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

Brain, Behavior, and Immunity 81 (2019) 111–121

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

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Yun Xiaa,1, Ye Weia,b,c,1, Zhen-Yu Lia, Xian-Yi Caid, Li-Ling Zhanga, Xiao-Rong Donga, ⁎ ⁎ Sheng Zhanga, Rui-Guang Zhanga, Rui Menga, Fang Zhua, , Gang Wua, a

Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai 200032, China c Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China d Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Sympathetic nervous system Catecholaminergic neurotransmitters Adrenergic receptor Tumor-associated macrophages Tumor angiogenesis

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.

1. 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

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Corresponding authors. E-mail addresses: [email protected] (F. Zhu), [email protected] (G. Wu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbi.2019.06.004 Received 29 November 2018; Received in revised form 3 June 2019; Accepted 4 June 2019 Available online 05 June 2019 0889-1591/ © 2019 Elsevier Inc. All rights reserved.

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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.

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 cancernerve 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 proand 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 βadrenergic signaling induced by prolonged stress can regulate macrophage activity and density in tumor growth (Qiao et al., 2018; ArmaizPena et al., 2015). More recent studies have shown that β-adrenergic-

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 instructions. 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. 2.3. Drug treatment To examine the effect of adrenergic receptor agonist or antagonist on the function of macrophages in the M1–M2 spectrum, BMDMs were incubated with the adrenergic receptor agonist NE (10 μM), EPI (10 nM) or non-selective adrenergic receptor antagonist propranolol (10 μM) for further tests. 2.4. Xenograft models of lung cancer All animal for the experiments were approved by and conformed to the relevant regulatory standards of the Ethical Committee of Huazhong University of Science and Technology. 4-week-old male BALB/c nu/nu mice were housed under pathogen-free conditions according to the animal care guideline. 5 × 106 lung cancer cells suspended in medium were subcutaneously injected in the right upper flank (HCC827) or right hind limb (H446) of mice. Tumor-bearing mice were randomly subdivided into two groups: control group (phosphate buffer, PBS) and 6-hydroxydopamine (6OHDA) group. Mice in the 6OHDA groups were injected intraperitoneally with 6OHDA on the 10th day (100 mg/kg) and 12th day (250 mg/kg) to deplete natural catecholamine stock 112

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and 6OHDA groups of both HCC827 and H446 cells were dewaxed in xylene and dehydrated through alcohols, followed by blocked for endogenous peroxidases and alkaline phosphatases if necessary. After antigen retrieval by microwaving, the slides were tested for CD31, CD11b, CD163, and iNOS. Primary antibody was applied at a 1:100 dilution factor in 1% BSA for 1 h and then washed and incubated in the appropriate fluorescent or secondary antibody for 1 h. To assess the distribution of all markers in each sample, serial sections were colored differently and co-located in the same area.

chemically, while mice in the control groups received an intraperitoneal injection of PBS at the same time points. Tumor dimensions were measured with vernier calipers and tumor volumes were estimated using the following formula: 0.5 × length × width2. After 2 weeks of the injection, In Vivo Fluorescence Imaging (In vivo FX PRO, Bruker, Germany) was used to monitor tumor growth (exposure times: 20 s). Before killing mice, blood samples were collected into tubes containing EDTA for flow cytometry by retro-orbital bleeding. Then, tumors, spleens and lungs were removed from mice and divided into 3 sections (fresh, frozen, fixed). Some tissues were frozen in liquid nitrogen, while others were fixed and paraffin-embedded for histological analysis.

2.10. Real-time PCR analysis RNA was extracted from whole cell lysates and reverse transcribed to cDNA using a reverse-transcriptional kit (TaKaRa). RT-PCR was performed in a triplicate with the SYBR Prime Script RT-PCR kit (TaKaRa) on the Step One Plus Real-Time PCR system (Applied Biosystems). The sequences are described in the Supporting Information for Materials and Methods.

