The TGF-βR1 inhibitor galunisertib re-shapes the PDAC-TME by limiting decidual-like natural killer cells polarization

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality worldwide. Natural Killer (NKs) cells are pivotal for tumor surveillance but are dysfunctional in PDAC. We evaluated whether pharmacological blockade of TGF-β1/TGF-βR1 axis in PDAC cells and cancer-associated fibroblasts (CAFs) could modulate NK polarization via soluble factors. The phenotype/functions of NKs from PDAC patients versus healthy controls (HC) were compared, and the polarization state of NKs exposed to the conditioned media of PDAC cells and fibroblasts was evaluated by flow cytometry. The ability of galunisertib (GAL) to reverse NK dysfunction in immunocompetent mice orthotopically implanted with FC1199 PDAC cells was evaluated. PDAC patients showed higher TGF-β1/ TGF-βR1 levels than HC, with worse outcomes in TGF-β1^high/TGF-βR1^high patients. Circulating CD9⁺ NKs were expanded in PDAC patients compared with HC and exhibited a pro-angiogenic secretome and higher pro-angiogenic activities in vitro and in vivo (leech Hirudo verbana), compared to the CD9^- NK cells. PDAC cells and CAF induced a CD9⁺-decidual-like phenotype, also impairing NK degranulation. GAL treatment restrains PDAC cell/CAF-induced NK anergy, restoring their cytotoxicity. Also, TGFβ-R1 knockdown in PDAC cells exhibited the capability to limit the generation of decidual-like NKs, while restoring their antitumor ability, via soluble factors. Secretome profiling of BxPC3 and MIAPaCA2 PDAC cell lines and CAFs showed that GAL downregulated the release of several growth, angiogenic, and immunoregulatory factors, including FGF2, HGF, IL11, PLGF, EGFR, and VEGF. In vivo in orthotopic tumors formed by FC1199 cells GAL decreased CD9⁺-NK frequency, promoted M1-macrophage polarization, and activated NK and CD8⁺T-cells, together with a significant reduction of tumor weight, fibrosis and inhibition of angiogenesis. Our study identifies CD9⁺NKs as a novel cell subset expanded in PDAC and underscores the role of TGF-β1/TGF-βR1 signalling in promoting a pro-tumoral NKs GAL-treatment emerges as immunomodulator able in re-educating pro-tumor NKs cells in PDAC.