A circulating subpopulation of monocytic myeloid-derived suppressor cells as an independent prognostic/predictive factor in untreated non-small lung cancer patients

Abstract

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of cells with immunosuppressive properties and might confer to worse prognosis in cancer patients. The presence of phenotypically newly described subpopulations of MDSCs and their association with the clinical outcome were investigated in non-small cell lung cancer (NSCLC) patients. The percentages and correlation between MDSCs and distinct immune cells in the peripheral blood of 110 chemotherapy-naive patients before treatment and healthy controls were investigated using flow cytometry. Two monocytic [CD14(+)CD15(-)CD11b(+)CD33(+)HLA-DR(-)Lin(-) and CD14(+)CD15(+)CD11b(+)CD33(+)HLA-DR(-)Lin(-)] and a granulocytic [CD14(-)CD15(+)CD11b(+)CD33(+)HLA-DR(-)Lin(-)] subpopulations of MDSCs were identified, expressing inducible nitric oxide synthase, and reactive oxygen species, respectively. Increased percentages of both monocytic-MDSCs' subpopulations were inversely correlated to dendritic/monocyte levels (P ≤ 0.04), while granulocytic-MDSCs were inversely correlated to CD4(+) T cells (P = 0.006). Increased percentages of monocytic-MDSCs were associated with worse response to treatment (P = 0.02) and patients with normal levels of CD14(+)CD15(+)CD11b(+)CD33(+)HLA-DR(-)Lin(-) had longer overall survival and progression free-survival compared to those with high levels (P = 0.008 and P = 0.005, resp.). Multivariate analysis revealed that the increased percentages of CD14(+)CD15(+)CD11b(+)CD33(+)HLA-DR(-)Lin(-) MDSCs were independently associated with decreased progression free-survival and overall survival. The data provide evidence that increased percentages of new monocytic-MDSCs' subpopulations in advanced NSCLC patients are associated with an unfavourable clinical outcome.

Figure 1

Phenotypic analysis of MDSC subpopulations in NSCLC patients. Representative dot plots, as well as the gating strategy for identification and quantification of MDSCs. Arrows indicate the sequence of gating. The gates for each dot plot and histogram are presented on the top of each box. The positive expression of markers compared to cells without Ab staining. Purple color represents the CD11b⁺CD14⁻ population whereas bright light green represents the CD11b⁺CD14⁺ population.

Figure 2

M-MDSC and G-MDSC subpopulations in NSCLC patients and normal controls. Percentage of monocytic (a), (b), and granulocytic (c) subpopulations of MDSCs in whole blood. Each point corresponds to an individual patient or healthy controls. The medians, 75 percentile (box), and max and min (whiskers) are represented. P values are determined by Mann-Whitney test (d). Comparison of the percentages between MDSCs subpopulations in the whole blood of the patients. Percentages indicated in the plots represent the percentages of phenotypic marker expression in the parental population, which are presented inside the brackets. The bars denote mean values ± SEM and the P values are determined by Wilcoxon matched-pairs signed rank test.

Figure 3

Representative histograms of flow cytometry analysis of (a) iNOS and (d) intracellular oxidative stress by the DCF method. Percentages of (b) iNOS and (e) ROS producing cells from healthy and NSCLC patients. Intracellular levels of (c) iNOS and (f) ROS in all tested subpopulations. The data are the mean fluorescence intensity (MFI). Intracellular ROS levels in subpopulations of MDSCs before and after PMA stimulation. Green bars, healthy controls; red bars, NSCLC patients. The gates for each dot plot and histogram are presented on the top of each box. The positive expression of markers is compared to cells without Ab staining. Colours in histograms represent the different subpopulations; pink colour, CD11b⁺CD14⁺CD15⁺CD33⁺HLA-DR⁻Lin⁻ population; black colour, CD11b⁺CD14⁺CD15⁻CD33⁺HLA-DR⁻Lin⁻, and bright blue, CD11b⁺CD14⁻CD15⁺CD33⁺HLA-DR⁻Lin⁻. Percentages indicated in the plots represent the percentages of phenotypic marker expression in the parental population, which are presented inside the brackets. The data are represented as the mean ± SEM and the P values are determined by Mann-Whitney test.

Figure 4

Response to 1st line treatment in patients according to the MDSC expression at baseline. The percentages of both monocytic (a) and (b), but not the granulocytic (c), subpopulations of MDSCs were increased in patients with disease progression (PD) compared to those with disease control after therapy. Each point corresponds to an individual patient or healthy controls. The medians, 75 percentile (box), and max and min (whiskers) are represented. Groups were compared by Mann-Whitney test.

Figure 5

Kaplan-Meier plots of OS and PFS in patients according to the percentages of monocytic subpopulation (CD14⁺CD15⁺CD11b⁺CD33⁺HLA-DR⁻Lin⁻) of MDSCs before any systemic treatment. Comparison of (a) progression-free survival (PFS) and (b) overall survival (OS) between normal (≤2.2%) and increased (>2.2%) percentages of CD14⁺CD15⁺CD11b⁺CD33⁺HLA-DR⁻Lin⁻MDSC.