Spatial and molecular profiling of multinucleated giant macrophages in pancreatic ductal adenocarcinoma

Macrophages constitute a dominant and heterogeneous immune population within the microenvironment of pancreatic ductal adenocarcinoma (PDAC), but how specific macrophage states contribute to tumor behavior remains poorly understood. In an institutional series of 145 PDAC specimens, we identified a distinct subset of multinucleated giant cells (MGCs) of macrophage origin, an entity well described in chronic inflammation but rarely characterized in cancer. CD68⁺MGCs were found in about 28% of cases, enriched in squamous, non-glandular regions, and more frequent after neoadjuvant chemotherapy. Spatial and high-dimensional profiling, including NanoString GeoMx® Digital Spatial Profiling, Hyperion Imaging System, and AI-guided histopathology, defined the morphological, phenotypic, and transcriptional features of these cells. PDAC-associated MGCs lacked canonical polarization markers (HLA-DR, CD163) and instead displayed a unique transcriptional program involving POLR2K, TUBA8, COX5B, and VDAC1, genes linked to oxidative stress, DNA repair, and MYC signaling. Gene set enrichment and spatial analyses indicated that MGC-rich regions coincide with hypoxic and extracellular matrix-remodeling niches. Experimental hypoxia promoted MGC formation in vitro . Morphometric assessment revealed abnormal nuclear architecture and increased 53BP1 + /Ki67 + nuclei in MGCs, suggesting proliferative activity despite DNA damage. A macrophage MGC gene signature was enriched in the squamous subtype of PDAC and correlated with shorter overall survival in TCGA datasets (p = 0.018). Collectively, these data identify multinucleated macrophages as a previously unrecognized immune cell state driven by microenvironmental stress and hypoxia. Their distinctive transcriptional and spatial profiles associate with aggressive tumor phenotypes, highlighting potential diagnostic and prognostic relevance in pancreatic cancer.