2.5. Flow cytometry Erythrocyte lysis buffer was added into blood samples to isolate the peripheral mononuclear cells. Fresh spleens and tumors were digested with type IV collagenase, hyaluronidase and DNase I for 1 h at 37 °C and then were gently dissociated through a 200-mesh stainless sieve to prepare the single-cell suspensions. Dead cells were excluded with zombie dye and leukocytes were labeled with anti-CD45. For the phenotypic analysis of TAMs, cell suspensions made from blood and tissues were stained with the following surface antibodies: anti-CD11b, antiF4/80, anti-CD86. Then, the cells were stained with anti-CD206 after fixation and permeabilization. For the analysis of the MDSCs, cell suspensions were stained with anti-CD11b, anti-GR1. For the analysis of the NKs, cell suspensions were stained with anti-CD3, anti-CD49b. And for the analysis of the active DCs, cell suspensions were stained with anti-CD11c, anti-CD86. All antibodies were purchased from Biolegend and labeled according to the manufacturer’s protocols. Based on the previous reports (Martinez et al., 2008; Yao et al., 2018), CD11b+F4/ 80+ population was designated as M0, CD11b+F4/80+CD86+ as M1, and CD11b+F4/80+CD206+ as M2 in our study.

2.11. Western blot analysis Lysates were prepared from tumor tissues and cells and then centrifuged at 14,000 rpm for 15 min. Protein concentration in each cell lysate was determined using BCA assay kit, and all samples were electrophoresed through 10% SDS-PAGE gel and transferred to PVDF membranes. The blots were probed with appropriate primary antibodies. After incubation with secondary antibodies, membranes were washed and stained with ECL according to the manufacturer’s protocol. 2.12. Statistical analysis Data are expressed as mean ± SEM. Each value is the mean of at least three separate experiments in each group. The statistical significance between groups was determined by the Student’s t-test or oneway ANOVA using the GraphPad Prism 6.0 software (GraphPad Software Inc., USA). P < 0.05 was considered as the statistically significant difference.

2.6. ELISa To evaluate the level of NE and EPI, frozen tumors, spleens and lungs were collected into tubes containing EDTA and sodium metabisulphite and homogenized in 0.01 N HCl solution at 4 °C. All homogenized samples were centrifuged and the supernatants were stored at −80 °C before analysis. Catecholamines were measured within 1 week using 2-CAT (A-N) Research ELISA Kit (Labor Diagnostika Nord GmbH/ Rocky Mountain Diagnostics) according to the manufacturer’s protocols.

3. Results 3.1. Blocking the sympathetic signaling reduces the release of catecholamines and delays tumor growth To investigate the interplay between adrenergic nerves and cancer growth, BALB/c nu/nu mice were injected with human non-small cell lung cancer cells HCC827 in the right upper flanks or small cell lung cancer cells H446 in the right hind limbs. These mice of each cancer model were randomly divided into two groups: the control group injected with PBS, and the experimental group chemically sympathectomized with 6OHDA to suppress secretion of catecholaminergic neurotransmitter in vivo. In both the H446 and HCC827 xenograft models, tumor development was delayed significantly, with mice in the 6OHDA group having smaller tumor sizes and lower tumor weights (Fig. 1A). On the 24th day, the tumor weights of control groups were 4.61 and 2.30-fold in H446 mice and in HCC827 mice, respectively, greater than those in the 6OHDA groups. To confirm the connection between tumor growth and the level of catecholamines, we assessed the concentration of EPI and NE in removed lungs, spleens, and tumors by ELISA. The results showed significant reductions in levels of catecholamines in 6OHDA group mice compared with controls (Fig. 1B). Consistent with the secretion characteristics of catecholamines, we found that the inhibited extent of transmitters varied with tissues. Although the downtrend of EPI and NE were in synchrony, the release of EPI was predominantly suppressed in spleens and lungs, while NE release was largely inhibited in tumor tissues. Especially, the relative NE concentrations of 6OHDA groups were 16.0% and 31.3% in H446 mice and

