Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: EphA2 antibody, anti-mouse, APC, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-109-187 RRID: AB_2651638.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: Theranostics

Article Title: EphA2-targeted alpha-particle theranostics for enhancing PDAC treatment

doi: 10.7150/thno.106948

Figure Lengend Snippet: CCLE and TCGA database of mRNA expression of EphA2. A) EphA2 expression in cell lines in CCLE. B) EphA2 mRNA expression in different types of tumors in TCGA. C) Comparison of mRNA expression of EphA2 in different normal tissues and corresponding tumors. D) Expression of different EPH receptors in pancreatic tumors. PAAD = pancreatic cancer; BLCA = Bladder cancer; READ = Rectal cancer; LUSC = Lung squamous cell carcinoma; THCA = Thyroid cancer; KIRP = Kidney papillary cell carcinoma; LUAD = Lung adenocarcinoma; KIRC = Kidney clear cell carcinoma; CHOL = Bile duct cancer; SKCM = melanoma; LIHC = Liver cancer; KICH = Kidney chromophobe; BRCA = Breast cancer; PRAD = Prostate cancer; DLBC = Large-B-cell lymphoma; LAML = Acute myeloid leukemia.

Article Snippet: Similarly mouse pancreatic cell lines were stained with FITC labeled anti-mouse EphA2 monoclonal antibody (SinoBiological Cat # 50586-R301-F).

Techniques: Expressing, Comparison

Journal: Theranostics

Article Title: EphA2-targeted alpha-particle theranostics for enhancing PDAC treatment

doi: 10.7150/thno.106948

Figure Lengend Snippet: Structure and in vitro characterization of AJ201. A) Structure of bicyclic peptide AJ201 having NOTA as bifunctional chelator for 68 Ga-labeling. In the structure, black color represents binding moiety, parakeet color represents the linker and red color represents the radiometal B) Surface plasmon resonance (SPR) analysis showing affinity of AJ201 for EphA2 using recombinant human (solid) and mouse (dashed) EphA2 proteins.

Article Snippet: Similarly mouse pancreatic cell lines were stained with FITC labeled anti-mouse EphA2 monoclonal antibody (SinoBiological Cat # 50586-R301-F).

Techniques: In Vitro, Labeling, Binding Assay, SPR Assay, Recombinant

Journal: Theranostics

Article Title: EphA2-targeted alpha-particle theranostics for enhancing PDAC treatment

doi: 10.7150/thno.106948

Figure Lengend Snippet: In vitro specificity of [ 68 Ga]AJ201 for EphA2 in human pancreatic cancer cells. A) Quantification of EphA2-mRNA using RT-PCR. B) Flow cytometry analysis of EphA2 receptor expression on cell surface. C) A representative plot of EphA2 receptor density in PDAC cells measured by quantibrite assay. D) [ 68 Ga]AJ201 binding (percent incubated activity, %IA) to different cells. Cells were incubated with 1 µCi [ 68 Ga]AJ201 at 4 °C for 1 h. [ 68 Ga]AJ201 uptake is EphA2 expression dependent, and co-incubation with 2 μM of non-radioactive AJ201 (0.2 nmol, blocking dose) significantly reduced radiotracer uptake confirming EphA2 specificity. E) In vitro uptake of [ 68 Ga]AJ201 over 60 min in Panc1 cells at 4 ˚C and 37 ˚C. F) Correlation of [ 68 Ga]AJ201 uptake with surface EphA2 receptor density. Data in panels A , D , E and F are presented as mean ± SD (n = 3-4). Significance was calculated using multiple unpaired t test in D ; ns, P ≥ 0.05; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001. Simple linear regression and Pearson coefficient were used in F . All in vitro experiments were performed three times and representatives are shown.

Article Snippet: Similarly mouse pancreatic cell lines were stained with FITC labeled anti-mouse EphA2 monoclonal antibody (SinoBiological Cat # 50586-R301-F).

