Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: GB cell line screening for target selection and generation of OE and KO cell line models. (a) Expression of glioma-associated membrane antigens in primary and conventional GB cell lines by RT-qPCR. N = 2 technical replicates (two independent qPCR reactions) per gene per cell line. (b) Protein expression levels of EPHA2, CD276, IL13Ra2 and CD70 (blue histograms) in primary GB cell lines, measured by flow cytometry. (c) Evaluation of EPHA2, CD276, IL13Ra2 and CD70 protein levels (blue histograms) on the surface of generated tumor cell models by flow cytometry. For (b and c), isotype control antibodies (red histograms) were used, and data were gated on live single cells. For (b and c), indicative histograms from N = 3 biological replicates per marker per cell line and N = 3 independent experimental repeats. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: Selection, Expressing, Membrane, Quantitative RT-PCR, Flow Cytometry, Generated, Control, Marker

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vitro evaluation of the mSA2 CAR-T cell specificity and killing potency. (a) In vitro experimental pipeline. (b) mSA2 CAR-T cell activation after co-culture with GB models, determined by flow cytometry. N = 3 biological replicates (three independent co-cultures) per group. Data gated on live single CD3 + cells (NT) or live single CD3 + /EGFP + cells (SFG, mSA2_h28z, mSA2_hBBz). An isotype control antibody was used for gating. An unpaired two-tailed student’s t-test was used to evaluate statistical significance. N = 2 independent T-Cell donors. Representative results from N = 3 independent experimental repeats. (c) Confocal IF images of P3/CD70 (CD70 + /CD276 + /EPHA2 + ) cells co-cultured with mSA2_hBBz cells. For a-EPHA2: t 0 = 0 min, t 1 = 140 min, t 2 = 280 min, t 3 = 420 min, t 4 = 560 min. For a-CD276: t 0 = 0 min, t 1 = 220 min, t 2 = 440 min, t 3 = 660 min, t 4 = 880 min. For a-CD70: t 0 = 0 min, t 1 = 350 min, t 2 = 700 min, t 3 = 1050 min, t 4 = 1400 min. N = 2 biological replicates (two separate co-cultures) per group. N = 1 T-Cell donor. (d) Quantification of tumor cell signal from (c). A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was performed to assess statistical significance at the t = 560 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Flow Cytometry, Control, Two Tailed Test, Cell Culture

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Investigation of the mSA2 CAR-T cell capacity to address tumor heterogeneity in vitro . (a) Co-culture pipeline. (b) Apoptotic tumor cell fraction after co-culture with mSA2 CAR-T cells, measured by flow cytometry. Data gated on live single EGFP − cells. N = 1 T-Cell donor. N = 3 biological replicates (three independent co-cultures) per group. A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was used for statistical significance. (c) Analysis of the Annexin-V-incorporating fraction from (b) by flow cytometry. Data gated on live single EGFP − /Annexin-V high tumor cells. (d) Quantification of Annexin-V incorporation from (c). (e) Confocal live cell if images of P3/CD276_KO : P3/EPHA2_KO cells (left panel) and P3/CD70 : P3/IL13Ra2 cells (right panel) after 48 h co-culture with mSA2_h28z cells, after incubation with combinations of biotinylated antibodies, or a biotinylated isotype control antibody. Indicative images from N = 2 biological replicates (two independent co-cultures) and N = 2 independent T-Cell donors per group. (f) Quantification of tumor cell signal over time from the co-culture in (e). For (d and f), an unpaired two-tailed student’s t-test was used to evaluate statistical significance. For (f), statistical significance was assessed at the t = 750 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Incubation, Control, Two Tailed Test