2.7. Tube formation assay To distinguish between HUVECs and macrophage subsets, HUVECs were labeled with DiO (green) and macrophages were labeled with DiR (red) according to the manufacturer’s protocol, prior to co-culture. HUVECs (2 × 104 cells/100 μl) and macrophages subsets (1 × 104 cells/100 μl) were gently co-incubated on top of Matrigel in 96-wells plates. 6 h later, capillary-like tubular structures were analyzed and photographed. Tube length, junctions, branches, and tubules were quantified in three random microscopic fields using the ImageJ software. 2.8. Cytokine detection Tissue and supernatant samples were isolated from each group and analyzed using RayBiotech mouse cytokine kit Q1 according to the vendor’s protocols. 2.9. Immunohistochemistry in formalin-fixed, paraffin-embedded sections Serial paraffin-embedded sections of xenografts from control groups 113

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Fig. 1. Decreasing catecholamine levels delays lung cancer growth. (A) Subcutaneous inoculation with H446 cells (upper, left) and HCC827 cells (upper, right) was established in mice and observed using in vivo imaging. The tumor volume was measured for 2 weeks consecutively after injecting H446 xenograft (lower, left) or HCC827 xenograft (lower, right) mice with 6OHDA; n = 5 or 6. Representative pictures of xenograft models are shown. (B) Tumor tissues, spleens, and lungs were removed from mice to detect the concentrations of EPI and NE. n = 3. *p < 0.05. **p < 0.01. ***p < 0.001. Abbreviations: N.S.: non-statistical significance.

pronounced than EPI after 6OHDA treatment. There were also some interesting differences between H446 and HCC827 models for the former achieved a greater inhibition of tumor growth with a lower NE concentrations in tumors, spleens and lungs. These results indicate that peripheral adrenergic nerves might positively play a role in regulating tumor progress, by releasing catecholaminergic neurotransmitters, through which the effect mediated by NE might be dominant in tumors.

in HCC827 mice, respectively, lower than those in control groups. However, similar levels of EPI were observed in H446 (P = 0.9738) or HCC827 (P = 0.2905) tumor tissues. EPI comes from the adrenal gland predominately, and may reach the tumor through blood circulation. In general, the only catecholamine released into the tumor microenvironment directly by SNS nerves would be NE. That may be the reason that why the decline of NE in tumor tissues is even more 114

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Fig. 2. Reduction of catecholamine levels affects macrophage polarization. (A) Spleens (upper) and tumor tissues (lower) were removed to detect the activation of macrophages using flow cytometry after 6OHDA injection. n = 3. (B) Typical cytokines secreted by activated macrophages were tested in tumors. *p < 0.05. ** p < 0.01. Abbreviations: N.S.: non-statistical significance.

3.2. Decreasing the levels of catecholamines reduces M2-polarized macrophages

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

To evaluate the interaction between catecholamines and the immune system, we further explored the activation states of splenic and intratumoral macrophages in the 6OHDA-treated mice. Although there was little impact on the polarization of splenic macrophages (Fig. 2A, upper, P = 0.19 for H446, P = 0.95 for HCC827), flow cytometry results suggested that the reduction in catecholamine levels increased the percentage of M1-polarized macrophages significantly and maintained a higher ratio of M1/M2 in tumors (Fig. 2A, lower). The mean M1/M2 ratios reversed from 0.28 to 1.14 in H446 mice and from 0.24 to 1.02 in HCC827 mice after 6OHDA treatment. In order to verify the influence of increased M1/M2 ratio on the tumor microenvironment, we assessed the levels of several classical cytokines of activated macrophages in removed tumor tissues. IL-6, IL10, and IL-12 are typical macrophage-associated factors and can reflect different activation states. While IL-6 and IL-12 are secreted by M1polarized macrophages and regarded as the pro-inflammatory factors, IL-10 is discharged by M2-polarized macrophages as an immunosuppressive molecule. As expected, we observed a remarkable decrease in IL-10 levels in tumor tissues, but the drop in IL-6 (P = 0.21 for H446, P = 0.06 for HCC827) and IL-12 (P = 0.42 for H446, P = 0.17 for HCC827) levels was not significant (Fig. 2B). These observations seem to support the idea that catecholamines participate in re-educating the phenotypes and activation states of tumor-associated macrophages and decreased catecholamine levels could trigger a reduction in the percentage of M2-polarized macrophages.

