Immunotherapy has revolutionized the field of tumor immunology. Recent evidences have revealed the clinical efficacy of programmed cell death-1/programmed death ligand-1 (PD-1/PD-L1) antibodies in patients with metastatic breast cancer, melanoma and non-small-cell lung cancer. The therapeutic efficacy of PD-1/PD-L1 inhibitors is high in patients with high PD-L1 expression. Recently, Tseng et collaborators showed that targeting Plasminogen Activator Inhibitor (PAI-1) by its inhibitor tiplaxtinin (TPX) synergizes with anti-PD-L1 checkpoint blockade in a model of murine melanoma. PAI-1 induced the internalization of surface PD-L1, resulting in the reduction of PD-L1 at membrane level. Binding of PAI-1 to uPA/uPAR complex results in the recruitment of low-density lipoprotein receptor protein 1 (LRP1) and triggers the endocytosis process leading to the internalization of PAI-1-uPA-uPAR-LRP1 quaternary complex. The endocytosed PAI-1 and uPA then undergoes lysosomal degradation, whereas uPAR and LRP1 are transported back to the plasma membrane by recycling endosomes. Another limiting factor of ICIs is the PD‐L1 packaging within specific membrane‐enclosed extracellular vesicles (EVs), the so‐called exosomal PD‐L1. We propose to inhibit PDL-1 endocytosis by uPAR inhibitors to maintain high-cell-surface levels of PD-L1 and, at the same time, to reduce the expression of exosomal PD-L1. Moreover, we propose to set up 3D co-culture system between non-small cell lung cancer cells and T cells to assess efficacy of uPAR inhibitors on immunotherapy responses.

2D and 3D cultures from A549 (non-small cell lung cancer cells) were treated with TPX and uPAR antagonist (IPR803) to evaluate the effect of TPX and IPR803 treatment on PD-L1 modulation. Conditioned media from untreated and treated TPX tumor cells were used for exosomes isolation to evaluate the effect of TPX treatment on exosomal PD-L1. 3D co-cultures were seeded between A549 previously treated with anti-human PD-L1 and IPR803 and T cells CD8+ CD4+ to asses cytotoxic effect of T cells on tumor cells.

Our result evidenced that in 2D and 3D cultures of A549 TPX and uPAR antagonist peptides are able to block the PD-L1 internalization and, consequently, to increase PD-L1 membrane levels. Moreover, we demonstrated that exosomes from TPX-treated A549 show a decrease of exosomal PD-L1 levels, compared to untreated cancer cells. In parallel, our data highlighted in 3D co-cultures an improvement of cytotoxic effect of T cells on A549 treated with uPAR antagonists and anti-PD-L1 antibodies

Our results evidenced that blockade of PD-L1 endocytosis induced a decrease of exosomal PD-L1 levels. Furthermore, our data demonstrated that uPAR inhibition by uPAR antagonist peptides result in a significant increase in surface PD-L1 levels, opening the way for new combined therapeutic strategies with uPAR inhibitors and anti-PD-1/PD-L1.