Abstract 2113: The effects of enzymatic digestion on epitope detection by flow cytometry
Authors: Middlebrook Aaron J. , Austin Caitlin , Santos Daniel , Snowden Eileen , Porter Warren , Hahn Friedrich , Ferguson Mitchell , Blaesius Rainer , Ghanekar Smita 
Published: August 2018

Abstract
The heterogeneous nature of solid tumors, coupled with the relatively small sample size of available biopsies, has led to an emerging need to glean as much information as possible from these valuable specimens. Current approaches to solid tumor analysis fail to completely reveal the diverse range of cellular compartments that comprise the tumor microenvironment. A comprehensive approach to tumor interrogation requires efficient tissue dissociation to facilitate analysis at the single-cell level. In contrast to current methods, single-cell analysis of tumor derived cell suspensions by flow cytometry has the potential to provide a more complete understanding of the many subpopulations within the tumor microenvironment and the cell to cell interactions that govern this space. In order to prepare a cell suspension from solid tumor tissue, most investigators subject the tumor sample to a combination of mechanical and enzymatic dissociation modalities. Regardless of the combination of enzymes used for dissociation, there exists a degree of non-specific proteolytic cleavage. In the work presented here, we set out to quantify the non-specific cleavage associated with a proprietary enzyme mixture developed for tumor dissociation. Using a mixed sample that included peripheral blood mononuclear cells and several cell lines, we were able to generate a sample that expressed approximately 90% of our current antibody catalog. By staining our sample with a rudimentary 3 color panel, we were able to identify 10 individual cell populations based on marker expression and/or scatter. This composite sample was left untreated or exposed to our propriety mixture of dissociation enzymes and then subsequently screened for all 251 antigens in our catalog. The impact of non-specific proteolytic cleavage on surface marker detection was gauged in terms of median fluorescence intensity of positive population and stain index. Our results indicate that of the 251 surface markers tested in our experimental series, less than 10 percent were significantly affected by enzymatic digestion. Of those markers that were affected only a fraction of them were affected to such an extent that it was impossible to detect positive expression. These results underscore the importance of understanding the factors that can influence surface marker detection and bolster our confidence that this particular mixture of enzymes is imparting minimal impact on surface marker detection.
Citation Format: Aaron J. Middlebrook, Caitlin Austin, Daniel Santos, Eileen Snowden, Warren Porter, Friedrich Hahn, Mitchell Ferguson, Rainer Blaesius, Smita Ghanekar. The effects of enzymatic digestion on epitope detection by flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2113.