Chronic stress reshapes bone marrow microenvironment to facilitate breast cancer bone metastasis

Chronic stress reshapes bone marrow microenvironment to facilitate breast cancer bone metastasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chronic stress reshapes bone marrow microenvironment to facilitate breast cancer bone metastasis Jinbo Li, Xing Li, Xinyu Liu, Xiaokuan Zhang, Jinxiao Fan, Yaning Xing, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7760970/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Cancer bone metastasis is associated with poor prognosis and resistance to immune checkpoint inhibitors. Although chronic stress promotes cancer progression, its role in reshaping the bone marrow (BM) immune microenvironment to facilitate metastasis remains poorly understood. We demonstrate that chronic stress promotes breast cancer bone metastasis primarily by reshaping the BM immune microenvironment. Specifically, chronic stress increases BM neutrophils, and inducible, specific clearance of these neutrophils effectively prevented stress-induced bone metastasis. In mice exposed to chronic stress, abundant BM neutrophils excessively consume arginine by upregulating iNOS, creating a low-arginine, pro-metastatic niche that impairs functional CD8⁺ cytotoxic T cells. Mechanistically, chronic stress stimulates norepinephrine (NE) release in the BM, which boosts osteopontin (OPN) expression by monocytes/macrophages. The elevated OPN then interacts with its receptor CD44 on neutrophils to trigger this excessive arginine consumption. This cascade was shut down in mice with a global knockout of the NE receptor ADRβ2. To therapeutically target this process, we developed a bone-targeting conjugate of propranolol (an ADRβ antagonist) and alendronate (AP). Local accumulation of AP in the bone reduced OPN-positive monocytes/macrophages and iNOS-positive neutrophils, restoring BM arginine levels and CD8⁺ T cell populations, and effectively inhibiting chronic stress-induced breast cancer bone metastasis. Our findings reveal a novel neuro-immune-metabolic axis through which chronic stress promotes breast cancer bone metastasis and highlight AP as a potential therapeutic strategy. Biological sciences/Cancer/Metastasis/Bone metastases Biological sciences/Cancer/Breast cancer Chronic immobilization stress norepinephrine ADRβ osteopontin monocytes neutrophils arginine breast cancer bone metastasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer, the most common cancer among women, is diagnosed approximately 47.8 per 100,000 women globally 1 . The invasive ductal carcinoma (IDC), the most common breast cancer 2 , often metastasizes to bone. Especially Luminal A breast cancer (ER/PR-positive, HER2-negative) has a higher propensity to metastasize to bone than other organs including brain, liver and lung 3 , results in approximately 70 percent of patients with advanced breast cancer develop bone metastasis 4 , 5 . Bone metastasis is generally incurable and significantly impact prognosis and outcomes, with affected patients having a median survival of 2 to 5 years 6 . Clinical evidence supports that the cancer patients with bone metastasis derive less benefit from immune checkpoint inhibitor (ICI) therapies 7 . In addition, skeletal-related events (SREs), including pathological fracture and spinal cord compression associated with bone metastasis, cause a significant increase in morbidity, hospitalization and even mortality 8 . Breast cancer patients often experience chronic stress, driven by concerns about disease progression and the burden of prolonged therapies, which can persist for weeks and years. Such chronic stress is associated with a higher risk of metastasis and poorer survival outcomes 9 , 10 . Chronic stress activates key neuroendocrine systems, including the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) 11 , disrupting whole-body homeostasis—particularly affecting cardiovascular and immune systems 12 . Chronic stress promotes primary tumor growth and remote metastasis, potentially by enhancing cancer cell proliferation, migrate, and dissemination 13 , as well as by establishing a pro-metastatic niche in distant organs 14 . A critical unresolved question is: for bone metastasis, which plays a more significant role—chronic-stress-induced intrinsic changes in primary tumor cells or extrinsic modifications to the pro-metastatic niche? Under chronic stress, elevated levels of stress hormones (e.g., cortisol and catecholamines) may stimulate osteoblastic cells to produce RANKL, thereby promoting osteoclastic bone resorption 15 , 16 . Currently, denosumab, an anti-RANKL monoclonal antibody, and bisphosphonates are clinically used to prevent SREs in breast cancer patients with bone metastasis 17 , 18 . Denosumab has also been reported to enhance efficacy of ICI therapy in treating bone metastasis in mice 7 . However, whether anti-resorptive drugs alone can prevent metastasis in cancer patients controversial 17 , 19 , highlighting the need to develop therapeutic strategies targeting bone erosion-independent mechanism. A growing body of mechanistic evidence has been provided to explain how chronic stress contributes to cancer metastasis. In the primary breast cancer environment, chronic stress can reduce natural killer (NK) cell activity, suppress T cells function, increase production of inflammatory cytokines such as TNFα, activate SNS to release norepinephrine (NE), and upregulate matrix metalloproteinases (MMPs) that degrade the extracellular matrix 20 , 21 . These alterations promote the growth, migration, invasion and dissemination of primary cancer cells 21 . Chronic stress also disrupts hematopoietic stem cell activity by inhibiting the environmental CXCL12 and induces monocytosis and neutrophilia in humans 22 . Monocytes from stressed mice and humans exhibit a characteristic inflammatory transcriptomic signature, which skews them to a primed hyperinflammatory phenotype 23 . Most recently, it is reported that chronic stress significantly alters the lung microenvironment by shifting normal circadian rhythm of neutrophils and elevating neutrophil extracellular trap (NET) formation 24 . However, far less is known about how chronic stress induces immune modifications to establish a pro-metastasis niche that supports the colonialization of disseminated cancer cells. In this study, we generated a chronic stress mouse model via chronic immobilization stress (CIS), in which CIS-exposed mice exhibited depression-like behaviors and increased release of NE in the bone marrow (BM). Our findings revealed that CIS enhanced osteopontin (OPN) expression in BM monocytes and macrophages, and that environmental OPN stimulated an excessive arginine metabolism in neutrophils through OPN/CD44 interaction, leading to a low-arginine bone microenvironment that impairs CD8 + cytotoxic T cell function. To intercept this pathological cascade at its onset, we developed a bone-targeting delivery strategy for propranolol to block NE/ADRβ signaling. Specifically, we synthesized alendronate-conjugated propranolol (AP) compound and evaluated its efficacy in preventing breast cancer bone metastasis. Results Chronic stress remodels the BM microenvironment and promoted breast cancer bone metastasis To investigate the impact of chronic stress on breast cancer bone metastasis, we generated a mouse model of chronic immobilization stress (CIS). BALB/c mice were subjected to chronic immobilization stress by confining them in 50 ml tubes for 2 hours per day, 6 days per week, for 6 consecutive weeks (Suppl. Figure 1A). We confirmed that CIS-exposed mice exhibited anxiety-like behavior (Suppl. Figure 1B-D) and accelerated orthotopic breast cancer growth compared to control (Ctrl) mice (Suppl. Figure 1E-H). To discern whether CIS primarily affects the intrinsic metastatic potential of breast cancer cells or the systemic microenvironment, a primary-secondary transplantation strategy was employed. 4T1 breast cancer cells, initially implanted orthotopically into the mammary fat pads of BALB/c mice previously exposed to CIS, were subsequently isolated from the orthotopic breast tumors and secondarily transplanted into untreated BALB/c mice via intracardiac injection (Fig. 1 A). In the primary implantation experiment, CIS-exposed mice demonstrated a higher incidence of remote metastasis to the lung, liver and bone (Fig. 1 B). Correspondingly, the size of bone metastatic lesions in the leg bones was significantly larger in CIS-exposed mice compared to Ctrl mice, as were lung metastases (Fig. 1 F, G). Notably, in the secondary transplantation model, the incidence (Fig. 1 B) and size of remote metastases to bone (Fig. 1 C, E) and lung (Fig. 1 F, H) were comparable between recipients of CIS-derived and Ctrl-derived cancer cells. These results indicate that CIS primarily promotes breast cancer remote metastasis through alterations in the microenvironment of target organs, rather than inducing intrinsic changes in cancer cells. To further confirm the role of CIS in enhancing bone metastasis across different mouse strains, luciferase-expression E0771 breast cancer cells were intracardially injected into Ctrl- and CIS-exposed C57BL6/J mice. Consistent with observations in BALB/c mice, CIS-exposed C57BL6/J mice exhibited a higher incidence of hind limb bone metastasis three weeks post cancer cell injection (Fig. 1 I). Luciferin flux, a quantifiable indicator of metastasis burden, was comparable between CIS and Ctrl groups at 1 and 2 weeks, but significantly elevated in CIS-exposed by week 3 (Fig. 1 I-J). Histological analysis of tibial paraffin sections further revealed significantly larger metastatic lesions in CIS-exposed C57BL6/J mice compared to Ctrl mice (Fig. 1 K-L). Increased bone turnover and osteoclastic bone resorption are known to promote breast cancer bone metastasis 25 , 26 , while serum levels of osteocalcin (OCN, an osteoblastic activity marker) and TRACP-5b (an osteoclastic bone resorption marker) were similar in CIS-exposed and Ctrl C57BL6/J mice (Fig. 1 M; Suppl. Figure 2A). Furthermore, the trabecular bone volume and the numbers of osteoblasts and osteoclasts in the tibial metaphysis were comparable between CIS-exposed and Ctrl mice (Suppl. Figure 2B-E). Interestingly, bone erosion activity, particularly osteoclast number and surface, was significantly reduced on trabecular and cortical bone surfaces adjacent to cancer cells (CC-adjacent) compared to non-CC-adjacent bone surfaces (Suppl. Figure 2F-I). These results indicate that CIS-promoted breast cancer bone metastasis does not rely on any increase in bone erosion. Of note, analysis of bone marrow (BM) immune cell composition revealed that CIS-exposed C57BL6/J mice displayed a reduced proportion of lymphoid lineage cells and an increased proportion of myeloid lineage cells in the BM compared to Ctrl mice (Fig. 1 N). Specifically, percentages of B220 + B cells and CD8 + T cells were significantly lower in CIS-exposed mice, while percentages of CD4 + T cells remained unchanged (Fig. 1 O-R). Conversely, percentages of CD11b + leukocytes, including Ly6C⁺ monocytes and Ly6G⁺ neutrophils, were significantly higher in CIS-exposed mice, whereas F4/80⁺ macrophages showed no significant difference (Fig. 1 O and 1 S-V). These findings collectively suggest that CIS promotes breast cancer bone metastasis, associated with a distinct remodeling of the BM immune cell landscape. Elevated norepinephrine release stimulates osteopontin expression by monocytes/macrophages in stress-exposed mice Chronic stress is known to activate the sympathetic nervous system, leading to increased norepinephrine (NE) release in mice and humans 27 , 28 . Given the presence of sympathetic nerves within the BM 29 , NE levels in the interstitial fluid of hindlimb BM were measured and found to be significantly higher in CIS-exposed C57BL6/J mice compared to Ctrl mice (Fig. 2 A). To investigate the role of sympathetic nervous system activation in mediating CIS-induced BM microenvironment changes, a denervation mouse model was generated by dissociating the sciatic and femoral nerves 30 . Denervated mice exhibited significantly reduced BM NE levels at 12 weeks post-surgery (Fig. 2 B) and a gradual reduction in trabecular bone volume and osteoclast number compared to sham-operated control mice (Suppl. Figure 3A-B). Proteomic studies were next carried out to identify significantly changed BM proteins associated with CIS-exposure and sympathetic activation. KEGG analysis ranked “ECM-receptor interaction” as the most significantly altered pathway in both CIS-exposed versus Ctrl mice (with 18 significantly upregulated and 14 downregulated proteins; Fig. 2 C-E) and in denervated versus sham mice (among Top 10 enriched pathways; with 322 upregulated and 162 downregulated proteins; Fig. 2 D-F). GO analysis further confirmed the upregulation of ECM-related pathways in BM of CIS-exposed mice compared to Ctrl mice (Suppl. Figure 3C-D). A comparative analysis revealed five proteins — COL11a1 (collagen type XI a1), COL1a1 (collagen type I a1), FAM207a (nucleolar protein regulating ribosome biogenesis), OPN (osteopontin) and GIT1 (ARF GTPase-activating protein) — that were differentially expressed in both CIS-exposed versus Ctrl mice, and in denervated versus sham mice (Fig. 2 G). Protein levels of COL11a1, COL1a1 and FAM207a were lower in CIS-exposed mice and higher in denervated mice compared to their respective controls. Notably, OPN exhibited an inverse expression pattern, being higher in CIS-exposed mice and lower in denervated mice compared to their respective controls (Fig. 2 G). OPN, a glycoprotein involved in ECM-receptor interactions through binding with collagens, integrins and CD44, et al 31, 32 , was confirmed to be significantly higher in the BM of CIS-exposed mice and lower in denervated mice by ELISA (Fig. 2 H-I), suggesting a positive correlation between BM NE and OPN. Immunofluorescence (IF) staining was carried out to determine cellular source of OPN and indicated clustered OPN + cell populations in the BM, which were significantly increased in CIS-exposed mice (Fig. 2 J). Flow cytometry corroborated this increase (Fig. 2 K; Suppl. Figure 3E), and identified monocytes (CD11b + Ly6C + ) and macrophages (F4/80 + ) as the primary OPN-expression cells in the BM, and these cells, but not neutrophils (CD11b + Ly6G + ), were significantly increased in the BM of CIS-exposed compared to Ctrl mice (Fig. 2 L- 2 N; Suppl. Figure 3F). To determine whether NE directly stimulates OPN expression in monocytes/macrophages, BM Ly6C + monocytes were isolated from Ctrl and CIS-exposed mice and treated with increasing doses of NE in vitro . NE significantly stimulated Opn gene transcription in monocytes in a dose-dependent manner, with a stronger response observed in monocytes from CIS-exposed mice (Fig. 2 O). These findings demonstrate that CIS-induced elevated NE release enhances OPN expression in BM monocytic cells. Excessive OPN targets CD44 neutrophils to promote bone metastasis in stress-exposed mice To elucidate the role of OPN in bone metastasis, a protein array comparing serum from breast cancer patients with newly diagnosed bone metastasis versus those without bone metastasis was performed. Patients with bone metastasis exhibited increased serum levels of OPN and IL-4, alongside reduced levels of CCL24, OPG and MIP-1b (Fig. 3 A). Notably, breast cancer patients with higher expression of OPN receptors, including ITGB5, ITGA5 and CD44, had significantly shorter bone metastasis-free survival (Fig. 3 B- 3 D), suggesting a role for environmental OPN-receptor interactions in promoting metastasis. Transwell cell migration assay demonstrated that NE-treated BM cells recruited significantly more E0771 cells compared to vehicle-treated controls, and this enhanced migration was partially abrogated by a CD44-blocking antibody in BM cells, implying a critical role for CD44 + BM cells in promoting E0771 cell migration (Fig. 3 E and 3 F). In CIS-exposed mice, CD44 + BM cells were primarily enriched within the CD11b + myeloid cell population. Sorted CD44 + cells from CD11b + myeloid cells showed significantly higher transcriptional levels of Itgα5 and Itgβ5 compared to CD44 − myeloid cells (Fig. 3 G), suggesting preferential co-expression of ITGA5 and ITGB5 with CD44 in these cells. Phenotypic analysis of CD44 + BM cells revealed that approximately 70% of CD44 + CD11b + myeloid BM cells were Ly6C − Ly6G + neutrophils in both CIS-exposed and Ctrl