Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: EGFRvIII expression in WT HNSCC cell lines. A Schematic structure of EGFR and EGFRvIII. The EGFRvIII variant is characterized by an in-frame deletion of exons 2–7 in the full-length EGFR gene. The domains of EGFR are shown, labeled domains L1 (also known as domain 1), CR1 (domain 2), L2 (domain 3) and CR2 (domain 4), respectively (adapted from Gan et al. 2009 ). B RT-PCR analysis of EGFRvIII mRNA expression. The presence of EGFRvIII mRNA was assessed in HNSCC cell lines by RT-PCR. A representative ethidium bromide-stained agarose gel shows PCR products for EGFR (1037 bp) and EGFRvIII (236 bp). Primary human fibroblasts (Fibro) served as negative control for EGFRvIII, while DK-MG cells were used as a positive control. Multiple HNSCC cell lines were analyzed. C and D Flow cytometric analysis of EGFRvIII protein expression. Cell lines were stained using a recombinant rabbit monoclonal EGFRvIII antibody followed by a PE-conjugated anti-rabbit IgG (H + L) F(ab’)₂ secondary antibody. EGFR expression was detected using APC- or PE-conjugated human EGFR antibodies. C Relative mean fluorescence intensity (MFI; y-axis) of EGFRvIII and EGFR expression in HNSCC cell lines. EGFRvIII data represent the mean ± SEM of three independent experiments. D Representative histograms of EGFRvIII (red), EGFR (blue), isotype staining with secondary antibody or isotype alone (dark green), and unstained cells (black). Normalized cell count is plotted against the fluorescence intensity

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Expressing, Variant Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, Analysis, Staining, Agarose Gel Electrophoresis, Negative Control, Positive Control, Recombinant, Fluorescence, Cell Characterization

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Artificial overexpression of EGFRvIII at different levels in HNSCC cell lines. A Schematic representation of the lentiviral EGFRvIII expression vector. The internal promoters (iP: hPGK, hEF1α and SFFV) drive expression of the EGFRvIII transgene and via an IRES site also the Geneticin resistance gene nptII (neoR). B - D UM-14C KO and UT-24A KO cells were transduced with LV EGFRvIII expression vectors. EGFRvIII-positive EGFR KO cells were further purified by flow sorting, generating new EGFRvIII-expressing cell lines. B and C Flow cytometric analysis of EGFRvIII surface expression after sorting. UM-14C and UT-24A cells were stained with ( B ) a PE-conjugated EGFR antibody for EGFR and EGFRvIII expression. Left: EGFR expression is shown by histogram. Right: Means ± SEM of MFI values of EGFRvIII-positive EGFR KO UM-14C and UT-24A cells as well as the corresponding UM-14C and UT-24A KO, WT and control cells transduced with an empty vector (data from three independent experiments) are shown. Differences between promoter groups within each cell line were analyzed using one-way repeated-measures ANOVA followed by Tukey’s multiple-comparisons test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); ns, not significant. C EGFRvIII expression was additionally determined using an EGFRvIII-specific recombinant rabbit monoclonal antibody and a PE-conjugated anti-rabbit IgG (H + L) F(ab’)₂ secondary antibody. Data are presented as mean ± SEM from two independent experiments. D RT-PCR analysis of EGFRvIII mRNA expression. Presence of EGFRvIII mRNA was assessed in HNSCC cell lines artificially overexpressing EGFRvIII by RT-PCR. A representative ethidium bromide-stained agarose gel shows PCR products for EGFR (1037 bp) and EGFRvIII (236 bp). WT cell lines (UM-14C WT and UT-24A WT ), their corresponding EGFR KO derivatives, and the three EGFRvIII-overexpressing cell lines generated using different internal promoters were analyzed

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Over Expression, Expressing, Plasmid Preparation, Transduction, Purification, Analysis, Staining, Control, Recombinant, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Generated

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Design, enrichment, and cytotoxicity of CAR-positive T-cells. A Lentiviral CAR expression vector expressing the blue fluorescent protein TagBFP and a 2nd generation CAR construct. The vector contains either CD19, EGFR, or EGFRvIII A–E -specific scFvs, a CD34-derived hinge region (c6) followed by the CD28 transmembrane (TM) and cytoplasmic (CP) domains and the CD3ζ signaling chain. A BFP reporter gene is co-expressed via a T2A sequence. Transgene expression is driven by the MPSV promoter. B Transduction and enrichment efficiency of CAR-positive T-cells. Transduced T-cells were analyzed by flow cytometry before magnetic separation (Pre-MACS), in the flow-through fraction and after MACS selection (Post-MACS). CAR-positive cells were quantified based on BFP co-expression. Representative histograms show CAR expression (x-axis) plotted against normalized cell count (y-axis). CAR-positive cells are shown as mean ± SEM ( n = 3). C CAR T-cell cytotoxicity. The target cell lines UM-14C KO +EGFRvIII high and DK-MG were co-cultured with CD19, EGFR and EGFRvIII A–E CAR T-cells or untransduced T-cells (no virus) for 16 to 20 h and target cell viability was assessed using a luciferase-based viability assay. Target cell lysis according to the E: T ratio (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from independent healthy donors, with n = 5 for UM-14C KO +EGFRvIII high and n = 3 for DK-MG. Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD19 CAR control

