Journal: bioRxiv

Article Title: Skeletal Metastasis of Prostate Cancer Is Augmented by Activation of EphA2 Noncanonical Signaling and Ligand-Deficient Bone Microenvironment

doi: 10.1101/2024.09.25.615079

Figure Lengend Snippet: A) Analysis of log 2 transformed FPKM gene expression values extracted from bulk RNA seq published previously (Smith R. et al. Scientific Reports 2020 ) shows high EphA2 expression in castration resistant, commercially available human PCa cell lines. B) Immunoblot characterization of select human PCa cell lines show increased EphA2 expression and S897 phosphorylation in mCRPC derived cell lines. C) EphA2 expression in representative human prostate cancer cell lines. D) Lysate of human prostate cancer tissues reveal increased expression of EphA2 in bone metastasis samples. Lysates were prepared from micro-dissected human prostate cancer tissues under the Rapid Autopsy Program at the Univ. Mich. PCa SPORE.

Article Snippet: Other antibodies were purchased as follows: Rabbit monoclonal anti-GAPDH (Cell Signaling Technology #2118S), rabbit monoclonal anti-pS473-Akt (Cell Signaling Technology #4060), rabbit polyclonal anti-pErk1/pErk2 (Cell Signaling Technology #9101S), rabbit monoclonal anti-EphA2 (Cell Signaling Technology #6697S), rat monoclonal anti-EphA2-PE (R&D Systems FAB639P), rabbit monoclonal anti-AR (Abcam ab133273), rabbit polyclonal anti-pY772-EphA2 (Cell Signaling Technology #8244S), rabbit monoclonal anti-pY588-EphA2 (Cell Signaling Technology #12677S), mouse monoclonal anti-Tubulin (R&D Systems MAB9344) Goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and from Biorad (#1706515, and #1706516 respectively).

Techniques: Transformation Assay, Expressing, RNA Sequencing Assay, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Skeletal Metastasis of Prostate Cancer Is Augmented by Activation of EphA2 Noncanonical Signaling and Ligand-Deficient Bone Microenvironment

doi: 10.1101/2024.09.25.615079

Figure Lengend Snippet: A) Diagram depicting the serial propagation of TRAMP-C1 cells in syngeneic mice to select for cells with increased tumorigenic capacity. B) The in vivo selection of cells resulted in accelerated tumor growth and reduced latency. C) Immunoblot analyses of the in vivo selected (IV1) and the parental TRAMP-C1 cells using the indicated antibodies. D,E,F,G,H,I,J) Quantification of relative band intensity (n = 4). All bands normalized to tubulin, except for pY588 and pS897 which are normalized to total EphA2 expression. K) Immunoblot of TRAMP-C1 parental and IV1 cells after stimulation with Ephrin-A1-Fc ligand for the indicated times. L,M) MPC3 and MycCaP cells were propagated in syngeneic C57Bl/6 and FVB mice, respectively. Lysates of the parental and the in vivo selected cells (IV1) were subject to immunoblot using the indicated antibodies.

Article Snippet: Other antibodies were purchased as follows: Rabbit monoclonal anti-GAPDH (Cell Signaling Technology #2118S), rabbit monoclonal anti-pS473-Akt (Cell Signaling Technology #4060), rabbit polyclonal anti-pErk1/pErk2 (Cell Signaling Technology #9101S), rabbit monoclonal anti-EphA2 (Cell Signaling Technology #6697S), rat monoclonal anti-EphA2-PE (R&D Systems FAB639P), rabbit monoclonal anti-AR (Abcam ab133273), rabbit polyclonal anti-pY772-EphA2 (Cell Signaling Technology #8244S), rabbit monoclonal anti-pY588-EphA2 (Cell Signaling Technology #12677S), mouse monoclonal anti-Tubulin (R&D Systems MAB9344) Goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and from Biorad (#1706515, and #1706516 respectively).

Techniques: In Vivo, Selection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Skeletal Metastasis of Prostate Cancer Is Augmented by Activation of EphA2 Noncanonical Signaling and Ligand-Deficient Bone Microenvironment

doi: 10.1101/2024.09.25.615079

Figure Lengend Snippet: A) Immunohistochemical (IHC) analyses of tissue microarray (TMA) prepared from primary, lymph node metastatic (LN), and bone metastatic human PCa tissues from the Rapid Autopsy Program of the Pacific Northwest PCa SPORE spotted. B and C) The relative IHC staining strengths of EphA2 and pS897-EphA2 on TMA was scored using 0-3 scale (0, absent; 1, weak; 2, moderate; 3, strong) . Data consists of 27 bone metastatic samples, and 18 soft tissue samples including 10 lymph node, 5 liver, and 3 lung metastatic samples. Comparisons were made using an unpaired t-test (B, p = 0.0003 C, p < 0.0001) D) mRNA expression analysis of human PCa metastatic biopsies revealed that bone metastases have the highest EphA2 expression compared to other sites of disease dissemination. Data from 208 samples from the SU2C/PCF Dream Team. mRNA Expression z-scores relative to all samples (log FPKM Capture) accessed via cBioportal. E) Kaplan-Meier curve of patients with bone metastases according to the levels of EphA2 expression.