mice (Fig. 3 H and 3 I). Furthermore, the percentage of CD44 + Ly6G + neutrophils in BM was significantly elevated in CIS-exposed mice, and over 90% of total neutrophils expressed CD44 (Fig. 3 K). To investigate the functional role of CD44 + Ly6G + neutrophils in CIS-induced breast cancer bone metastasis, Ly6G CreERT2 ROSA26 DTA mice (hereafter referred to as Ly6G-DTA mice), allowing for inducible neutrophil depletion, were utilized. Tamoxifen administration effectively depleted > 99% neutrophils in the BM of Ly6G-DTA mice (Suppl. Figure 4A-C), in which osteoclastic activity was slightly increased (Suppl. Figure 4D). Following 3 weeks of CIS treatment and concurrent tamoxifen-induced neutrophil clearance, E0771 cells were transferred into the left ventricle, and metastasis was monitored for an additional 4 weeks (Fig. 3 L). Notably, bone metastasis was markedly reduced in Ly6G-DTA mice compared to ROSA26 DTA (wild-type, WT) controls (Fig. 3 M-N), and Ly6G-DTA mice exhibited significantly longer overall survival (Fig. 3 O). Histomorphometry of tibial paraffin sections consistently showed significantly smaller metastatic breast cancer lesions in Ly6G-DTA mice compared to WT mice (Fig. 3 P-Q). These findings suggest that neutrophils are a major BM immune cell population targeted by OPN, thereby contributing to the promotion of bone metastasis. Chronic stress stimulates excessive arginine metabolism in neutrophils To determine how CIS models the BM microenvironment via neutrophils, Ly6G + BM neutrophils were magnetically sorted and subjected to bulk mRNA sequencing (RNA-Seq). KEGG analysis identified "Arginine and proline metabolism" as the sole metabolism-related pathway among the top 10 enriched pathways (Fig. 4 A). Consistently, transcriptional levels of arginase-2 ( Arg2 ) and inducible nitric oxide synthase (iNOS, Nos2 ), key enzymes in arginine metabolism, were significantly higher in BM neutrophils from CIS-exposed mice compared to Ctrl mice, regardless of tumor burden (Fig. 4 B). Metabolic profiling further validated these findings. Principal component analysis (PCA) revealed strong internal consistency within CIS and Ctrl groups (Fig. 4 C). KEGG analysis of differentially abundant metabolites highlighted "D-arginine and D-ornithine metabolism" as the most significantly altered pathway, with "Arginine biosynthesis" and "Arginine and proline metabolism" also ranking among the top 10 metabolism-related pathways (Fig. 4 D). Among 1,135 identified metabolites, 5 were upregulated and 32 were downregulated (FC > 1.5 or FC < 0.67, p < 0.05) (Fig. 4 E). Notably, arginine and DL-arginine levels were significantly lower in neutrophils from CIS-exposed mice compared to Ctrl mice (Fig. 4 E-F). Additionally, arginine-containing di- and tri-peptides were reduced in CIS-exposed mice (Fig. 4 F), suggesting that enhanced arginine metabolism and consumption drive the depletion of arginine in the BM microenvironment. To determine whether neutrophils contribute to arginine consumption under CIS, arginine levels were measured in the interstitial fluid of CIS-exposed WT and Ly6G-DTA mice bearing breast tumors. Arginine levels were significantly lower in CIS WT mice compared to Ctrl WT mice, but this reduction was completely reversed in CIS-exposed Ly6G-DTA mice with specific neutrophil depletion (Fig. 4 G). To further investigate whether OPN promotes arginine consumption by neutrophils through upregulating Arg2 or Nos2 expression, BM cells from WT and Ly6G-DTA mice were cultured with vehicle, OPN plus vehicle or arginine. Subsequent qPCR analysis of sorted neutrophils revealed that OPN treatment significantly increased transcriptional levels of Nos2 and Arg1 (but not Tnfα , Tgfβ1 and Ifnγ ) in WT neutrophils compared to vehicle-treated controls (Fig. 4 H-I). Consistently, flow cytometry analysis revealed a significantly higher frequency of iNOS + neutrophils in OPN-treated WT BM cells (Fig. 4 J; Suppl. Figure 5A-B). Moreover, OPN treatment significantly reduced the frequencies of CD8 + cytotoxic T cells (Fig. 4 K) and IFNγ + CD8 + cytotoxic T cells (Fig. 4 L; Suppl. Figure 5C-D), and this reduction was effectively prevented by arginine supplementation or neutrophil depletion in Ly6G-DTA BM cells. These findings indicate that OPN stimulates arginine consumption by neutrophils, establishing a low-arginine BM microenvironment that suppresses CD8 + cytotoxic T cells. NE/ADRβ2 axis sustains OPN-expressing monocytes/macrophages and neutrophil arginine metabolism To investigate the mechanism by which NE induces OPN expression in monocytes/macrophages, transcription levels of NE receptors ( Adrβ1 , Adrβ2 and Adrβ3 ) in the BM cells from CIS-exposed and Ctrl mice were compared. Adrβ2 mRNA expression were significantly more abundant than other isoforms in both of CIS-exposed and Ctrl mice (Fig. 5 A), suggesting ADRβ2 as the dominant NE receptor in BM. In global ADRβ2 knockout (Adrβ2-KO) mice, the percentages of myeloid lineage cells, such as neutrophils, monocytes and macrophages, were comparable to those in WT mice (Suppl. Figure 6A-D). However, the percentage of OPN + BM cells in Adrβ2-KO mice was significantly lower than in WT littermates (Fig. 5 B), with marked reductions observed particularly in OPN + monocytes (Ly6C + ) and macrophages (F4/80 + ), the two major OPN-expressing cell populations in BM (Fig. 5 C). In addition, the percentages of total neutrophils and CD44 + neutrophils in the BM of Adrβ2-KO mice were comparable to WT mice, while the percentage of iNOS + neutrophils was significantly lower in Adrβ2-KO mice than WT mice (Fig. 5 D- 5 F). Consistently, BM arginine levels were higher in Adrβ2-KO mice (Fig. 5 G), reflecting reduced neutrophil-mediated arginine consumption. Immune cell profiling revealed that Adrβ2-KO mice exhibited significantly higher percentage of CD8 + cytotoxic and CD4 + T cells in BM (Fig. 5 H, 5 K-L), with no significant differences observed in CD11b + leukocytes or B220 + B cells, compared to WT mice (Fig. 5 H-J). Transwell cell migration assay confirmed that ADRβ2 mediates NE-dependent tumor cell attraction: NE-treated WT BM cells attracted more E0771 cells than vehicle-treated controls, but this effect was abolished in NE-treated Adrβ2-KO BM cells (Fig. 5 M-N). Furthermore, NE-induced Opn transcription in WT BM cells was largely blocked by propranolol, an FDA-approved non-selective adrenergic β receptor antagonist (Fig. 5 O). These findings collectively indicate that ADRβ2 knockout or blockade inhibits OPN expression in BM cells, thereby disrupting the NE-driven cascade that promotes breast cancer cell migration. Bone-targeted delivery of propranolol inhibits chronic stress-induced bone metastasis Above results suggest that blocking ADRβ2 in bone microenvironment represents a potential therapeutic strategy for preventing CIS-induced metastasis. To investigate this, we endeavored to synthesize a bone-targeting propranolol through generating an a lendronate- p ropranolol (AP) conjugate, as illustrated in Fig. 6 A. Assessment of the toxicity and bone-targeting efficiency of AP confirmed that it exhibited high bone-specific accumulation (Suppl. Figure 7A-D) and no overt liver and kidney toxicity (Suppl. Figure 7E). To assess the therapeutic efficiency of AP, CIS-exposed, tumor-bearing mice with intratibial injection of E0771 cells were treated with vehicle and AP twice weekly throughout the entire CIS-exposure period (Fig. 6 B). Notably, the percentage of OPN + cells in the BM from AP-treated CIS-exposed mice was decreased in a dose-dependent pattern (Fig. 6 C). To compare the therapeutic potential of AP with propranolol and alendronate, CIS-exposed, tumor-bearing mice were treated with AP at an optimized dose of 9.05 mg/kg body weight (Fig. 6 C; Suppl. Figure 7E), or with equimolar concentrations of β-cyclodextrin (β-CD), propranolol, or alendronate as controls. Mice treated with alendronate or AP exhibited increased trabecular bone mass and a reduction in osteoclast number and surface compared to other two groups (Suppl. Figure 7F-G). In addition, the survival rate of AP-treated mice was significantly higher than that of CIS-exposed mice treated with vehicle, propranolol alone, or alendronate alone (Fig. 6 D). Luciferin flux, a quantifiable indicator of metastasis burden, was significantly lower in the tibiae of AP-treated mice compared to vehicle- or propranolol-treated controls (Fig. 6 E-F). AP treatment also effectively suppressed tumor growth for up to 5 weeks post-intratibial injection of E0771 cells (Fig. 6 G). Histomorphometry analysis revealed markedly smaller metastatic breast cancer lesions in the tibiae of AP-treated mice compared to vehicle- and propranolol-treated mice (Fig. 6 H-I). Immunophenotyping of the BM revealed that AP-treated CIS-exposed, tumor-bearing mice exhibited significantly lower frequencies of OPN + monocytes (Fig. 6 J), OPN + macrophages (Fig. 6 K) and iNOS + neutrophils (Fig. 6 M) in the BM compared to vehicle- and propranolol-treated mice. No significant differences were observed in the frequencies of total monocytes, macrophages, neutrophils or CD44 + neutrophils (Fig. 6 L; Suppl. Figure 8A-D). Consistent with these findings, BM arginine levels were elevated in AP-treated mice (Fig. 6 N), in which the frequency of CD8 + T cells in the BM was significantly increased compared to vehicle-treated controls (Fig. 6 O). Dendritic cells and B lymphocyte populations did not show a similar increase (Suppl. Figure 8F). In summary, the bone-targeted AP delivery effectively inhibited CIS-induced breast cancer bone metastasis. This effect was associated with reduced frequencies of OPN + monotypes/macrophages and iNOS + neutrophils, decreased arginine consumption, and a restored cytotoxic CD8 + T cell population. Discussion Chronic stress, a pervasive factor in the lives of cancer patients, is increasingly recognized as a significant driver of tumor progression and distant metastasis, notably in bone-metastasis-prone cancers like breast cancer. The BM microenvironment, in particular, may offer a protective niche for disseminated cancer cells, potentially contributing to increased resistance to immune checkpoint inhibitors. Therefore, a comprehensive understanding of the mechanisms by which chronic stress promotes cancer bone metastasis and the development of targeted therapeutic strategies to counteract this process are imperative. This study unveils a novel neuro-immune-metabolic axis, by which chronic immobilization stress reshapes the BM niche, creating a low-arginine, pro-metastatic environment that impairs CD8 + cytotoxic T cells and ultimately promotes breast cancer bone metastasis. Mechanistically, OPN emerges as a central mediator in this process. Elevated environmental NE, a consequence of chronic stress, stimulates OPN expression in BM monocytes/macrophages through ADRβ2 signaling. Subsequently, this increased OPN binds to its receptor, CD44, on neutrophils, which in turn upregulates NOS2 expression and accelerates arginine metabolism within these cells. This excessive arginine consumption by neutrophils and an exhaustion of CD8 + cytotoxic T cells consequently establishes a low-arginine, pro-metastatic BM microenvironment (Fig. 7 ). This novel neuro-immune-metabolic axis was further validated using neutrophil-specific depletion or global Adrβ2 knockout mouse models. To therapeutically prevent this NE-ADRβ-initiated cascade, we developed alendronate-conjugated propranolol (AP), a bone-targeting ADRβ antagonist. AP administration effectively restored BM arginine levels and prevented CIS-induced breast cancer bone metastasis (Fig. 7 ). Taken together, our findings not only offer critical insights into how chronic stress reshapes the BM microenvironment through neural-immune interactions but also propose a promising therapeutic strategy for mitigating this cascade in bone metastasis. Cancer patients frequently experience profound psychological stress and sympathetic nerve system (SNS) activation, a response exacerbated by factors such as diagnosis, treatment implications, and disease progression. Tumors themselves can also initiate stress responses, activating anxiety-related circuits and further stimulating SNS 33 . Elevated SNS activity, mediated by catecholamines like NE and epinephrine (EPI), profoundly alters the tumor microenvironment, potentially enhancing primary tumor cell proliferation, survival, migration, invasion 34 , and neoangiogenesis 35 , 36 . Moreover, SNS activation significantly modulates the tumor immune microenvironment, notably by suppressing anti-tumor immune responses through catecholamine release 34 , 37 . The bidirectional interaction between the tumor and SNS establishes a positive-feedback loop that accelerates cancer progression and metastasis. Intervening this loop could involve either suppressing the intrinsic invasive capacity of primary tumor cells or preventing the formation of pro-metastatic niche in distant organs. However, the relative contribution of these mechanisms to distant metastasis remains a critical area of investigation. Our orthotopic breast cancer model revealed that CIS significantly promoted primary tumor growth and enhanced breast cancer cell metastasis to bone, liver and lung. Crucially, in vitro analyses demonstrated that cancer cells isolated from primary tumors in CIS-exposed mice exhibited comparable metastatic potential to those from Ctrl mice upon secondary transplantation. These findings collectively indicate that CIS primarily drives remote metastasis by remodeling pro-metastatic niches in distant organs, such as bone, via peripheral SNS activation or other stress-related factors, rather than by increasing the intrinsic invasive capacity of tumor cells. Persistent elevation of catecholamines, such as NE and EPI, due to chronic stress exerts several detrimental effects on the immune system. This includes impairing the proliferation and function of T and B lymphocytes, which compromises T-cell-mediated immunity and antibody responses 38 . Elevated catecholamine disrupts the normal function of hematopoietic stem and progenitor cells (HSPCs) and natural killer (NK) cells, also drives the production of pro-inflammatory cytokines like IL-6 and IL-1β, which contributes to the pathogenesis of neurodegenerative and neuroinflammatory diseases 39 , 40 . In our study, we observed that CIS-exposed mice exhibited a significantly higher proportion of myeloid lineage cells (including monocytes and neutrophils) and a significantly lower proportion of lymphoid lineage cells (including CD8 + cytotoxic T cells and B cells) in the BM compared to Ctrl mice. These findings indicate that CIS exposure increases the neutrophil-to-lymphocyte ratio in the BM, which may contribute to chronic stress-induced bone metastasis. Our study identified OPN as a key mediator of bone metastasis associated with alterations in the BM immune system. OPN is a multifunctional glycoprotein known to promote cancer progression and therapy resistance via multiple mechanisms 41 , 42 . These include enhanced angiogenesis, increased cancer cell survival, proliferation, invasion, and migration, recruitment of tumor-associated macrophages (TAMs), and PD-L1 upregulation to suppress anti-tumor T cell activation 43 , 44 . OPN has been reported to stimulate osteoclast differentiation, and it is also produced by osteoclasts to reprogram the extraosseous tumor microenvironment 45 . While the role of OPN in primary tumor progression and invasion is well-documented, its function in forming distant pro-metastatic niches is less understood. In contrast to prior research, our results identified BM monocytes and macrophages, not cancer cells, as the primary source of OPN in the BM of metastatic bones. Given the scarcity of breast cancer cells anchored in the BM during early metastasis, cancer cells are unlikely to be the major source of OPN for establishing a pro-metastatic niche. The limited presence of TRAP + osteoclasts around breast cancer lesions in metastatic bones, suggesting osteoclasts might play a limited role in mediating CIS-induced bone metastasis. We demonstrate that chronic stress induces OPN upregulation specifically in BM monocytes and macrophages. This upregulation is mediated by NE activation of Adrβ2 isoform, as deletion of Adrβ2 resulted in significantly fewer OPN + monocytes/macrophages, and BM cells from Adrβ2 -KO mice failed to mediate tumor cell attraction in response to NE. Innate and adaptive immunity are both essential for the body’s defense against