ABSTRACT: Macrophages constitute a dominant and functionally diverse immune population within the microenvironment of pancreatic ductal adenocarcinoma (PDAC), yet how this heterogeneity contributes to the tumor remains poorly defined. In an institutional cohort of 145 PDAC specimens, we identified a population of multinucleated giant cells (MGCs) of macrophage origin, an entity previously described in chronic inflammation but rarely in cancer. CD68⁺ MGCs were present in 28% of tumors, enriched in squamous, non-glandular regions, and more frequent after neoadjuvant chemotherapy. By integrating spatial transcriptomics and quantitative imaging, we defined the features of these cells, which, compared with MGCs in non-neoplastic inflammatory lesions, lacked canonical polarization markers (HLA-DR, CD163) and displayed a distinctive transcriptional program characterized by upregulation of POLR2K, TUBA8, COX5B, and VDAC1 genes, involved in DNA repair, oxidative stress, and MYC signaling. Spatial analyses revealed activation of hypoxia and extracellular matrix–remodeling pathways in MGC-associated niches, and experimental hypoxia promoted MGC formation in vitro. Consistently, a macrophage MGC gene signature was enriched in the squamous PDAC subtype and correlated with poorer overall survival in TCGA (p = 0.018). Morphometric and immunofluorescence analyses further showed increased 53BP1⁺/Ki67⁺ nuclei and nuclear atypia in MGCs, indicating ongoing proliferation despite DNA damage. Together, these data identify multinucleated macrophages as a previously unrecognized immune cell state shaped by hypoxia and stress signaling, associated with aggressive tumor phenotypes, and potentially exploitable as an immune classifier in pancreatic cancer.
