Chronic stress induces pulmonary epithelial cells to produce acetylcholine that remodels lung pre-metastatic niche of breast cancer by enhancing NETosis

Abstract

Background: Chronic stress promotes most hallmarks of cancer through impacting the malignant tissues, their microenvironment, immunity, lymphatic flow, etc. Existing studies mainly focused on the roles of stress-induced activation of systemic sympathetic nervous system and other stress-induced hormones, the organ specificity of chronic stress in shaping the pre-metastatic niche remains largely unknown. This study investigated the role of chronic stress in remodeling lung pre-metastatic niche of breast cancer.

Methods: Breast cancer mouse models with chronic stress were constructed by restraint or unpredictable stress. Expressions of tyrosine hydroxylase, vesicular acetylcholine transporter (VAChT), EpCAM and NETosis were examined by immunofluorescence and confocal microscopy. mRNA and protein levels of choline acetyltransferase (ChAT), VAChT, and peptidylarginine deiminase 4 were detected by qRT-PCR and Western blotting, respectively. Immune cell subsets were analyzed by flow cytometry. Acetylcholine (ACh) and chemokines were detected by ELISA and multi chemokine array, respectively. ChAT in lung tissues from patients was examined by immunohistochemistry.

Results: Breast cancer-bearing mice suffered chronic stress metastasized earlier and showed more severe lung metastasis than did mice in control group. VAChT, ChAT and ChAT⁺ epithelial cells were increased significantly in lung of model mice undergone chronic stress. ACh and chemokines especially CXCL2 in lung culture supernatants from model mice with chronic stress were profoundly increased. Chronic stress remodeled lung immune cell subsets with striking increase of neutrophils, enhanced NETosis in lung and promoted NETotic neutrophils to capture cancer cells. ACh treatment resulted in enhanced NETosis of neutrophils. The expression of ChAT in lung tissues from breast cancer patients with lung metastasis was significantly higher than that in patients with non-tumor pulmonary diseases.

Conclusions: Chronic stress promotes production of CXCL2 that recruits neutrophils into lung, and induces pulmonary epithelial cells to produce ACh that enhances NETosis of neutrophils. Our findings demonstrate for the first time that chronic stress induced epithelial cell derived ACh plays a key role in remodeling lung pre-metastatic niche of breast cancer.

Keywords: Acetycholine; Breast cancer; Chronic stress; Lung metastasis; NETosis; Neutrophils.

Fig. 1

Chronic stress promotes lung metastasis of breast cancer. a. Flow chart of the establishment of breast cancer mouse model with chronic restraint stress. b. Dynamic observation of tumor burdens by biofluorescence imaging in metastasis model mice. c. Fluorescence images of lung tissues in model mice with chronic stress for 3 weeks (left) and the corresponding fluorescence intensity in each group (right). d. Photo of primary tumors at 2 weeks (left) and dynamic observation of primary tumor volumes in orthotopic injection mouse model (right). e. Histological examination of lungs from orthotopic inoculation model mice at 6 weeks (left) and the percentage of metastatic area to the total lung area (right). Red arrows point to metastatic loci. f. Flow chart for the establishment of breast cancer mouse model with chronic unpredictable stress. g. Photo of primary tumors (left) and the tumor volumes (right) in orthotopic inoculation mice model with or without unpredictable stress for 2 weeks. h. Histological examination of lung tissues from breast cancer mouse model with or without unpredictable stress for 6 weeks (left) and the percentage of metastasis area to total lung area (right). Red arrows point to metastasis of breast cancer in the lung. ns: no sense, *p < 0.05, ***p < 0.001, ****p < 0.0001