cancer progression, with their interplay being crucial for effective anti-tumor response. For example, tumor-activated dendritic cells present tumor antigens to T cells— a process vital for initiating anti-tumor immunity. A key question is how chronic stress disrupts this interplay between innate and adaptive immunity. Neutrophils, a distinctive type of innate immune cells, possess several defining features: they are the most abundant type of leukocytes, they rapidly response to physiological perturbations like infection, they have a short half-life ranging from hours to days, and they defend against pathogens through mechanisms such as neutrophil extracellular trap (NET) formation. Neutrophils also play a complex role in cancer. They can also release NETs to trap circulating cancer cells and promote their adhesion to distant organs 46 . Conversely, neutrophils may generate reactive oxidative species (ROS) and granule proteins like elastase and MPO to kill cancer cells 47 . While the role of neutrophils in stress-induced metastasis remained largely unknown until recently, when it was reported that glucocorticoids released during chronic stress induce NET formation, establishing a lung metastasis-promoting microenvironment in breast cancer-bearing mouse models 17 . In our study, we first demonstrated that inducible depletion of Ly6G + neutrophils effectively prevented bone metastasis in breast cancer-bearing mice. Through metabolomics analysis and bulk mRNA sequencing, we further found that chronic stress significantly enhanced arginine metabolism in neutrophils. Notably, over 90% neutrophils express CD44 on their surface, suggesting an intimate interplay between CD44 + neutrophils and OPN + monocytes/macrophages, though the mechanisms by which OPN promotes arginine metabolism in neutrophil requires further investigation. Clinical evidence indicates that lower plasma arginine levels correlate with higher tumor burden and poor prognosis 48 . T cells are vulnerable to a low-arginine microenvironment due to their low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC) 49 . In our model, excessive arginine consumption by neutrophils drives a low-arginine BM microenvironment in CIS-exposed mice, and this effect was effectively reversed in Ly6G-DTA mice with a specific depletion of neutrophils. This reduction in BM arginine is associated with a significant decrease in CD8 + cytotoxic T cells, likely because arginine is essential for T cell activation, survival and proliferation, and its deficiency induces T cell anergy 50 . We also observed that the genes Arg2 and Nos2 (but not Arg1 ) were significantly upregulated in BM neutrophils from CIS-exposed mice, regardless of tumor burden. Our findings, therefore, address how chronic stress disrupts the interplay between innate and adaptive immunity. Chronic stress activates a cascade involving innate immunity cells—specifically OPN + monocytes/macrophages and CD44 + neutrophils—leading to the generation of a low-arginine BM microenvironment. This microenvironment subsequently suppresses CD8 + cytotoxic T cells, disrupting the effective anti-tumor immune response. The conjugation of therapeutic agents to bisphosphonates (BPs) is a highly effective strategy for achieving selective drug accumulation in bone 51 . Due to their stable P-C-P backbone and strong affinity for hydroxyapatite in bone, bisphosphonate-drug conjugates have been extensively explored for applications in chemotherapy, radiotherapy, immune modulation, and nanoparticle delivery for the treatment of bone-related conditions, including osteoporosis and bone cancers (primary and metastatic tumors) 51 . Propranolol, a β-adrenergic receptor blocker, is widely used to treat various cardiovascular and neurological conditions by blocking β1 and β2 adrenergic receptors. In our study, we observed that Adrβ2 transcription levels were higher than those of the other two isotypes ( Adrβ1 and Adrβ3 ). Global Adrβ2 knockout mice had fewer OPN-expressing monocytes/macrophages and iNOS-expressing neutrophils, along with higher BM arginine levels, compared to WT mice. Propranolol was found to effectively prevented NE-induced OPN expression, supporting its potential to inhibit the OPN/arginine metabolism axis and CIS-induced bone metastasis. To enable bone-specific delivery of propranolol, we conjugated it to alendronate via a urethane (carbamate) bond to generate the conjugate AP. We hypothesized that this conjugate would be cleaved in the acidic BM microenvironment, achieving bone-specific delivery of propranolol. Consistent with our hypothesis, CIS-exposed mice treated with AP exhibited significantly fewer OPN-expressing monocytes/macrophages and iNOS-expressing neutrophils, higher BM arginine levels, and an increase in CD8 + cytotoxic T cells, compared to vehicle-treated, CIS-exposed mice. Notably, bone metastatic lesions were significantly smaller in AP-treated, CIS-exposed mice. Basing on these findings, we propose that AP could effectively block the CIS-induced NE/ADRβ axis activation, thereby inhibiting bone metastasis not only in breast cancer but also in other cancers prone to bone metastasis under chronic stress conditions. This study acknowledges certain limitations. Direct evidence for the causal role of OPN + monocytes and macrophages in chronic stress-induced bone metastasis would benefit from a conditional knockout mouse model with inducible Opn deletion in the monocyte/macrophage lineage (e.g., LyzM CreER Opn fl/fl mice). Additionally, given the systemic distribution of sympathetic nerves and neutrophils, the mechanisms identified here for bone metastasis may also contribute to stress-induced metastasis in other organs or in other bone-metastasis-prone cancers (e.g., lung and prostate cancers), warranting further investigation beyond the scope of the current study. Methods Experimental animals Eight- to ten-week-old BALB/c and C57BL/6J female mice were purchased from Beijing Huafukang Biotechnology Co., LTD. and Beijing Charles River Laboratories (CRL), respectively. Mice were kept in an SPF barrier animal center. Except under special experimental conditions, mice had free access to sterilized food and water. Ly6G CreER ROSA26 DTA (Ly6G-DTA) mice were generated by crossing Ly6G CreER mice with ROSA26 DTA mice. Four-month-old Ly6G-DTA female mice and wildtype (Ly6G CreER ) control female mice were restricted for 3 weeks and then implanted with tumor cells. To deplete Ly6G-expressing neutrophils, tamoxifen (100 mg/Kg body weight) in corn oil were intraperitoneally injected into Ly6G-DTA mice and, 3 doses per week for another 4 weeks. Subsequently, the mice were sacrificed and the efficiency and specificity of Ly6G-expressing neutrophil depletion was confirmed. During the period of observation, the survival curve was recorded and the growth of metastatic tumors were analyzed. Eight-week-old C57BL/6J female mice were purchased from Charles River Laboratories. Adrenal β2 and β3 receptor knockout (Adrb2- and Adrb3-KO) mice were purchased from Shanghai Model Organisms Center, Inc. Mice were raised in a SPF-level barrier animal center. Except under special experimental conditions, mice had free access to food and water, which were sterilized. The feeding environment conditions: 12-hour light-dark cycle, ambient temperature of 22 °C -26 °C, and relative humidity of 50%-60%. The bedding was changed twice a week, and the cages, water bottles and bedding were sterilized by high temperature and high pressure. All animal experiments were carried out according to the protocol approved by the Experimental Animal Ethics Committee of Hebei Medical University. Reagents The breast cancer cell lines used in this experiment were 4T1-Luciferase, derived from BALB/c mice, and E0771-Luciferase, derived from C57BL/6J mice. Both cell lines were purchased from Yuanjing Biotechnology Co., Ltd. (Wuhan, China) and cryopreserved in liquid nitrogen storage upon receipt and initial amplification. Fluorophore-conjugated primary antibodies were used to detect cell-surface antigens. The following antibodies were purchased from eBioscience (San Diego, CA, USA): FITC-conjugated anti-mouse CD4 (Catalog #11-0041-82), Violet 421-conjugated anti-mouse B220 (Catalog #62-0452-82), Violet 650-conjugated anti-mouse CD41a (Catalog #64-0411-82), PE-conjugated anti-mouse CD11b (Catalog #12-0012-82). The following antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA): PE/CF594-conjugated anti-mouse IgM (Catalog #562565), PerCP/Cy5.5-conjugated anti-mouse CD44 (Catalog #560570), Violet 510-conjugated anti-mouse CD8 (Catalog #740155), Violet 605-conjugated anti-mouse Ly6C (Catalog #563011), Violet 786-conjugated anti-mouse CD11c (Catalog #568973), APC-conjugated anti-mouse Ly6G (Catalog #560599), APC/R700-conjugated anti-mouse F4/80 (Catalog #565787), APC/Cy7-conjugated anti-mouse Ter119 (Catalog #560509). Ly6G-Biotin antibody (Catalog #127603) was also purchased from eBioscience (San Diego, CA, USA). Streptavidin microbeads (Catalog #130-048-101) for cell sorting were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). For flow cytometry analysis, bone marrow cells were stained using the fluorophore-conjugated antibodies listed above. Antibody solutions were prepared at a 1:100 dilution in FACS buffer (Phosphate-Buffered Saline (PBS) containing 2% fetal bovine serum). The staining was performed on ice and in the dark, with antibody solutions prepared and used immediately. The primary antibody for Osteopontin (OPN) (Catalog #AF808) was purchased from R&D Systems (Minneapolis, MN, USA), and the FITC-conjugated secondary antibody (Catalog #ab150129) was acquired from Abcam (Cambridge, UK). Mouse L-Arginine (L-Arg) ELISA Kit (Catalog #FT-P9S1390X) for the measurement of arginine levels in bone marrow supernatant was purchased from Fantaibio (Shanghai, China). Solutions Preparation of sugar water preference experiment for mouse behavior experiment: weigh 10 g of sucrose and dissolve it in 1 L of mouse drinking water. Shake it well to fully dissolve it, and get 1% sucrose drinking water. Preparation of matrix gel solution for mouse breast cancer orthotopic tumor implantation: the ABW high-concentration matrix gel used in the construction of mouse orthotopic breast cancer model is stored in a refrigerator at -20 °C. Take it out and put it in an ice box the day before use, and put the ice box in a refrigerator at 4 °C to thaw overnight. The matrix gel is in liquid state on the day of use. Please note that it should be operated on ice during use. When the temperature exceeds 10 °C, the matrix gel will solidify. Preparation of mouse breast cancer tumor tissue digestion solution: prepare 100× tissue digestion solution stock solution: 100 mg/ml collagenase IV, 20 mg/ml DNase I, and then dilute 100 times with RPMI-1640 medium with 5% fetal bovine serum to make the working concentration 1 mg/ml collagenase IV and 200 μg/ml DNase I. Preheat the 1× digestion solution in a 37 °C water bath for use. Preparation of mouse bone tissue decalcification solution: first, dissolve 100 g EDTA powder in 800 ml PBS solution, and use a magnetic stirrer to assist dissolution. At this time, the EDTA powder cannot be completely dissolved. Add sodium hydroxide powder to the solution in small amounts and multiple times, and measure the pH value of the solution at the same time. When the pH value is adjusted to 7.2, the EDTA powder is completely dissolved. Finally, use PBS solution to make the volume 1 L. Store at room temperature for use. Construction of mouse restraint model and behavioral tests Put the mouse into the barrel of a 50 ml syringe and insert the needle core to fix it at the 50 ml mark. Before the experiment, drill about 20 small holes with a diameter of 5 μm on the side wall of the syringe so that the mouse can fully ventilate in the syringe. The mouse can move freely in this space but cannot run or jump. The mice were deprived of food and water during the restraint process. The mice in the control group were also deprived of food and water during the same period, but were not restrained. CIS model: restraint for 2 hours at a fixed time every day, restrain for 6 consecutive days a week, and not restrain on the 7th day. Restrain for 6 consecutive weeks to complete the construction of a complete model. After the entire restraint period, the mice were subjected to behavioral tests. RS model: restraint for 6 hours at a fixed time period every day, and a complete model is constructed for 8 consecutive days. After the restraint period is over, the mice are subjected to behavioral tests. The experimental steps of the behavioral test are briefly described as follows: Sucrose preference test (SPT): The first 24 hours are the experimental adaptation phase. Two water bottles are filled with ordinary drinking water and 1% sucrose drinking water respectively. The initial weight of the two bottles of water is recorded before placement. The positions of the two water bottles are changed at the 12th hour (to avoid differences in drinking sugar water due to horizontal position preference), and the weight of the remaining water in the two bottles is finally recorded after 24 hours. After the adaptation phase, the formal experimental phase of 24 hours begins. The positions of the water bottles are also changed once every 12 hours in the middle. The weight of the initial and 24 hours after the water bottles are recorded respectively, and then the amount of water consumed by the mice is calculated according to the formula. Finally, the data of the formal experimental phase are used as the results for statistics. Sugar water preference rate calculation formula: sugar water intake / (sugar water intake + ordinary drinking water intake) × 100%. Tail suspension test (TST): 2 h before the experiment, transfer mice from the animal breeding room to the behavioral testing laboratory to allow them to adapt to the environment in advance. Before starting the experiment, adjust the equipment such as the hanging rod, camera, and recording system software. Use medical tape to fix the mouse tail on the hanging rod so that its head hangs down, and the fixed position is about 1 cm away from the tip of the mouse tail. The tail suspension time of each mouse is recorded for 6 min, and the immobility time of the mouse in the last 4 min is counted. Forced swimming test (FST): Prepare a transparent swimming bucket with a diameter of 10 cm and a height of 25 cm, and inject 22 °C-25 °C sterilized water into the bucket so that the water surface reaches 15 cm. 2 h before the formal start of the experiment, transfer mice from the animal breeding room to the behavioral testing laboratory to allow them to adapt to the environment in advance. During the experiment, gently put the mouse into the water and start recording the image and time. Each mouse swims for a total of 6 min, and the immobility time of the mouse in the water in the last 4 min is counted. ELISA assay for norepinephrine (NE) After mouse sacrifice, the leg bones of the left hind limb including tibiae and femora from the CIS-exposed mice, the denervation mice and their respective control mice, were harvested and the muscle tissue were cleaned off from the bones. Remove off the knee joint cartilage from the femora and tibiae, and flush the bone marrow cells out from a tibia and a femur from an individual mouse using 300 μl of pre-cooled PBS in 1 ml syringe. Peptide up and down the bone marrow cell clots for several times, and mildly push the cells through a 25G needle to make single-cell suspension. The cells were next centrifuged at 4 °C and 3000 rpm for 5 min. The supernatant obtained was the bone marrow interstitial fluid. According to the instructions of the ELISA kit, the bone marrow interstitial fluid sample was diluted by 2.5 times for final measurement. For example, 40 μl bone marrow interstitial fluid was mixed with 60 μl sample diluent provided by the ELISA kit for further assay. The assay procedure was strictly carried out according to the manufactory instructions of the kit. In brief, we first extracted and acetylated NE in the samples and the standard. Add the standard and diluted samples to a specific well plate, add TE buffer, cover with film, and shake on a shaker (20 °C to 25 °C, 200 rpm) for 60 min. After washing, add 100 μl Acylation buffer and 25 μl Acylation reagent to each well, shake the plate and then add 100 ul HCl and shake the plate for 10 min. To perform the enzyme catalysis, mixed 90 μl acetylated NE solution above with 25 μl enzyme solution, and incubated at 37 °C for 2 hours. After this incubation, we next added 100 μl of this incubated liquid into designed plates and then added 50 μl NE antiserum, incubated at 4 °C overnight. After washing, add 100 μl Enzyme conjugate, cover the plate with a membrane and shake the plate for 30 min. After washing, add 100 μl substrate and shake the plate in the dark for 20-30 min, then add 100 μl stop solution. Within 10 min after the reaction, use a microplate reader to measure the absorbance (optical density, OD) value of each well at 450 nm and 620 nm. Draw a standard curve based on the OD value of the standard, according to which, the concentrations of NE in each sample were calculated. Duplicate wells were set up for the all standards and samples. Breast cancer cell culture and in situ model 4T1- and E0771-Luciferase cells were cultured in RPMI-1640 medium and DMEM high-glucose medium (containing 10% FBS and 1% penicillin-streptomycin), respectively. These cells were seeded in T75 culture flasks and maintained in 37 °C incubator with 5% CO2, and 70% humidity. To confirmed the expression and activity of luciferase in 4T1- and E0771-Luciferase cells, these cells were counted and seeded in a 96-well cell culture plate at 5×10 4 , 2.5×10 4 , 1.2×10 4 , 0.6×10 4 , 0.3×10 4 cells per well, and the wells with no cell seeded as blank control. Four replicates were set up for each cell concentration. After the cells were plated, they were placed in an incubator for 2-3 hours to allow the cells to adhere to the wall. Thereafter, 10 μl of substrate potassium luciferin (15 mg/ml) was added to each well and incubated for 2 minutes before imaging using the IVIS imaging system under the cell shooting mode. The luminescence values of individual cells were calculated. It is generally believed that cells with a luminescence value of >175 photons/s/cell can be used for in vivo transplanted tumor experiments. 