Abstract 2187: A multiplexed staining method to quantify EMT derived tumor heterogeneity presents a means of predicting patient prognosis based on EMT state
Authors: Brown Meredith S. 
Published: June 2022

Abstract
Intra-tumoral heterogeneity is a key contributor to poor prognosis and metastatic disease in breast cancer. This study seeks to detect and quantify intra-tumoral heterogeneity deriving from the Epithelial-to-Mesenchymal transition using a multi-round, multiplexed immunofluorescence staining approach to delineate EMT states at a single cell resolution in primary tumor specimens to aid in pathological patient diagnosis. Despite major advances in our understanding, the contributions of EMT research to improvements in diagnostic pathology or cancer therapy have been minimal. One reason for this gap stems from our inability to accurately detect and quantify epithelial-mesenchymal heterogeneity in primary tumor specimens. Secondly, the significance of recently identified intermediate or partial EMT states to predicting tumor prognosis and therapy response are unclear. To study the role of various states within the EMT spectrum and their regulatory networks, the heterogeneous breast cancer cell line, SUM149PT, was used to derive six single cell clones encompassing the spectrum of EMT states, from epithelial to mesenchymal. Interrogation of this model system in vivo has revealed increased tumor growth and metastatic potential in the intermediate EMT states when compared to the extreme epithelial and mesenchymal states. To further elucidate EMT states in vivo, we employ a 6-marker multi-round immunofluorescence-based staining approach to identify cells that reside in various states along the EMT spectrum. We subsequently used cell-segmented, entropy-based approach and weighted phenotype analysis on these tumors with the purpose of scoring heterogeneity and overall EMT state. Notably, this analysis segregated stromal infiltrates and their contributions to aggregate EMT scoring, which has been a major hurdle in using EMT as a scoring metric in patient samples. Overall, SUM149 clone-derived tumors held true to the relative EMT states of the starting cell populations; intermediate-derived tumors displayed high heterogeneity while epithelial and mesenchymal clone-derived tumors had lower levels of heterogeneity, despite retaining different EMT scores. Decoupling of heterogeneity and EMT state in this way provides two metrics to assess potential metastatic ability of a tumor. This staining method and analysis has been successfully applied in a preliminary set of patient tumors, showing promise for these two factors, E-M Heterogeneity and EMT score, as a tumor prognostic indicator to inform therapeutic decision-making. In the future, this staining and quantification shows promise as a means of predicting patient prognosis and informing potential treatment options based on targeting EMT states.
Citation Format: Meredith S. Brown. A multiplexed staining method to quantify EMT derived tumor heterogeneity presents a means of predicting patient prognosis based on EMT state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2187.