Fig. 2

Chronic stress activates parasympathetic nervous system and promotes acetylcholine secretion in lungs. a-b. Immunofluorescence examinations of TH (a) and VAChT (b) expression in lung tissues from breast cancer model mice with or without chronic restraint stress for 2 weeks. c. Immunofluorescence examination of VAChT expression in lung tissues from breast cancer model mice with or without unpredictable stress for 2 weeks. d. qRT-PCR (top) and Western blot analyses of ChAT (middle and bottom) in lung tissues from breast cancer model mice with or without chronic restraint stress for 2 weeks. e. qRT-PCR (left) and Western blot analyses (middle and right) of VAChT in lung tissues of mice in control and chronic restraint stress groups at 2 weeks. f. ACh concentrations in serum and lung cultural supernatant of breast cancer model mice in control group and chronic restraint stress group for 2 weeks. ns: no sense, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Chronic stress activates acetylcholinergic pathway of pulmonary epithelial cells with neuroendocrine functions. a. Gating strategy of flow cytometry analyses of ChAT⁺ epithelial cells and CD4⁺ T cells. b-c. FACS analyses of ChAT expression in EpCAM⁺ epithelial cells (b) and CD4⁺ T cells (c) in lungs of breast cancer model mice with or without chronic restraint stress for 2 weeks and their corresponding ChAT positive percentages. d. Immunofluorescence examinations of EpCAM and VAChT in lung tissues of breast cancer model mice in control group and chronic restraint stress group at 2 weeks. e. Immunofluorescence examinations of EpCAM and VAChT in lung tissues of breast cancer model mice in control group and chronic unpredictable stress group. f-g. Percentage (left) and fluorescence intensity (right) of EpCAM-VAChT colocalization in experiments as described in d and e, respectively. ns: no sense, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Chronic stress induces infiltration of neutrophils into lung. a. Gating strategy of flow cytometry analyses of immune cell subsets. b. FACS analyses of immune cell subsets in lungs from orthotopic inoculation murine breast cancer model with or without restraint stress. c. FACS analyses of Ly6G^hiCD11b⁺ neutrophils in lungs from murine breast cancer model with or without chronic restraint stress for 2 weeks. d. Percentages of Ly6G^hi/CD11b⁺ neutrophils in lungs from breast cancer model mice with or without chronic unpredictable stress. e. FACS analyses of neutrophils in lungs from model mice with or without chronic unpredictable stress for 2 weeks. f. FACS analyses of neutrophils in lungs from 14-week-old PyMT-MMTV spontaneous breast cancer model mice with or without chronic restraint stress for 6 weeks (upper) and their corresponding statistical results (bottom). ns: no sense, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Chronic stress induces the chemotaxis of neutrophils into lung via CXCL2-CXCR2 axis. a. Transwell chemotaxis assay to detect the chemotaxis activity of lung culture supernatants to peripheral blood neutrophils of model mice in the control group at 1 week. CLCS: lung culture supernatant of model mice in control group; SLCS: lung culture supernatant of model mice in chronic restraint stress group at 2 weeks. b. Transwell chemotaxis assay to detect the chemotaxis activity of ACh to peripheral blood neutrophils from model mice in the control group at 1 week. c. Chemokine array to determine the contents of different chemokines in lung culture supernatants from orthotopic inoculation breast cancer mice with or without chronic restraint stress for 2 weeks. d. Transwell chemotaxis assay to examine the influence of CXCL2 neutralizing antibody on neutrophil chemotactic activity of SLCS. DMEM: culture medium to serve as the blank control; CLCS: lung culture supernatant of mice in control group; SLCS: lung culture supernatant of mice in chronic restraint stress group at 2 weeks; SLCS + iCXCL2: SLCS + 1 μg/ml CXCL2 neutralizing antibody; CXCL2: 2ng/ml. e. Cell counts of experiments as described in d. f. qRT-PCR to dynamically detect the mRNA levels of CXCR2 in lung neutrophils from breast cancer model mice with or without chronic restraint stress. ns: no sense, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Chronic stress enhances NETosis of neutrophils in the lung of model mice. a. Immunofluorescence analyses of in situ NETosis of neutrophils in lung tissues of breast cancer model mice with or without chronic restraint stress for 2 weeks. b. NETosis percentages of neutrophils in experiments as described in a. c. Immunofluorescence to examine NETs structures of neutrophils isolated from lungs of model mice in control group and in chronic restraint stress group at 2 weeks. d. NETosis percentages of neutrophils in experiments as described in c. e-f. qRT-PCR and Western blot to detect mRNA (e) and protein (f) levels of PADI4 in neutrophils isolated from lungs of model mice in control group and in chronic restraint stress group at 2 weeks. **p < 0.01, ***p < 0.001

Fig. 7

Chronic stress promotes NETotic neutrophils to capture breast cancer cells. a. Confocal analyses to examine the ability to capture cancer cells of neutrophils isolated from lungs of model mice with or without chronic restraint stress for 2 weeks. b. Percentage (left) and fluorescence intensity (fold change, right) of DAP-CitH3-4T1-GFP colocalization in experiments as described in a. c. Number of 4T1-GFP cells captured by NETotic neutrophils in experiments as described in a. ***p < 0.001, ****p < 0.0001

Fig. 8

ACh promotes NETosis of neutrophils from healthy human and mice. a-b. Confocal analyses to examine the effects of ACh on NETosis of healthy human peripheral blood neutrophils (a) and mouse bone marrow derived neutrophils (b). c-d. NETosis percentage of human (c) and mouse (d) neutrophils in different treatment groups, respectively. ns: no sense, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 9

Immunohistochemistry analyses of ChAT expression in lung tissues of breast cancer patients with lung metastasis. a. Representative immunohistochemistry images of ChAT expression in lung tissues from patients with non-tumor bearing pulmonary diseases and from breast cancer patients with lung metastasis. b. Average optical density (upper) and percentage of ChAT positive area to total area (bottom) analyzed by ImageJ software. **p < 0.01

Fig. 10

Schematic diagram of the mechanisms by which chronic stress remodels lung pre-metastatic niche of breast cancer. Chronic stress remodels lung pre-metastatic niche of breast cancer mainly by two ways. On one hand, chronic stress promotes production of CXCL2 chemokine in the lung, which recruits neutrophils. On the other hand, chronic stress motivates lung epithelial cells to secrete ACh that enhances NETosis of the aggregated neutrophils in the lung. The NETotic neutrophils capture the circulating tumor cells, hereby favoring lung metastasis of breast cancer. This image (AARIT33ae3) was generated by Figdraw (https://www.figdraw.com)