4T1- and E0771-Luciferase cell lines were seeded in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin in T75 culture flasks under conditions of 37 °C, 5% CO2, and 70% humidity. 4T1 cells in the exponential growth phase as they reached 70%-80% of coverage, were rinsed with 4 ml PBS once and then digested using 2 ml 0.25% trypsin solution. When 4T1 and E0771 cells are digested in culture incubator for 2-3 minutes in trypsin, 4 ml complete culture medium was added to terminate the digestion, collected 4T1 and E0771 cells in 15 ml centrifuge tube, and span down in a centrifuge at 1500 rpm for 5 min. The collected tumor cells were next counted and resuspended evenly in appropriate amount of liquid ABW high-concentration matrix gel on ice. The procedure of an in-situ breast cancer model construction presented as following: a small animal gas anesthesia machine to inhale isoflurane through the mask of the mouse for gas anesthesia after 2 weeks of chronic restraint treatment. After the mouse enters deep anesthesia, fix it in a supine position on the operating table, and remove the hair and disinfect it with iodine on the skin of the fourth breast area on the left side. Use a micro-syringe (range 50 μl) to draw 20 μl of matrix gel cell suspension containing 5×10 5 cells and slowly inject it into the fat pad of the fourth mammary gland on the left side of the mouse. After the operation, the mice were continually maintained in the SPF mouse feeding room and imaged using in-vivo IVIS system to observe the growth of the in-situ tumor and the metastasis to distant organs every week. At the same time, the tumor volume was measured using a vernier caliper and recorded every week. To the estimate the volume of tumor, the longest diameter of the tumor as the long diameter was calculated, and the short diameter perpendicular to the long diameter of the tumor was measured as well, and the tumor volume was calculated according to the formula: tumor volume = 1/2 × long diameter × short diameter 2 . The tumor growth was observed after tumor cell in-situ implantation for 4-5 weeks when the size of in-situ tumors reached 1 cm 3 , and the mice were sacrificed and the required tumor samples were obtained. To observe the tumor cell bone metastasis, 100 μl of cell suspension containing 2×10 5 cells and slowly inject it into the left ventricle of recipient mice. When bright red arterial blood is observed to rush into the syringe in a pulsed manner, it proves that the needle tip has entered the left ventricle, then stop inserting the needle and slowly inject the tumor cell suspension. The recipient mice were continually maintained in the SPF mouse feeding room and imaged using in-vivo IVIS system to observe the metastasis to bones and other distant organs weekly for 3 weeks in total. Assay of cell migration using Transwell system Bone marrow cell preparation: Five-month-old Adrb2 global knockout (KO) female mice and wildtype female littermates were sacrificed and the leg bons from these mice were moved off. Maximally cleaned the excess skin and muscle tissue and flushed out the bone marrow cells with pre-cooled PBS, lysed the red blood cells, and counted the bone marrow cells. The Adrb2 -KO or WT bone marrow cells were next suspended in three different culture mediums as following: 1) Control medium: RPMI-1640 medium containing 1% penicillin-streptomycin, 1% NEAA and 20 ng/ml M-CSF; 2) NE medium: Control medium containing extra 50 μM NE; 3) NE+anti-CD44 medium: Control medium containing extra 50 μM NE and 10 μg/ml anti-CD44 antibody. 5×10 5 bone marrow cells in 600 μl different culture medium were next evenly seeded in each well of 24-well plate, and duplicate wells were set up for each condition. Tumor cell preparation: As the growth density of E0771-Luc cells reached about 70%-80%, the cells were digested with 2 ml 0.25% trypsin solution per culture flask at 37 °C incubator for 3 minutes, and then 4 ml complete culture medium (containing 10% FBS) was added to terminate the digestion. The cells were span down, counted, and resuspended in DMEM culture medium. Subsequently, gently put the transwell chambers into the 24-well-plate wells seeded with bone marrow cells, and add 3×10 4 E0771-Luc cells in 200 μl culture medium to each chamber, and put the Transwell culture system into the incubator (37 °C, 5% CO2, and 70% humidity) for 24 hours. Plate staining and observation: As the incubation completed, discarded the culture medium, rinsed the chambers and bottom wells with PBS for two times, fixed the cells with 4% PFA at room temperature for 10 minutes, and discarded the fixation solution and rinsed with PBS for 3 times. Subsequently, add 100 μl 0.1% crystal violet stain into each the upper chamber and incubate at room temperature for 10 minutes, then discarded the staining solution and rinsed the chambers with ddH 2 O for 3 times. The cells on the bottom surface of the chamber were gently wiped clean with cotton swabs, and the E0771-Luc cells that migrated to the outer layer of the chamber were observed under an inverted microscope and photographed for further statistical analysis. Secondary cardiac transplantation of orthotopic tumor cells The orthotopic tumor lesions were aseptically stripped and excised, removed of non-tumor cell tissues such as necrotic sites and fascia, and then approximate 5 mm 3 tumor tissues were removed from the orthotopic tumor lesions of each mouse. The tumor tissues were dipped in 0.5 ml RPMI-1640 culture medium in 1.5 ml centrifuge tube and cut into tissue slurry on ice as much as possible with sterile ophthalmic scissors. The tissue slurry was transferred to the pre-prepared tissue digestion solution in 15 ml tubes and placed in an incubator at 37 °C for enzymatic digestion, and vortexed every 10 minutes for 3 times to sufficient isolation of tumor cells. To terminate enzymatic digestion, the tubes containing tumor cells were added 5 ml RPMI-1640 medium containing 10% FBS, mixed well and stored on ice. The isolated tumor cells were next filtered using a 70 μm cell strainer and washed with pre-cooled RPMI-1640 basic medium for 2 times. The isolated tumor cells were span down in a centrifuge at 4 °C, 1500 rpm for 5 min, counted and taken the required cells and placed on ice for later intracardiac injection. To anesthetize recipient mice, the isoflurane gas was pumped and constantly supplied to an inhale mouse mask using a small animal gas anesthesia machine from RWD. After the mouse enters deep anesthesia, fix it on the operating table in a supine position, and remove the hair and disinfect the skin of the precardiac area with iodine. Use an insulin syringe (range 1 ml) to draw 100 μl of cell suspension containing 2×10 5 cells and slowly inject it into the left ventricle. When bright red arterial blood is observed to rush into the syringe in a pulsed manner, it proves that the needle tip has entered the left ventricle, then stop inserting the needle and slowly inject the tumor cell suspension. The recipient mice were continually maintained in the SPF mouse feeding room and imaged using in-vivo IVIS system to observe the metastasis to bones and other distant organs weekly for 3 weeks in total. Monitoring tumor growth using in-vivo imaging system (IVIS) Before the IVIS test, 15 mg/ml potassium luciferin solution should be made by dissolving potassium luciferin powder in sterile PBS and filtering with a 0.2 μm filter. This solution could be aliquoted and stored in dark at -80 °C, and thaw it in dark at RT before use. In terms of imaging by IVIS, injected potassium luciferin solution into the mouse intraperitoneally at a dose of 100 μl/10 g body weight, and wait for 15 min until the luminescence value entering the plateau phase, and the mice were imaged using IVIS. During this process, the mice are anesthetized by inhalation of isoflurane in the induction box and then continuously inhaled by mask. After in-vivo imaging, the results are quantitatively analyzed using Living Image software. Briefly, the in-situ lesions or bone metastasis lesions of each mouse were circled first, then a unified quantitative analysis of the luminescence value was performed. The in-situ lesions were located at the fourth mammary gland on the right side of the mouse. The most common bone metastasis lesions were the proximal end of the tibia and the distal end of the femur. Therefore, the fluorescence around the knee joint was determined to be bone metastasis. Histomorphometry analysis After the mice being sacrificed, mouse tissues, such as tibia, femur, lung and liver, were harvested immediately and placed in sufficient 4% paraformaldehyde (4% PFA) fixative solution at 4 °C for 48 hours. After fixation, the tissues were rinsed 3 times with PBS solution to clean off the fixative. The soft tissues including the mouse lung and liver were dehydrated overnight using the soft tissue dehydration program of the dehydrator, and then embedded in paraffin blocks for further section. After fixation, the leg bones were then proceeded to decalcification using 10% EDTA solution at 4 °C for 2-3 weeks, and this solution were changed with fresh decalcification solution weekly. After decalcification, the leg bones were gently rinsed 3 times with PBS solution and dehydrated following a hard tissue dehydration program, and then embedded in paraffin blocks for further section. All paraffin blocks were sectioned with a thickness of 4 μm using Minux S700 slicing machine from RWD. Before staining, the paraffin slices were baked in an oven at 65 °C for 30 minutes to fully melt the paraffin on the glass slices. For H&E staining, the procedure as following: Briefly, the sections were soaked in xylene for 4 minutes × 3 times, then in 100% ethanol, 95% ethanol, 90% ethanol, 85% ethanol, and 75% ethanol in order for 2 minutes for each, respectively. The sections were next rinsed gently in tap water for 2 min, stained in hematoxylin staining solution for 30-60 s (adjust the staining time according to the degree of staining of the cell nucleus), rinsed in tap water for 5 minutes, then soaked in ammonium hydroxide solution for 15 seconds and washed with deionized water for 2 minutes. The sections were next stained in eosin staining solution for about 30 seconds, decolorize with 95% ethanol for 3 minutes, with 100% ethanol for 1 minute × 2 times, and soaked in xylene for 4 minutes × 3 times, and finally sealed with permount mounting medium. Immunofluorescence staining Tibial samples of CIS model and control mice were fixed with 4% PFA for 48 hours, and transferred into 10% EDTA solution at 4 °C for decalcification for 21 days. After decalcification, the samples were dehydrated in a tissue dehydrator, embedded in paraffin and sectioned with a thickness of 4 μm using a rotary paraffin microtome from RWD (Shenzhen, China). Before staining, the paraffin sections were baked at 65 °C for 30 minutes, rehydrated with xylene and gradient alcohol step by step. The sections were slowly rinsed with running water for 5 minutes and washed three times with ddH2O to check whether the dewaxing was complete. Antigen retrieval: Set the water bath to 98 °C, add 200 ml Tris-EDTA retrieval solution (pH=8.0) to a slide staining box, gently place the dewaxed slides into the staining box, and dipped the box in the water bath. As the temperature of retrieval solution in the staining box reached 90 °C, start to count for 10 minutes, thereafter lifted the staining box and dipped into running tap water to cool down gradually to room temperature. Washing the slides with PBS for three times, drew a wax circle on the slice using a wax circle pen, incubated tissue on slides with 0.3% Triton-PBS for 15 minutes to punch the cell membrane. Added 20% donkey serum in 0.3% Triton-PBS to block at room temperature for 1 hour. After 3 washes with PBS, added OPN primary antibody solution (AF808; R&D system, 1:100 diluted), and incubate overnight at 4 °C in a wet box. On the second day, the slides in the wet box were continually incubated at room temperature for 30 minutes, and washed with 0.5% PBST for three times, 5 minutes/time. Subsequently, FITC-labeled donkey anti-Goat secondary antibody (A16000; Novex by life technologies, 1:100 diluted) was added and incubated at room temperature for 1 hour. After three washes with 0.5% PBST, the slides were sealed with mounting medium with DAPI (H1200-10; Vector laboratories) and kept in dark at 4 °C, and scanned them for further histomorphometry analysis. ELISA assay for osteocalcin (OCN) and tartrate-resistant acid phosphatase 5b (TRAP5b) To prepare mouse serum, mouse peripheral blood was collected into 1.5 ml EP tubes and stand at room temperature over 4 hours until it coagulated naturally. The serum, light yellow supernatant liquid, was slowly aspirated and moved to a new EP tube and stored in a -80°C refrigerator for long-term storage until it is used for testing. Before ELISA tests, thaw the serum sample on ice and warm up the required reagents in the ELISA kit to room temperature. After sufficient vortex, 70 μl of serum samples were mixed with 140 μl sample diluent, and add 100 μl of this diluted serum samples to each well of antibody pre-coated ELISA plates. Standard preparation: centrifuged the powder in Standard tube at 12000 rpm for 1 minute, added 1 ml of standard diluent, vortexed gently and let it stand for 10 minutes. To draw standard curve, dilute 50 ng/ml standard to 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.13 ng/ml, 1.57 ng/ml, 0.78 ng/ml in order, and add equal volume of standard diluent as a blank control. As the various standard and diluted samples added, the ELISA plate was covered with film firmly, and incubated at 37 °C for 90 minutes. Shake dry the sample solutions in the wells, add 100 μl of biotinylated antibody working solution, cover the plate with film, and incubate at 37 °C for 60 minutes. Drain the biotinylated antibody solution in the wells, pat dry on absorbent paper, then add 350 μl washing solution, soak for 1 min, shake off the washing solution and pat dry, repeat the above washing steps 3 times. Then add 100 ul enzyme conjugate working solution to the each well, cover the plate with film, incubate at 37 °C for 30 min. Drain the liquid and wash 5 times with washing solution. Add 90 μl of substrate solution to each well, cover the plate with film, incubate at 37 °C in the dark for 15 min, then add 50 μl of reaction stop solution to each well, and immediately measure the absorbance value at 450 nm with an enzyme reader. Draw a standard curve based on the OD value of the standard and obtain the calculation formula. Then