Abstract PR03: Subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer
Authors: Winter Peter S. , Raghavan Srivatsan , Navia Andrew , Williams Hannah , DenAdel Alan , Kalekar Radha , Galvez-Reyes Jennyfer , Lowder Kristen , Mulugeta Nolawit , Raghavan Manisha , Borah Ashir , Ng Raymond , Wang Junning , Reilly Emma , Ragon Dorisanne , Brais Lauren , Ng Kimmie , Cleary James , Crawford Lorin , Manalis Scott , Nowak Jonathan , Wolpin Brian , Hahn William , Aguirre Andrew , Shalek Alex 
Published: December 2020

Abstract
The majority of patients with pancreatic ductal adenocarcinoma (PDAC) present at diagnosis with metastatic disease and have median survival times of less than 12 months. Recent studies have demonstrated that PDAC tumors with distinct transcriptional phenotypes are associated with different clinical outcomes. However, the mechanisms underlying this survival difference, the degree of cellular heterogeneity within a given tumor, and the subtype-specific contributions from the local immune microenvironment are not understood. In addition, there are ongoing efforts to understand if patient-derived organoid models can be used as functional surrogates for an individual patient’s disease. It remains unclear if patient transcriptional phenotypes are preserved in their matched organoid models. Here, we describe a pipeline that permits both direct characterization of the PDAC liver metastatic niche via single-cell RNA-sequencing and functional assessment of PDAC tumor biology in patient-matched organoid models. Starting from core needle biopsies of metastatic PDAC lesions containing 50-100k viable cells, we simultaneously perform: (1) single-cell RNA-sequencing using Seq-Well and (2) three-dimensional organoid culture generation. We have applied this approach to profile 23 patients and their matched early passage organoid models. Our pipeline yields high-quality single-cell measurements across diverse cell types—both malignant and non-malignant—enabling a principled dissection of tumor intrinsic and extrinsic factors. Evaluation of clinically relevant transcriptional signatures (e.g., Basal-like vs Classical) revealed extensive heterogeneity at the single-cell level. Single malignant cells are capable of co-expressing markers of both Basal-like and Classical states suggesting these phenotypes lie on a continuum rather than as discrete types. Basal cells express more stem-like features and inhabit a distinct microenvironment compared to their Classical counterparts. Microenvironmental composition differed on several levels between the two types, most notably their T/NK cell and macrophage populations with specific implications for subtype-specific microenvironmental directed therapy. Finally, we found that the microenvironment in traditional organoid culture selects against the Basal-like subtype and that these tumors are capable of significant phenotypic plasticity in vitro. We are able to recover Basal-like features by altering the organoid growth conditions. These findings suggest the need for distinct environments to support specific transcriptional subtypes in PDAC. Overall, our work provides a framework for the analysis of human cancers and their matched models using single-cell methods, and reveals novel, actionable insights into the heterogeneity and plasticity underlying survival in transcriptionally distinct forms of PDAC.
Citation Format: Peter S. Winter, Srivatsan Raghavan, Andrew Navia, Hannah Williams, Alan DenAdel, Radha Kalekar, Jennyfer Galvez-Reyes, Kristen Lowder, Nolawit Mulugeta, Manisha Raghavan, Ashir Borah, Raymond Ng, Junning Wang, Emma Reilly, Dorisanne Ragon, Lauren Brais, Kimmie Ng, James Cleary, Lorin Crawford, Scott Manalis, Jonathan Nowak, Brian Wolpin, William Hahn, Andrew Aguirre, Alex Shalek. Subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR03.