Article Snippet: Other antibodies were purchased as follows: Rabbit monoclonal anti-GAPDH (Cell Signaling Technology #2118S), rabbit monoclonal anti-pS473-Akt (Cell Signaling Technology #4060), rabbit polyclonal anti-pErk1/pErk2 (Cell Signaling Technology #9101S), rabbit monoclonal anti-EphA2 (Cell Signaling Technology #6697S), rat monoclonal anti-EphA2-PE (R&D Systems FAB639P), rabbit monoclonal anti-AR (Abcam ab133273), rabbit polyclonal anti-pY772-EphA2 (Cell Signaling Technology #8244S), rabbit monoclonal anti-pY588-EphA2 (Cell Signaling Technology #12677S), mouse monoclonal anti-Tubulin (R&D Systems MAB9344) Goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and from Biorad (#1706515, and #1706516 respectively).

Techniques: Immunohistochemical staining, Microarray, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Skeletal Metastasis of Prostate Cancer Is Augmented by Activation of EphA2 Noncanonical Signaling and Ligand-Deficient Bone Microenvironment

doi: 10.1101/2024.09.25.615079

Figure Lengend Snippet: A) Immunoblot analysis of PC3 cells transduced with lentivirus to express WT-EphA2 or S897A-EphA2. Empty vector-transduced cells were used as control. Cells were starved for 24 hours in serum-free medium and stimulated with either FBS alone or FBS togethr with ephrin-A1-Fc. B) PC3 cells overexpressing WT-EphA2 or S897A-EphA2 mutant were xenografted subcutaneously into nude mice (n = 10 per group). Vector alone cells were used as control. Comparison between WT-EphA2 and S897A-EphA2 made with an unpaired t-test at day 46 post-injection (p = 0.0402).

Article Snippet: Other antibodies were purchased as follows: Rabbit monoclonal anti-GAPDH (Cell Signaling Technology #2118S), rabbit monoclonal anti-pS473-Akt (Cell Signaling Technology #4060), rabbit polyclonal anti-pErk1/pErk2 (Cell Signaling Technology #9101S), rabbit monoclonal anti-EphA2 (Cell Signaling Technology #6697S), rat monoclonal anti-EphA2-PE (R&D Systems FAB639P), rabbit monoclonal anti-AR (Abcam ab133273), rabbit polyclonal anti-pY772-EphA2 (Cell Signaling Technology #8244S), rabbit monoclonal anti-pY588-EphA2 (Cell Signaling Technology #12677S), mouse monoclonal anti-Tubulin (R&D Systems MAB9344) Goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and from Biorad (#1706515, and #1706516 respectively).

Techniques: Western Blot, Transduction, Plasmid Preparation, Control, Mutagenesis, Comparison, Injection

Journal: bioRxiv

Article Title: Skeletal Metastasis of Prostate Cancer Is Augmented by Activation of EphA2 Noncanonical Signaling and Ligand-Deficient Bone Microenvironment

doi: 10.1101/2024.09.25.615079

Figure Lengend Snippet: A) Analysis of log 2 transformed FPKM gene expression values extracted from bulk RNA seq published previously (Smith R. et al. Scientific Reports 2020 ) showing low Efna1-5 gene expression in commercially available castration resistant and androgen sensitive human PCa cell lines. B) Immunoblot characterization of select PCa cell lines reveals simultaneous high EphA2 expression and low EphrinA1 expression in metastatic castration resistant cells compared with androgen sensitive cells. C ) Quantitative analysis of data in (B). D) Kaplan-Meier survival curve from the MSK dataset ( Cancer Cell 2010 ). Samples were aggregated into the top 25 percent EFNA1 expressing ( EFNA1 High), and lower 75 percent EFNA1 expressing ( EFNA1 Low). Data were accessed via cBioportal.

Article Snippet: Other antibodies were purchased as follows: Rabbit monoclonal anti-GAPDH (Cell Signaling Technology #2118S), rabbit monoclonal anti-pS473-Akt (Cell Signaling Technology #4060), rabbit polyclonal anti-pErk1/pErk2 (Cell Signaling Technology #9101S), rabbit monoclonal anti-EphA2 (Cell Signaling Technology #6697S), rat monoclonal anti-EphA2-PE (R&D Systems FAB639P), rabbit monoclonal anti-AR (Abcam ab133273), rabbit polyclonal anti-pY772-EphA2 (Cell Signaling Technology #8244S), rabbit monoclonal anti-pY588-EphA2 (Cell Signaling Technology #12677S), mouse monoclonal anti-Tubulin (R&D Systems MAB9344) Goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and from Biorad (#1706515, and #1706516 respectively).

