. Nanoplasmonic chip is illuminated with a tunable light source and imaged with a CMOS camera to acquire high-resolution spectral information from each camera pixel. (b) Hyperspectral datacube enables flexible probing wavelength ( λ λ p ) selection for optimized intensity interrogation of the plasmonic resonance peak shifts that are induced by antibody binding to the viral antigens immobilized in a microarray on the sensor surface. Note that spectral information is not necessary, as detection information can be retrieved by probing wavelength ( λ λ p ) in the form of intensity contrast, Δ Δ I(λ λ p ), which enables simpler optical reader setups. (c) Single-step label-free biorecognition assay to quantify multiple antigen-specific antibodies simultaneously. Plasmonic sensor chips are functionalized with SARS-CoV-2 Nucleocapsid (N), Spike (S) proteins, and anti-bovine antibodies for negative control in a microarray pattern. (d) A large-area nanoplasmonic sensor chip functionalized with high-throughput microarray printing technology. (e) A single image showing a representative field-of-view that can capture 49 detection spots at once. (f) Schematic and photographs demonstrate the plasmonic sensor integrated fluidic cartridge that can take low-volume blood samples, filter blood, and transfer plasma into biosensing chamber rapidly using a low-cost plunger system, which is critical for point-of-care deployment.

Article Snippet: SARS-CoV-2 Nucleocapsid (N) and Spike (S) proteins (Sinobiological, Beijing, People’s Republic of China) were prepared through dilution from 1 mg/mL stock solution to 150 µg/mL with 1x phosphate buffer saline (PBS), and Milli-Q Water, respectively; both solvents contain 0.5% trehalose (m/v) and 0.005% Tween20 (v/v).

Techniques: Imaging, Optical Imaging, Microarray, Selection, Binding Assay, Negative Control, High Throughput Screening Assay

Journal: Cells

Article Title: Identification and Characterization of Fully Human FOLR1-Targeting CAR T Cells for the Treatment of Ovarian Cancer

doi: 10.3390/cells13221880

Figure Lengend Snippet: Overview of the identification and selection workflow for novel, fully human CAR T cell lead candidates. Initial panning and ELISA screening of two naïve human scFv phage display libraries resulted in >2266 candidates. After sequence analysis, 189 human/mouse cross-reactive anti-FOLR1 candidate binders were thereby identified. Flow cytometric screening of the binder candidates against human/murine FOLR variant-expressing cells was performed to select FOLR1-specific candidates. The 20 functional binders were characterized in two parallel approaches. In the first approach, the 20 functional binders were converted into CAR sequences and used to assess functionality, specificity, and cell expansion via a high-throughput CAR T cell screening approach. Finally, four candidates were selected for further advanced in vitro analysis. In the second approach, the 20 functional binders were used in a scFv-Fc format in a tissue cross-reactivity study employing ultra-high content imaging to assess potential on-target and off-target binding on human tissue samples. Finally, one anti-FOLR1 CAR lead candidate was selected based on the performance in both approaches.

Article Snippet: On the day of assay use, the number of viable CAR T cells was determined by staining them with 7-AAD, biotinylated human FOLR1 Protein (Miltenyi Biotec, Bergisch Gladbach, Germany), and an anti-biotin antibody conjugated to PE.

Techniques: Selection, Enzyme-linked Immunosorbent Assay, Sequencing, Variant Assay, Expressing, Functional Assay, High Throughput Screening Assay, In Vitro, Imaging, Binding Assay

Journal: Cells

Article Title: Identification and Characterization of Fully Human FOLR1-Targeting CAR T Cells for the Treatment of Ovarian Cancer

doi: 10.3390/cells13221880

Figure Lengend Snippet: Flow cytometric analysis of binders in the scFv-Fc format identifies candidates enabling specific staining of FOLR1-expressing cells compared to other FOLR variants. ( A ) Following generation of candidates in scFv-Fc format, binders were validated on Jurkat cells expressing hFOLR1 or mFOLR1, respectively. Flow cytometric measurements were performed in triplicates and are displayed as mean frequency of stained cells. Lead candidates are marked in red. Binders that do not meet the selection criteria are marked in grey. ( B ) Venn diagram indicating the number of clones meeting the respective criteria for each cell line staining. Staining of hFOLR1- and mFOLR1-expressing Jurkat cells was considered positive if frequencies >20% were detected. Staining of non-target cells expressing either hFOLR2, mFOLR2, hFOLR4, or mFOLR4, respectively, was considered negative if frequencies <1% were detected. In total, 87 binders were analyzed.

Article Snippet: On the day of assay use, the number of viable CAR T cells was determined by staining them with 7-AAD, biotinylated human FOLR1 Protein (Miltenyi Biotec, Bergisch Gladbach, Germany), and an anti-biotin antibody conjugated to PE.

