Abstract A015: Defining the spatial distribution of n-glycans and ECM peptides in primary pancreatic ductal adenocarcinoma, metastases and premalignant disease

Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an extensive desmoplastic stromal reaction and an altered N-glycome. Stromal collagens serve to modulate the immune microenvironment, limit nutrient and oxygen diffusion, influence chemotherapy resistance, and promote tumor progression and metastasis. Dysregulated cancer cell N-glycosylation also promotes immune evasion and neoplastic spread. Despite their important roles in cancer pathogenesis, little is known about the spatial localization and organization of the various stromal collagens, other extracellular matrix proteins, and N-glycans within the PDAC tumor microenvironment. In this study, Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) with Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) was utilized to characterize the extracellular matrix proteome and glycome of 10 primary PDAC patient tumors, 28 unmatched lung and liver metastases and a 1,060 patient pancreatic disease tissue microarray in collaboration with the Washington University SPORE in Pancreatic Cancer at the Siteman Cancer Center. This tissue microarray contained samples from control, pancreatitis, various premalignancies, and PDAC. Following PNGaseF glycosidase digestion of PDAC tissues, 214 released N-glycans and their spatial distributions were detected by MALDI-MSI. These tissues were then digested with Collagenase III for detection and localization via peptide MALDI-MSI. Tissues were scraped from the slide and further digested with Collagenase III in solution for peptide ID analysis by LC-MS/MS, with 4,187 putative identified ECM peptides. Spatially, both ECM peptides and N-glycans uniquely colocalize with various histopathologic features according to multiplex immunohistochemistry. N-glycans with bisecting N-acetylglucosamine structures associated specifically with the invasive tumor front and clusters of immune cell infiltrate, while distinct collagen species from Collagen IV and Collagen VI encased tumor cell regions and formed stromal nests surrounding immune cells. N-glycosylation patterns in liver and lung metastases matched those of primary tumors, while the ECM proteome of metastases was distinct and more similar to their in-situ tissue niches. The degree of proline hydroxylation and other post translational modifications also varied across confirmed ECM peptides and may impact stability and deposition. From this analysis we can visualize the molecular progression of pancreatic disease from benign to advanced metastatic PDAC. Additionally, we may begin to link the N-glycome and ECM proteome of PDAC with patient survival, treatment response and other clinical variables. Citation Format: Caroline Kittrell, Jade Macdonald, Blake Sells, Lyndsay E.A. Young, David DeNardo, Peggi M. Angel, Richard R. Drake. Defining the spatial distribution of n-glycans and ECM peptides in primary pancreatic ductal adenocarcinoma, metastases and premalignant disease [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 A015.