Multi-Compartment Reprogramming And Spatially-Resolved Interactions In Frozen Pancreatic Cancer With And Without Neoadjuvant Chemotherapy And Radiotherapy At Single-Cell Resolution.

Abstract A molecular classification of pancreatic ductal adenocarcinoma (PDAC) that informs clinical management remains elusive. Previously identified bulk expression subtypes in the untreated setting were influenced by contaminating stroma whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Two consensus subtypes have arisen from these prior efforts: (1) classical-like, and (2) basal-like. Basal-like tumors were associated with worse survival in the metastatic setting but attempts to refine this binary classification have failed to further stratify patient survival. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for banked frozen PDAC specimens and studied a cohort of untreated resected primary tumors (n ~ 20). Gene expression programs learned across malignant cell and cancer-associated fibroblast (CAF) profiles uncovered a clinically-relevant molecular taxonomy with improved prognostic stratification compared to prior classifications. Digital spatial profiling revealed an association between malignant cells expressing basal-like programs and greater immune infiltration with relatively fewer macrophages, whereas those exhibiting classical-like programs were linked to inflammatory CAFs and macrophage-predominant microniches. Recent clinical trials have supported the increasing adoption of neoadjuvant therapy to aggressively address the risk of micro-metastatic spread and to circumvent concerns of treatment tolerance in the postoperative setting. There is an urgent need to understand how preoperative treatment impacts residual tumor cells and their interactions with other cell types in the tumor microenvironment to identify additional therapeutic vulnerabilities that can be exploited. Towards this end, we performed snRNA-seq on an unmatched cohort of neoadjuvant-treated resected primary tumors (n ~ 25) with most cases involving FOLFIRINOX chemotherapy followed by chemoradiation. Remarkably, the quality of single-nucleus mRNA profiles was comparable between heavily pre-treated and untreated specimens. We identified differentially expressed genes between treated and untreated samples to infer cell-type specific reprogramming in the residual tumor. This analysis revealed that in the neoadjuvant treatment context, there was lower expression of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states. Our refined molecular taxonomy and spatial resolution may help advance precision oncology in PDAC through informative stratification in clinical trials and insights into compartment-specific therapies. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Payman Yadollahpour, Jason Reeves, Eugene Drokhlyansky, Nicholas Van Wittenberghe, Samouil Farhi, Denis Schapiro, George Eng, Jason M. Schenkel, William A. Freed-Pastor, Orr Ashenberg, Clifton Rodrigues, Domenic Abbondanza, Toni Delorey, Devan Phillips, Jorge Roldan, Debora Ciprani, Marina Kern, Jaimie L. Barth, Daniel R. Zollinger, Kit Fuhrman, Robin Fropf, Joseph Beechem, Colin Weekes, Cristina R. Ferrone, Jennifer Y. Wo, Theodore S. Hong, Orit Rozenblatt-Rosen, Andrew J. Aguirre, Mari Mino-Kenudson, Carlos Fernandez-del- Castillo, Andrew S. Liss, David T. Ting, Tyler Jacks, Aviv Regev. Multi-compartment reprogramming and spatially-resolved interactions in frozen pancreatic cancer with and without neoadjuvant chemotherapy and radiotherapy at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 94.