Techniques: Transformation Assay, Expressing, RNA Sequencing Assay, Western Blot

Journal: bioRxiv

Article Title: Skeletal Metastasis of Prostate Cancer Is Augmented by Activation of EphA2 Noncanonical Signaling and Ligand-Deficient Bone Microenvironment

doi: 10.1101/2024.09.25.615079

Figure Lengend Snippet: A) Lysates of PC-3 cells transduced by lentiviral vector expressing EFNA1 (EA1) or empty control vector were subject to immunoblot with the indicated antibodies. B,C) Quantification of pY588 marking canonical signaling and pS897 marking noncanonical signaling by EphA2, relative to total EphA2 expression. D) Vec. and EA1-expressing PC-3 cells were stimulated with recombinant exogenous EA1-Fc ligand in vitro for the indicated times, and lysates were subject to immunoblot with the indicated antibodies. E) PY99, a monoclonal antibody that recognizes most pY motifs, was used to probe overall pY profiles using lysates from Vec. and EA1-expressing PC-3 cells stimulated with EA1-Fc. N-cadherin and pY416-Src were also probed. F) PC-3 cells expressing EA1 exhibit reduced cell growth in an in vitro cell proliferation assay. G) Vec. control and EA1-expressing cells were implanted subcutaneously into nude mice. Paired t-test to be used to monitor (p = 0.0319). G) Bioluminescence imaging of mice four and six weeks after intratibial injection of PC3-vec. or PC-3-EA1 expressing cells tagged with luciferase. H) X-ray imaging of mice 6 weeks post intratibial injection of PC3-Vec and PC3-EA1 cells.

Article Snippet: Other antibodies were purchased as follows: Rabbit monoclonal anti-GAPDH (Cell Signaling Technology #2118S), rabbit monoclonal anti-pS473-Akt (Cell Signaling Technology #4060), rabbit polyclonal anti-pErk1/pErk2 (Cell Signaling Technology #9101S), rabbit monoclonal anti-EphA2 (Cell Signaling Technology #6697S), rat monoclonal anti-EphA2-PE (R&D Systems FAB639P), rabbit monoclonal anti-AR (Abcam ab133273), rabbit polyclonal anti-pY772-EphA2 (Cell Signaling Technology #8244S), rabbit monoclonal anti-pY588-EphA2 (Cell Signaling Technology #12677S), mouse monoclonal anti-Tubulin (R&D Systems MAB9344) Goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and from Biorad (#1706515, and #1706516 respectively).

Techniques: Plasmid Preparation, Expressing, Control, Western Blot, Recombinant, In Vitro, Proliferation Assay, Imaging, Injection, Luciferase

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: EPHA2 is expressed on the cell surface of undifferentiated mouse ESCs and downregulated during differentiation. (A) Relative gene expression of Epha2 mRNA in mouse ESCs (D3) cultured without LIF for 0, 3, 5, and 7 days. (B) Immunoblotting of EPHA2 protein of mouse ESCs cultured as in (A). (C) EPHA2 protein levels in (B) normalized to α-Tubulin. (D) Immunofluorescent staining of mouse ESCs. The cells were cultured with or without LIF for 7 days. Bars; 200 μm. (E, F) Flow cytometric analysis of EPHA2 protein on the cell surface of mouse ESCs. Living cells were stained with an EPHA2 antibody. The propidium iodide + dead cells were removed from the analysis. FSC and SSC profiles (E) and the histogram of EPHA2-AF488 levels (F) of the gated cells in (E) were shown. Statistical analyses in (A) and (C) were calculated by Dunnett’s test. Data were graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01; *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Cell Culture, Western Blot, Staining

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Knock-down of Epha2 induces spontaneous differentiation of mouse ESCs. (A) qRT-PCR analysis of Epha2 mRNA after knock-down in mouse ESCs (D3). The cells were infected with Epha2 shRNA retrovirus and cultured with or without LIF for 7 days. (B) Images of phase contrast and alkaline phosphatase (AP) staining of Epha2 KD mouse ESCs cultured in ES maintenance medium with LIF for 5 days. Bars; 200 μm. (C) qRT-PCR analysis of undifferentiated state-specific marker genes in Epha2 KD mouse ESCs. (D) Immunofluorescent staining of Epha2 KD mouse ESCs cultured with LIF for 7 days. Bars; 100 μm. (E) Culture conditions of Epha2 KD mouse ESCs after infection with Epha2 shRNA retrovirus. One day after infection with Epha2 shRNA virus, mouse ESCs were cultured with or without 2i in the presence of G418 and LIF for 4 days. (F) Phase contrast images of Epha2 KD mouse ESCs cultured as in (E). Bar; 200 μm. (G) qRT-PCR analysis of Epha2 and undifferentiated state-specific marker genes in Epha2 KD mouse ESCs cultured with 2i. All qRT-PCR analyses were performed with 3 independent biological replicates and graphed as means ± SE. The significant differences were calculated by Tukey test in (A and C) and t -test in (G). * P < .05; ** P < .01, *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Knockdown, Quantitative RT-PCR, Infection, shRNA, Cell Culture, Staining, Marker, Virus

