Mechanism Of Chronic Stress Promoting Lung Metastasis In Breast Cancer

Stress is an organism's unspecific adaptive response to a stressor.Stressor stimulus response causes enhanced activities of the hypothalamus-pituitary-adrenal axis(HPA)and the sympathetic nervous system(SNS),leading to release of catecholamine,glucocorticoid,and other stress-related hormones.Particularly,chronic stress results in sustained release of stress-related hormones,which profoundly influence on the progress and prognosis of disease,especially of cancer.Recently,numerous studies have indicated that the factors secreted by primary tumors induce metastasis-promoting microenvironments(pre-metastatic niche)at distant organs(pre-metastatic organs)for subsequent metastatic colonization by circulating tumor cells(CTCs)in the autocrine and paracrine manners.Considering that chronic stress leads to disorder of nerve-endocrine-immune network,we assume that the disturbance of host macroenvironmental homeostasis may impact on remodeling of future metastatic organs.However,the influences imposed by host-derived factors on pre-metastatic organs have been largely disregarded.Little is known about the host response at the critical stage of metastatic development.In this study,we utilized a mouse model of chronic unpredictable stress(CUS)to explore the effects of chronic stress/?-adrenergic signal on pulmonary metastasis of breast cancer.The main results are as follows:1.Chronic psychological stress promotes spontaneous lung metastasis of breast cancer through activating ?-adrenergic signaling.In order to explore the impact of chronic psychological stress on spontaneous metastasis of breast cancer,Balb/C mice were exposed to CUS daily for five consecutive days before injecting 4T1 murine mammary carcinoma cells subcutaneously.Then the mice were subjected to CUS again for 21 successive days.The formation of tumor metastases in the lung tissue of mice was observed.The results show that the numbers of lung metastases were significantly more in the mice exposed to CUS than in the control mice,suggesting that CUS promoted spontaneous lung metastases of breast cancer.Ablation of sympathetic nerve function by 6-OHDA or blocking of ?-adrenergic signaling by propranolol remarkably reversed CUS/ISO-induced lung metastasis of breast cancer in mice,suggesting that activation of the ?-adrenergic signal induced by sustained release of catecholamine promotes pulmonary metastasis of breast cancer under chronic stress.2.Activation of ?-adrenergic signal promotes metastatic colonization by circulating tumor cellsMetastatic colonization and proliferation by circulating tumor cells at the future metastasis site is a critical limiting step of tumor metastasis.We utilized an experimental metastasis model by tail vein injection,which results primarily in pulmonary metastasis,to explore the influence of the ?-adrenergic signaling on metastatic colonization.We observed that only three days of ISO pre-treatment significantly promoted the colonization of circulating breast cancer cells in the lungs.6-OHDA and propranolol effectively inhibited CUS or ISO-induced lung metastases.By using the MMTV-PyMT transgenic mouse,which spontaneously develops palpable mammary tumors and the lung metastasis,we confirmed that CUS or ISO stimulation in the pre-metastatic phase accelerated the formation of lung metastases.Our results suggest that activation of the ?-adrenergic signaling plays a critical role in orchestrating pre-metastatic niche formation and accelerating lung metastasis of breast cancer under chronic psychological stress.3.Adrenergic signaling-induced metastatic lung colonization by breast cancer cells relies on infiltration of macrophages in the lungPre-metastatic niche harbors various types of cells,among which immune cells exert critical proinflammatory functions.To define the cell populations influenced by catecholamine,we employed Balb/C nude mice(mutant mice with congenital thymus defect),NSG mice(lacking mature T,B,and functional NK cells)to repeat the above experiments.The results show that T,B,NK cells are not the target cells affected by catecholamine in the pre-metastatic phase.However,when we treated the mice with clodronate-loaded liposomes to remove monocytes/macrophages in the circulation and lung,the effects of ISO were completely abrogated.The pro-metastatic effects of ISO were also suppressed by selective depletion of the macrophages in the lung through intranasal administration with clodronate-loaded liposomes.These results suggest that adrenergic signaling-induced metastatic lung colonization by breast cancer cells relies on infiltration of macrophages in the lung.4.Activation of adrenergic signaling induces accumulation of macrophages in the lungIn order to further study the role of macrophages in pre-metastatic lungs under chronic stress,we first observed the dynamic changes of macrophages in lung tissues.The results show that the number of CD11b+F4/80+ macrophages in lung tissue was significantly increased 5 days after stimulation with ISO,suggesting that elevated levels of catecholamines can promote the accumulation of macrophages in lung tissue.Subcutaneous implantation