Macrophage phenotype and function in primary lung cancer

Background: Lung cancer is one of the most commonly reported cancers, and is known to be associated with a poor prognosis. The role of macrophages in lung cancer, including alveolar macrophages (AMs), tumour-associated macrophages (TAMs) and monocytes, is multifaceted and the literature shows conflicting roles. Aims: (I) To characterise the M1/M2 macrophage populations within the AM population in patients with non-small lung carcinoma (NSCLC) compared to controls; (II) To evaluate the M1/M2 monocyte populations and Th1/Th2 cytokine serum levels in patients with NSCLC compared to controls; (III) To use quantitative proteomics to investigate the up-regulation of novel proteins in bronchoalveolar lavage (BAL) fluid from patients with lung adenocarcinoma to identify new potential biomarkers expressed by AMs; and (IV) To characterise the M1/M2 macrophage populations within TAMs in different subtypes of NSCLC compared to non-tumour tissues. Methods and materials: AMs were obtained from patients with NSCLC and controls and analysed for surface marker differences using flow cytometry.<br><br> The mRNA expression of different cytokines was measured using qPCR. Freshly prepared peripheral blood mononuclear cell (PBMC) samples were also obtained from patients with NSCLC and from controls. Flow cytometry was performed to investigate the expression of M1/M2 markers on classical monocytes. The Th1/Th2 cytokine levels were analysed in serum samples using the cytometric bead array (CBA) and the Bio-Plex systems. In addition, BAL fluid samples from subjects with and without lung adenocarcinoma were analysed using quantitative proteomics. Finally, TAM subsets from non-tumour and tumour tissues were analysed using immunohistochemistry (IHC). Results: The expression of M2 markers (CD163), CD71 and CD44 was greater on AMs from NSCLC patients compared to controls. However, there were no significant differences in the expression of M1 markers (HLA-DR) and CD11b. The mRNA expression levels of IL-10 and MMP-9 were increased in AMs from NSCLC patients compared to controls. There were no significant differences in the expression of HLA-DR, CD163, CD11b, CD11c, CD71 and CD44 markers on classical monocytes in NSCLC patients compared to controls. The Th1/Th2 cytokine levels in serum revealed no significant difference between NSCLC patients and controls. However, the level of IL-1β, IL-4, IL-6 and IL-8 was significantly increased in serum of large cell lung carcinoma patients compared to controls. In addition, 1,100 proteins were identified and 33 of these were found to be consistently over-expressed in the BAL fluid of adenocarcinoma samples compared to controls. <br><br>Finally, the expression of CD68 and CD163 (M2 marker) was significantly increased in all NSCLC subtypes compared to non-tumour tissues. In contrast, the expression of iNOS (M1 marker) was significantly decreased in the tumour tissue of patients with adenocarcinoma and squamous cell lung carcinoma compared to non-tumour tissue. Conclusion: the outcome of this work suggests that the presence of NSCLC is associated with an alteration in macrophage phenotype and function. This study revealed a number of specific proteins (e.g. CD163) and cytokines (e.g. IL-1β) that were identified to be associated with NSCLC patients and we suggest may be used as novel prognostic and/or diagnostic biomarkers in patients with NSCLC.