Abstract A106: Tracing the immune landscape: Temporal and spatial remodeling during pancreatic cancer progression

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and immunologically "cold" cancer, with a 5-year survival rate of 13%. Despite the success of immunotherapy in other malignancies, its efficacy in PDAC remains limited due to complex immune evasion mechanisms and a pro-tumorigenic immune microenvironment. Several lines of evidence suggest that PDAC progresses from acinar-to-ductal metaplasia (ADM) to pancreatic intraepithelial neoplasia (PanIN) lesions to invasive cancer, accompanied by increasing immunosuppression. We hypothesize that understanding the spatial and temporal transitions of immune cell states during tumor evolution is key to uncovering mechanisms of immune dysfunction and guiding new therapeutic strategies. Using single-cell RNA sequencing (scRNA-seq) and multispectral imaging, we aim to uncover comprehensive, large-scale, and longitudinal changes in the immune microenvironment during PDAC progression. Here, we utilized the KrasG12D/+; Trp53R172H/+; Ptf1a-Cre (KPC) genetically engineered mouse model, and through longitudinal histological analysis of pancreas, we delineated the trajectory of disease progression, with PanIN I-II lesions appearing by 8–9 weeks, PanIN III by 11–12 weeks, and locally invasive PDAC by 16 weeks of age. To study the temporal evolution of immune cell-state transitions, we performed scRNA-seq on CD45+ enriched immune cells isolated from KPC mouse pancreas collected at 4, 8, 12, and 16 weeks using the 10x Genomics Chromium platform. To examine the spatial dynamics of immune cells, we performed multispectral imaging at defined time points using the Lunaphore COMET platform with a panel of over 15 antibodies, enabling simultaneous visualization of several markers on a single tissue section. In our analysis to date, we identified subsets of pro-tumorigenic macrophages emerging during PDAC development, along with a marked increase in myeloid-derived suppressor cells (MDSCs) in late-stage tumors. We also observed dynamic shifts in other immune cell populations evolving alongside tumor progression. Notably, we uncovered distinct subsets of monocytes, macrophages, and T cells exhibiting tumor-promoting potential, progressively enriched during the advanced stages of PDAC. Using multispectral imaging, we generated a comprehensive spatial map of the immune microenvironment across PDAC progression, capturing patterns of immune exclusion, infiltration, and cell–cell neighborhood interactions. Our study highlights the multi-layered immune dysfunction that evolves from early to late stages of PDAC - features that are challenging to capture in human samples. CD45+ enrichment improved the detection of rare and transient immune subsets by mitigating the masking effects caused by abundant cancer and stromal cells. Together, these findings provide an unbiased and detailed view of the evolving immune landscape in PDAC, laying the foundation for identifying microenvironmental and cell-intrinsic factors that either constrain or promote malignant progression, as well as for developing immune-based therapeutic strategies. Citation Format: Atul Verma, Doreen Klingler, Linda Bergmayr, Tanvi V. Inamdar, Jonas Rosendahl, Michael Böttcher, Stefan Hüttelmaier, Patrick Michl, Philipp Jurmeister, Sebastian Krug. Tracing the immune landscape: Temporal and spatial remodeling during pancreatic cancer progression [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): nr A106.