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Expression of EPHA2 in heterogenous subpopulation of human PSCs. (A) qRT-PCR analysis of EPHA2 and undifferentiated state-specific marker genes during EB-based random differentiation of human iPSCs (201B7) without basic FGF. Statistical analysis was done by Dunnett’s test comparing to day 0 and graphed as means ± SE of 3 independent experiments. * P < .05; ** P < .01. (B) Representative flow cytometric plots of living human iPSCs stained with EPHA2 antibody-conjugated with AFF488. Gate3 and Gate4 were sorted as EPHA2 − and EPHA2 + cell populations, respectively. See also . (C) Feature plots of EPHA2 and undifferentiated state-specific genes in publicly available undifferentiated human ESC H1 and H9 data subsets from GSE75748 scRNA-seq dataset. Note that EPHA2 expression in hESCs was heterogeneous. Normalized expression levels were plotted. (D, E) Immunofluorescent staining of human iPSC cultured on SyntheMax II-coated plate with StemFit medium. The cells were fixed with paraformaldehyde in PBS and permeabilized. Bars; 200 μm. (F) qRT-PCR analysis of fractioned EPHA2 + and EPHA2 - subpopulations. EPHA2 + cells express higher OCT4 and NANOG , than EPHA2 − cells. Means ± SE of 3 independent experiments were shown. Statistical significance was defined as * P < .05 by t -test.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Quantitative RT-PCR, Marker, Staining, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Transplantation of EPHA2 + cells into immune-deficient mice formed tumors in vivo. (A) Immunofluorescent staining of mouse EBs differentiated into hepatocyte lineages. Expression of an early hepatocyte marker AFP at day 10 and a mature marker ALB at day 14 were detected. Bars; 200 μm. (B) Depletion of undifferentiated ES colonies after removal of EPHA2 + cells from EBs. Oct4-egfp ESCs were differentiated by EB formation for 10 and 14 days. EPHA2 + cells were removed from EBs using anti-EPHA2 antibody-bound MACS after dissociation with trypsin/EDTA. The residual cells were cultured in ES maintenance medium with LIF for 7 days. The alkaline phosphatase (AP) activity was visualized by incubating with AP substrate. Bar; 2 cm. (C) The number of EGFP + cell colonies in (B). Statistical analysis was done by Tukey test and graphed as means ± SE of 4 independent experiments. (D) Scheme of in vivo transplantation experiment. Mouse ESCs (D3) were differentiated into hepatocyte linages and the EBs were dissociated by EDTA treatment. The cells were transplanted into SCID mice after depletion of EPHA2 + cells by MACS. (E) Decreased teratoma formation after transplantation of EPHA2 − cells. White arrowheads indicate teratomas. (F) H&E staining of teratomas formed in the testicular subcutaneous tissue without MACS procedure. Typical cell types of 3 germ layers were confirmed. Bar; 500 μm. (G) Typical teratoma formation after transplantation of dissociated EB at day 10 through hepatic portal vein. White arrowheads indicate teratomas. (H) Quantification of teratoma formation in (E) and (G). Statistical analysis was done by Chi-square test, * P < .05, ** P < .01.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Transplantation Assay, In Vivo, Staining, Expressing, Marker, Cell Culture, Activity Assay

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Co-expression of EPHA2 with OCT4 in EBs during human iPSC differentiation into hepatocyte. (A, B) Relative gene expression of undifferentiation and differentiation markers during hepatic induction. Statistical analysis was done by Dunnett’s test against day 0 and graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01, *** P < .001. (C) Immunofluorescent staining of EBs at days 5, 8, and 10. Bars; 200 μm. (D) Enlarged images of EB at day 5 in (C). Bars; 50 μm. (E, F) Quantification of immune-positive cells in . Box plot showing the percentage of EPHA2 + cells among SOX17 + or OCT4 + cells (E). Box plot showing the percentage of EPHA2 + and TRA1-81 + cells among OCT4 + cells (F). Each box represents 1st quartile, median, and 3rd quartile, and whiskers show the minimum and maximum values. Ten images of independent EBs were analyzed. Total count of DAPI + nuclei per image were between 1 × 10 3 and 2 × 10 3 . Statistical significance was defined by Tukey test of SOX17 and OCT4, respectively in (E) and t -test between EPHA2 and TRA1-81 in (F). **P p < .01, *** P < .001, N.S; no significance between 3 with P > .05.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Staining

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: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet:

Article Snippet: Rabbit Anti human/mouse Phospho-EphA2 (Ser897) (D9A1) , Cell Signaling Technology , Cat#6347; RRID: AB_11220420.

