Journal: Molecular Oncology

Article Title: Multi‐omic molecular profiling guide’s efficacious treatment selection in refractory metastatic breast cancer: a prospective phase II clinical trial

doi: 10.1002/1878-0261.13091

Figure Lengend Snippet: Summary of molecular findings in the Side Out 2 trial based on patients’ GMIs. Frequencies of gene expression, protein expression by immunohistochemistry and protein activation by RPPA (Panel A, Panel B, and Panel C, respectively) for 22 of the 25 patients enrolled in the Side Out 2 trial.

Article Snippet: Patient‐matched fresh frozen material was used by the Center for Applied Proteomics and Molecular Medicine at George Mason University for protein pathway activation mapping using laser capture microdissection (LCM) to isolate tumor epithelia followed by Reverse Phase Protein Microarray (RPPA) analysis as previously described [ ].

Techniques: Gene Expression, Expressing, Immunohistochemistry, Activation Assay

Journal: Expert opinion on medical diagnostics

Article Title: Antibody Profiling with Protein Antigen Microarrays in Early Stage Cancer

doi: 10.1517/17530059.2012.672969

Figure Lengend Snippet: A schematic of the native protein antigen “reverse capture” microarray platform. Well-characterized monoclonal antibodies are printed onto a glass slide. Native protein antigens derived from cell lines or cancer tissues are immobilized onto the array by the printed monoclonal antibodies, with the corresponding antigen captured by a specific monoclonal antibody. Using the immobilized antigens as bait, labeled autoantibodies from a patient and control are then allowed to react with the immobilized antigen. The amount of fluorescent dye-labeled autoantibody between test and control will determine reactivity with the immobilized antigen. We termed this array a “reverse capture” microarray since the standard nomenclature for “capture” refers to the analyte/antigen and not the antibody.

Article Snippet: Declaration of interest: Brian Liu serves on the Scientific Advisory Board of Inanovate, Inc., and is one of the inventors and has licensing rights to the “reverse capture” native protein antigen microarray platform described in the article.

Techniques: Microarray, Bioprocessing, Derivative Assay, Labeling, Control

Journal: Expert opinion on medical diagnostics

Article Title: Antibody Profiling with Protein Antigen Microarrays in Early Stage Cancer

doi: 10.1517/17530059.2012.672969

Figure Lengend Snippet: Whole proteome lysate fractionation microarray. Tumor or cell lysates are separated into distinct fractions using a 2-D liquid chromatography fractionation strategy where the 1st dimension is separation by isoelectric points and the 2nd dimension is separation by hydrophobicity. Following the 2nd dimension, the fractions are arrayed onto nitrocellulose coated microscope slides. The slides are then incubated with fluorescent dye-labeled serum antibody isolated from cases and controls. The informative spots on the arrays and their corresponding fractions containing the associated antigens are then validated and the protein antigens subsequently identified by mass spectrometry.

Article Snippet: Declaration of interest: Brian Liu serves on the Scientific Advisory Board of Inanovate, Inc., and is one of the inventors and has licensing rights to the “reverse capture” native protein antigen microarray platform described in the article.

Techniques: Fractionation, Microarray, Liquid Chromatography, Microscopy, Incubation, Labeling, Isolation, Mass Spectrometry

Journal: Expert opinion on medical diagnostics

Article Title: Antibody Profiling with Protein Antigen Microarrays in Early Stage Cancer

doi: 10.1517/17530059.2012.672969

Figure Lengend Snippet: This figure is an image of a whole proteome breast cancer lysate microarray slide scanned for fluorescence after incubating with antibodies isolated from serum from a patient with late stage breast cancer that have underwent autologous tumor vaccination. Post-vaccination antibodies were labeled with Cy3 (green) fluorescent dye, and pre-vaccination antibodies from the same patient were labeled with Cy5 (red) fluorescent dye. Note that there was little reactivity from the pre-vaccination antibodies, but numerous post-vaccination reactive “spots” are seen.

Article Snippet: Declaration of interest: Brian Liu serves on the Scientific Advisory Board of Inanovate, Inc., and is one of the inventors and has licensing rights to the “reverse capture” native protein antigen microarray platform described in the article.

Techniques: Microarray, Fluorescence, Isolation, Labeling

Journal: Scientific Reports

Article Title: Combined PI3Kα-mTOR Targeting of Glioma Stem Cells

doi: 10.1038/s41598-020-78788-z

Figure Lengend Snippet: A discrete role for the alpha catalytic PI3K isoform in proneural (PN) GSCs. ( A ) Expression z-score data from TCGA (Extended Verhaak) for PIK3CA (left panel), PIK3CB (middle panel) and PIK3CD (right panel) were downloaded from the GBM-BioDP ( http://gbm-biodp.nci.nih.gov/ ) for CL (n = 105), MES (n = 124) and PN (n = 113) subtype GBM from the HT Human Genome U133 (HT_HG-U133A) array and analysed using GraphPad Prism 8. As recent evidence suggests the neural subtype represents excessive contamination with normal brain, it has been excluded from the analysis. Unpaired one-way ANOVA, * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001. ( B ) Survival analysis based on the impact of the multi-gene prognostic index for coexpression of PIK3CA and NES (upper panels), PIK3CB and NES (middle panels) or PIK3CD and NES (lower panels) for CL (n = 53, left panels), MES (n = 57, middle panels) and PN (n = 56, right panels) subtype. TCGA gene expression data (Verhaak Core) for PIK3CA and NES , PIK3CB and NES or PIK3CD and NES from the HT_HG-U133A array were used for multigene prognostic index. Figure was generated using the GBM-BioDP software. ( C ) GSC lines JK16, 83Mes, 157PN and AC17PN were analysed by Western blot using antibodies against NF1, p110α and GAPDH. Membrane was stripped and reprobed with an antibody against p110β. Lysates from the same experiment were run in parallel and probed for p110 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\updelta $$\end{document} δ and GAPDH. ( D ) Reverse-phase protein microarray (RPPA) data from TCGA GBM samples. Boxplots of RPPA analytes for phospho-AKT(Ser 473 ) measured in CL (n = 28), MES (n = 29) and PN (n = 41) samples from the cohort of GBM tumours (Extended Verhaak) that was subjected to RPPA analysis within the TCGA consortium. RPPA data were downloaded from the GBM-BioDP and analysed using GraphPad Prism 8. Unpaired one-way ANOVA: * p ≤ 0.05. ( E ) Survival analysis based on RPPA analysis for phospho-AKT(Ser 473 ) using the Extended Verhaak cohort submitted to the TCGA consortium for CL (n = 28, left panel), MES (n = 29, middle panel) and PN (n = 41, right panel) subtypes. Figure was generated using the GBM-BioDP software. ( F ) GSC lines JK16, 83Mes, 157PN and AC17PN were analysed by Western blot using antibodies against AKT. Membrane was stripped and re-probed for p-AKT(Thr 308 ), then stripped and reprobed for p-AKT(Ser 473 ), then stripped and re-probed for HSP90.

Article Snippet: Similarly, protein Reverse Phase Protein Microarray (RPPA) data of the Extended Verhaak dataset for p-AKT (S473) were downloaded and analysed using GraphPad Prism 8.

Techniques: Expressing, Gene Expression, Generated, Software, Western Blot, Membrane, Microarray