Journal: PLoS ONE

Article Title: Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

doi: 10.1371/journal.pone.0018128

Figure Lengend Snippet: 96 well ELISA high binding plates were incubated O/N with EphA2-Fc and the following day washed and blocked with PBS +0.5% BSA for 1 hour at 37°C. Compounds were added in the wells at proper concentrations 1 hour before the addition of biotinylated ephrinA1-Fc. After 4 hours wells were washed and incubated with a streptavidin-HRP solution for 20 minutes at room temperature. Wells were washed again and incubated with tetra-methylbenzidine. The reaction was stopped with 3N HCl and the absorbance was measured at 450 nm. A, lithocholic acid dose-dependently displaced binding of ephrin-A1-Fc ectodomain from immobilized EphA2-Fc ectodomain. B, binding of ephrin-A1-Fc ectodomain to immobilized EphA2-Fc ectodomain in presence of different concentration of lithocholic acid. C, The dissociation constants (Kd) from the previous plot were used to calculate Log (Dose-ratio - 1) and to graph the Schild plot. pKi of lithocholic acid was estimated by the intersection of the interpolated line with the X-axis. The slope of the interpolated line can be related to the nature of the binding. A slope between 0.8 and 1.2 is related to a competitive binding whereas higher numbers are related to non-specific interactions. D, EphA2-ephrinA1 binding in presence of 200 µM LCA with or without washing three times with PBS.

Article Snippet: EphA2-, EphB4- and EGFR-phosphorylation were measured in cell lysates using DuoSet®IC Sandwich ELISA (RnD Systems, #DYC4056, #DYC4057 and #DYC1095, respectively) following manufacturer's protocol.

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

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 mRNA expression profiles of various tumor samples and paired normal tissues. The height of the bar represents the median expression level (in transcripts per million (TPM)) in each tumor or normal tissue type.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 expression patterns in pancreatic adenocarcinoma (PAAD) patients. (a) EphA2 transcriptional levels in PAAD and normal pancreatic tissues and (b) EphA2 mRNA levels in PAAD primary tumors and normal tissues.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Associations between pancreatic adenocarcinoma (PAAD) clinical pathological stage and EphA2 expression.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: The prognostic value of EphA2 expression in pancreatic adenocarcinoma (PAAD) patients. Kaplan–Meier plots are shown for (a) overall survival and (b) disease-free survival.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 promoter DNA methylation patterns in pancreatic adenocarcinoma (PAAD) primary tumors and normal tissues.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: DNA Methylation Assay

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Correlations of immune cell infiltration and EphA2 expression in pancreatic adenocarcinoma (PAAD) patients.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Multivariate Cox proportional risk model of pancreatic adenocarcinoma (PAAD).

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Protein-protein interaction (PPI) analysis of EphA2 in pancreatic adenocarcinoma (PAAD) patients. (a) PPI network of EphA2. (b) Potential functions of the proteins of interest and (c) Molecular complex detection components identified in the gene lists.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques:

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Functional enrichment analysis of EphA2 in pancreatic adenocarcinoma (PAAD) patients.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Functional Assay

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Analysis of EphA2 protein expression using immunohistochemistry (IHC) in tissue samples from pancreatic adenocarcinoma (PAAD) patients. Representative images are shown at 100x magnification. (a) High EphA2 protein expression in pancreatic cancer tissue and (b) Low EphA2 protein expression in paracancerous tissue.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing, Immunohistochemistry

Journal: OncoImmunology

Article Title: Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

doi: 10.1080/2162402x.2021.1960728

Figure Lengend Snippet: Figure 1. Developing EphA2-Specific CAR-T Cells (a) Schematic diagram of two different chimeric antigen receptors (CARs) targeting EphA2. It consists of EphA2 scFv, the hinge, transmembrane (TM) region of CD28, CD28 and 4–1BB signaling domain, and human CD3ζ chain. (b) CAR-T cells were subjected to immunoblotting to detect the expression of full-length EphA2-CARs by using CD3ζ antibody. (c) Different EphA2-CAR-T cells were labeled with CFSE and then co-cultured with U251 cells for 72 h. The extent of T cell proliferation is reflected by the loss of incorporated CFSE. (d) CAR-T cells were co-cultured with U251 cells for 15 days at an E:T ratio of 2:1. T cells were stimulated every three days with fresh U215 cells, and T cells were counted before the addition of U251 cells. Results were analyzed by student’s t-test, and a p < .05 was considered significant. *p < .05, ***p < .001. SD, splice donor; SA, splice acceptor.

Article Snippet: GBM cells was stained with Alexa fluor 700 labeled mouse anti-human EphA2 (R&D System) and then subjected to flow cytometry to examine the cell surface EphA2 expression.

Techniques: Western Blot, Expressing, Labeling, Cell Culture

Journal: OncoImmunology

Article Title: Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

doi: 10.1080/2162402x.2021.1960728

Figure Lengend Snippet: Figure 2. Comparison of antitumor effects of different EphA2 CAR-T cells In vitro. NT or EphA2 CAR-T cells were co-cultured with different target cells at different E:T ratios (1:1, 2.5:1, 5:1, and 10:1) for 24 h. (a) The cell lysis rate was quantified by examining the luciferase activity. (b) The supernatants were collected to evaluate IFN-γ levels by ELISA (E:T = 10:1). (c) Continuous graphical output of cell index values up to the 50 h time point was monitored using the xCELLigence impedance system. Results were analyzed by one-way ANOVA, and statistical significance was set at *p < .05, **P < .01, ***p < .001.