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A–C) Epha2 homozygous deletion in mice causes development of progressive cataract. (A) Cataracts were visible by gross inspection in homozygous Epha2 knockout mice ( Epha2 −/− ) between 5 to 8 months of age, but not in heterozygous or wild type mice. Shown are slit lamp images confirming development of cataract in Epha2 −/− but not Epha2 +/+ mice. (B) Dark field imaging of the dissected lens. Although not readily detectable by visual inspection, cataracts were found on dissected lens by 3 months of age. This lens was tilted to show denser opacity near the equator (arrow). Enucleation frequently occurred during dissection of mature cataract after 8 months (far right). (C) Retroillumination examination revealed clusters of small vacuoles by one month of age. Scale bars: 1 mm for middle panel; 150 µm for right panel. (D) Immunoblot of total lens lysates showing decreasing EPHA2 expression with aging. (E–M) Compartmentalized and gradient expression of EPHA2 (red) in mouse lens. Blue: DAPI nuclear staining. (E–I) Midsagittal sections of lens from 14-day-old wild type mice were stained for EPHA2. (E) Low power view of an entire lens revealed dense expression of EPHA2 in subcortical lens fiber cells. Dotted arrows indicate gradient expression in lens epithelial cells near the equator. Scale bar: 1 mm. (F) Low EPHA2 expression in anterior lens epithelial cells (arrow head, sandwiched between dotted lines). (G) Inset from (F) showing high EPHA2 expression in lens fiber cells. (H) High level of EPHA2 expression at the bow. (I) Inset from (H) showing dense expression at modulus (arrow). Scale bars: 5 µm for F–I. (J–M) Coronal sections through the bow region of lens co-stained for EPHA2 and N-cadherin. (J) Note the spatially regulated expression pattern in subcortical lens fiber cells. (K) Inset from (J) showing “honey-comb” membrane staining pattern of EPHA2 in the cross sections of fiber cells at high magnifications. (L) N-cadherin from the same section show overlapping but distinct expression pattern compared with that of EPHA2. (M) Merged images of EPHA2/N-cadherin. 10 µm for J, L, and M; 2 µm for K.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Knock-Out, Imaging, Dissection, Western Blot, Expressing, Staining, Membrane

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: Incidence of Visible Cataracts in Wild-Type and Epha2 -null Mice.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A–D) Expression of ephrin-A1, a ligand for EPHA2. Note the disorganized ephrin-A1 expression and formation of vacuoles in the Epha2 −/− lens (arrow heads). Scale bars: 1 mm for A and C; 10 µm for B and D. (E,F) N-cadherin staining showing disorganization of lens fiber cells. Scale bars: 5 µm. (G) Overexpression of HSP25 but not HSP90 in Epha2 −/− lens which was quantified in (H), and confirmed by immunofluorescence staining (I). Scale bars: 40 µm. (J) Immunoblot for phosphorylated HSP25 revealed relatively low degree of phosphorylation in Epha2 −/− lens.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Expressing, Staining, Over Expression, Immunofluorescence, Western Blot, Phospho-proteomics

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: Characteristics of SNPs in the EPHA2 gene.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: P values from rank transformed traits and effect sizes from the quantitative cortical scores (β) of the risk allele at markers under the dominant model in EPHA2 for each separate study and for the joint analysis of all studies.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Transformation Assay

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A) Examination of crystal structure of EPHA2 kinase domain reveals that Arg721 in αE forms a salt bridge with Asp872 in αI. (B) Concordant conservation of Arg721 and Asp872 in different members of human Eph kinases. Note that EPHA9 and EPHB5 are not present in human genome and are not shown. (C) The same residues are also concordantly conserved across different species.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A,B) Arg721Gln mutation causes an increased basal activation of EPHA2 kinase, which was correlated with dramatically reduced basal ERK1/2 activities. In a kinetic study (A), HEK 293 cells expressing WT-, Arg721Gln -EPHA2 or vector control were stimulated with 2 µg/ml ephrin-A1-Fc for the indicated times. In a separate experiment, a dose-response study was carried out (B), where different doses of ephrin-A1-Fc were used to stimulate cells expressing WT- or Arg721Gln -EPHA2 for 10 min. Cell lysates from both experiments were blotted with the indicated antibodies as described previously . (C) HEK 293 cells expressing Arg721-Gln mutant EPHA2 but not WT-EPHA2 were growth-inhibited by ephrin-A1 in a clonal growth assay as described previously . About 200 cells/well were seeded in a 24-well culture dish and cultured for 10 days in the presence or absence of ephrin-A1. (D) Stochastic intracellular trapping of Arg721Gln mutant in MEF cells derived from Epha2 knockout embryos. Shown is a cluster of cells with the mutant EPHA2 trapped inside the cells. In contract, WT-EPHA2 was primarily expressed on the cytoplasmic membrane. Scale bar: 5 µm.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Mutagenesis, Activation Assay, Expressing, Plasmid Preparation, Control, Growth Assay, Cell Culture, Derivative Assay, Knock-Out, Membrane

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) EphA2 and EphA4 receptor expression in normal and cancer cell lines. Bar graph shows the fold expression of both EphA2 and EphA4 receptors in MDA-MD-231, PC-3, and LNCaP cancer cell lines compared to cultured human dermal fibroblast (HDF) and to a total human prostate RNA sample (Prostate). (B) Correlation between EphA2 mRNA expression and EphA2 protein levels in 30 selected human cancer cell lines. (C) Box and Whisker plots of the 10–90th percentile of EphA2 expression for the RNA samples grouped according to their tissue of origin. Dotted lines depict cutoffs for 7 fold, and 14 fold the expression levels observed in the reference HDF cell line, respectively.