Techniques: Recombinant, Modification, Membrane, Transfection, In Vivo, Software

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Treatment failure and evaluation of the underlying mechanism of non-response in TNBC recurrence, post treatment with 5FU (A) Survival curve of DFS in TNBC patients with differential treatment regimens, one SOC containing 5FU and the other cohort received SOC without 5FU. (B) Differential expression of phospho-EphA2 and phospho- c -MET in recurrent TNBC tissue. (C) Schematic representation of ex vivo explants culture. (D and E) Differential expression of Ki67 and Caspase 3c in TNBC patient derived ex vivo explants cultures, stratifying the cohort into responders and non-responder population. (F) Expressional status of phopsho-EphA2 and phospho- c -MET in the non-responder population from explants cultured tissues. Data are representative of triplicate experiments. ∗p < 0.05 represents statistically significant difference and was calculated by the log-rank test. Scale bars: 200 μm and 100 μm (in inset). HGF = hepatocyte growth factor; 5FU = 5-fluorouracil; SOC = standard of care; TNBC = triple negative breast cancer; HE = Hematoxylin & Eosin staining. Also see, Figures S1 and ; Tables S1–S6 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Quantitative Proteomics, Ex Vivo, Derivative Assay, Cell Culture, Staining

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Evaluation of the effect of individual and dual knock down of c-MET and EphA2 genes on MD-MB-231 in the presence of HGF (A) c-MET and EphA2 knockdown/inhibition and its effect on downstream effectors on MDA-MB-231 cells was analyzed by western blot; NTP = non-target pool control. (B) Transwell migration assay post knockdown/inhibition of c-MET or EphA2 or both and HGF as a chemotactic factor on MDA-MB-231 cells. (C) Transwell invasion assay post knockdown/inhibition of EphA2 and c-MET or both and HGF as a chemotactic factor on MDA-MB-231 cells. (D) A schematic representation of the mammosphere formation assay. (E) Mammosphere formation assay post knockdown/inhibition of c-MET or EphA2 or both in the presence of HGF. (F) Matrigel tube formation assay post knockdown/inhibition of c-MET or EphA2 or both in the presence of HGF. (G) Representative photomicrograph of freshly harvested breast tumors with a ruler (below) for scale. (H) Graph representing the tumor volume vs. the various groups. Data are representative of triplicate experiments (mean ± SD). ∗p < 0.05 statistically significant difference compared to corresponding control by one-way ANOVA. Scale bars: 100 μm. Also see, Figures S3 and .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Knockdown, Inhibition, Western Blot, Control, Transwell Migration Assay, Transwell Invasion Assay, Tube Formation Assay

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Evaluation of the combinatorial effect of Lupeol and 5FU on MDA-MB 231 cells (A) Western blot analysis of MDA-MB-231 cells treated with 5FU, Lupeol, or both in the presence of HGF and probed for phospho-EphA2 (S897), EphA2, phospho- c -MET, total c-MET, pERK1/2, total ERK1/2, MMP2 and Laminin-5Ƴ2 protein expression. β- Actin was used as a loading control. (B) Protein-protein interaction (PPI) network analysis using input proteins from the present study using STRING platform showing interacting molecules and edge confidence levels in the network. (C and D) Transwell migration and invasion of MDA-MB-231 cells upon treatment with Lupeol, 5FU or both, while HGF was used as a chemotactic factor (Scale bars: 100 μm). (E) Matrigel tube formation of MDA-MB-231 cells upon treatment with Lupeol, 5FU, or both in the presence of HGF (Scale bars: 500 μm and 100 μm in inset). (F) Primary and secondary Mammosphere formation of MDA-MB-231 cells in the presence of HGF and treated with 5FU, Lupeol, or both (Scale 200 μm in white and 100 μm in red). (G) Immunofluorescence staining of MDA-MB-231 cells for the detection of E-cadherin (green) and Vimentin (red) post treatment with Lupeol or 5FU or both in the presence of HGF (Scale bars: 100 μm). (H) Western blot analysis of MDA-MB-231 cells treated with Lupeol, 5FU, or both in the presence of HGF and probed for E-cadherin, Vimentin, SNAI1, SLUG, and TWIST. Data are representative of triplicate experiments (mean ± SD). HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Also see, Figure S5 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Western Blot, Expressing, Control, Migration, Immunofluorescence, Staining

