Tumor-infiltrating lymphocytes (TIL)-derived Natural Killer (NK) cells from Pancreas Ductal Adenocarcinoma (PDAC)

Pancreatic ductal adenocarcinoma (PDAC) presents with high mortality rates, poor diagnosis, and limited therapeutic options, with most patients relapsing after surgery. Natural Killer (NK) cells from tumor-infiltrating lymphocytes (TIL) are a promising strategy for cancer treatment. They can recognize tumor cells independently of major histocompatibility complex class I (MHC-I) molecules and display a safe profile with a low risk of inducing severe side effects. Additionally, TIL-NK cells present superior tumor infiltration capacity since they are found in the tumor microenvironment (TME). Despite their potential, expanding functional ex vivo NK cells remains challenging. Here, we describe a protocol to culture and characterize TIL-NK cells from central and peripheral tumor regions of PDAC patients and respective peripheral blood mononuclear cells (PBMC). A distinct distribution of NK cell infiltration was observed within the PDAC TME, with superior numbers in peripheral tumor regions. While the cytotoxic NK cell subset is predominant in the TME, central tumor regions display an enrichment of the CD16- NK cells. Moreover, IL-2, IL-15, and short IL-12 stimulation improved NK cell activation and cytotoxicity in PBMC- and TIL-NK cells, exhibiting superior expansion, activation, and cytotoxicity. Importantly, even in an immunosuppressive TME, NK cells from central tumor regions showed higher expansion rates compared to those infiltrating peripheral tumor regions and from PBMC. This study presents relevant insights into tumor-infiltrating NK cells from PDAC patients, providing a foundation for exploring and developing NK cell-based immunotherapies tailored for solid tumors.