Enhancing Non-Small Cell Lung Cancer Treatment Utilizing Natural Killer Cell-Derived Extracellular Vesicles (NKEVS) As A Novel Adoptive Cellular Therapeutic [EV RNA]
An established mechanism contributing to Immune Checkpoint Inhibitor (ICI) therapy failure is tumor evasion of T-cell responses via downregulation of human leukocyte antigen (HLA). Conversely, the effector function of Natural Killer (NK) cells is enhanced in the absence of HLA expression, making NK-based cellular therapies an attractive option for tumors that are resistant to ICIs. We investigated the preclinical efficacy of using selective isolation of NK cells and harvesting of NKEVs in NSCLC. Single-cell RNAseq (scRNAseq) was used to examine the cellular landscape in PBMCs and tumor tissue. NK cells were isolated from PBMCs, expanded in vitro, and NK-derived EVs (NKEV) were collected and the EV RNA and protein cargo was characterized using proteomics and transcriptomics. The functional capabilities of patient derived NKEVs were assayed with 3-dimensional organoid-like structures derived from patient tumor cells.  This work described the cellular landscape in NSCLC circulating and tumor infiltrating immune cells. Additionally, it demonstrated that NKEVs can be successfully harvested from patient derived, expanded NK cells, and highlighted their anti-tumor properties in combination with standard-of-care therapies. Overall design: Samples from NSCLC patients (n=10) enrolled in the study were collected at Baylor Scott and White, by trained medical personnel. Specifically, a blood sample was collected pre- and post- tumor resection surgery, and PBMCs were isolated via density gradient separation. From the tumor resection, whatever tissue was left behind after pathology and diagnostic tests, was snap frozen in preparation for scRNAseq. Everything was collected, and kept frozen at Baylor Scott and White, and shipped to the Translational Genomics Research Institute(TGen) for analysis. At TGen, PBMCs were thawed, the NK cells were isolated via negative selection and magnetic beads, and cultured in suspension for 2-4 weeks. Cell culture medium was retrieved every 48 hours, and size exclusion chromatography was used to isolate the extracellular vesicles.