Techniques: Staining, Expressing, Selection, Clone Assay

Journal: Cells

Article Title: Identification and Characterization of Fully Human FOLR1-Targeting CAR T Cells for the Treatment of Ovarian Cancer

doi: 10.3390/cells13221880

Figure Lengend Snippet: CAR T cell screening reveals functional, FOLR1-specific CAR T cell candidates in vitro. ( A ) Workflow scheme to identify functional and specific CAR T cells in serial in vitro co-culture assays. Read-outs included real-time live cell analysis, quantification of cytokine secretion after repeated addition of target cells, and flow cytometric quantification of transduced T cells at the end of each round of co-culture. ( B ) Lysis of ovarian cancer cells (OV-90) expressing FOLR1 by four CAR candidates of three different donors. GFP-expressing OV-90 target cells were seeded and co-cultured with anti-FOLR1 CAR T cells to measure antigen-dependent lysis of ovarian cancer cells. After 92 h of co-culture, fresh OV-90 cells were added, followed by a second addition of OV-90 cells after 164 h. Target cell lysis by the CAR T cell candidates was analyzed by decrease in GFP signal over time by measuring the Green Calibrated Units (GCU) per µm 2 /image for 11 days. As negative controls, OV-90 target cells were either cultured without addition of CAR T cells or co-cultured with untransduced T cells. Two positive control CAR T cell constructs, known to successfully induce FOLR1-specific lysis of target cells, were included. Error bars indicate standard deviation (SD) of respective mean values. ( C ) CAR T cell expansion of all candidates from three different donors as CD3 + LNGFR + cells/well after 92 h, 164 h, as well as 260 h of co-culture, respectively. Cell counts were determined by flow cytometry before each addition of target cells, and at the endpoint, respectively. Dotted line indicates 50,000 transduced T cells used as CAR T cell input on day 0. No significant differences were found amongst CAR T cell preparations at the end of round 1 and 2. However, at the end of round 3, anti-FOLR1 CARs 6, 9, 10, 11, 13, 17, 19, and 20 resulted in significantly lower proliferative responses compared to positive control 1. ( D ) IFN-γ secretion in repeated co-cultures 24 h after each addition of OV-90 cells to candidate CAR T cells. No significant differences in IFN-γ secretion amongst CAR T cell preparations were observed for anti-FOLR1 CARs 1, 2, 4, 5, 7, 12, 14, 16, and 18 compared to positive control 1 in round 1. All other CAR T samples expressed significantly lower IFN-γ. In round 2, amounts of IFN-γ secreted by anti-FOLR1 CAR T cells 4, 12, 14, and 18 were not significant compared to positive control 1. All other CAR T cell preparations produced significantly lower IFN-γ. Finally, in round 3, no differences were identified for anti-FOLR1 CARs 1, 2, 4, 5, 7, 12, 14, and 18, whereas all other CAR constructs mediated significantly lower cytokine production by T cells. Statistical analysis was performed by Dunnett post hoc analysis after 1-way ANOVA. Abbreviations in figure: untransduced (UTD) and positive control (PC).

Article Snippet: On the day of assay use, the number of viable CAR T cells was determined by staining them with 7-AAD, biotinylated human FOLR1 Protein (Miltenyi Biotec, Bergisch Gladbach, Germany), and an anti-biotin antibody conjugated to PE.

Techniques: Functional Assay, In Vitro, Co-Culture Assay, Cell Analysis, Lysis, Expressing, Cell Culture, Positive Control, Construct, Standard Deviation, Flow Cytometry, Produced

Journal: Cells

Article Title: Identification and Characterization of Fully Human FOLR1-Targeting CAR T Cells for the Treatment of Ovarian Cancer

doi: 10.3390/cells13221880

Figure Lengend Snippet: Advanced in vitro CAR T cell assays identify functional lead anti-FOLR1 CAR T cell candidate. ( A ) Schematic CAR architecture of the anti-FOLR1 CAR T cell candidates consisting of a FOLR1-directed scFv, CD8 hinge, CD8 transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ activation domain, respectively. ( B ) Workflow scheme of advanced in vitro assays. GFP-expressing ovarian cancer cells were seeded and co-cultured with anti-FOLR1 CAR T cells (effector cell-to-target cell ratio of 1:2) to measure antigen-dependent lysis of ovarian cancer cells for five days. After 48 h of co-culture, fresh ovarian cancer cells were added to the co-culture. Cell lysis by the CAR T cell candidates was measured by changes in the green area confluency. After 48 h and 108 h, respectively, activation and exhaustion marker expression were analyzed by flow cytometry and cytokine secretion was assessed with the MACSPlex Cytokine 12 Kit. ( C ) Representative killing assay of four anti-FOLR1 CAR candidates co-cultured with FOLR1-proficient, GFP-expressing ovarian cancer cells (OV-90, left) and FOLR1-deficient, GFP-expressing ovarian cancer cells (OV-90 FOLR1 KO, right). Data points represent mean values and standard deviation (SD) is indicated ( n = 3). ( D ) Expression of activation markers CD25, CD69, and 4-1BB at the indicated time points was analyzed by flow cytometry (three donors). Bars represent mean values and SD is depicted ( n = 3). Statistical Analysis: Two-way ANOVA a = 0.05, ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. ( E ) Concentration of secreted cytokines in supernatant from co-culture with OV-90 cells at the indicated time points was analyzed with the MACSPlex Cytokine 12 Kit. Bars represent mean values, and SD is depicted ( n = 3). Crosses indicate values above the upper detection limit.

