Evidence of different subsets in circulating and in low-density neutrophils in patients with non-small cell lung cancer (NSCLC) - 17/02/23

Lung cancer remains the leading cause of cancer mortality in France, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of lung cancer cases. Several studies have highlighted the role of neutrophils (PMN) in immune tolerance and tumor progression in NSCLC, but their functions and phenotypes are poorly characterized. Our hypothesis is that defined PMN subpopulations endowed with specific activities are involved in NSCLC progression.

Blood from 25 patients with metastatic NSCLC was collected. Ficoll gradient separates low-density neutrophils (LDN) from conventional normal density neutrophils. Membrane phenotype was examined using a 24 markers panel with an Aurora spectral cytometer. Functional analysis of NADPH oxidase-dependent ROS production has been determined in whole blood PMN by luminol-amplified luminescence. We recruited 24 healthy donors (HD) from the Établissement Français du Sang. All data have been collected according to ethics rules (CPP number 22.04.07).

PMN counts showed a significant increase in NSCLC patients compared to HD. Analysis of markers in whole blood shows a significant decrease in surface antigen related to differentiation and maturity, especially the expression of CD10, CD13 and CD16 in PMN from NSCLCs compared to HD. No significant difference was observed in activation markers such as CD11b, CD66b and FPR1 between NSCLC PMN compared to HD. Regarding metabolic markers, a significant increase in the membrane expression of GLUT1 was observed in NSCLC PMN compared to HD. Analysis of PMN subsets was next performed according to the double labelling CD16/CD62L which allows discriminating nuclear morphology, function and phenotype of neutrophils according to an established method. In HD, the subset of CD16high/CD62Lhigh represents the majority of the PMN (Fig. 1). A new subset characterized by CD16high/CD62Llow was observed in NSCLC patients, indicative of chronic inflammatory state. Interestingly, the percentage of LDN was significantly increased in NSCLC patients compared to HD. In addition, the major difference between HD and NSCLC patients was the presence of subsets within the LDN fraction with an immature (CD16low/CD62Llow) phenotype. Concerning PMN function, NADPH oxidase-dependent ROS production measured in whole blood was significantly increased in NSCLC patients compared to HD.

We discovered new subpopulations in NSCLC normal and low density neutrophils which represent a disturbance in PMN phenotype, function, and metabolism which could open to new therapeutic or prognostic strategies.