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: In vivo evaluation of the combinatorial effect of Lupeol and 5FU in a TNBC syngeneic mice model (A) Schematic representation of dosing schedule (all compounds were administered intraperitoneally). (B) In vivo live animal images of the 4T1 luc2 induced tumors in various treatment arms in BALB/c mice at day 5 (initiation of treatment) and at day 15 (experimentation endpoint). (C) Graph representing the log of average radiance vs. Time of the tumor growth. (D) Representative photomicrograph of freshly harvested breast tumors with a ruler (below) for scale. (E) Graph representing the tumor volume vs. the various treatment arms. (F) Representative images of Hematoxylin and Eosin stain followed by IHC staining for Ki67 and Caspase 3c in the sections of harvested tumors developed in BALB/c mice along with their quantitative graphs. (G) Representative images of the IHC staining to evaluate the differential expression of phospho-EphA2 and phospho-cMET in the tumors of the various treatment arms along with their quantitative graphs. ∗p < 0.05 and ∗∗∗p < 0.001 statistically significant difference compared to corresponding control by one-way ANOVA. Data are representative of triplicate experiments (mean ± SD) ns = not significant; HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Scale bars: 200 μm. Also see, Figure S6 and Table S8 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: In Vivo, H&E Stain, Immunohistochemistry, Quantitative Proteomics, Control

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Evaluation of the combinatorial effect of 5FU and Lupeol in ex vivo explant culture model (A) Graphical representation of the patient data used in this experiment, pertaining to the nodal status and tumor stage. (B) Representative images of IHC staining of TNBC tissue fragments, post exposure to HGF, 5FU, Lupeol alone or in combination cells for the detection of Ki67 and Caspase 3c protein expression (Scale bars: 200 μm). (C) Graph representing the percentage of cells expressing Ki67 vs. the various treatment arms. (D) Graph representing the IHC score of Caspase 3c vs. various treatment arms. (E) Representative images of IHC staining for the evaluation of phospho-EphA2 and phospho-cMET in various treatment arms (Scale bars: 200 μm and 100 μm in inset). (F) Graph representing the various IHC scores of phospho-EphA2 in various treatment arms. (G) Graph representing the various IHC scores of phospho-cMET in various treatment arms. (H) Graph representing the various IHC scores of Caspase 3c expression in TNBC tumors (N = 7) after treating them with various SOC and 5FU and Lupeol combination. Data are representative of triplicate experiments (mean ± SD). ∗p < 0.05 statistically significant difference compared to corresponding control by one-way ANOVA. HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Also see, Table S9 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Ex Vivo, Immunohistochemistry, Expressing, Control

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet:

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Recombinant, Modification, Membrane, Transfection, In Vivo, Software

Journal: PLoS ONE

Article Title: A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

doi: 10.1371/journal.pone.0017649

Figure Lengend Snippet: Interference with eHsp90-LRP1 signaling inhibits GBM cell motility and invasion. (A, B) Treatment of cells with NPGA (1 µM) or anti-Hsp90 antibodies (20 ug/ml) (A), or suppression of LRP1 (B) similarly impaired G48a cell motility in wound healing assays. NS shRNA represents a nonspecifc shRNA control sequence. Percent migration is normalized to the 16 hr control and values represent the mean ± SD from 3 independent experiments (*p<0.001). (C) Serum starved parental or LRP1 silenced cells were added to top chambers of a Boyden assay and serum induced chemotaxis initiated in the presence of vehicle or NPGA. Cell numbers represent the mean ± SD from five random fields (*p<0.001). (D) The effects of eHsp90 targeting upon cell invasion were assessed by a Matrigel assay in the presence or absence of NPGA. Data is represented as the mean (± SD) of three replicates. *p<0.001. (E–F) Interference with eHsp90 function does not further inhibit cell motility or invasion in tandem with EphA2 silencing. G48a cells were transduced with either nonspecific (NS shRNA) or shEphA2 and effects of NPGA upon cell motility assessed in Boyden (right panel), and invasion assessed by Matrigel (left panel).

Article Snippet: Antibodies to P-src Y416 (2101), src (2108), P-AKT S473 (4058), and AKT (9272) were purchased from Cell Signaling; goat and mouse EphA2 antibodies (AF3035, MAB3035) were from R&D Biosystems; Rabbit P-EphA2 S897 anitbody was produced in Dr. Bingcheng Wang's laboratory HA conjugated beads (11815016001) were from Roche; Protein G agarose beads (15920-010) were from Invitrogen, anti-phosphotyrosine antibody (PY20) was from Santa Cruz; mouse and rabbit Hsp90 antibodies (ADI-SPA-830, ADI-SPS-771) were from Assay Designs, and anti- alpha tubulin antibody (T6074) was from Sigma.

