Small Extracellular Vesicles in Pre-Therapy Plasma Predict Clinical Outcome in Non-Small-Cell Lung Cancer Patients

SIMPLE SUMMARY: Since re-biopsy of the primary tumor or metastatic lesions is not an option in most NSCLC patients, the use of the “liquid tumor biopsy” represents a promising non-invasive diagnostic/prognostic approach. Small extracellular vesicles (sEV) are currently emerging as a promising source for liquid tumor biopsies. The present study reports that pre-therapy total sEV protein (TEP) levels are associated with patient clinical outcome. We show that the high sEV TEP levels in pre-treatment plasma are a significant negative predictor of PFS and OS in treatment-naïve NSCLC patients. We also observed a positive correlation between the sEV TEP levels and the percentages of circulating CD8(+)PD-1(+) and CD8(+)PD-L1(+)T cells at baseline prior to any therapy. This suggested that sEV in plasma hinders anti-tumor immune responses via the PD-L1/PD-1 pathway. The data suggest that pre-therapy plasma sEV levels could be useful as non-invasive predictors of NSCLC progression and outcome after conventional therapy. ABSTRACT: The potential use of plasma-derived small extracellular vesicles (sEV) as predictors of response to therapy and clinical outcome in chemotherapy-naïve patients with non-small-cell lung cancer (NSCLC) was explored. sEV were isolated by size-exclusion chromatography from the plasma of 79 chemotherapy-naïve NSCLC patients and 12 healthy donors (HD). sEV were characterized with regard to protein content, particle size, counts by qNano, morphology by transmission electron microscopy, and molecular profiles by Western blots. PD-1 and PD-L1 expression on circulating immune cells was analysed by flow cytometry. Pre-treatment levels of total sEV protein (TEP) were correlated with overall (OS) and progression-free survival (PFS). The sEV numbers and protein levels were significantly elevated in the plasma of NSCLC patients compared to HD (p = 0.009 and 0.0001, respectively). Baseline TEP levels were higher in patients who developed progressive disease compared to patients with stable disease (p = 0.007 and 0.001, stage III and IV, respectively). Patient-derived sEV were enriched in immunosuppressive proteins as compared to proteins carried by sEV from HD. TEP levels were positively correlated with CD8(+)PD-1(+) and CD8(+)PD-L1(+) circulating T cell percentages and were independently associated with poorer PFS (p < 0.00001) and OS (p < 0.00001). Pre-therapy sEV could be useful as non-invasive biomarkers of response to therapy and clinical outcome in NSCLC.