The finding that decreased catecholamine levels correlated with increased M1/M2 radio led us to hypothesize that catecholamines might promote macrophages’ shift toward the M2-side. To explore the role of catecholamines in macrophage polarization, BMDMs were isolated from mice and stimulated toward M0 or M1, and then incubated with EPI or NE. Flow cytometry results revealed that catecholamines not only increased the percentage of M2-polarized macrophages in M0 cells slightly to 5.2% in EPI groups and 8.4% in NE groups (Fig. 3A, upper), but also strongly promoted a shift in M1 cells toward M2 because the percentage rose to 16.4% and 23.5% respectively (Fig. 3A, lower). To highlight a stronger switch to M2 by M1 cells than by M0 cells, we examined the impact of catecholamines on M1 macrophages in the following experiments. Consistent with the above data, the transcription of M1-associated gene iNOS was inhibited (Fig. 3B, upper), concomitant with the increased M2-associated gene Arg-1 in catecholamine-treated M1 macrophages (Fig. 3B, lower). An analysis of cytokines also consolidated our hypothesis, M1-associated IL-6, IL-12, TNF-a reduced significantly and M2-associated IL-10 markedly increased after catecholamine treatment (Fig. 3C). Taken together, these findings support the notion that adrenergic-stimulated macrophages display an M2-polarized phenotype.

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Fig. 3. Catecholamine stimulation leads to an M2-like polarization. (A) M2 marker CD206 was detected in M0 macrophages (upper) and M1 macrophages (lower) after treatment with 10 nM EPI or 10 μM NE, respectively. n = 3. (B) Typical genes of M1 macrophages (iNOS, upper) and M2 macrophages (Arg-1, lower) were examined by real-time PCR at the transcriptional level. n = 3. (C) Typical cytokines of M1 macrophages (IL-6, IL-12, TNF-a) and M2 macrophages (IL-10) were examined. n = 3. *p < 0.05. **p < 0.01. ***p < 0.001.

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Fig. 4. Catecholamine stimulation promotes tube formation in vitro and tumor neovascularization in vivo. (A) HUVECs (green) were co-incubated with M1 macrophages, EPI- or NE-treated M1 macrophages, and M2 macrophages (red). Tube formation assays were conducted after 6 h to measure their capillary-like tubular structures. Representative photomicrographs are shown (scale bar: 200 μm); n = 3. The number of junctions, branches and tubules, total length, and total tubules area was measured in M1-, M2-polarized macrophages and catecholamine-treated M1 cells. (B) In vivo, the density of microvessels and M2 macrophages were obtained from the immunohistochemistry of serial tumor sections (scale bar: 100 μm); n = 3. *p < 0.05. **p < 0.01. ***p < 0.001. Abbreviations: N.S.: non-statistical significance; A.U.: arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

around the CD31-stained neo-vasculatures. Compared with control, the deficiency in catecholamines caused a significant reduction in MVD and M2 density in lung cancer models. After 6OHDA treatment, the MVD dropped to less than half of those in control groups in both the H446 and HCC827 models with a greater decline of M2 density in H446 (61.8%) mice than in HCC827 (46.9%) mice (Fig. 4B). These analyses collectively reveal that adrenergic signals mediated by macrophages could shift their polarization and modulate tumor angiogenesis.

3.4. Adrenergic-stimulated macrophages contribute to angiogenesis Considering that M2-polarized macrophages are often connected with tumor angiogenesis, we investigated the role of adrenergic-stimulated macrophages on vascular density and vasculature patterns in vitro and in vivo. Firstly, the M1 and M2 cells were set as two controls. Next, M1 cells treated with catecholamines for 24 h were collected and co-incubated with HUVECs to evaluate the tube formation capacity in vitro. From M1, EPI-treated M1, and NE-treated M1 to M2 cells, statistical analyses of capillary patterning revealed a gradually increased tendency in tube junctions, branches, length, and lumens (Fig. 4A), consistent with their position in the M1–M2 spectrum. In vivo, the distributions of M1 and M2 macrophage cells were compared and linked to microvascular density (MVD). We found that there were few iNOS-stained M1 cells in tumor areas but lots of CD163-stained M2 cells

3.5. Catecholamine-stimulated macrophages contribute to angiogenesis The findings that catecholamine-treated macrophages promote tube formation led us to believe an underlying link exists between catecholamines and macrophage-associated angiogenesis in lung cancer. To explore the underlying mechanisms, the key factor, VEGF, was 117