Techniques: shRNA, Sequencing, Migration, Boyden Assay, Chemotaxis Assay, Matrigel Assay, Transduction

Journal: PLoS ONE

Article Title: A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

doi: 10.1371/journal.pone.0017649

Figure Lengend Snippet: (A) Control or LRP1 silenced G48a cells were treated with NPGA, ephrin A1, or the combination, and the effects upon P-AKT S473 and P-EphA2 S897 were evaluated. The effect of src silencing was included for relative comparison. (B) Interference with eHsp90 signaling (NPGA or LRP1 silencing), or AKT activation (src silencing or treatment with ephrin A1) disrupts EphA2-AKT protein complexes. (C) G48a cells grown were fixed 4 hr after wounding, followed by immunostaining with the indicated antibodies. Arrows indicate the leading edge localization of P-EphA2 S897 (a–c). Stimulation of cells with ephrinA1 was included as positive control for suppression of EphA2 S897 phosphorylation (d–f). Lamellipodia formation and concomitant localization of P-EphA2 S897 is similarly suppressed by NPGA (g–i) or LRP1 silencing (j–l), or by src silencing (m–o). Scale bars 25 µm.

Article Snippet: Antibodies to P-src Y416 (2101), src (2108), P-AKT S473 (4058), and AKT (9272) were purchased from Cell Signaling; goat and mouse EphA2 antibodies (AF3035, MAB3035) were from R&D Biosystems; Rabbit P-EphA2 S897 anitbody was produced in Dr. Bingcheng Wang's laboratory HA conjugated beads (11815016001) were from Roche; Protein G agarose beads (15920-010) were from Invitrogen, anti-phosphotyrosine antibody (PY20) was from Santa Cruz; mouse and rabbit Hsp90 antibodies (ADI-SPA-830, ADI-SPS-771) were from Assay Designs, and anti- alpha tubulin antibody (T6074) was from Sigma.

Techniques: Activation Assay, Immunostaining, Positive Control

Journal: PLoS ONE

Article Title: A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

doi: 10.1371/journal.pone.0017649

Figure Lengend Snippet: (A) Serum starved (8 hr) G48a cells were exposed to either native or Hsp90 ΔATPase proteins (3 µg/ml) for 15 min in the presence or absence of NPGA, and the indicated signaling molecules evaluated by immunoblot. The effect of these treatments upon EphA2-AKT interaction was also evaluated. (B) A Matrigel invasion assay was utilized to evaluate the ability of native or Hsp90 ΔATPase proteins to sustain cell invasion in the presence of NPGA. Values represent the mean (± SD) of 3 independent experiments. (C) G48a cells stably transduced with the indicated HA-tagged myristolyated AKT constructs were exposed to NPGA and HA immunopurified complexes were evaluated for P-AKT and P-EphA2 S897 . (D) The pro-motility function of the indicated AKT proteins was evaluated in the presence or absence of NPGA using a scratch wound assay.

Article Snippet: Antibodies to P-src Y416 (2101), src (2108), P-AKT S473 (4058), and AKT (9272) were purchased from Cell Signaling; goat and mouse EphA2 antibodies (AF3035, MAB3035) were from R&D Biosystems; Rabbit P-EphA2 S897 anitbody was produced in Dr. Bingcheng Wang's laboratory HA conjugated beads (11815016001) were from Roche; Protein G agarose beads (15920-010) were from Invitrogen, anti-phosphotyrosine antibody (PY20) was from Santa Cruz; mouse and rabbit Hsp90 antibodies (ADI-SPA-830, ADI-SPS-771) were from Assay Designs, and anti- alpha tubulin antibody (T6074) was from Sigma.

Techniques: Western Blot, Invasion Assay, Stable Transfection, Transduction, Construct, Scratch Wound Assay Assay

Journal: PLoS ONE

Article Title: A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

doi: 10.1371/journal.pone.0017649

Figure Lengend Snippet: (A) The ability of hypoxia to modulate Hsp90α secretion was determined by ELISA, as in Supplementary . (B) G48a cells cultured in 1% serum were exposed to hypoxia (1% O 2 ) for 36 hr and surface expression of Hsp90α and LRP1 was determined in intact cells by flow cytometry. A corresponding immunoblot shows total cellular expression of LRP1 and Hsp90α. (C) Representative immunoblot demonstrating effects of hypoxia upon activation of src, AKT, and EphA2 in the presence or absence of NPGA. (D) A Matrigel invasion assay was utilized to evaluate the effects of hypoxia (16 hr) upon cell invasion in the presence or absence of NPGA.

