Abstract
Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.
Highlights
Protein extracts from matched fresh frozen (FF) and formalin fixation and paraffin embedding (FFPE) tissues derived from 20 xenograft individual tumors (40 samples) were evaluated by Reverse phase protein arrays (RPPA) using a panel of 300 markers
For the BxPC-3 and FaDubearing xenograft tumors, protein extracts were prepared from untreated mice (n ϭ 3 biological replicates each) and from mice treated with the anti- human epidermal growth factor receptor 3 (HER3) antibody (n ϭ 3 each)
coefficient of variation (CV) values were summarized to a median CV of 11% for FF samples and a median CV of 18% for FFPE samples
Summary
Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)*□S. Relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The expression and activation status of proteins from these deregulated pathways has traditionally been analyzed using single marker techniques such as immunohistochemistry and Western blotting These techniques have provided valuable information on the molecular abnormalities underlying human disease, they are labor intensive, have a low throughput, and often require high sample volume.
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