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Fig. 5. The inhibition of adrenergic signaling restrains the catecholamine-induced VEGF secretion. (A) Release of VEGF was measured in the supernatant of EPI- and NE-treated M1 macrophages. n = 3. (B) Expression of VEGF was measured in tumor tissues after injection with 6OHDA or not. n = 3. (C) Expression of VEGF was measured in catecholamine-stimulated M1 cells and cells pretreated with propranolol. n = 3. (D) The expression of CD86 and CD206 were assessed in EPI- or NEtreated M1 macrophages preincubated with propranolol or not. n = 3. (E) The formation of capillary-like tubular structures in the presence of propranolol or not was observed in catecholamine-treated M1 macrophages. n = 3. *p < 0.05. **p < 0.01. ***p < 0.001. Abbreviations: N.S.: non-statistical significance; A.U.: arbitrary units.

cells, 93.3% to 7.4% in NE-treated cells. Results of tube formation assay also reinforced the fact that the inhibition of adrenergic signaling weakened the building of capillary-like structures (Fig. 5E), leading to reduced junctions, branches, length, and lumens. Taken together, these findings suggest that catecholamine-induced macrophages polarize toward M2-like phenotype to promote angiogenesis by secreting VEGF, which is regulated through adrenergic signaling.

evaluated in cells and in tissues. In vitro, the VEGF secretion of macrophages increased significantly after incubation with catecholamines (Fig. 5A). In vivo, the concentration of VEGF reduced remarkably due to the diminished levels of catecholamines (Fig. 5B). To assess the relationship between VEGF and the chief adrenergic signaling of macrophages, adrenergic signaling was suppressed with the use of nonselective β-blocker propranolol to pretreat M1 macrophages before catecholamine stimulation. As expected, co-incubation with propranolol caused a significant inhibition of the catecholamine-induced increase of VEGF secretion in macrophages (Fig. 5C) and partly neutralized the effect of catecholamine on M2-like polarization (Fig. 5D). Inhibiting adrenergic signaling decreased the proportion of M2 cells in polarized macrophages from 90.8% to 13.2% in EPI-treated

3.6. Catecholamines alter innate immune cell distribution within the tumor microenvironment Macrophages regulate the turnover of immune response not only by the release of pro-or anti-tumor factors, but also by the communication 118

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Fig. 6. The effect of catecholamines on MDSCs, NKs, and DCs. MDSCs (A), NKs (B), and DCs (C) in peripheral blood, spleens, and tumors were observed in mice treated with 6OHDA or not. n = 4 or 5. *p < 0.05.

percentage of intratumoral active DCs from 12.2% to 34.6% in the former and kept roughly equal in the latter (Fig. 6C, P = 0.038 for H446, P = 0.55 for HCC827). Despite no significance in the rare NKs (P = 0.30 for H446, P = 0.09 for HCC827), which only accounted for 1–2% or less in the tumor microenvironment, the average proportion was still higher in H446 mice, the more effective model after 6OHDA treatment (Fig. 6B). These data collectively imply that inhibition of adrenergic signaling impacts on multiple immune cells, remodeling a more defensive tumor microenvironment via the neuro-immune axis.

with immune cell subsets. To reflect the impact of catecholamines on overall immune response, we tested the density of several innate immune cells, such as MDSCs, NKs, and DCs, in mice treated with 6OHDA. Blood, spleens, and tumor tissues were collected to evaluate the peripheral, splenic, and intratumoral immune cell types. MDSCs were marked as the immunosuppressive cell subset to inhibit the activation of antitumor immunity, DCs as the crucial antigen presenting cells, and NKs as the key component in innate immune to exert positive and collaborative functions in immune defense. In our study, although catecholamine deficiency had no influence on MDSCs (In blood, P = 0.87 for H446, P = 0.44 for HCC827; In spleens, P = 0.71 for H446, P = 0.56 for HCC827), NKs (In blood, P = 0.06 for H446, P = 0.65 for HCC827; In spleens, P = 0.46 for H446, P = 0.92 for HCC827), and DCs (In spleens, P = 0.052 for H446, P = 0.46 for HCC827) in circulation and in spleens, blocking adrenergic signaling led to a decrease in MDSCs and enhanced DC activation in tumor tissues. In H446 and HCC827 models, 6OHDA significantly reduced the percentage of intratumoral MDSCs from 60.8% to 32.4%, 83.3% to 60.1% respectively (Fig. 6A, P = 0.014 for H446, P = 0.012 for HCC827), increased the