Article Snippet: Antibodies to P-src Y416 (2101), src (2108), P-AKT S473 (4058), and AKT (9272) were purchased from Cell Signaling; goat and mouse EphA2 antibodies (AF3035, MAB3035) were from R&D Biosystems; Rabbit P-EphA2 S897 anitbody was produced in Dr. Bingcheng Wang's laboratory HA conjugated beads (11815016001) were from Roche; Protein G agarose beads (15920-010) were from Invitrogen, anti-phosphotyrosine antibody (PY20) was from Santa Cruz; mouse and rabbit Hsp90 antibodies (ADI-SPA-830, ADI-SPS-771) were from Assay Designs, and anti- alpha tubulin antibody (T6074) was from Sigma.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Flow Cytometry, Western Blot, Activation Assay, Invasion Assay

Journal: PLoS ONE

Article Title: A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

doi: 10.1371/journal.pone.0017649

Figure Lengend Snippet: (A) HA-EphA2 transfected U87 cells were treated with NPGA, ephrin A1, or the src inhibitor PP2 for 16 hr. LRP1 was detected from HA immunoprecipitates. (B) U87 cells were transfected with HA-tagged wild type or point mutant (S897G) EphA2 plasmid, and LRP1 was detected from HA immunoprecipitates. (C) U87 cells were immunostained with indicated antibodies showing that LRP1 co-localized with EphA2. Scale bar 25 µm. The bottom panels represent magnified areas of confocal images derived from the respective upper panels, as delineated by the boxed region. Scale bar 5 µm. (D) Detection of P-EphA2 S897 and LRP1 in human GBM specimens. Panels a–f, and m–o are paraffin sections, panels g–l are frozen specimens. Magnification ×200. Lower panels ( a–l ) are paraffin sections from the recurrent GBM specimens. Magnification ×400.

Article Snippet: Antibodies to P-src Y416 (2101), src (2108), P-AKT S473 (4058), and AKT (9272) were purchased from Cell Signaling; goat and mouse EphA2 antibodies (AF3035, MAB3035) were from R&D Biosystems; Rabbit P-EphA2 S897 anitbody was produced in Dr. Bingcheng Wang's laboratory HA conjugated beads (11815016001) were from Roche; Protein G agarose beads (15920-010) were from Invitrogen, anti-phosphotyrosine antibody (PY20) was from Santa Cruz; mouse and rabbit Hsp90 antibodies (ADI-SPA-830, ADI-SPS-771) were from Assay Designs, and anti- alpha tubulin antibody (T6074) was from Sigma.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Derivative Assay

Journal: PLoS ONE

Article Title: A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

doi: 10.1371/journal.pone.0017649

Figure Lengend Snippet: An eHSP90/LRP1 signaling axis is required to sustain src directed AKT activation, AKT dependent P-EphA2 S897 , and LRP1 recruitment to EphA2. These signaling events facilitate lamellipodia formation and support GBM cell motility and invasion. Hypoxia amplifies eHsp90 signaling and corresponding motility via enhanced LRP1 expression and Hsp90 secretion. NPGA inhibits eHsp90 signaling, with consequent inhibition of AKT, disruption of EphA2 and LRP1 complexes, and blockade of cell motility. ephrin A1 ligand similarly suppresses AKT activation, P-EphA2 S897 , EphA2-LRP1 complexes, and elicits comparable inhibitory effects upon GBM cell motility and invasion.

Article Snippet: Antibodies to P-src Y416 (2101), src (2108), P-AKT S473 (4058), and AKT (9272) were purchased from Cell Signaling; goat and mouse EphA2 antibodies (AF3035, MAB3035) were from R&D Biosystems; Rabbit P-EphA2 S897 anitbody was produced in Dr. Bingcheng Wang's laboratory HA conjugated beads (11815016001) were from Roche; Protein G agarose beads (15920-010) were from Invitrogen, anti-phosphotyrosine antibody (PY20) was from Santa Cruz; mouse and rabbit Hsp90 antibodies (ADI-SPA-830, ADI-SPS-771) were from Assay Designs, and anti- alpha tubulin antibody (T6074) was from Sigma.

Techniques: Activation Assay, Expressing, Inhibition

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: 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: 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: Comparison of antitumor effects of different EphA2 CAR-T cells in xenograft mouse models. (a) 5 × 10 6 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 × 10 7 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/cm 2 /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 × 10 5 cells were injected into the left striatum through a burr hole in the skull. Two weeks after tumor cells injection, 3 × 10 7 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/cm 2 /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: 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: 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: 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 × 10 7 U251.eGFP.Luc cells to construct a xenograft mouse model. Five days post-tumor xenograft, mice were treated with a total of 3 × 10 7 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: 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:

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:

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: Cell Analysis, Variant Assay