Article Snippet: On the day of assay use, the number of viable CAR T cells was determined by staining them with 7-AAD, biotinylated human FOLR1 Protein (Miltenyi Biotec, Bergisch Gladbach, Germany), and an anti-biotin antibody conjugated to PE.

Techniques: In Vitro, Functional Assay, Activation Assay, Expressing, Cell Culture, Lysis, Co-Culture Assay, Marker, Flow Cytometry, Standard Deviation, Concentration Assay

Journal: Cells

Article Title: Identification and Characterization of Fully Human FOLR1-Targeting CAR T Cells for the Treatment of Ovarian Cancer

doi: 10.3390/cells13221880

Figure Lengend Snippet: Tissue cross-reactivity workflow identifies anti-FOLR1 binder candidates’ on-/off-target and on-tumor binding profiles. ( A ) Overview of the tissue cross-reactivity workflow and the individual steps. ( B ) Gating strategy for on-/off-target binding analysis of new scFv-Fc binders. Thresholds are based on the mean intensity value of the negative reference. Cells color-coded cyan are defined as on-target binding, whereas red-labeled cells are defined as off-target binding. ( C ) Example of cell segmentation workflow for on-/off-target binding analysis according to gating strategy in ( B ). Nuclear borders identified from DAPI signal are marked in blue, whereas cell cytoplasms are filled in grey. The color-code for cell gating is adapted from gating strategy shown in ( B ). ( D ) Exemplary representation of cell segmentation and on-/off-target gating of binder candidates 8 and 12 (scFv-Fc 8 and scFv-Fc 12, respectively). The staining was performed on FOLR1-proficient cell lines ( FOLR1 knock-in (Jurkat FOLR1 KI), top row), and FOLR1-deficient cell lines (FOLR1 knock-out (OV-90 FOLR1 KO, middle row). Additionally, primary OvCa tissue (bottom row) was segmented, and binders were analyzed for on-tumor reactivity. ( E ) Quantification of the on- and off-target binding frequency of all binder candidates on cell lines (Jurkat FOLR1 KI and OV-90 FOLR1 KO, respectively) and primary OvCa tissue. Thresholds (dotted line) for evaluation of binder candidate specificity was set to >50% for on-target binding, and <10% for off-target binding. ( F ) Venn diagram summarizes on-target (>50% staining frequency of Jurkat FOLR1 KI cells), off-target (<10% staining frequency of OV-90 FOLR1 KO cells), and on-tumor reactivity (>50% staining frequency of tumor cells within primary ovarian cancer tissue) of new CAR binder candidates. Taken together, six candidates match the requirements. All scale bars represent 100 µm.

Article Snippet: On the day of assay use, the number of viable CAR T cells was determined by staining them with 7-AAD, biotinylated human FOLR1 Protein (Miltenyi Biotec, Bergisch Gladbach, Germany), and an anti-biotin antibody conjugated to PE.

Techniques: Binding Assay, Labeling, Staining, Knock-In, Knock-Out

Journal: Cells

Article Title: Identification and Characterization of Fully Human FOLR1-Targeting CAR T Cells for the Treatment of Ovarian Cancer

doi: 10.3390/cells13221880

Figure Lengend Snippet: Tissue cross-reactivity workflow identifies anti-FOLR1 binder candidates’ off-tumor binding profile. ( A ) Exemplary representation of selected healthy tissues (breast, lung, kidney, brain) from tissue micro array analysis employing anti-FOLR1 scFv-Fc candidates 5 and 12. Cell segmentation workflow and gating for on-/off-target binding analysis was applied according to B. ( B ) Quantification of the off-tumor binding frequency of all binder candidates on breast, lung, kidney, and brain tissue, with total numbers of analyzed cells 1576, 6202, 7494, and 1324, accordingly. Thresholds (dotted line) for evaluation of binder candidate specificity was set to <10% off-tumor binding. ( C ) Venn diagram summarizes off-tumor reactivity on healthy tissues (<10% staining frequency of cells) of new binder candidates. Taken together, 11 candidates match the requirements. Scale bar represents 100 µm.

Article Snippet: On the day of assay use, the number of viable CAR T cells was determined by staining them with 7-AAD, biotinylated human FOLR1 Protein (Miltenyi Biotec, Bergisch Gladbach, Germany), and an anti-biotin antibody conjugated to PE.

Techniques: Binding Assay, Microarray, Staining