4. Discussion In this study, we reported that catecholamines could reshape the TAM phenotype and reeducate them toward tumor-supportive M2-polarized macrophages by stimulating adrenergic signaling. These findings emphasize the plasticity of the TAM phenotype induced by catecholamines and the possibility to revert their antitumor properties through dampening of adrenergic nerve activity. Consequently, adrenergic receptors on macrophages have emerged as an interest in cancer 119

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biology due to the findings linking neurotransmitters and macrophage functions. Consistent with previous reports about the pro-tumoral effects of M2-polarized macrophages (Hughes et al., 2015; Sica et al., 2006), our data also revealed that the stimulation of ARs on macrophages resulted in the production of pro-angiogenic molecules and immunosuppressive cytokines, while suppressing proinflammatory cytokines, leading to a switch of immune dysfunction. Immunosuppressive IL-10 were potently upregulated after catecholamine treatment with a decreased release of proinflammatory IL-6 and IL-12, facilitating the immune escape of tumor cells. As tumors need to re-develop a vascular network to ensure nutrition and communication, neural input may provide a critical set of signals that coordinate cancer progression (Sica et al., 2008). Numerous studies have shown that stress-related catecholamines might act as a crucial factor in regulating tumor growth by promoting angiogenesis (Chen et al., 2018). Animal tumor models suggest that TAMs’ depletion inhibits angiogenesis and tumor growth, whereas increased amounts of TAMs exhibit the opposite effects. Preclinical trials report that ARs have critical roles in NE-induced VEGF expression in cancer cells, resulting in the stimulation of angiogenesis (Park et al., 2011; Yang et al., 2009). Our work also supported an adrenergic signaling-dependent mechanism where NE and EPI potently acted on the adrenergic receptors of macrophages to stimulate the secretion of VEGF, which promoted angiogenesis for lung cancer growth. In addition, the ablation of the sympathetic function by 6OHDA or blockage of the adrenergic signaling by propranolol in competition with adrenergic receptor-stimulating agents suppressed stress-induced lung cancer growth remarkably. These results provide a new insight into the dynamic nature of TAMs during tumor growth and support the claim that catecholamines can function as a key factor in macrophages polarization, tumor angiogenesis, and immune defense. Interactions between neurons and immune cells are multifactorial and multidimensional. Stimulation of adrenergic signaling not only regulates cell development, trafficking for immune surveillance, but also directs the cell-to-cell interactions necessary for a coordinated immune response. The expression of receptors for neurotransmitters has been identified on TAMs, MDSCs, NKs, DCs as well as other immune cells (Herve et al., 2013; Jin et al., 2013), which collectively facilitate the neural regulation of immune responses in a more complex neuroimmunomodulatory circuitry (Chavan et al., 2017). In order to elucidate the impact of catecholamines on localized and systematic immune systems, peripheral blood and sympathetic target organs, including tumor tissues and the best-defined lymphoid organs spleen, were analyzed in our study. Although there was no significant difference in peripheral blood and spleens, our results revealed a clear effect at localized targets. As the critical immunocytes in innate and adaptive immunity, NKs, DCs and another major immunosuppressive myeloid cell population MDSCs were monitored in our investigation. Inhibition of adrenergic signaling could facilitate the immunologically active conversion of tumors with the decreased immunosuppressive MDSCs and increased active DCs, suggesting a shift from passive response to positive defense. Although blocking the adrenergic signaling could delay tumor growth in both types of lung cancers, the difference in the inhibition rates between SCLC and NSCLC should be noted. Based on our results, H446, the SCLC cell line responded better to the treatment targeting adrenergic signaling for a greater tumor regression, a lower secretion of VEGF, and a higher radio of M1/M2 after 6OHDA injection. Supporting this observation were the lower catecholamine concentrations, more remarkable decreased MVD of tumors, as well as the extent of increased active DCs and decreased MDSCs. Some works notice that catecholamines have a widely negative impact on immune cells’ activity, including the mobilization and recruitment of macrophages, NKs, and DCs in circulation and in lymphoid tissues (Herve et al., 2013; Nissen et al., 2018). Such results were not observed in the peripheral blood and spleens in our study probably due to the incomplete clearance of the

