br To examine the e ect of adrenergic receptor agonist
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 SQ-109 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
chemically, while mice in the control groups received an in-traperitoneal injection of PBS at the same time points. Tumor dimen-sions 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 ni-trogen, while others were fixed and paraﬃn-embedded for histological analysis.
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 phe-notypic analysis of TAMs, cell suspensions made from blood and tissues were stained with the following surface antibodies: anti-CD11b, anti-F4/80, anti-CD86. Then, the cells were stained with anti-CD206 after fixation and permeabilization. For the analysis of the MDSCs, cell sus-pensions 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.
To evaluate the level of NE and EPI, frozen tumors, spleens and lungs were collected into tubes containing EDTA and sodium metabi-sulphite and homogenized in 0.01 N HCl solution at 4 °C. All homo-genized 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 proto-cols.
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 ana-lyzed 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 Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
and 6OHDA groups of both HCC827 and H446 cells were dewaxed in xylene and dehydrated through alcohols, followed by blocked for en-dogenous 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.