Abstract A035: Stromal composition, fibroblast heterogeneity and spatial organization in pancreatic adenocarcinoma

Abstract

Abstract Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is characterized by highly abundant stromal fibroblasts that are hypothesized to influence tumor behavior. While recent studies have identified multiple populations of stromal fibroblasts with divergent roles in PDAC biology, stromal composition remains incompletely characterized in human tumors, including the landscape of fibroblast subtypes and their spatial organization. Design: We designed multiplex immunofluorescence assays to quantify tumor cells (cytokeratin), immune cells (CD45), fibroblasts (αSMA, CD74, FAP, LaminA), other cells (CD31, CD56, CALD1, NG2), and collagen I in fixed tissue sections. Combinatorial fibroblast marker expression identified myofibroblasts (myCAF), inflammatory fibroblasts (iCAF), antigen-presenting fibroblasts (apCAF), and other hybrid fibroblast subtypes. We characterized fibroblast populations in up-front resected and neoadjuvant-treated cohorts of patients with localized PDAC and in patients with biopsies of metastatic lesions. Using digital imaging, supervised machine learning, and spatial proximity metrics, we quantified cell phenotypes, fibroblast subtype composition and spatial organization at the single-cell level and in the context of tumor clinicopathologic features and patient outcomes. Results: Across 301 up-front resected tumors, we cumulatively analyzed 1.7 million cells, including 611,000 fibroblasts. Stromal fibroblast density ranged from 632 to 4150 cells/mm2. Five main fibroblast subtypes constituted more than 85% of the fibroblast population in these tumors: αSMA+ (myCAF), FAP+αSMA+ (FAP+ myCAF), LaminA+ (iCAF), αSMA+ LaminA+ (myCAF/iCAF), and FAP+ αSMA+ LaminA+ (FAP+ myCAF/iCAF). Unexpectedly, 39% of fibroblasts showed co-expression of markers previously considered distinct for major fibroblast subtypes. By unsupervised clustering of fibroblast subtype proportions, we identified 3 fibroblast-defined tumor subgroups, including tumors rich in FAP+ fibroblasts, hybrid fibroblasts, and single positive fibroblasts (myCAFs, iCAFs, and apCAFs), which were associated with patient survival. Furthermore, fibroblast subtypes were spatially organized, with preferential localization of FAP+ myCAFs near tumor cells and iCAFs more distant from tumor cells yet closer to immune cells. FAP+ myCAFs were more abundant in basal-like tumors than in classical tumors, and FAP+ myCAFs were located closer to basal-like tumor cells than classical tumor cells within individual tumors. Compared to up-front resected primary tumors, neoadjuvant FOLFIRINOX treated tumors and metastatic lesions harbored distinct fibroblast profiles. Conclusion: Fibroblasts in the PDAC microenvironment are heterogeneous both in abundance and subtype composition, exhibit distinct spatial organization and are associated with patient outcomes. Treatment with FOLFIRINOX alters the composition of fibroblast subtypes in primary PDAC, and metastatic PDAC lesions have a different fibroblast composition compared to primary tumors. Citation Format: Dalia Elganainy, Andressa Dias Costa, Alexander Jordan, Suryun Kim, Sara A. Väyrynen, Hannah L. Williams, Chen Yuan, Douglas A. Rubinson, Kimberly Perez, Harshabad Singh, Richard F. Dunne, Thomas E. Clancy, David C. Linehan, Daniel T. Chang, Aram F. Hezel, Albert C. Koong, Andrew Aguirre, Brian M. Wolpin, Jonathan A. Nowak. Stromal composition, fibroblast heterogeneity and spatial organization in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A035.