Abstract 1241: A single cell landscape of malignant pancreas cancer ascites
Authors: Umeda Shigeaki , Karnoub Elias- Ramzey , Ziv Etay , Wu Jean , Cruz Cristian , Park Wungki , Balogun Fiyinfolu , Sánchez Alejandro Jiménez- , Chaligné Ronan , O'Reilly Eileen M. , Donahue Christine A. Iacobuzio- 
Published: June 2023

Abstract
Despite advancement in surgical and medical management of pancreatic ductal adenocarcinoma (PDAC), this tumor type is currently the 3rd leading cause of cancer-related death in the United States and expected to become the 2nd by 2030. The development of ascites in PDAC patients generally indicates a poor prognosis although the reasons for this largely remain unknown. The exact mechanism of peritoneal spread of the cancer is still largely unknown. Herein, we collected and processed sterile ascites samples of 20 PDAC samples from 19 patients and performed single-cell RNA-seq analysis. Sixteen ascites samples were obtained from abdominal paracentesis and two cases in the postmortem period. For one patient ascites was collected both pre and postmortem. All but one patient received chemotherapy prior to ascites collection. Routine clinical cytology evaluation of ascites samples from 17 patients indicated it was malignant in 11 patients and nonmalignant in six patients. All ascites samples were purified by gradient centrifuge using Ficoll-Paque Plus to remove erythrocytes and necrotic debris. DAPI negative and Calcein positive cells were selected to enrich live cells from the cell suspension by flow sorting. We adopted the 10x Genomics Chromium Single Cell Gene Expression platform for single-cell RNA-seq library preparation. Scanpy was used for computational analysis of expression data after Cell Ranger pipelines. After QC check, normalization and doublet elimination, a total of 248,263 cells were analyzed. UMAP projection accurately clustered cells from each patient. Cell type annotation identified large numbers of immune cells compared to cancer cells and mesothelial cells with the most dominant population being macrophages. A subpopulation (0.23% of all cells) representing plasmacytoid dendritic cells was also found. Cancer cells were detected in twelve samples whereas no cancer cells were detected in eight samples after exhaustive marker gene exploration, and these findings largely matched the clinical diagnoses for each sample. In the ongoing period we will compare malignant versus non-malignant ascites by differential gene expression analysis, gene set enrichment analysis and trajectory inference to determine a) the extent the immunological clusters are enriched in malignant versus benign ascites, b) the distinct phenotypes and cell states of PDAC cells in immune rich versus immune poor ascites, c) role of plasmacytoid dendritic cells or mesothelial cells contribution to formation of malignant ascites. We believe our results will shed light on the dynamics of ascites formation and in turn identify novel targets for intervention.
Citation Format: Shigeaki Umeda, Elias- Ramzey Karnoub, Etay Ziv, Jean Wu, Cristian Cruz, Wungki Park, Fiyinfolu Balogun, Alejandro Jiménez- Sánchez, Ronan Chaligné, Eileen M. O'Reilly, Christine A. Iacobuzio- Donahue. A single cell landscape of malignant pancreas cancer ascites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1241.