Article Snippet: GBM cells was stained with Alexa fluor 700 labeled mouse anti-human EphA2 (R&D System) and then subjected to flow cytometry to examine the cell surface EphA2 expression.

Techniques: Comparison, In Vitro, Cell Culture, Lysis, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: OncoImmunology

Article Title: Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

doi: 10.1080/2162402x.2021.1960728

Figure Lengend Snippet: Figure 3. Comparison of antitumor effects of different EphA2 CAR-T cells in xenograft mouse models. (a) 5 × 106 eGFP-Luc-U251 cells were injected subcutaneously into the left flank of 6–8 week-old female NOD-SCID mice. Ten days after injection, the tumor bearing mice were treated with 3 × 107 EphA2 CAR T cells through direct injection into the tumor, with un-transduced T cells (NT) as control. The tumor growth was monitored using the IVIS system with a tumor diameter of 2 cm as the endpoint. Quantitative bioluminescence (radiance = photons/cm2/sr) imaging data for all mice are shown. Overall survival of mice with GBM xenografts was measured using the Kaplan–Meier method, with Cox proportional hazard regression analysis for group comparison. A p-value less than 0.05 was considered significant. (b) Six to eight week-old NOD-SCID mice were anesthetized and then 2 × 105 cells were injected into the left striatum through a burr hole in the skull. Two weeks after tumor cells injection, 3 × 107 CAR-T cells were injected through the tail vein. Thereafter, the tumor growth was monitored using in vivo imaging system IVIS. Quantitative bioluminescence (radiance = photons/cm2/sr) imaging data for all mice are shown. Overall survival of mice with GBM xenografts were measured using the Kaplan–Meier method, with Cox proportional hazard regression analysis for group comparison. A p-value less than 0.05 was considered significant.

Article Snippet: GBM cells was stained with Alexa fluor 700 labeled mouse anti-human EphA2 (R&D System) and then subjected to flow cytometry to examine the cell surface EphA2 expression.

Techniques: Comparison, Injection, Control, Imaging, In Vivo Imaging

Journal: OncoImmunology

Article Title: Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

doi: 10.1080/2162402x.2021.1960728

Figure Lengend Snippet: Figure 5. Low IFN-γ and CXCL8 levels relate to better anti-tumor activity in EphA2-CAR-T cells. EphA2 CAR-T cells before or after co-culture with GBM cells were subjected to RT-qPCR to examine the expression of IFN-γ, CXCL8, IL-21, CXCR1, and CXCR2. Results were analyzed by one-way ANOVA and statistical significance was set at p < .05. *p < .05, **P < .01.

Article Snippet: GBM cells was stained with Alexa fluor 700 labeled mouse anti-human EphA2 (R&D System) and then subjected to flow cytometry to examine the cell surface EphA2 expression.

Techniques: Activity Assay, Co-Culture Assay, Quantitative RT-PCR, Expressing

Journal: OncoImmunology

Article Title: Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

doi: 10.1080/2162402x.2021.1960728

Figure Lengend Snippet: Figure 6. EphA2-b-CAR-T cells induced upregulated PD-L1 expression in GBM cells. (a) GBM cells were co-cultured with CAR-T cells at an E:T ratio of 2:1 for 30 min and 4 h. The GBM cells were then subjected to RT-qPCR to determine the PD-L1 level. (b) The PD-L1 and Ki-67 levels in the tumors from CAR-T cells treated mice were detected by immunohistochemistry. The positive signal was quantified by Image J. Results were analyzed One-way ANOVA and statistical significance was set at p < .05. *p < .05, **P < .01, ***p < .001.

Article Snippet: GBM cells was stained with Alexa fluor 700 labeled mouse anti-human EphA2 (R&D System) and then subjected to flow cytometry to examine the cell surface EphA2 expression.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Immunohistochemistry

Journal: OncoImmunology

Article Title: Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

doi: 10.1080/2162402x.2021.1960728

Figure Lengend Snippet: Figure 7. EphA2-b-CAR-T treatment combined with PD1 blockade exhibited enhanced antitumor activity. U251 and U373 cells were transiently transfected with one of the siRNAs to knock down the expression of IFNGR1, IFNGR2 or PG-L1 separately (a and b), and then the cells were subjected to an in vitro killing assay at an E:T = 1:1. The cell lysis rates were determined by detecting luciferase activity (c). NOD-SCID mice were injected subcutaneously with 1 × 107 U251.eGFP.Luc cells to construct a xenograft mouse model. Five days post-tumor xenograft, mice were treated with a total of 3 × 107 CAR-T cells or non-transduced control T cells peritumorally, followed by peritumoral (p.t.) administration of 200 μg PD1 antibody. On day 13, the mice were administered a second dose of PD1antibody, and tumor growth was monitored using IVIS (D, E, and F).

Article Snippet: GBM cells was stained with Alexa fluor 700 labeled mouse anti-human EphA2 (R&D System) and then subjected to flow cytometry to examine the cell surface EphA2 expression.

Techniques: Activity Assay, Transfection, Knockdown, Expressing, In Vitro, Lysis, Luciferase, Injection, Construct, Control

Journal: OncoImmunology

Article Title: Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

doi: 10.1080/2162402x.2021.1960728

Figure Lengend Snippet: Figure 8. Experiment workflow and proposed model of better anti-tumor efficacy after EphA2-a-CAR-T cells treatment.

Article Snippet: GBM cells was stained with Alexa fluor 700 labeled mouse anti-human EphA2 (R&D System) and then subjected to flow cytometry to examine the cell surface EphA2 expression.

Techniques: