Analytical Validation of Digital Spatial Profiling - a novel approach for multiplexed characterization of protein distribution and abundance in FFPE tissue sections

Nanostring Technologies’ Digital Spatial Profiling (DSP) technology allows for the simultaneous analysis of 10’s to 100’s of proteins from discrete regions of interest (ROI), providing a morphological context to multiplexed protein analysis. The purpose of this study is to validate an antibody panel designed to characterize key immunology and tumor markers on the DSP platform. IHC was performed on human FFPE tissues and human cell line pellets to evaluate binding specificity in both unconjugated and oligo-conjugated antibodies. The dynamic range of antibodies was tested using positive and negative FFPE cell pellets at different ratios. Interaction screens were performed to evaluate potential effects of multiplexing antibodies. A tissue microarray (TMA) containing normal and cancer tissues was employed to assess assay robustness. IHC analysis of antibodies displayed indistinguishable staining patterns for both unconjugated and oligoconjugated antibodies. Mixed-proportion cell pellet assays revealed strong correlations between positive counts and positive cell numbers in an ROI. For example, CD3 displayed a LOD of 4% when assayed using cell pellet mixtures containing CD3+ CCRF-CEM cells and CD3− HEK293T cells. Interaction studies showed similar values for antibodies alone or in combination (R2 > 0.8). TMA hierarchical clustering analysis demonstrated expected patterns for immune and tumor cells across different tissues. These results demonstrate that indexing oligo conjugation does not interfere with antibody specificity and that these conjugated antibodies are robust reagents for quantification of protein abundance. Continued work on the DSP platform will expand the library of antibodies accessible for profiling.