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Cell Culture, Whisker Assay

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) The YSA peptide targets quantum dots to EphA2 on the cell surface. COS cells transfected with either the extracellular and transmembrane portions of EphA2 fused to enhanced green fluorescent protein (EphA2-EGFP) or membrane targeted EGFP-F were incubated with YSA bound to red fluorescent quantum dots (YSA-Qdots). YSA-Qdots only bind to cells transfected with EphA2-EGFP. EGFP fluorescence is green; nuclei are stained in blue with DAPI. (B) YSA targets quantum dots to endogenous EphA2 on the cell surface. HUVE cells were incubated at 4ºC with YSA conjugated Qdots (YSA-Qdots), an unrelated 12-mer control peptide that does not bind to EphA2 also conjugated to Qdots (Ctrl-Qdots), or unconjugated Qdots (Qdots). Only quantum dots conjugated to the YSA peptide bind to the HUVE cells. (C) YSA targets Qdots to lysosomes. MDA-MB-231 cells, which express high levels of endogenous EphA2, were incubated at 4ºC with YSA-Qdots followed by incubation at 37 ºC for 0, 15, and 60 min. YSA-Qdots are seen on the cell surface at 0 min but become concentrated in structures near the nucleus at 15 and 60 min (arrow). Double labeling with the lysosomal marker Lamp1 (green) shows colocalization of the quantum dots in lysosomes at 60 min (arrows).

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Transfection, Membrane, Incubation, Fluorescence, Staining, Control, Labeling, Marker

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) General chemical structures of YSA and DYP peptide-drug conjugates. (B) YSA, YSA-PTX and control DYP-PTX were incubated at the indicated concentrations together with a constant concentration of ephrin-A5 AP in ELISA wells pre-coated with EphA2 Fc. The ratio of ephrin-A5 AP bound in the presence and in the absence of peptide is shown. (C) Biotinylated YSA-PTX and control DYP-PTX were incubated at the indicated concentrations in EphA2 Fc-coated ELISA wells and were then detected with streptavidin-HRP. The graphs show averages ± SE from quadruplicate measurements in representative experiments, while the KD and IC50 values are calculated from 3 to 5 experiments. (D) ATP-Lite analysis of viability of PC-3 cells 72 hours after the addition of DYP, DYP-PTX, YSA, YSA-PTX or PTX at 10 nM (n = 3).

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Control, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: EphA2 inhibition

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Sequencing

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) YSA-PTX coupled to fluorescent quantum dots, but not DYP-PTX, is internalized with EphA2 into lysosomes of prostate cancer cells expressing EphA2. PC3 prostate cancer cells, which express high levels of EphA2, or LNCaP prostate cancer cells, which do not detectably express EphA2 protein, were treated for 20 min with 100 μM YSA-PTX or control DYP-PTX, followed by a 20 min incubation with streptavidin-conjugated quantum dots (Qdots). After removing the solution containing the peptides and the quantum dots, the cells were incubated for 2 h at 37°C to allow EphA2 internalization induced by YSA-PTX binding. The cells were stained for EphA2 (left) or the lysosomal marker Lamp1 (right). Qdots were also imaged and nuclei were labelled with DAPI in blue. Representative fluorescence micrographs are shown. Scale bar = 25 μM. (B) Ephrin-A1 Fc and YSA-PTX, but not DYP-PTX, causes EphA2 internalization into lysosomes. PC3 cells were treated for 2 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA-PTX or 100 μM control DYP-PTX. The cells were stained for Lamp1 (red) and EphA2 (green). Nuclei were labelled with DAPI (blue). Representative confocal micrographs are shown. Scale bar = 25 μm. (C) Ephrin-A1 Fc and YSA-PTX, but not DYP-PTX, cause EphA2 internalization into cells. PC3 prostate cancer and HUVE endothelial cells were treated for 1 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA-PTX or 100 μM control DYP-PTX. Proteins present on the cell surface were then labeled with biotin. EphA2 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr), streptavidin-HRP (biotin) and EphA2 antibody. The amount of GAPDH in the cell lysates used for the immune-precipitations is also shown as a control for equal amounts of protein in the lysates used for immunoprecipitation.

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Control, Incubation, Binding Assay, Staining, Marker, Fluorescence, Labeling, Immunoprecipitation