catecholamine stock with 6OHDA. Despite our promising findings, it is hard to say whether the stricter control of local catecholamines in tumors or the biological differences within lung cancer types decides the better efficiency in H446. Further research is required to distinguish the dynamic tumor microenvironment and to explore the underlying mechanisms between SCLC and NSCLC. Additionally, there are some limitations in our experiments. For the systematic effects of catecholamines, more examinations focused on the sites beyond tumors need to be tested, especially some critical statistical significances were found in splenic active DCs (P = 0.06 for H446) and in circulating NKs (P = 0.06 for H446). Enzymatic digestion was used to isolate macrophages from tumor, and this procedure has been reported to help macrophages produce more cytokines such as IL6, IL-10 and TNF-a, indicating a change of their functional activity (Bryniarski et al., 2005; Bryniarski et al., 2005). Although the digestion time and the whole experiment was controlled to minimize the impact, whether collagenase treatment influences the macrophage phenotype is still unknown, more critical evaluation of the method should be required. Besides, we lacked the orthotopic injection models and the functional verification of infiltrating immune cells to investigate the natural environment in lungs. Despite these limitations, our findings still demonstrate that SNS can impact tumor progression substantially and that such effects are associated with significant alterations in host immune function. These finding set preclinical foundations to the potential utility of anti-neurogenic therapies. There is increasing evidence that TAM accumulation is associated with poor clinical prognosis and resistance to cancer therapy, which is in part due to the immunosuppressive and tumor-promoting activities of TAMs (Cassetta and Pollard, 2018). Recently, several studies on TAM-targeting cancer therapy have concentrated on the following strategies: the inhibition of macrophage recruitment, conversion of protumorigenic M2 to the antitumor M1 phenotype, and suppression of TAM survival (Cassetta and Pollard, 2018). Because the classical M1 macrophage possesses antitumor activity, the polarization from tumorpromoting M2 to tumoricidal M1 macrophages appears to be a better potential target for cancer therapy. Thus, the re-education of TAMs plasticity via adrenergic signaling could block the pro-tumorigenic effects of TAMs. Recent clinical research has supported the hypothesis of using β-blockers to reduce the rates of disease progression and improve overall survival in locally advanced NSCLC (Wang et al., 2013). More recently, a targetable mechanism of EGFR inhibitor resistance elucidated that chronic stress hormones promote EGFR TKI resistance via βAR signaling by an LKB1/CREB/IL-6-dependent mechanism and suggested that the combination of β-blockers with EGFR TKIs merit further investigation as a strategy to abrogate resistance (Nilsson et al., 2017). In rodent animal models, reducing β-adrenergic signaling facilitates the conversion of tumors to an immunologically active tumor microenvironment with an increased intratumoral frequency of CD8+ T cells and a decreased expression of programmed death receptor-1 (PD-1), inducing a shift in antitumor immunity (Bucsek et al., 2017). Accumulating evidence supports the assertion that catecholaminemediated adrenergic pathways potentiate the neuro-immune-tumor interactions. Our results show that macrophages play a crucial role in connecting neural signals and immune response, wherein the inhibition of adrenergic nerves shifts the active state of macrophages to block angiogenesis and reshapes the immunosuppressive microenvironment that supports aggressive lung cancer. Identifying and targeting the neuro-immune axis that improve therapeutic efficacy by bolstering anti-tumor responses lays the groundwork for the translation of adrenergic blockade as a promising adjuvant to existing therapeutic strategies in clinical oncology. 5. Financial support This work is supported by the National Natural Science Fund, Grant No. 81672979 and Grant No. 81472201. 120

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Declaration of Competing Interest

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