Abstract A009: Characterizing the pre-metastatic liver in PDAC patients using scRNA-seq of FFPE tissue

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains an essentially incurable disease. Even among patients considered for curative-intent surgery and chemotherapy, recurrence persists in nearly all patients, and 5-year PDAC survival remains at only 8%. Hepatic metastases tend to occur earlier than metastases to other sites, and patients with liver involvement have the worst survival outcomes, even when compared to those with multisite disease. A critical need exists for novel, scalable diagnostic metrics capable of identifying and characterizing patients at high risk for metastatic spread of PDAC, particularly to the liver. The tumor microenvironment (TME) plays a critical role in tumorigenesis, progression, and metastasis, and the varied immune and stromal cell populations within the TME represent promising targets to serve as biomarkers for the early diagnosis of cancer, as they undergo characteristic phenotypic changes during disease onset and progression. With this in mind, we built a unique biorepository to bank pre-metastatic liver samples collected at the time of curative-intent surgery, aiming to identify features of the microenvironment present in the liver of PDAC patients prior to metastatic spread. Clinical follow-up for patients in this biorepository has since matured, allowing us to bin patients based on time to hepatic recurrence. We utilized a recently developed Flex assay from 10X Genomics for performing single-cell RNA sequencing (scRNA-seq) on formalin-fixed, paraffin-embedded (FFPE) tissues, an approach well-suited for retrospective analysis. Herein, we demonstrate the feasibility of applying this method to FFPE liver samples from PDAC patients across various disease states by profiling the immune landscape of the pre-metastatic liver. Specifically, we profiled liver samples from patients with no recurrence, early recurrence, or confirmed PDAC liver metastases, and conducted comparative transcriptional analysis against healthy liver samples. This analysis revealed multiple distinct immune cell populations within the liver, including myeloid cells such as Kupffer cells, macrophages, dendritic cells, and granulocytes, lymphoid cells such as CD4+ and CD8+ T cells, B cells, and NK cells, and relevant stromal populations such as fibroblasts and sinusoidal endothelial cells. These populations exhibited unique, patient-specific, and disease state-specific expression patterns. Additionally, we identified distinct zonal hepatocyte populations, which will allow us to query transcriptional changes in fundamental liver cell types. Furthermore, through gene set enrichment analysis (GSEA), we observed pathway-level changes characteristic of these cell populations within the liver. Together, these findings demonstrate the applicability of this scalable approach and its potential to meaningfully supplement existing PDAC diagnostics by enabling earlier characterization of the liver’s pre-metastatic niche to assess recurrence risk. Citation Format: Ryan Humphrey, Ash Fletcher, Julia Button, Daniel Nussbaum, Erika Crosby. Characterizing the pre-metastatic liver in PDAC patients using scRNA-seq of FFPE tissue [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A009.