of 4T1 cells also led to an increase in the number of CD11b+F4/80+ macrophages in the lung tissue,indicating that tumor cells may promote macrophage infiltration into lung in a paracrine manner.However,pulmonary infiltration of CD11b+F4/80+ positive macrophages was the most prominent in the mice implanted with 4T1 cells and treated with ISO,suggesting double effects of catecholamines and tumor-related factors on lung pre-metastatic microenvironment.As determined by flow cytometric analysis,a significant increase in the percentage of Ly6C+CD11b+CD115+ monocytes in peripheral blood,spleen,and bone marrow was observed in the mice treated with ISO or subcutaneously implanted with 4T1 cells.However,when the tumor-bearing mice were given ISO,the percentage of the population was higher in the lungs of the tumor-bearing mice treated with ISO.The results were confirmed by immunohistochemical analysis.F4/80+ Macrophages accumulated around the terminal bronchioles of the lungs and the number of macrophages was further increased with time extension.Our data confirm that catecholamine-induced adrenergic signal activation induces bone marrow monocyte mobilization and macrophage aggregation in lung tissue before the arrival of metastatic tumor cells.5.Chronic psychological stress enhances macrophage chemokine CCL2 expression in the lungIn order to explore the molecular mechanism of macrophage infiltration induced by catecholamine in lung tissue,we analyzed the expression of monocyte/macrophage chemokine CCL-2 in lung tissue in response to CUS or ISO by ELISA.The results showed that the expression of CCL2 in lung tissue was significantly increased after 5 days of CUS or ISO stimulation.CUS-induced CCL2 expression up-regulation could be effectively reversed by propranolol,suggesting that chronic stress could promote the expression of CCL2 in lung tissue by activating the ?-adrenergic signal.The expression of CCL2 was also observed in the lung tissues of the mice implanted with 4T1 cells subcutaneously.However,the CCL2 expression was significantly higher in the lung of tumor-bearing mice that were treated with ISO,suggesting that elevation of catecholamine level in the pre-metastatic phase is an important factor affecting the pre-metastatic lung microenvironment.In vitro studies demonstrate that ISO upregulated the expression of CCL2 in primary pulmonary microvascular endothelial cells and that ?-AR/PKA pathway inhibitors propranolol and H89 effectively inhibited ISO-induced CCL2 expression.The results of ELISA showed that the NE level in lung tissue of the tumor-bearing or CUS-exposed mice was significantly increased.CUS exposure further promoted the release of NE in the lungs of the tumor-bearing mice.These results suggest that sustained release of catecholamine under chronic stress induce the CCL2 expression in mouse pulmonary microvascular endothelial cells by activating the ?-AR/PKA pathway,thereby inducing circulating monocytes into lung tissue.6.ISO induces monocyte / macrophage expression of CCR2CCR2 is a receptor for CCL2.Chemokines bind to chemokine receptors and mediate cellular migration,proliferation,and function by activating multiple signaling pathways.In order to explore whether the expression of chemokine receptors in macrophages is altered in response to the stimulation of catecholamines or whether the expression level of chemokine receptors is associated with macrophage infiltration into lung tissues,we analyzed the CCR2 expression on the surface of monocytes.The results showed that ISO stimulation during tumor pre-metastasis phase resulted in a significant increase in the CCR2 expression in peripheral blood monocytes of mice.The administration of mice with the CCL2 neutralizing antibody significantly inhibited CUS or ISO-induced macrophage infiltration in lung tissues.The results were verified by in vitro experiments utilizing human monocyte cell line THP-1 and mouse monocyte cell line Raw264.7,confirming that ISO stimulation significantly promotes the CCR2 expression in human and mouse monocytes.H89 or propranolol markedly reversed the effect of ISO,indicating that the activation of ?-AR/PKA signaling pathway is involved in regulating the expression of CCR2 in monocytes/macrophages.Furthermore,intraperitoneal injection of propranolol significantly reduced the number of metastases in mouse lungs up to 90%,suggesting that blocking the ?-adrenergic signaling pathway during pre-metastatic phase may control an important rate-limiting step in tumor metastasis.Taken together,we elucidated a novel mechanism by which the ?-adrenergic signaling alters the lung microenvironment under chronic psychological stress.Activation of the ?-adrenergic signaling during the pre-metastatic phase induces mobilization and release of bone marrow-derived monocytes and upregulates the expression of CCL2 and CCR2 in the lung microvascular endothelial cells and monocytes/macrophages,respectively,resulting in the recruitment and accumulation of macrophages in the pre-metastatic lung.Macrophages as the main components dominantly contribute to the modulation of local microenvironment and metastatic colonization of CTCs in mouse breast cancer models.