substitute the OD value of the sample into the formula to calculate the concentration of the samples for statistical analysis. Denervation mouse model The anesthetized mice were placed on the operating table in a prone position, maintained in anesthesia via inhalation of isoflurane, depilated the fur at the junction of the left hind limb and the back and disinfected this site with iodine. About 0.5 cm longitudinal incision was cut at the root of the left hind limb using ophthalmic scissors. The muscles were peeled off along the muscle texture and fascia, and the sciatic nerve was easily found underneath. To sever the sciatic nerve, it was cut off about 0.3 cm using ophthalmic scissors. The muscle position was restored, the incision skin was sutured and disinfected with iodine. The mice were then placed in a supine position, and the junction of the left hind limb and the abdomen was depilated and disinfected with iodine. About 0.5 cm oblique incision was cut at the junction of the abdomen and the left hind limb (roughly at the left groin). The muscles were peeled off at the groin to find the femoral nerve underneath, and a 0.3 cm-long section was cut off from the femoral nerve. The muscle position was restored, the incision skin was sutured and disinfected with iodine. After the operation, turn off the gas anesthesia machine, remove the mouse mask, and wait for the mouse to wake up and observe if there is no abnormality before putting them back to the feeding room. In order to reduce the impact of the operation on the mouse's mobility, we only performed nerve severance on the left hind limb to ensure that the mouse's eating and drinking activities were not affected. Protein mass spectrum As described above, the bone marrow cells of one leg bone from each mouse were flushed out and centrifuged at 4 °C, 3000 rpm for 3 minutes. During the operation, special attention was paid to prevent the sample contamination from mouse hairs to avoid of the introduction of high-abundance proteins such as keratin in this assay. Briefly, lyse the red blood cells in the bone marrow cells efficiently using 1 ml lysis buffer, on ice for 2 minutes, to avoid of the potential influence of hemoglobin. The cells were next centrifuged at 4 °C and 3000 rpm for 3 minutes, discarded the supernatant, and resuspended in 250 μl of 8M urea solution (containing 1:100 diluted protease inhibitor cocktail). Use an ultrasonic cell crusher with a small probe to break the cells with parameters as following: the power was 100 w, it worked for every 5 s and followed by paused for 5 s, and lasted for 1 minute. During the process of protein extraction, the EP tubes containing cells were placed in an ice-water mixture to continuously cool down to prevent the protein from degradation. After protein extraction, the protein concentration was determined using BCA method. Before mass spectrum assay, SDS-PAGE gel electrophoresis was typically performed. Briefly, 10 μg of each protein sample was loaded and separated in 10% concentration SDS-PAGE gel. As the electrophoresis was done, stain the gel with 1× Coomassie brilliant blue dye, and observe the bands of each sample, to preliminarily determine the molecular weight of high-abundance proteins and the protein degradation, and to confirm the protein quantification. Finally, 30 μg protein solution of each sample was applied in the protein mass spectrum assay. Before detection, the isobaric chemical tag (TMT) of the tandem mass spectrometer of Thermo Fisher was used for protein labeling. After obtaining the results, the data was imported into the Lianchuan Biological Cloud Platform (https://www.omicstudio.cn/) to analyze the different expression of proteins between the experimental and the control groups. Correlation analysis on GEO data The GSE2603 gene expression dataset (sequencing platform: Affymetrix Human Genome U133A Array) was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The GSE2603 dataset contains gene expression information of surgical resection specimens of breast cancer patients and corresponding clinical information of patients, including age, tumor volume, estrogen receptor expression status, progesterone receptor expression status, human epidermal growth factor receptor 2 (HER2) expression status, metastasis status including bone and lung metastasis, metastasis-free survival, et. al. The patients were grouped according to the median value of gene expression. The high and low expression groups were defined as above and below the median value, respectively. The correlation between Opn , integrin α and β family proteins, such as Cd44 , Itgb1 , Itgb3 , Itgb5 , Itgb7 and Itga5, and the bone metastasis-free survival of breast cancer patients. R (version: 4.0.3) software and "Survival" and "Survminer" packages were applied in this analysis. Metabolomic assay The bone marrow cells flushed out from a tibia plus a femur with 300 μl pre-cooled PBS. After red blood cell lysis, 5×10 6 cells from each mouse were aliquoted and sent to Shanghai APTBIO Co., Ltd. for non-targeted metabolomic assay. After the frozen cell samples were slowly thawed at 4 °C, appropriate amounts of samples were added to pre-cooled methanol/acetonitrile/water solution (2:2:1, v/v/v), vortexed, sonicated at low temperature for 30 minutes, left at -20 °C for 10 minutes, centrifuged at 14,000 g for 20 minutes at 4 °C, and the supernatant was vacuum dried. For mass spectrometry analysis, 100 μl of acetonitrile aqueous solution (acetonitrile: water = 1:1, v/v) was added to resolute the sample, vortexed, centrifuged at 14,000 g for 15 minutes at 4 °C, and took the supernatant for analysis of the different expression of metabolites from bone marrow cells. Neutrophil sorting and RNASeq Eight-week-old C57BL/6J female CIS and control mice were sacrificed after 6 weeks of CIS treatment. Bone marrow cells from a femur plus a tibia from these mice were collected and resuspended in FACS buffer (PBS containing 2% FBS) after red blood cell lysis. 4×10 7 cells from each mouse were used for neutrophil magnetic sorting. 2 μl of anti-Ly6G primary antibody was added to every 1×10 7 cells in 100 μl staining solution. Incubated at 4 °C for 30 minutes, centrifuged at 1500 rpm for 5 minutes, and washed twice with FACS buffer. Resuspended the cells with 380 μl FACS buffer + 20 μl beads (5 μl beads/1×10 7 cells), incubated at 4 °C for 20 minutes, washed twice with FACS buffer, and resuspended in 1 ml FACS buffer for sorting, according to the instructions of the commercial Miltenyi LS separation columns. Briefly, install the magnetic separation column on the magnetic pole, rinse it with 3 ml pre-cooled PBS, and add the prepared cell suspension in when the PBS is about to drip off. As the cell suspension is about to drip off, add 3 ml FACS buffer into the column to rinse it, and repeat this rinse twice. During the separation process, be careful to avoid air drying of the column, otherwise bubbles will be generated. After the rinse buffer drips off, take off the separation column away from the magnetic pole, change a new 15 ml tube for cell collection, fill the column with 5 ml FACS buffer, and use the tube core to push out the cells. These collected cells are positively selected neutrophils. Count the Ly6G-positive cells, dissolve 2×10 6 sorted cells in 1 ml Trizol, and store it in a -80 °C refrigerator for further transcriptome sequencing. Flow cytometry As the mice being sacrificed, the hind limbs were removed and optimally cleaned off the soft tissue, the ends of the femora and tibiae were cut using ophthalmic scissors to expose the bone marrow cavity. The bone marrow in these bones were flushed out from bone cavity in 300 μl pre-cooled PBS solution using a 1 ml sterile syringe. Pulse-flush the bone cavity for 3 times to ensure maximally harvest the bone marrow cells, spin down the cells at 4 °C, 3000 rpm for 3 minutes, and carefully aspirate the supernatant and transfer it into a new EP tube as bone marrow interstitial fluid and freeze it in a -80 °C refrigerator for later use. Add 1 ml of red blood cell lysis buffer to the cell pellet and lyse it on ice for 2 minutes, then add 5 ml PBS containing 2% FBS (so-called FACS buffer) and spin down these cells at 4 °C, 3000 rpm for 3 minutes, discard the supernatant, and resuspend the cell pellet in 3-5 ml FACS buffer. After counting the cells, take 5×10 6 cells in each 1.5 ml EP tube for further flow antibody staining. Fluorophore-conjugated antibodies were diluted by 1:100 in antibody diluent, and 100ul of the antibody mixed solution were added into each sample and incubated at 4 °C in the dark for 30 minutes. For OPN staining, after the staining for cell surface markers, the stained cells were permeabilized and stained with 0.2 μg/μl primary anti-mouse OPN antibody (AF808; R&D system) at room temperature for 1 hour, and then stained with FITC-conjugated donkey anti-goat IgG (secondary antibody) was diluted by 1:200 and incubated at 37 °C in the dark for 60 minutes. A sample incubated with FITC-conjugated secondary antibody only, but not primary antibody, were applied as a negative control for OPN expression. Another sample with all surface antibodies stained, but not OPN, was applied as FMO (full minus one) control for OPN measurement. After primary and secondary antibody staining, the bone marrow cells were washed 2 times with 1 ml FACS buffer, centrifuge at 4 °C, 3000 rpm for 3 minutes, discard the supernatant, resuspend the cell pellet with 200 μl FACS buffer and stored at 4°C in dark for further analysis using Beckman Cytoflex. Data were analyzed using FlowJo. Primary monocyte culture After lysing red blood cells, bone marrow cells were resuspended in FACS buffer, add 2 μl anti-Ly6C antibody in every 1×10 7 cells, incubate at 4 °C for 30 minutes, and wash the cells twice with FACS buffer. Resuspend cells with 250 μl FACS buffer containing 12.5 μl microbeads for every 1×10 7 cells, incubate at 4 °C for 20 minutes, wash the cells twice with FACS buffer and finally resuspend the cells with 1 ml FACS buffer for further magnetic sorting. The procedure of cell sorting was the same with neutrophil sorting mentioned above. Count the sorted Ly6C-positive cells, and seed 1×10 6 cells in each well of 12-well plate. Every 1×10 6 cells resuspended in 2 ml α-MEM medium containing 10% FBS, 1% penicillin-streptomycin, 1% NEAA, and 20 ng/ml M-CSF. Three duplicated wells were set up for each treatment condition. mRNA extraction and real-time qPCR Add 1 ml TRIzol to the bone marrow cells or cultured monocytes and mix them by pipetting and place on ice in a fume hood for 5 minutes. Then add 0.2 ml chloroform to each sample, shake vigorously for 15 seconds, and let it stand at room temperature for 5 minutes. Centrifuge at 13000 rpm for 15 min at 4 °C, take 0.4 ml of the upper layer of the aqueous liquid and transfer it to a new 1.5 ml EP tube. Add 0.4 ml of isopropanol to each sample, gently invert and mix for about 10 times, and place still at room temperature for 10 minutes. Centrifuge at 13000 rpm for 10 min at 4 °C to see white RNA precipitate, discard the supernatant and invert the tubes on filter paper to air dry the RNA precipitates. Add 1 ml of 75% ethanol and gently invert to wash away impurities, centrifuge at 7500 rpm for 5 min at 4 °C, discard the supernatant and invert the tubes on filter paper to air dry the RNA precipitates. Add 20-50 μl of DEPC water to dissolve RNA according to the amount of RNA precipitates, measure the concentration using Nanodrop, and judge the RNA contamination and degradation according to the parameters such as OD260/280 value. After digestion with DNase I, 1 μg extracted mRNA was proceeded to reverse transcription to obtain cDNA using a reverse transcription kit (RR047A; Takara). Subsequently, the expression levels of target genes were tested in the cDNA in 25 μl reaction system using a Takara qPCR kit (820A; Takara). The qPCR primers are listed as following: Adrb1 : forward, 5’-CTCATCGTGGTGGGTAACGTG-3’ and reverse, 5’-ACACACAGCACATCTACCGAA-3’; Adrb2 : forward, 5’-GGGAACGACAGCGACTTCTT-3’ and reverse, 5’-GCCAGGACGATAACCGACAT-3’; Adrb3 : forward, 5’-GGCCCTCTCTAGTTCCCAG-3’ and reverse, 5’-TAGCCATCAAACCTGTTGAGC-3’; Opn : forward, 5’-CCCGGTGAAAGTGACTGATTC-3’ and reverse, 5’-ATGGCTTTCATTGGAATTGC-3’; and Gapdh: forward,5’-GGTCGGTGTGAACGGATTTG-3’ and reverse, 5’-ATGAGCCCTTCCACAATG-3’. The qPCR assay was performed using Thermal Cycler Dice Real Time System from Bio-Rad. The reaction was amplified according to the instrument standard program, with the denaturation at 95 °C and the annealing at 55 °C for 40 cycles. The 2 - △△ CT method was used to calculate gene expression levels. Metastatic model treated with bone-targeted propranolol Cell preparation: E0771-Luc cells in the exponential growth phase were digested with 0.25% trypsin, span down to obtain cell precipitates, resuspended with sterile PBS, counted the cells and made single-cell suspension solution with a concentration of 5×10 6 E0771-Luc cells/ml. The cell solution was placed on ice for immediate use. Eight-week-old female C57BL/6J CIS-exposed mice were anesthetized and fixed in a supine position, and disinfected with iodine on both knee joints. 20 μl prepared E0771-Luc cell solution (containing 1×10 5 cells) was in situ injected into bilateral tibial bone marrow cavity using an insulin syringe. During the injection, kept the tibia and femur perpendicular to each other, and slowly inserted the needle tip of the insulin syringe into the tibia in a direction perpendicular to the tibial plateau. A clear breakthrough feeling was the sign that the needle has successfully entered the bone marrow cavity. After that, slowly injected the cell solution into the bone marrow cavity and slowly withdrawn the needle tip after 2 seconds to prevent tumor cells from overflowing from the needle channel. After tumor cell implantation, the recipient mice were continuously treated with CIS, and intraperitoneally injected with alendronic acid-propranolol (9.05 mg/kg body weight) and vehicle (equal volume of solvent) twice a week [5, 6] (on the 1st and 4th days of each week) for 5 weeks. The tumor growth was monitored via IVIS imaging once a week. At the end of observation, mice were killed and tissue samples were collected for subsequent analysis. Statistical analysis The experimental data were processed using GraphPad Prism 9.0 software, and the results were presented as mean ± standard deviation. The unpaired t-test was used for statistical analysis of the two groups, and One-way ANOVA analysis with Tukey repeated measures were applied for the comparison of one parameter among multiple groups, and the Two-way repeated measures ANOVA was used for repeated detection of multiple time points in the two groups. P<0.05 indicated statistical difference. For the analysis of flow cytometry, the raw data were exported from the instrument, analyzed and exported with FlowJo software, and then statistical analysis was performed. Declarations Conflict of interests: The authors declare that they have no conflicts of interest with the contents of this article. Acknowledgements Research reported in this publication was supported by the National Natural Science Foundation of China under Award Numbers 81702865 (to X.L.), 82172469, 82472481, 32100916 (to J.L.), 82071533 and 82374058 (to H.Z.), and by the Natural Science Foundation of Hebei Province under Award Number H2025206343 (to J.L.). Author contributions X. Li coordinated the studies, performed and analyzed the experiments shown in all Figures except Figs. 3B-D, 4G-L, 5G, 6A-G and 6N, and wrote the paper; X. Liu generated the bone-targeting propranolol, tested the delivery specificity and toxicity, and performed and analyzed the experiments shown in Figures in Figs. 6A-G; X. Zhang performed and analyzed the experiments shown in Figures in Figs. 3B-D, 4G-L, 5G and 6N; J. Fan sectioned all paraffin blocks and blindly analyzed the histological parameters shown in Figs. 1C-E, 1K-L, 3P-Q and 5H-I. J. Fan and Y. Xing generated Ly6G CreER R26 DTA mice and ADRβ2 knockout mice, respectively, and analyze the mouse phenotype; H. Jiao analyzed the data generated by flow cytometry; P. Li drew the Fig. 7 and all experimental design sketches; D. Kong carried out the protein mass spectrum assay and instructed the data analysis; J. Qi instructed the process of alendronate-propranolol conjugation; X. Du supervised and coordinated the studies, and revised the paper. J. Li. and H. Zhang supervised, conceived, and coordinated the studies, and wrote the paper. All authors reviewed the results and approved the final version of the manuscript. Data availability All relevant data are available from the authors upon reasonable request. References Lei, S.; Zheng, R.; Zhang, S.; Wang, S.; Chen, R.; Sun, K.; Zeng, H.; Zhou, J.; Wei, W. Global patterns of breast cancer incidence and mortality: A population-based cancer registry data analysis from 2000 to 2020. Cancer Commun (Lond) 2021 , 41 (11), 1183-1194. DOI: 10.1002/cac2.12207. Onkar, S.; Cui, J.; Zou, J.; Cardello, C.; Cillo, A. R.; Uddin, M. R.; Sagan, A.; Joy, M.; Osmanbeyoglu, H. U.; Pogue-Geile, K. L.; et al. Immune landscape in invasive ductal and lobular breast cancer reveals a divergent macrophage-driven microenvironment. Nat Cancer 2023 , 4 (4), 516-534. DOI: 10.1038/s43018-023-00527-w. Parkes, A.; Clifton, K.; Al-Awadhi, A.; Oke, O.; Warneke, C. L.; Litton, J. K.; Hortobagyi, G. N. Characterization of bone only metastasis patients with respect to tumor subtypes. NPJ Breast Cancer 2018 , 4 , 2. DOI: 10.1038/s41523-018-0054-x. Buijs, J. T.; van der Pluijm, G. Osteotropic cancers: from primary tumor to bone. Cancer Lett 2009 , 273 (2), 177-193. DOI: 10.1016/j.canlet.2008.05.044. Gkikopoulou, E.; Syrigos, C. C.; Mantogiannakou, I.; Petraki, C. E.; Stathopoulou, M.; Dragolia, M.; Rinotas, V.; Ntafis, V.; Rauner, M.; Douni, E. RANKL Drives Bone Metastasis in Mammary Cancer: Protective Effects of Anti-Resorptive Treatments. Int J Mol Sci 2025 , 26 (11). DOI: 10.3390/ijms26114990. Wang, R.; Zhu, Y.; Liu, X.; Liao, X.; He, J.; Niu, L. The Clinicopathological features and survival outcomes of patients with different metastatic sites in stage IV breast cancer. BMC Cancer 2019 , 19 (1), 1091. DOI: 10.1186/s12885-019-6311-z. Jiao, S.; Subudhi, S. K.; Aparicio, A.; Ge, Z.; Guan, B.; Miura, Y.; Sharma, P. Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy. Cell 2019 , 179 (5), 1177-1190 e1113. DOI: 10.1016/j.cell.2019.10.029 From NLM Medline. Huang, J. F.; Shen, J.; Li, X.; Rengan, R.; Silvestris, N.; Wang, M.; Derosa, L.; Zheng, X.; Belli, A.; Zhang, X. L.; et al. Incidence of patients with bone metastases at diagnosis of solid tumors in adults: a large population-based study. Ann Transl Med 2020 , 8 (7), 482. DOI: 10.21037/atm.2020.03.55. Chida, Y.; Hamer, M.; Wardle, J.; Steptoe, A. Do stress-related psychosocial factors contribute to cancer incidence and survival? Nat Clin Pract Oncol 2008 , 5 (8), 466-475. DOI: 10.1038/ncponc1134 From NLM Medline. Moreno-Smith, M.; Lutgendorf, S. K.; Sood, A. K. Impact of stress on cancer metastasis. Future Oncol 2010 , 6 (12), 1863-1881. DOI: 10.2217/fon.10.142 From NLM Medline. Dai, S.; Mo, Y.; Wang, Y.; Xiang, B.; Liao, Q.; Zhou, M.; Li, X.; Li, Y.; Xiong, W.; Li, G.; et al. Chronic Stress Promotes Cancer Development. Front Oncol 2020 , 10 , 1492. DOI: 10.3389/fonc.2020.01492 From NLM PubMed-not-MEDLINE. Yaribeygi, H.; Panahi, Y.; Sahraei, H.; Johnston, T. P.; Sahebkar, A. The impact of stress on body function: A review. EXCLI J 2017 , 16 , 1057-1072. DOI: 10.17179/excli2017-480 From NLM PubMed-not-MEDLINE. Obradovic, M. M. S.; Hamelin, B.; Manevski, N.; Couto, J. P.; Sethi, A.; Coissieux, M. M.; Munst, S.; Okamoto, R.; Kohler, H.; Schmidt, A.; et al. Glucocorticoids promote breast cancer metastasis. Nature 2019 , 567 (7749), 540-544. DOI: 10.1038/s41586-019-1019-4 From NLM Medline. Peinado, H.; Zhang, H.; Matei, I. R.; Costa-Silva, B.; Hoshino, A.; Rodrigues, G.; Psaila, B.; Kaplan, R. N.; Bromberg, J. F.; Kang, Y.; et al. Pre-metastatic niches: organ-specific homes for metastases. Nat Rev Cancer 2017 , 17 (5), 302-317. DOI: 10.1038/nrc.2017.6 From NLM Medline. Yirmiya, R.; Goshen, I.; Bajayo, A.; Kreisel, T.; Feldman, S.; Tam, J.; Trembovler, V.; Csernus, V.; Shohami, E.; Bab, I. Depression induces bone loss through stimulation of the sympathetic nervous system. Proc Natl Acad Sci U S A 2006 , 103 (45), 16876-16881. DOI: 10.1073/pnas.0604234103 From NLM Medline. Guo, Q.; Chen, N.; Qian, C.; Qi, C.; Noller, K.; Wan, M.; Liu, X.; Zhang, W.; Cahan, P.; Cao, X. Sympathetic Innervation Regulates Osteocyte-Mediated Cortical Bone Resorption during Lactation. Adv Sci (Weinh) 2023 , 10 (18), e2207602. DOI: 10.1002/advs.202207602 From NLM Medline. Coleman, R.; Finkelstein, D. M.; Barrios, C.; Martin, M.; Iwata, H.; Hegg, R.; Glaspy, J.; Perianez, A. M.; Tonkin, K.; Deleu, I.; et al. Adjuvant denosumab in early breast cancer (D-CARE): an international, multicentre, randomised, controlled, phase 3 trial. Lancet Oncol 2020 , 21 (1), 60-72. DOI: 10.1016/S1470-2045(19)30687-4 From NLM Medline. Stopeck, A. T.; Lipton, A.; Body, J. J.; Steger, G. G.; Tonkin, K.; de Boer, R. H.; Lichinitser, M.; Fujiwara, Y.; Yardley, D. A.; Viniegra, M.; et al. Denosumab compared with zoledronic acid for the treatment of bone metastases in patients with advanced breast cancer: a randomized, double-blind study. J Clin Oncol 2010 , 28 (35), 5132-5139. DOI: 10.1200/JCO.2010.29.7101 From NLM Medline. Terpos, E.; Confavreux, C. B.; Clezardin, P. Bone antiresorptive agents in the treatment of bone metastases associated with solid tumours or multiple myeloma. Bonekey Rep 2015 , 4 , 744. DOI: 10.1038/bonekey.2015.113 From NLM PubMed-not-MEDLINE. Cathomas, F.; Lin, H. Y.; Chan, K. L.; Li, L.; Parise, L. F.; Alvarez, J.; Durand-de Cuttoli, R.; Aubry, A. V.; Muhareb, S.; Desland, F.; et al. Circulating myeloid-derived MMP8 in stress susceptibility and depression. Nature 2024 , 626 (8001), 1108-1115. DOI: 10.1038/s41586-023-07015-2 From NLM Medline. Khan, A.; Song, M.; Dong, Z. Chronic stress: a fourth etiology in tumorigenesis? Mol Cancer 2025 , 24 (1), 196. DOI: 10.1186/s12943-025-02402-x From NLM In-Process. Heidt, T.; Sager, H. B.; Courties, G.; Dutta, P.; Iwamoto, Y.; Zaltsman, A.; von Zur Muhlen, C.; Bode, C.; Fricchione, G. L.; Denninger, J.; et al. Chronic variable stress activates hematopoietic stem cells. Nat Med 2014 , 20 (7), 754-758. DOI: 10.1038/nm.3589 From NLM Medline. Barrett, T. J.; Corr, E. M.; van Solingen, C.; Schlamp, F.; Brown, E. J.; Koelwyn, G. J.; Lee, A. H.; Shanley, L. C.; Spruill, T. M.; Bozal, F.; et al. Chronic stress primes innate immune responses in mice and humans. Cell Rep 2021 , 36 (10), 109595. DOI: 10.1016/j.celrep.2021.109595 From NLM Medline. He, X. Y.; Gao, Y.; Ng, D.; Michalopoulou, E.; George, S.; Adrover, J. M.; Sun, L.; Albrengues, J.; Dassler-Plenker, J.; Han, X.; et al. Chronic stress increases metastasis via neutrophil-mediated changes to the microenvironment. Cancer Cell 2024 , 42 (3), 474-486 e412. DOI: 10.1016/j.ccell.2024.01.013 From NLM Medline. Clezardin, P.; Coleman, R.; Puppo, M.; Ottewell, P.; Bonnelye, E.; Paycha, F.; Confavreux, C. B.; Holen, I. Bone metastasis: mechanisms, therapies, and biomarkers. Physiol Rev 2021 , 101 (3), 797-855. DOI: 10.1152/physrev.00012.2019 From NLM Medline. Wang, L.; Fang, D.; Xu, J.; Luo, R. Various pathways of zoledronic acid against osteoclasts and bone cancer metastasis: a brief review. BMC Cancer 2020 , 20 (1), 1059. DOI: 10.1186/s12885-020-07568-9 From NLM Medline. Chu, B.; Marwaha, K.; Sanvictores, T.; Awosika, A. O.; Ayers, D. Physiology, Stress Reaction. In StatPearls , 2025. Hansen, J. L.; Carroll, J. E.; Seeman, T. E.; Cole, S. W.; Rentscher, K. E. Lifetime chronic stress exposures, stress hormones, and biological aging: Results from the Midlife in the United States (MIDUS) study. Brain Behav Immun 2025 , 123 , 1159-1168. DOI: 10.1016/j.bbi.2024.10.022 From NLM Medline. Katayama, Y.; Battista, M.; Kao, W. M.; Hidalgo, A.; Peired, A. J.; Thomas, S. A.; Frenette, P. S. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 2006 , 124 (2), 407-421. DOI: 10.1016/j.cell.2005.10.041 From NLM Medline. Kamiya, A.; Hayama, Y.; Kato, S.; Shimomura, A.; Shimomura, T.; Irie, K.; Kaneko, R.; Yanagawa, Y.; Kobayashi, K.; Ochiya, T. Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression. Nat Neurosci 2019 , 22 (8), 1289-1305. DOI: 10.1038/s41593-019-0430-3 From NLM Medline. Song, Z.; Chen, W.; Athavale, D.; Ge, X.; Desert, R.; Das, S.; Han, H.; Nieto, N. Osteopontin Takes Center Stage in Chronic Liver Disease. Hepatology 2021 , 73 (4), 1594-1608. DOI: 10.1002/hep.31582 From NLM Medline. Yim, A.; Smith, C.; Brown, A. M. Osteopontin/secreted phosphoprotein-1 harnesses glial-, immune-, and neuronal cell ligand-receptor interactions to sense and regulate acute and chronic neuroinflammation. Immunol Rev 2022 , 311 (1), 224-233. DOI: 10.1111/imr.13081 From NLM Medline. Hong, Y.; Zhang, L.; Liu, N.; Xu, X.; Liu, D.; Tu, J. The Central Nervous Mechanism of Stress-Promoting Cancer Progression. Int J Mol Sci 2022 , 23 (20). DOI: 10.3390/ijms232012653. Cole, S. W.; Nagaraja, A. S.; Lutgendorf, S. K.; Green, P. A.; Sood, A. K. Sympathetic nervous system regulation of the tumour microenvironment. Nat Rev Cancer 2015 , 15 (9), 563-572. DOI: 10.1038/nrc3978 From NLM Medline. Thaker, P. H.; Han, L. Y.; Kamat, A. A.; Arevalo, J. M.; Takahashi, R.; Lu, C.; Jennings, N. B.; Armaiz-Pena, G.; Bankson, J. A.; Ravoori, M.; et al. Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat Med 2006 , 12 (8), 939-944. DOI: 10.1038/nm1447 From NLM Medline. Lu, Y.; Zhao, H.; Liu, Y.; Zuo, Y.; Xu, Q.; Liu, L.; Li, X.; Zhu, H.; Zhang, Y.; Zhang, S.; et al. Chronic Stress Activates PlexinA1/VEGFR2-JAK2-STAT3 in Vascular Endothelial Cells to Promote Angiogenesis. Front Oncol 2021 , 11 , 709057. DOI: 10.3389/fonc.2021.709057 From NLM PubMed-not-MEDLINE. Globig, A. M.; Zhao, S.; Roginsky, J.; Maltez, V. I.; Guiza, J.; Avina-Ochoa, N.; Heeg, M.; Araujo Hoffmann, F.; Chaudhary, O.; Wang, J.; et al. The beta(1)-adrenergic receptor links sympathetic nerves to T cell exhaustion. Nature 2023 , 622 (7982), 383-392. DOI: 10.1038/s41586-023-06568-6 From NLM Medline. Delgado-Benito, V.; Berruezo-Llacuna, M.; Altwasser, R.; Winkler, W.; Sundaravinayagam, D.; Balasubramanian, S.; Caganova, M.; Graf, R.; Rahjouei, A.; Henke, M. T.; et al. PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification. J Exp Med 2020 , 217 (10). DOI: 10.1084/jem.20200137 From NLM Medline. Pons-Espinal, M.; Blasco-Agell, L.; Fernandez-Carasa, I.; Andres-Benito, P.; di Domenico, A.; Richaud-Patin, Y.; Baruffi, V.; Marruecos, L.; Espinosa, L.; Garrido, A.; et al. Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson's disease. JCI Insight 2024 , 9 (3). DOI: 10.1172/jci.insight.163359 From NLM Medline. Lopez-Rodriguez, A. B.; Hennessy, E.; Murray, C. L.; Nazmi, A.; Delaney, H. J.; Healy, D.; Fagan, S. G.; Rooney, M.; Stewart, E.; Lewis, A.; et al. Acute systemic inflammation exacerbates neuroinflammation in Alzheimer's disease: IL-1beta drives amplified responses in primed astrocytes and neuronal network dysfunction. Alzheimers Dement 2021 , 17 (10), 1735-1755. DOI: 10.1002/alz.12341 From NLM Medline. Bill, R.; Wirapati, P.; Messemaker, M.; Roh, W.; Zitti, B.; Duval, F.; Kiss, M.; Park, J. C.; Saal, T. M.; Hoelzl, J.; et al. CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers. Science 2023 , 381 (6657), 515-524. DOI: 10.1126/science.ade2292 From NLM Medline. Eun, J. W.; Yoon, J. H.; Ahn, H. R.; Kim, S.; Kim, Y. B.; Lim, S. B.; Park, W.; Kang, T. W.; Baek, G. O.; Yoon, M. G.; et al. Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib. Cancer Commun (Lond) 2023 , 43 (4), 455-479. DOI: 10.1002/cac2.12414 From NLM Medline. Li, Y.; Liu, H.; Zhao, Y.; Yue, D.; Chen, C.; Li, C.; Zhang, Z.; Wang, C. Tumor-associated macrophages (TAMs)-derived osteopontin (OPN) upregulates PD-L1 expression and predicts poor prognosis in non-small cell lung cancer (NSCLC). Thorac Cancer 2021 , 12 (20), 2698-2709. DOI: 10.1111/1759-7714.14108 From NLM Medline. Zhao, L.; Wang, Z.; Tan, Y.; Ma, J.; Huang, W.; Zhang, X.; Jin, C.; Zhang, T.; Liu, W.; Yang, Y. G. IL-17A/CEBPbeta/OPN/LYVE-1 axis inhibits anti-tumor immunity by promoting tumor-associated tissue-resident macrophages. Cell Rep 2024 , 43 (12), 115039. DOI: 10.1016/j.celrep.2024.115039 From NLM Medline. Cheng, J. N.; Jin, Z.; Su, C.; Jiang, T.; Zheng, X.; Guo, J.; Li, X.; Chu, H.; Jia, J.; Zhou, Q.; et al. Bone metastases diminish extraosseous response to checkpoint blockade immunotherapy through osteopontin-producing osteoclasts. Cancer Cell 2025 , 43 (6), 1093-1107 e1099. DOI: 10.1016/j.ccell.2025.03.036 From NLM Medline. Cools-Lartigue, J.; Spicer, J.; McDonald, B.; Gowing, S.; Chow, S.; Giannias, B.; Bourdeau, F.; Kubes, P.; Ferri, L. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest 2013 , 123 (8), 3446-3458. DOI: 10.1172/JCI67484 From NLM Publisher. Hedrick, C. C.; Malanchi, I. Neutrophils in cancer: heterogeneous and multifaceted. Nat Rev Immunol 2022 , 22 (3), 173-187. DOI: 10.1038/s41577-021-00571-6 From NLM Medline. Ma, L. X.; Titmuss, E.; Loree, J. M.; Jonker, D. J.; Kennecke, H. F.; Berry, S.; Couture, F.; Ahmad, C. E.; Goffin, J. R.; Kavan, P.; et al. Plasma arginine as a predictive biomarker for outcomes with immune checkpoint inhibition in metastatic colorectal cancer: a correlative analysis of the CCTG CO.26 trial. J Immunother Cancer 2024 , 12 (12). DOI: 10.1136/jitc-2024-010094 From NLM Medline. Fultang, L.; Booth, S.; Yogev, O.; Martins da Costa, B.; Tubb, V.; Panetti, S.; Stavrou, V.; Scarpa, U.; Jankevics, A.; Lloyd, G.; et al. Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity. Blood 2020 , 136 (10), 1155-1160. DOI: 10.1182/blood.2019004500 From NLM Medline. Geiger, R.; Rieckmann, J. C.; Wolf, T.; Basso, C.; Feng, Y.; Fuhrer, T.; Kogadeeva, M.; Picotti, P.; Meissner, F.; Mann, M.; et al. L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity. Cell 2016 , 167 (3), 829-842 e813. DOI: 10.1016/j.cell.2016.09.031 From NLM Medline. Hu, B.; Zhang, Y.; Zhang, G.; Li, Z.; Jing, Y.; Yao, J.; Sun, S. Research progress of bone-targeted drug delivery system on metastatic bone tumors. J Control Release 2022 , 350 , 377-388. DOI: 10.1016/j.jconrel.2022.08.034 From NLM Medline. Additional Declarations There is NO Competing Interest. Supplementary Files Suppl.FigsX.Li.etal.pdf Supplementary Figure 1, 2, 3, 4, 5, 6, 7 and 8 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7760970","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":526624479,"identity":"8b7264db-9c6b-4f61-8db0-9c3eef71f313","order_by":0,"name":"Jinbo 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1","display":"","copyAsset":false,"role":"figure","size":511983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced breast cancer bone metastasis and altered BM microenvironment in CIS-exposed mice\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Experimental design for primary orthotopic implantation of 4T1 breast cancer cells in CIS-exposed BALB/c mice and secondary intracardiac injection of orthotopic cancer cells in BALB/c mice without CIS treatment. (\u003cstrong\u003eB\u003c/strong\u003e) The incidence rate of remote metastasis to organs including lung, liver and bone in the recipient BALB/c mice following primary and secondary breast cancer cell implantation. n=6-10 mice in these groups. (\u003cstrong\u003eC\u003c/strong\u003e) Hematoxylin and eosin (H\u0026amp;E)-stained tibial paraffin sections showing the metastatic breast cancers in the metaphyseal area. The tumor tissue (T) is outlined with yellow dotted lines. (\u003cstrong\u003eD-E\u003c/strong\u003e) The percentages of metastatic tumor area in total bone area counted in both primary and secondary implantation mouse models. (\u003cstrong\u003eF\u003c/strong\u003e) H\u0026amp;E-stained lung paraffin sections showing the metastatic breast cancers, and (\u003cstrong\u003eG-H\u003c/strong\u003e) the percentages of metastatic tumor area in total lung area counted in both primary and secondary implantation mouse models. (\u003cstrong\u003eI\u003c/strong\u003e) Representative IVIS images showing the luciferin signal of the Ctrl and CIS-exposed mice on 7-, 14- and 21-days post intracardiac injection of luciferase-expressing E0771 cells. n=5 mice per group. (\u003cstrong\u003eJ\u003c/strong\u003e) Analysis of the total flux of the luciferin signal in the area of hind limb of Ctrl and CIS-exposed mice. n=3 to 9 mice per group. (\u003cstrong\u003eK\u003c/strong\u003e) H\u0026amp;E-stained tibial paraffin sections showing the metastatic breast cancers in the metaphyseal area. The tumor tissue (T) is distinguished from normal bone marrow and is outlined with yellow dotted lines. (\u003cstrong\u003eL\u003c/strong\u003e) The area of metastatic tumors on tibial sections were calculated. n=5 and 4 mice for Ctrl and CIS groups, respectively. (\u003cstrong\u003eM\u003c/strong\u003e) The levels of TRACP-5b