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Expressing, Binding Assay, Incubation, Activity Assay, Blocking Assay

Journal: Theranostics

Article Title: EphA2-targeted alpha-particle theranostics for enhancing PDAC treatment

doi: 10.7150/thno.106948

Figure Lengend Snippet: In vivo specificity of [ 68 Ga]AJ201 for EphA2 in NSG mice with PDAC tumor xenografts. A) Whole-body PET/CT images of different human PDAC xenografts at 60 min after the injection of radiotracer. Mice were injected with ~ 7.4 MBq (~200 µCi) [ 68 Ga]AJ201; K, kidney. Panc1 mouse image is reproduced from figure E. B) [ 68 Ga]AJ201 uptake quantification (%ID/g) in different PDAC tumors by ex vivo biodistribution at 60 min after injection. Left: %ID/g of different tumor xenografts, tumor-to-blood (middle) and tumor-to-muscle (right) ratios in each individual mice harboring respective xenografts. C) IHC staining for EphA2 expression in PDAC xenografts (digitally scanned at 40x).; data in figure B is shown as box and whisker plots (median ± IQR) showing all data points (n = 4-5).

Article Snippet: Similarly mouse pancreatic cell lines were stained with FITC labeled anti-mouse EphA2 monoclonal antibody (SinoBiological Cat # 50586-R301-F).

Techniques: In Vivo, Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo, Immunohistochemistry, Expressing, Whisker Assay

Journal: Theranostics

Article Title: EphA2-targeted alpha-particle theranostics for enhancing PDAC treatment

doi: 10.7150/thno.106948

Figure Lengend Snippet: In vivo distribution of [ 68 Ga]AJ201 in Panc1-orthotopic tumor model. A) Coronal sections of the fused PET/MR images showing [ 68 Ga]AJ201 distribution. Primary tumor is indicated by red circle; K = Kidney, B = Bladder. B) Quantification of accumulated activity in tumor, muscle and pancreas of images shown in panel A (n = 4-5) C) tumor-to-muscle (T/M) and tumor-to-pancreas (T/P) ratios from the images shown in panel A (n = 4) D) EphA2 IHC of orthotopic Panc1 tumor and normal pancreas to validate the EphA2 expression. Panc1 orthotopic tumor model was generated by surgically implanting a 3-5 mm³ section of Panc1 tumor xenograft directly onto the pancreas. The implanted tumors were allowed to grow for 10 days, after which they were ready for imaging. At this stage, MRI measurements indicated that tumor sizes ranged from 10-50 mm 3 . data in figure B and C is shown as box and whisker plots (median ± IQR) showing all data points (n = 3-5).

Article Snippet: Similarly mouse pancreatic cell lines were stained with FITC labeled anti-mouse EphA2 monoclonal antibody (SinoBiological Cat # 50586-R301-F).

Techniques: In Vivo, Positron Emission Tomography-Magnetic Resonance Imaging, Activity Assay, Expressing, Generated, Imaging, Whisker Assay

Journal: MedComm – Oncology

Article Title: Ephrin A1 ligand‐based CAR‐T cells for immunotherapy of EphA2‐positive cancer

doi: 10.1002/mog2.70010

Figure Lengend Snippet: FIGURE 3 EFNA1 chimeric antigen receptor (CAR)‐T cells effectively suppressed stem‐like pancreatic cancer cells. (A, B) The expression levels of the CSC markers CD133, CXCR4, OCT4, CD44, and CD24 on parental (WT) cells, and induced‐PANC1 CSC (A) and induced‐MIA‐PaCa2 CSC (B) were detected by real‐time quantitative PCR. (C) The protein expression levels of EphA2 in parental (WT) cells and induced‐CSCs (PANC1‐CSC and MIA PaCa2‐CSC) were detected by western blot analysis. (D) The grayscale analysis of western blot analysis. (A–C) each include three technical replicates. The pictures showed are one example from these three technical replicates. (E) The cytotoxicity of NTD, MOCK, and EFNA1 CAR‐T cells against CSCs at an E:T ratio of 4:1 was measured using a luciferase cytotoxicity assay. (F–I) The release levels of IFN‐γ (F, G) and TNF‐α (H, I) were measured by enzyme‐linked immunosorbent assay after coculturing NTD, MOCK, and EFNA1 CAR‐T cells with CSCs for 24 h at an E:T ratio of 4:1. (E–I) comprises three biologic replicates and three technical replicates. An unpaired t‐test was used for the analysis of two groups, while a two‐tailed one‐way ANOVA was employed for three groups. Data are shown as mean ± SD; **p < 0.01, ***p < 0.001, ns, no significance.

