Abstract 2911: Single cell mass cytometry analysis of human lung adenocarcinoma
Authors: Doxie Deon B. , Lehman Jonathan M. , Zou Yong , Ortega Maria S. , Maier Caroline E. , Irish Jonathan M. , Massion Pierre P. 
Published: July 2017

Abstract
Introduction: Lung cancer is the leading cause of cancer-related mortality in the world. Lung adenocarcinoma is the most common subtype. Tumor heterogeneity among adenocarcinomas presents a challenge in the management of the disease. Understanding heterogeneity may have implications in understanding the biological processes driving progression. Single cell platforms like mass cytometry offer an opportunity to profile tumor heterogeneity and to identify populations of driven by the activation of signaling pathways.
Methods: Adenocarcinomas were collected at the time of surgery and dissociated into suspension and cryopreserved. Mass cytometry analysis of tumors and adenocarcinoma cell lines was performed with a 30 marker antibody panel and rhodium intercalator dye to identify dead cells. The antibody panel included markers to characterize identity, cell cycle status, and signaling events. Biaxial gating or unsupervised analysis approaches SPADE and viSNE were used to compare major populations of cells.
Results: Validation of mass cytometry panels was performed with adenocarcinoma cell lines and human tumors. Adenocarcinoma cell lines PC9 (lung), A549 (lung), H520 (squamous) and SW620 (colon) exhibited phenotypically distinct cells between samples by expression of cytokeratin, CK7, and EGFR. Furthermore, PD-L1 expression was expressed on subsets within cell lines. Disaggregation of tumors was optimized with dissociation that included collagenase and DNase to release viable cells within 1 hour. Detection of viable cells was optimized with rhodium intercalator viability dye and histone H3 to identify nucleated cells. Using the 30 marker panel and viSNE analysis four populations were identified within tumors as infiltrating leukocytes, endothelial cells, fibroblast, and epithelial tumor cells. Epithelial cells were found to exhibit cellular heterogeneity between patients based on the expression of cytokeratin, CK7, TTF1, EGFR, vimentin, CD44, and MET. Preliminary signaling data identified basal kinase activity active in infiltrating leukocytes and cancer cells. Cancer cells and infiltrating immune cells had basal p-STAT5 activation, and cancer cells had high basal p-AKT implicated in dysregulated cell growth.
Conclusions: Populations of infiltrating stromal cells and epithelial (cancer) cells were identified in lung adenocarcinoma. The single cell phenotyping from tumors was consistent with the profile found in two lung adenocarcinoma cell lines. Preliminary differences in basal signaling pathways responsible for growth were observed within adenocarcinoma cell populations. This work demonstrates the feasibility of mass cytometry to identify and characterize tumor heterogeneity. Work is underway to define phenotypes within the epithelial and leukocyte populations that could predict tumor behavior and immune response within the microenvironment. This work is supported by NCI CA196405 to PPM.
Citation Format: Deon B. Doxie, Jonathan M. Lehman, Yong Zou, Maria S. Ortega, Caroline E. Maier, Jonathan M. Irish, Pierre P. Massion. Single cell mass cytometry analysis of human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2911. doi:10.1158/1538-7445.AM2017-2911