in serum of Ctrl and CIS-exposed mice with breast cancer bearing were measured using ELISA. n=5 mice per group. (\u003cstrong\u003eN\u003c/strong\u003e) tSNE graphs of lymphoid and myeloid cells in BM cells tested using flow cytometry. (\u003cstrong\u003eO\u003c/strong\u003e) Representative graphs showing the gate strategies of CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells and B220\u003csup\u003e+ \u003c/sup\u003elymphocytes in BM of Ctrl and CIS-exposed mice. (\u003cstrong\u003eP-R\u003c/strong\u003e) Frequencies of lymphoid subpopulations including B220\u003csup\u003e+ \u003c/sup\u003eB lymphocytes, CD4\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells in BM of Ctrl and CIS-exposed mice. n=5 mice per group. (\u003cstrong\u003eS-V\u003c/strong\u003e) The frequencies of CD11b\u003csup\u003e+\u003c/sup\u003e leukocytes (\u003cstrong\u003eS\u003c/strong\u003e) and myeloid subpopulations including F4/80\u003csup\u003e+\u003c/sup\u003e macrophages (\u003cstrong\u003eT\u003c/strong\u003e), Ly6C\u003csup\u003e+\u003c/sup\u003e monocytes (\u003cstrong\u003eU\u003c/strong\u003e) and Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophils (\u003cstrong\u003eV\u003c/strong\u003e) in BM of Ctrl and CIS-exposed mice. n=5 mice per group. All data are presented as mean ± SD. Analysis: unpaired Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/ea3f35e76e795b8bf52ca74a.png"},{"id":93130857,"identity":"04d064ac-990f-406c-aac5-27c54a9c663a","added_by":"auto","created_at":"2025-10-09 11:29:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":395300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated NE release stimulated OPN expression by monocytes/macrophages in CIS-exposed mice\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Norepinephrine (NE) levels in interstitial fluid of BM of CIS and Ctrl mice tested by ELISA. n=3 mice per group. (\u003cstrong\u003eB\u003c/strong\u003e) NE) levels in interstitial fluid of BM of denervated and sham mice at 0, 2 and 12 weeks after surgery. n=4 to 5 mice per group. (\u003cstrong\u003eC\u003c/strong\u003e) The Top10 signaling pathways basing on KEGG analysis of significantly changed proteins in BM cells of CIS-exposed mice versus Ctrl mice using mass spectrum. (\u003cstrong\u003eD\u003c/strong\u003e) The Top10 signaling pathways basing on KEGG analysis of significantly changed proteins in BM cells of denervated mice versus sham mice. (\u003cstrong\u003eE-F\u003c/strong\u003e) Volcano plots represent proteomics analysis of BM cells of CIS versus Ctrl mice (E), and of denervated versus sham mice (F). (FC\u0026gt;1.33 or \u0026lt;0.67, p\u0026lt;0.05). n=4 mice per group. (\u003cstrong\u003eG\u003c/strong\u003e) Venn diagram and heatmap graphs represent an overlap of 5 significantly changed proteins, including COL11a1, COL1a1, FAM207a, OPN and GIT1, in both of CIS and denervated mouse groups. (\u003cstrong\u003eH\u003c/strong\u003e) Immunofluorescence staining for OPN protein expression on tibial sections, and the cell numbers of OPN\u003csup\u003e+\u003c/sup\u003e cells accounted using ImageJ. n=3 mice per group. (\u003cstrong\u003eI-J\u003c/strong\u003e) ELISA tests of OPN protein levels in BM cells from CIS and Ctrl mice (left panel; \u003cstrong\u003eI\u003c/strong\u003e), and from denervated and sham mice (right panel; \u003cstrong\u003eJ\u003c/strong\u003e). n=3 to 4 mice per group. (\u003cstrong\u003eK\u003c/strong\u003e) The frequencies of OPN-expressing cells in BM cells from CIS and Ctrl mice measured using flow cytometry. n=5 mice per group. (\u003cstrong\u003eL-N\u003c/strong\u003e) The frequencies of OPN-expressing monocytes (CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e), neutrophils (CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e) and macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e) in BM cells from CIS and Ctrl mice. n=5 mice per group. (\u003cstrong\u003eO\u003c/strong\u003e) Experimental design of BM Ly6C\u003csup\u003e+\u003c/sup\u003e monocytes sorted from WT and CIS-exposed mice and treated with increasing doses of NE in vitro, and \u003cem\u003eOpn\u003c/em\u003e gene expression by these cells measured using qPCR. n=3 samples for each treatment. All data are presented as mean ± SD. Analysis: One-way ANOVA test with Tukey post analysis in (H); unpaired Student’s \u003cem\u003et\u003c/em\u003e test in all others.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/def9ebb86ae05f26cd607e2d.png"},{"id":93131209,"identity":"d049bbc9-bbc0-429e-96de-01f3072032a6","added_by":"auto","created_at":"2025-10-09 11:37:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":474055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExcessive OPN targeted neutrophils to promote bone metastasis in CIS-exposed mice.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Volcano plot graph reveals the significantly changed proteins in serum from breast cancer patients with bone metastasis only versus that from breast cancer patients without any metastasis. n=3 patients per group. (\u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e) The K-M curves of bone metastasis free survival showing the difference on cumulative survival between CD44\u003csup\u003ehigh\u003c/sup\u003e versus CD44\u003csup\u003elow\u003c/sup\u003e, ITGA5\u003csup\u003ehigh\u003c/sup\u003e versus ITGA5\u003csup\u003elow\u003c/sup\u003e, and ITGB5\u003csup\u003ehigh\u003c/sup\u003e versus ITGB5\u003csup\u003elow\u003c/sup\u003e patients in MSKCC database. (\u003cstrong\u003eE\u003c/strong\u003e) Representative images showing the E0771 cancer cells migrating through the transwell membrane in response to attractive signals from BM cells treated with NE (50µM) plus vehicle or anti-CD44 blocking antibody (10µg/ml). (\u003cstrong\u003eF\u003c/strong\u003e) The numbers of E0771 cells migrated through transwell membrane were counted 24 hours later. n=5 wells per group. (\u003cstrong\u003eG\u003c/strong\u003e) CD44\u003csup\u003e-\u003c/sup\u003e and CD44\u003csup\u003e+\u003c/sup\u003e neutrophil (CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e-\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e) subpopulations were sorted and gene transcription levels of \u003cem\u003eItga5\u003c/em\u003e and\u003cem\u003e Itgb5 \u003c/em\u003ewere tested by qPCR. n=3 mice per group. (\u003cstrong\u003eH\u003c/strong\u003e) The flow gates for Ly6C\u003csup\u003e+\u003c/sup\u003e and Ly6G\u003csup\u003e+\u003c/sup\u003e cells in CD11b\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e BM cells from CIS and Ctrl mice. (\u003cstrong\u003eI\u003c/strong\u003e) Analysis of the constituent percentages of Ly6C\u003csup\u003e-\u003c/sup\u003eG\u003csup\u003e-\u003c/sup\u003e, Ly6C\u003csup\u003e+\u003c/sup\u003eG\u003csup\u003e-\u003c/sup\u003e and Ly6C\u003csup\u003e-\u003c/sup\u003eG\u003csup\u003e+\u003c/sup\u003e cells in CD11b\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e BM cells in (\u003cstrong\u003eH\u003c/strong\u003e). (\u003cstrong\u003eJ\u003c/strong\u003e) The frequencies of CD44\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e cells in BM from CIS and Ctrl mice. (\u003cstrong\u003eK\u003c/strong\u003e) The frequencies of CD44\u003csup\u003e+\u003c/sup\u003e cells in total neutrophils in BM from CIS and Ctrl mice. (\u003cstrong\u003eL\u003c/strong\u003e) A sketch graph shows that Ly6G-DTA and WT littermates were induced CIS for 7 weeks, and intracardiac injection of E0771 cells were performed at the end of 3\u003csup\u003erd\u003c/sup\u003e week, and then tumor growth and metastasis was observed lasting for 4 weeks. (\u003cstrong\u003eM\u003c/strong\u003e) Representative IVIS images showing the luciferin signal of Ly6G-DTA and WT mice at 7-, 14-, 21- and 28-days post intracardiac injection of luciferase-expressing E0771 cells. (\u003cstrong\u003eN\u003c/strong\u003e) Analysis of the total flux of the luciferin signal in the area of hind limb of Ly6G-DTA and WT mice. (\u003cstrong\u003eO\u003c/strong\u003e) Survival rates of Ly6G-DTA and WT CIS-exposed mice with breast cancer bearing. n=10 to 11 mice per group. (\u003cstrong\u003eP\u003c/strong\u003e) HE staining of tibial paraffin sections reveals the metastatic breast cancers in the metaphyseal area. The tumor tissue (T) is outlined with yellow dotted lines. (\u003cstrong\u003eQ\u003c/strong\u003e) The area of metastatic tumors on tibial sections were calculated. All data are presented as mean ± SD. Analysis: One-way ANOVA test with Tukey post analysis in (\u003cstrong\u003eG\u003c/strong\u003e), unpaired Student’s \u003cem\u003et\u003c/em\u003e test in all others.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/7bd60eab24059ff37919e36b.png"},{"id":93130859,"identity":"ce3b724e-5894-484f-853e-e28aef5c89d4","added_by":"auto","created_at":"2025-10-09 11:29:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":386446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChronic stress stimulated excessive arginine metabolism in neutrophils\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) The Top10 signaling pathways basing on KEGG analysis of significantly changed gene expression by neutrophils in BM from CIS and Ctrl mice. (\u003cstrong\u003eB\u003c/strong\u003e) Comparison of genes involved in ‘arginine and proline metabolism’ pathway in neutrophils from CIS and Ctrl mice with or without breast cancer bearing. (\u003cstrong\u003eC\u003c/strong\u003e) Principal component analysis of metabolites detected in BM neutrophils from CIS and Ctrl mice. (\u003cstrong\u003eD\u003c/strong\u003e) The histogram of KEGG analysis on metabolites in neutrophils reveals obvious alterations in various metabolic signaling pathways. (\u003cstrong\u003eE\u003c/strong\u003e) The Volcano plot showing significantly changed metabolites in neutrophils from CIS-exposed mice versus Ctrl mice. n=4 mice per group. (\u003cstrong\u003eF\u003c/strong\u003e) Heatmap of metabolites associated with arginine metabolism. n=4 mice per group. (\u003cstrong\u003eG\u003c/strong\u003e) The levels of arginine in BM from WT and Ly6G-DTA CIS-exposed mice were tested by ELISA. n=10, 14 and 10 mice in WT Ctrl, WT CIS and Ly6G-DTA CIS mouse groups, respectively. (\u003cstrong\u003eH\u003c/strong\u003e) Experimental design for treating BM cells from WT and Ly6G-DTA mice with vehicle, OPN plus arginine for 24 hours before further flow and qPCR analysis. (I) qPCR analysis of mRNA expression levels of \u003cem\u003eNos2\u003c/em\u003e, \u003cem\u003eTnfα\u003c/em\u003e, \u003cem\u003eArg1\u003c/em\u003e, \u003cem\u003eTgfβ1\u003c/em\u003e and \u003cem\u003eIfnγ\u003c/em\u003e by BM cells from WT mice in (H). (\u003cstrong\u003eJ\u003c/strong\u003e-\u003cstrong\u003eL\u003c/strong\u003e) Flow analysis of iNOS\u003csup\u003e+\u003c/sup\u003e neutrophils (\u003cstrong\u003eJ\u003c/strong\u003e), CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells (\u003cstrong\u003eK\u003c/strong\u003e) and IFNγ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (\u003cstrong\u003eL\u003c/strong\u003e) in BM cells from Ly6G-DTA and WT mice in (\u003cstrong\u003eH\u003c/strong\u003e). n=3 mice per treatment. All data are presented as mean ± SD. Analysis: One-way ANOVA test with Tukey post analysis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/51179ae354b1535d9019ceb4.png"},{"id":93130860,"identity":"b32423a6-7fa4-4ae2-9755-2a6ac51a778a","added_by":"auto","created_at":"2025-10-09 11:29:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":407253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAdrβ2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockout mice exhibited reduced OPN\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e monocytes/macrophages and neutrophil arginine metabolism. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) qPCR tests of the transcription levels of \u003cem\u003eAdrβ1\u003c/em\u003e, \u003cem\u003eAdrβ2\u003c/em\u003e and \u003cem\u003eAdrβ3\u003c/em\u003e genes in total BM cells from CIS and Ctrl mice. (\u003cstrong\u003eB\u003c/strong\u003e) The frequencies of OPN-expressing cells in BM from WT and Adrβ2-KO mice tested by flow cytometry. (\u003cstrong\u003eC\u003c/strong\u003e) The frequencies of OPN-expressing CD41a\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;and Ly6G\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells in BM from WT and Adrβ2-KO mice tested by flow cytometry.\u0026nbsp; (\u003cstrong\u003eD-F\u003c/strong\u003e) Representative graphs showing the strategies for gating CD44- and iNOS-expressing cells in BM cells (\u003cstrong\u003eD\u003c/strong\u003e), and the frequencies of CD44\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;(\u003cstrong\u003eE\u003c/strong\u003e) and iNOS\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;neutrophils (\u003cstrong\u003eF\u003c/strong\u003e) in BM from WT and Adrβ2-KO mice. (\u003cstrong\u003eG\u003c/strong\u003e) The levels of arginine in BM from WT and Adrβ2-KO mice tested by ELISA. (\u003cstrong\u003eH\u003c/strong\u003e) Representative graphs showing the strategies for gating various types of cells, including CD11b\u003csup\u003e+\u003c/sup\u003e, B220\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;and CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells, in BM cells. (\u003cstrong\u003eI\u003c/strong\u003e-\u003cstrong\u003eL\u003c/strong\u003e) The frequencies of B220\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;B cells, CD11b\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;myeloid cells, CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;and CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells in BM cells from WT and Adrβ2-KO mice. (\u003cstrong\u003eM\u003c/strong\u003e) Experimental design for testing E0771 cell migration toward WT and Adrβ2-KO BM cells treated with vehicle and NE, and representative images showing the E0771 cancer cells migrating through the transwell membrane in response to attractive signals from WT and Adrβ2-KO BM cells treated with NE (50 µM). (\u003cstrong\u003eN\u003c/strong\u003e) The numbers of E0771 cells migrated through transwell membrane in (\u003cstrong\u003eM\u003c/strong\u003e) 24 hours later. n=5 wells per group. (\u003cstrong\u003eO\u003c/strong\u003e) qPCR tests of the transcription levels of \u003cem\u003eOpn\u003c/em\u003e gene in total BM cells with NE (50 µM) plus vehicle or propranolol. All data are presented as mean ± SD. Analysis: One-way ANOVA test with Tukey post analysis in (\u003cstrong\u003eO\u003c/strong\u003e); unpaired Student’s \u003cem\u003et\u003c/em\u003e test in all others.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/22a2f3f7979dd1ceee8dd3e6.png"},{"id":93130863,"identity":"a4706815-11fe-4ca8-8261-ddb75a258a9c","added_by":"auto","created_at":"2025-10-09 11:29:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":319913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlendronate-conjugated propranolol prevented bone metastasis in CIS-exposed mice\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) The schematic route for synthesis of alendronic acid-propranolol (AP). (\u003cstrong\u003eB\u003c/strong\u003e) The diagram of experimental design for treating CIS-exposed mice with placebo (BCD) and AP, twice/week, intraperitoneal injection. (\u003cstrong\u003eC\u003c/strong\u003e) Flow analysis of the frequencies of OPN-expressing cells in\u0026nbsp; BM cells of C57 mice treated with increasing doses of AP. (\u003cstrong\u003eD\u003c/strong\u003e) Survival rates of C57 mice with breast cancer bearing following the treatment of vehicle, propranolol, alendronic acid and AP. (\u003cstrong\u003eE\u003c/strong\u003e) The representative IVIS images showing the luciferin signal of metastatic breast cancer cells in mice treated with β-CD (vehicle), propranolol (5 mg/Kg) and AP (9.05 mg/Kg), and (\u003cstrong\u003eF\u003c/strong\u003e) analysis of the total flux of luciferin signals of each mice. (\u003cstrong\u003eG\u003c/strong\u003e) Tumor growth rate calculated by normalizing the total luciferin influx of tumor at Day 12, 24, 35 post tumor cell intratibial injection to that influx value at Day 7. n=8, 10, 10 mice for βCD-, Pro- and AP-treated groups, respectively. (\u003cstrong\u003eH\u003c/strong\u003e) Hematoxylin and eosin (H\u0026amp;E)-stained tibial paraffin sections showing the breast cancers (outlined with yellow dotted lines) in the metaphyseal area. Scale bar: 1 mm. (\u003cstrong\u003eI\u003c/strong\u003e) The area of breast cancer tissue in tibial metaphysis. n=8, 10 and 13 mice for βCD-, Pro- and AP-treated groups, respectively. (\u003cstrong\u003eJ\u003c/strong\u003e-\u003cstrong\u003eK\u003c/strong\u003e) The frequencies of OPN\u003csup\u003e+\u003c/sup\u003e monocyte and macrophage in BM from βCD-, Pro- and AP-treated mice using flow cytometry. n=4, 5 and 7 mice for these groups, respectively. (\u003cstrong\u003eL\u003c/strong\u003e-\u003cstrong\u003eM\u003c/strong\u003e) The frequencies of CD44\u003csup\u003e+\u003c/sup\u003e and iNOS\u003csup\u003e+\u003c/sup\u003e neutrophils in BM from βCD-, Pro- and AP-treated mice. (N) The levels of arginine in BM from βCD-, Pro- and AP-treated mice tested by ELISA. (O) The frequencies of CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells in BM. n= 4, 5 and 7 mice for βCD-, Pro- and AP-treated mice, respectively. Analysis: unpaired Student’s \u003cem\u003et\u003c/em\u003e test in (N); One-way ANOVA test with Tukey post analysis in all others.