Article Snippet: The study utilized mouse β‐actin monoclonal antibody (Santa Cruz, 1:1000, SC‐47778), rabbit GAPDH monoclonal antibody (abcam, 1:1000, ab9484), and recombinant mouse EphA2 monoclonal antibody (Santa Cruz 1:1000, SC‐398832).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: MedComm – Oncology

Article Title: Ephrin A1 ligand‐based CAR‐T cells for immunotherapy of EphA2‐positive cancer

doi: 10.1002/mog2.70010

Figure Lengend Snippet: FIGURE 4 EFNA1 chimeric antigen receptor (CAR)‐T cells exhibited effective antitumor activity in PANC1 xenograft mouse model. (A) Diagram illustrating the PANC1 pancreatic cancer mouse model. (B) Six‐week NCG mice were injected with PANC1‐luciferase cells (3 × 105/mouse) subcutaneously. MOCK T cells and EFNA1 CAR‐T cells (1 × 107/mouse: total number of T cells) were injected via the tail vein 4 days after PANC1‐luciferase injection. Tumor growth was monitored and evaluated based on total bioluminescence signals, n = 4. (C) The statistics of total radiance of the mice in MOCK and EFNA1 CAR groups. (D) Representative confocal microscopy images of the EphA2 expression in the tumors of MOCK and EFNA1 CAR groups, detected by immunofluorescence staining (scale bar = 50 μm). (E) The DNA copy numbers of MOCK T cells and EFNA1 CAR‐T cells in the peripheral blood were measured by real‐time quantitative PCR at different time points (n = 3). The experiments were conducted twice independently, yielding consistent results. Two groups were compared using an unpaired t‐test. The results are presented as the mean ± SD. *p < 0.05, **p < 0.01, ns, no significance.

Article Snippet: The study utilized mouse β‐actin monoclonal antibody (Santa Cruz, 1:1000, SC‐47778), rabbit GAPDH monoclonal antibody (abcam, 1:1000, ab9484), and recombinant mouse EphA2 monoclonal antibody (Santa Cruz 1:1000, SC‐398832).

Techniques: Activity Assay, Injection, Luciferase, Confocal Microscopy, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

Journal: MedComm – Oncology

Article Title: Ephrin A1 ligand‐based CAR‐T cells for immunotherapy of EphA2‐positive cancer

doi: 10.1002/mog2.70010

Figure Lengend Snippet: FIGURE 5 Effectiveness and safety of EFNA1 chimeric antigen receptor (CAR)‐T cells in mice. (A) EphA2 expression in mouse tumor cell lines (4T‐1 and Hepa1‐6) was detected by immunofluorescence staining, isotype as a negative control; scale bar = 20 μm. (B) Mean fluorescence intensity (MFI) of EphA2 staining per cell, n = 3. (A) and (B) each include three technical replicates. The pictures showed are one example from these three replicates. (C) The cytotoxicity of EFNA1 CAR‐T cells against 4T1 and Hepa1‐6 at an E:T ratio of 4:1 for 24 h was detected by luciferase‐based assay. (D, E) The IFN‐γ (D) and TNF‐α (E) release levels in the cocultured supernatants were assessed by enzyme‐linked immunosorbent assay. (C–E) comprise three biological and technical replicates. (F) Pathological analysis of the indicated organs in NCG mice was assessed after T‐cell injection for 56 days by hematoxylin and eosin (H&E) staining; scale bars = 50 μm. Three groups were analyzed by two‐tailed one‐way ANOVA. Data were plotted and are shown as mean ± SD; ***p < 0.001.

Article Snippet: The study utilized mouse β‐actin monoclonal antibody (Santa Cruz, 1:1000, SC‐47778), rabbit GAPDH monoclonal antibody (abcam, 1:1000, ab9484), and recombinant mouse EphA2 monoclonal antibody (Santa Cruz 1:1000, SC‐398832).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control, Fluorescence, Luciferase, Enzyme-linked Immunosorbent Assay, Injection, Two Tailed Test

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: 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: 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: Staining was completed with EphA2-PE (anti-mEphA2 PE conjugated R&D Systems #FAB639P).

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: Staining was completed with EphA2-PE (anti-mEphA2 PE conjugated R&D Systems #FAB639P).

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: Staining was completed with EphA2-PE (anti-mEphA2 PE conjugated R&D Systems #FAB639P).

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: Staining was completed with EphA2-PE (anti-mEphA2 PE conjugated R&D Systems #FAB639P).

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: Staining was completed with EphA2-PE (anti-mEphA2 PE conjugated R&D Systems #FAB639P).

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: Staining was completed with EphA2-PE (anti-mEphA2 PE conjugated R&D Systems #FAB639P).

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

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