br Solid tumors are known to be strongly
Solid tumors are known to be strongly infiltrated by inflammatory leukocytes, and accumulating evidence has clearly established a link between increased density of macrophages and poor prognosis in both mouse and human malignancies (Ruﬀell and Coussens, 2015; Yang et al., 2018). Macrophages are recognized as playing a paradoxical role in cancer-immune cross-talk, owing to their potential to express pro-and anti-tumor cytokines, which results in polarized pro- or anti-tumor functions (Qian and Pollard, 2010). Due to their plasticity and flex-ibility, macrophages can shift functional phenotypes and activation states in response to various microenvironmental signals generated from tumor and stromal SQ 109 (Sica and Mantovani, 2012). Based on their activity, macrophages are divided primarily into two categories, ranging from the classical M1 to the alternative M2 macrophages, which represent the extremes of a continuum in a universe of activation states. M1 macrophages are involved in the inflammatory response, pathogen clearance, and antitumor immunity, whereas, M2 macro-phages forestall immune cell attack, promote malignant expansion, and build new vasculatures. Tumor-associated macrophages (TAMs) closely resemble M2-polarized macrophages and function as the pivotal mod-ulator of immunosuppressive and pro-tumorigenic properties (Chanmee et al., 2014); maintaining a favorable microenvironment to facilitate tumor development and progression (Chen et al., 2011; Noy and Pollard, 2014). Researches point out that TAMs are emerging as the key target of adrenergic regulation in several cancer contexts (Cole and Sood, 2012). As the most abundant immune cells in tumor tissues, TAMs aﬀect the homeostasis of microenvironments based on their functional skewing under physiological and/or pathological conditions. Dynamic changes drive an M1 toward an M2 switch during the tran-sition from early inflammatory response toward advanced im-munosuppression. Coexistence of macrophages in mixed phenotypes collectively decides the tendency of tumor development under the control of complex tissue-derived signals (Ruﬀell and Coussens, 2015; Lamkin et al., 2016).
As two integrative systems, the nerve system and the immune system work together to detect threats, provide host defense, and maintain homeostasis (Qiao et al., 2018). Recent eﬀorts have shed light on molecular and cellular pathways, linking the nerve system and in-flammation to cancer. Sympathetic nerve fibers deliver adrenergic signals to remodel the properties of immune cells via adrenergic re-ceptors, which are widely expressed on macrophages (Marino and Cosentino, 2013). Previous studies have found that the activation of β-adrenergic signaling induced by prolonged stress can regulate macro-phage activity and density in tumor growth (Qiao et al., 2018; Armaiz-Pena et al., 2015). More recent studies have shown that β-adrenergic- Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
stimulated macrophages trend firmly toward the M2 side of the M1-M2 spectrum and to some extent, reverse the M1-like macrophages to M2 (Lamkin et al., 2016; Grailer et al., 2014).
Given these reported findings, we are led to explore the complex mechanism by which stress-induced neurotransmitters promote mac-rophage-mediated tumor growth.
Our findings revealed that catecholamines stimulate macrophages through adrenergic signaling, leading to an M2-polarized phenotype and increasing VEGF production and angiogenesis in tumors. We also observed that lack of catecholamines could reverse the above eﬀect. Additionally, concentrations of pro-tumor cytokines and numbers of myeloid-derived suppressor cells (MDSCs) were on a decline, con-comitant with an increase in active dendritic cells (DCs), indicating a better antitumor microenvironment after chemical denervation. Thus, our work supports an existing communication between stress-induced neural signaling and inflammation, which consolidates the critical role of neural-immune crosstalk in tumor progression and provides a strategy to improve cancer outcomes.
2. Materials and methods
2.1. Cells and culture conditions
Human non-small cell lung cancer cell line HCC827 and human small cell lung cancer cell line H446 were cultured in an RPMI-1640 medium supplemented with 10% FBS at 37 °C in a humidified 5% CO2 atmosphere. For some experiments in vivo, HCC827 and H446 cells were infected by lentivirus containing green fluorescent protein (GFP, GeneChem, China) in advance according to the manufacturer’s in-structions. Bone marrow-derived macrophages (BMDMs) and human umbilical vein endothelial cells (HUVECs) were cultured in a DMEM medium supplemented with 10% FBS.
2.2. Isolation and polarization of BMDMs
To obtain bone marrow-derived macrophage population, bone marrow cells were isolated and characterized as described previously (Pineda-Torra et al., 2015). Cells were induced with M-CSF (20 ng/ml) and collected after 7 days. These BMDMs were further processed to become M0, M1, and M2. Macrophages cultured in the normal medium were defined as M0; macrophages that underwent a 24-h incubation with lipopolysaccharide (200 ng/ml) and IFN-gamma (10 ng/ml) were defined as M1; and macrophages that underwent a 24-h incubation with IL-4 (10 ng/ml) were defined as M2.