Catecholamines contribute to the neovascularization of lung cancer via tumor-associated macrophages

Highlights

• Blocking the sympathetic signaling arrests tumor growth of lung cancer cells.

• Adrenergic-stimulated macrophages tend to exhibit an M2-like phenotype.

• Catecholamine-stimulated macrophages contribute to angiogenesis.

• Catecholamines regulate tumor microenvironment by affecting the innate immune cells.

Abstract

Purpose

Elevated catecholamines in the tumor microenvironment often correlate with tumor development. However, the mechanisms by which catecholamines modulate lung cancer growth are still poorly understood. This study is aimed at examining the functions and mechanisms of catecholamine-induced macrophage polarization in angiogenesis and tumor development.

Experimental design

We established in vitro and in vivo models to investigate the relationship between catecholamines and macrophages in lung cancer. Flow cytometry, cytokine detection, tube formation assay, immunofluorescence, and western blot analysis were performed, and animal models were also used to explore the underlying mechanism of catecholamine-induced macrophage polarization and host immunological response.

Results

Catecholamines were shown to be secreted into tumor under the control of the sympathetic nerve system to maintain the pro-tumoral microenvironment. In vivo, the chemical depletion of the natural catecholamine stock with 6OHDA could reduce the release of catecholamines within tumor tissues, restrain the function of alternatively activated M2 macrophage, attenuate tumor neovascularization, and inhibit tumor growth. In vitro, catecholamine treatment triggered the M2 polarization of macrophages, enhanced the expression of VEGF, promoted tumor angiogenesis, and these catecholamine-stimulated effects could be reversed by the adrenergic receptor antagonist propranolol. In addition to regulating tumor-associated macrophages (TAM) recruitment, decreasing catecholamine levels could also shift the immunosuppressive microenvironment by decreasing myeloid-derived suppressor cells’ (MDSCs) recruitment and facilitating dendritic cells’ (DCs) activation, potentially resulting in a positive antitumor immune response.

Conclusion

Our study demonstrates the potential of adrenergic stress and catecholamine-driven adrenergic signaling of TAMs to regulate the immune status of a tumor microenvironment and provides promising targets for anticancer therapies.

Introduction

Nerve system is one important component of the tumor microenvironment. The local extension of cancer cells along nerves is a frequent clinical finding and denervation of the primary tumor is reported to suppress cancer metastasis, suggesting that the nerve system is not a bystander during tumorigenesis (Boilly et al., 2017, Zhao et al., 2014). Nerve-related cancer progression is also believed to be the result of elevated neurotransmitters and growth factors in local microenvironments, so that the neurochemical changes of continuous exposure to stress are able to create a setting conducive for tumor initiation up to progression (Amit et al., 2016). Chronic activation of the sympathetic nerve system (SNS) has become increasingly recognized as a critical factor associated with the poor survival in cancer patients (Cole et al., 2015). Stress-induced SNS activation leads to the elevated levels of catecholaminergic neurotransmitters, which is mainly norepinephrine (NE), contributing to the growth, dissemination, and metastasis of cancer by promoting tumor invasion and remodeling tumor microenvironment (Cole et al., 2015, Eng et al., 2014, Saloman et al., 2016, Magnon et al., 2013). Stress-induced neurotransmitters and hormones can enter tumor tissue through vasculature, innervation, or even indirectly through immune cell production to manipulate the behaviors of tumor cells (Eng et al., 2014, Saloman et al., 2016, Jobling et al., 2015, Gabanyi et al., 2016). Apart from the systematic effect of EPI through blood circulation, data from mounting studies also show that sympathetic nerves infiltrate into the vicinity of cancer and locally release NE to mediate the pro-tumoral effects of activated SNS by acting on the corresponding receptors expressed on tumoral and stromal cells (Scanzano and Cosentino, 2015). Finally, adrenergic stimulation of immune cells regulates the activation state of the immune system and modulates their functional capacity (Bellinger and Lorton, 2014). Conversely, nerve ablation has an inhibitory impact on disease progression, including the delayed development of precancerous lesions, decreased tumor growth, and metastasis. Inhibition of the neurotransmitter receptor seems to facilitate the anticancer effects of chemotherapy and targeted therapy. In addition, patients taking antagonists of adrenergic receptors have improved prognosis in various cancers (Barron et al., 2011, Wang et al., 2013, Jansen et al., 2014), and preclinical research have identified β-blockers as having the ability to potentiate the anti-tumor efficacy of chemotherapy agents (Pasquier et al., 2011). All these findings highlight a significant role of cancer-nerve communication in tumor growth and metastasis.

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 flexibility, macrophages can shift functional phenotypes and activation states in response to various microenvironmental signals generated from tumor and stromal cells (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 macrophages 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 modulator 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 transition from early inflammatory response toward advanced immunosuppression. 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 inflammation to cancer. Sympathetic nerve fibers deliver adrenergic signals to remodel the properties of immune cells via adrenergic receptors, 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 macrophage activity and density in tumor growth (Qiao et al., 2018, Armaiz-Pena et al., 2015). More recent studies have shown that β-adrenergic-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 macrophage-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, concomitant 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.