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Expressing, Plasmid Preparation, Construct, Derivative Assay, Sequencing, Transduction, Flow Cytometry, Selection, Cell Characterization, Cell Culture, Virus, Luciferase, Viability Assay, Lysis, Analysis, Control

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: CAR T-cell cytotoxicity against different HNSCC cell lines. Different WT and genetically engineered UM-14C/UT-24A cells were co-cultured with CD19, EGFR, EGFRvIII D or EGFRvIII E CAR T-cells or untransduced (no virus) T-cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD19 CAR control

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Cell Culture, Virus, Cytotoxicity Assay, Lysis, Analysis, Control

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Concept, design and cytotoxicity of CAR and nsCAR T-cells. A Schematic representation of CAR/nsCAR co-expression strategies. 2nd generation CARs were combined with non-signaling CARs, which lack signaling domains and provide additional antigen/epitope engagement. B Lentiviral expression vectors encoding CARs or nsCARs. The 2nd generation CAR vector contains either CD44v6, CD33, or EGFRvIII D−E -specific scFvs, the c6-hinge, CD28 transmembrane and cytoplasmic regions, and the CD3ζ chain. An mCherry reporter was co-expressed via a T2A sequence. The nsCAR contains EGFR- or CD19-specific scFvs, the n3-hinge, and the CD8 transmembrane domain. C Killing efficiency of CAR and nsCAR T-cells against WT and genetically engineered UM-14C cells. Primary human T-cells were singly transduced with lentiviral constructs encoding CAR or nsCARs and CAR- or nsCAR-positive T-cells (> 90%) were co-cultured with UM-14C cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD33 CAR control

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Expressing, Plasmid Preparation, Sequencing, Transduction, Construct, Cell Culture, Cytotoxicity Assay, Lysis, Analysis, Control

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Killing efficiency of CAR/nsCAR T-cells. Primary human T-cells were singly or doubly transduced, and CAR-positive T-cells, including CAR/nsCAR double-positive cells, were enriched using CD34 microbeads. Cells were co-cultured with UM-14C cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analyses were performed using two-way repeated-measures ANOVA across the tested E: T ratios, with donor as the matched experimental unit. Prespecified comparisons assessed the effect of EGFR nsCAR co-expression relative to the corresponding CD19 nsCAR control condition

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Cell Culture, Cytotoxicity Assay, Lysis, Expressing, Control

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Killing dynamics of CAR/nsCAR T-cells. Primary human T-cells were transduced with an EGFRvIII D/E or CD44v6-specific CAR alone or in combination with additional EGFR- or CD19-specific nsCAR constructs. CAR-positive T-cells, including CAR/nsCAR double-positive cells, were enriched using CD34 microbeads and co-cultured with UM-14C KO +EGFRvIII mid cells for 6, 10, and 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analyses were performed separately for each time point using two-way repeated-measures ANOVA across the tested E: T ratios, with donor as the matched experimental unit. Prespecified comparisons assessed the effect of EGFR nsCAR co-expression relative to the corresponding CD19 nsCAR control condition

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Transduction, Construct, Cell Culture, Cytotoxicity Assay, Lysis, Expressing, Control

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Visualization of the IS. The IS was analyzed by fluorescence microscopy, visualizing the EGFRvIII D CAR (FITC-conjugated CD34 antibody, green) and EGFR or CD19 nsCAR (PE-Vio770-conjugated NGFR antibody, red) proteins on MACS-enriched CAR/nsCAR T-cells (BFP expression, blue) in relation to UM-14C KO +EGFRvIII high cells (phase contrast). Contrast settings (channels: PH, 377/447 nm, 470/525 nm and 545/620 nm) were adjusted to optimize IS visualization

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Fluorescence, Microscopy, Expressing, Phase Contrast

Journal: Science Advances

Article Title: Renoprotective effects of tubular glucagon receptor activation mediated by V-ATPase

doi: 10.1126/sciadv.aeg2534

Figure Lengend Snippet: ( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO primary RTECs treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following EGF stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: RTECs were serum-starved for 4 hours and stimulated with recombinant human EGF (100 ng/ml, MCE HY-P7109) for 0–120 min at 37°C.

Techniques: Fluorescence, Derivative Assay, Western Blot, Viability Assay, Immunofluorescence, Staining, Two Tailed Test