TGF-β-driven NK Cells plasticity in hepatocellular carcinoma

Valentina Reverberi¹Anna Montali¹Andrea Vecchi²Marzia Rossi¹Alessio Pelagatti¹Sara Doselli¹Benedetta Farina¹Andrea Olivani²Giorgio Economopoulos¹Raffaele Dalla Valle¹Diletta Laccabue¹Francesca Ferraglia²Amalia Penna²Paola Fisicaro²Carolina Boni^1*Gabriele Missale¹

• ¹University of Parma, Parma, Italy
• ²Azienda Ospedaliero-Universitaria di Parma, Parma, Italy

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, with limited curative options for advanced disease. Natural Killer (NK) cells are critical innate immune effectors, but their anti-tumor function is severely compromised by the immunosuppressive tumor immune microenvironment (TIME), particularly through transforming growth factor-beta (TGF-β). This study investigates the pivotal role of TGF-β signaling in modulating NK cell phenotypes and functions within the HCC TIME. Methods: to comprehensively assess TGF-β pathway activation and its impact on NK cells, tumor-infiltrating lymphocytes (TILs) and liver-infiltrating lymphocytes (LILs) were isolated from HCC patients undergoing curative resection. Phenotypic and functional analyses were performed, along with functional restoration experiments targeting TGF-β signaling. Results: Tumor-infiltrating NK cells (TINKs) exhibited significant activation of both canonical (SMAD-dependent) and non-canonical (TAK1/p38 MAPK) TGF-β signaling, with a predominance of the non-canonical pathway. This activation was associated with the emergence of an ILC1-like NK subset (CD103⁺/CD49a⁺), which was nearly absent in non-tumor liver tissue. These ILC1-like cells maintained strong cytokine production and expressed high levels of inhibitory receptors (PD-1, TIM-3, TIGIT), whereas conventional NK cells (cNKs; CD103⁻/CD49a⁻/CD9⁻) were functionally impaired. Notably, blocking TGF-β receptor binding and SMAD3 activation restored cNK functionality. This is a provisional file, not the final typeset article Discussion: our findings suggest that while non-canonical TGF-β signaling drives phenotypic reprogramming and contributes to NK cell dysfunction, canonical SMAD-dependent signaling remains a key therapeutic target for functional restoration. These results highlight the dual role of TGF-β in immune modulation and suggest that targeted pathway inhibition could enhance innate anti-tumor responses, opening new avenues for combination therapies in HCC.