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/af522bcc79369e25fce409e5.png"},{"id":93131211,"identity":"9c0e2073-52b0-4adc-9cc8-0e954d78c9e8","added_by":"auto","created_at":"2025-10-09 11:37:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":294086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel showing how chronic stress reshapes the BM microenvironment via a neuro-immune-metabolic axis to facilitate breast cancer bone metastasis. \u003c/strong\u003eChronic stress activates the sympathetic nervous system, leading to the release of norepinephrine (NE) from nerve fibers in the bone marrow (BM) microenvironment. It triggers a pro-metastatic cascade involving the following steps: 1)\u003cstrong\u003e \u003c/strong\u003eActivation of monocytes/macrophages: elevated NE binds to adrenergic receptor β2 (ADRβ2) on the surface of monocytes/macrophages, which upregulates their expression of osteopontin (OPN). 2) Neutrophil-mediated immunosuppression: This elevated OPN then binds to its receptor, CD44, on neutrophils. This interaction boosts \u003cem\u003eNos2\u003c/em\u003e expression and accelerates arginine metabolism by neutrophils, thus generating a low-arginine, pro-metastatic BM microenvironment characterized by a loss of functional CD8+ cytotoxic T cells. This novel neuro-immune-metabolic axis is confirmed using mouse models with neutrophil specific depletion or global Adrβ2 knockout. To block the NE-ADRβ2 interaction, a bone-targeting conjugate of propranolol and alendronate (AP) was developed. AP administration effectively reduced OPN-positive monocytes/macrophages and iNOS-positive neutrophils, which restored BM arginine levels and CD8⁺ T cell populations, thereby inhibiting chronic stress-induced breast cancer bone metastasis.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/90e25fac1c7e755603e3ab0b.png"},{"id":93132320,"identity":"e95f4337-fca1-4148-8771-f39dc8b4240a","added_by":"auto","created_at":"2025-10-09 11:45:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4351253,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/0db2ed04-8ae5-4bd7-b781-c8ffe5fb94c6.pdf"},{"id":93131210,"identity":"62b2ed9a-f0e3-42c6-bdef-8523119e4aad","added_by":"auto","created_at":"2025-10-09 11:37:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6774262,"visible":true,"origin":"","legend":"Supplementary Figure 1, 2, 3, 4, 5, 6, 7 and 8","description":"","filename":"Suppl.FigsX.Li.etal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7760970/v1/0c7a6eaa5b8336b25d1cd23f.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Chronic stress reshapes bone marrow microenvironment to facilitate breast cancer bone metastasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer, the most common cancer among women, is diagnosed approximately 47.8 per 100,000 women globally\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The invasive ductal carcinoma (IDC), the most common breast cancer\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, often metastasizes to bone. Especially Luminal A breast cancer (ER/PR-positive, HER2-negative) has a higher propensity to metastasize to bone than other organs including brain, liver and lung\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, results in approximately 70 percent of patients with advanced breast cancer develop bone metastasis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Bone metastasis is generally incurable and significantly impact prognosis and outcomes, with affected patients having a median survival of 2 to 5 years\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Clinical evidence supports that the cancer patients with bone metastasis derive less benefit from immune checkpoint inhibitor (ICI) therapies\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In addition, skeletal-related events (SREs), including pathological fracture and spinal cord compression associated with bone metastasis, cause a significant increase in morbidity, hospitalization and even mortality\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBreast cancer patients often experience chronic stress, driven by concerns about disease progression and the burden of prolonged therapies, which can persist for weeks and years. Such chronic stress is associated with a higher risk of metastasis and poorer survival outcomes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Chronic stress activates key neuroendocrine systems, including the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, disrupting whole-body homeostasis\u0026mdash;particularly affecting cardiovascular and immune systems\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Chronic stress promotes primary tumor growth and remote metastasis, potentially by enhancing cancer cell proliferation, migrate, and dissemination\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, as well as by establishing a pro-metastatic niche in distant organs\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. A critical unresolved question is: for bone metastasis, which plays a more significant role\u0026mdash;chronic-stress-induced intrinsic changes in primary tumor cells or extrinsic modifications to the pro-metastatic niche? Under chronic stress, elevated levels of stress hormones (e.g., cortisol and catecholamines) may stimulate osteoblastic cells to produce RANKL, thereby promoting osteoclastic bone resorption\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Currently, denosumab, an anti-RANKL monoclonal antibody, and bisphosphonates are clinically used to prevent SREs in breast cancer patients with bone metastasis\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Denosumab has also been reported to enhance efficacy of ICI therapy in treating bone metastasis in mice\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, whether anti-resorptive drugs alone can prevent metastasis in cancer patients controversial\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, highlighting the need to develop therapeutic strategies targeting bone erosion-independent mechanism.\u003c/p\u003e\u003cp\u003eA growing body of mechanistic evidence has been provided to explain how chronic stress contributes to cancer metastasis. In the primary breast cancer environment, chronic stress can reduce natural killer (NK) cell activity, suppress T cells function, increase production of inflammatory cytokines such as TNFα, activate SNS to release norepinephrine (NE), and upregulate matrix metalloproteinases (MMPs) that degrade the extracellular matrix\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These alterations promote the growth, migration, invasion and dissemination of primary cancer cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Chronic stress also disrupts hematopoietic stem cell activity by inhibiting the environmental CXCL12 and induces monocytosis and neutrophilia in humans\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Monocytes from stressed mice and humans exhibit a characteristic inflammatory transcriptomic signature, which skews them to a primed hyperinflammatory phenotype\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Most recently, it is reported that chronic stress significantly alters the lung microenvironment by shifting normal circadian rhythm of neutrophils and elevating neutrophil extracellular trap (NET) formation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, far less is known about how chronic stress induces immune modifications to establish a pro-metastasis niche that supports the colonialization of disseminated cancer cells.\u003c/p\u003e\u003cp\u003eIn this study, we generated a chronic stress mouse model via chronic immobilization stress (CIS), in which CIS-exposed mice exhibited depression-like behaviors and increased release of NE in the bone marrow (BM). Our findings revealed that CIS enhanced osteopontin (OPN) expression in BM monocytes and macrophages, and that environmental OPN stimulated an excessive arginine metabolism in neutrophils through OPN/CD44 interaction, leading to a low-arginine bone microenvironment that impairs CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cell function. To intercept this pathological cascade at its onset, we developed a bone-targeting delivery strategy for propranolol to block NE/ADRβ signaling. Specifically, we synthesized alendronate-conjugated propranolol (AP) compound and evaluated its efficacy in preventing breast cancer bone metastasis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChronic stress remodels the BM microenvironment and promoted breast cancer bone metastasis\u003c/h2\u003e\u003cp\u003eTo investigate the impact of chronic stress on breast cancer bone metastasis, we generated a mouse model of chronic immobilization stress (CIS). BALB/c mice were subjected to chronic immobilization stress by confining them in 50 ml tubes for 2 hours per day, 6 days per week, for 6 consecutive weeks (Suppl. Figure\u0026nbsp;1A). We confirmed that CIS-exposed mice exhibited anxiety-like behavior (Suppl. Figure\u0026nbsp;1B-D) and accelerated orthotopic breast cancer growth compared to control (Ctrl) mice (Suppl. Figure\u0026nbsp;1E-H). To discern whether CIS primarily affects the intrinsic metastatic potential of breast cancer cells or the systemic microenvironment, a primary-secondary transplantation strategy was employed. 4T1 breast cancer cells, initially implanted orthotopically into the mammary fat pads of BALB/c mice previously exposed to CIS, were subsequently isolated from the orthotopic breast tumors and secondarily transplanted into untreated BALB/c mice via intracardiac injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In the primary implantation experiment, CIS-exposed mice demonstrated a higher incidence of remote metastasis to the lung, liver and bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Correspondingly, the size of bone metastatic lesions in the leg bones was significantly larger in CIS-exposed mice compared to Ctrl mice, as were lung metastases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Notably, in the secondary transplantation model, the incidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and size of remote metastases to bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, E) and lung (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, H) were comparable between recipients of CIS-derived and Ctrl-derived cancer cells. These results indicate that CIS primarily promotes breast cancer remote metastasis through alterations in the microenvironment of target organs, rather than inducing intrinsic changes in cancer cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm the role of CIS in enhancing bone metastasis across different mouse strains, luciferase-expression E0771 breast cancer cells were intracardially injected into Ctrl- and CIS-exposed C57BL6/J mice. Consistent with observations in BALB/c mice, CIS-exposed C57BL6/J mice exhibited a higher incidence of hind limb bone metastasis three weeks post cancer cell injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Luciferin flux, a quantifiable indicator of metastasis burden, was comparable between CIS and Ctrl groups at 1 and 2 weeks, but significantly elevated in CIS-exposed by week 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). Histological analysis of tibial paraffin sections further revealed significantly larger metastatic lesions in CIS-exposed C57BL6/J mice compared to Ctrl mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L).\u003c/p\u003e\u003cp\u003eIncreased bone turnover and osteoclastic bone resorption are known to promote breast cancer bone metastasis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, while serum levels of osteocalcin (OCN, an osteoblastic activity marker) and TRACP-5b (an osteoclastic bone resorption marker) were similar in CIS-exposed and Ctrl C57BL6/J mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM; Suppl. Figure\u0026nbsp;2A). Furthermore, the trabecular bone volume and the numbers of osteoblasts and osteoclasts in the tibial metaphysis were comparable between CIS-exposed and Ctrl mice (Suppl. Figure\u0026nbsp;2B-E). Interestingly, bone erosion activity, particularly osteoclast number and surface, was significantly reduced on trabecular and cortical bone surfaces adjacent to cancer cells (CC-adjacent) compared to non-CC-adjacent bone surfaces (Suppl. Figure\u0026nbsp;2F-I). These results indicate that CIS-promoted breast cancer bone metastasis does not rely on any increase in bone erosion. Of note, analysis of bone marrow (BM) immune cell composition revealed that CIS-exposed C57BL6/J mice displayed a reduced proportion of lymphoid lineage cells and an increased proportion of myeloid lineage cells in the BM compared to Ctrl mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). Specifically, percentages of B220\u003csup\u003e+\u003c/sup\u003e B cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells were significantly lower in CIS-exposed mice, while percentages of CD4\u0026thinsp;+\u0026thinsp;T cells remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO-R). Conversely, percentages of CD11b\u003csup\u003e+\u003c/sup\u003e leukocytes, including Ly6C⁺ monocytes and Ly6G⁺ neutrophils, were significantly higher in CIS-exposed mice, whereas F4/80⁺ macrophages showed no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS-V). These findings collectively suggest that CIS promotes breast cancer bone metastasis, associated with a distinct remodeling of the BM immune cell landscape.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eElevated norepinephrine release stimulates osteopontin expression by monocytes/macrophages in stress-exposed mice\u003c/h3\u003e\n\u003cp\u003eChronic stress is known to activate the sympathetic nervous system, leading to increased norepinephrine (NE) release in mice and humans\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Given the presence of sympathetic nerves within the BM\u003csup\u003e29\u003c/sup\u003e, NE levels in the interstitial fluid of hindlimb BM were measured and found to be significantly higher in CIS-exposed C57BL6/J mice compared to Ctrl mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To investigate the role of sympathetic nervous system activation in mediating CIS-induced BM microenvironment changes, a denervation mouse model was generated by dissociating the sciatic and femoral nerves\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Denervated mice exhibited significantly reduced BM NE levels at 12 weeks post-surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and a gradual reduction in trabecular bone volume and osteoclast number compared to sham-operated control mice (Suppl. Figure\u0026nbsp;3A-B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProteomic studies were next carried out to identify significantly changed BM proteins associated with CIS-exposure and sympathetic activation. KEGG analysis ranked \u0026ldquo;ECM-receptor interaction\u0026rdquo; as the most significantly altered pathway in both CIS-exposed versus Ctrl mice (with 18 significantly upregulated and 14 downregulated proteins; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E) and in denervated versus sham mice (among Top 10 enriched pathways; with 322 upregulated and 162 downregulated proteins; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). GO analysis further confirmed the upregulation of ECM-related pathways in BM of CIS-exposed mice compared to Ctrl mice (Suppl. Figure\u0026nbsp;3C-D). A comparative analysis revealed five proteins \u0026mdash; COL11a1 (collagen type XI a1), COL1a1 (collagen type I a1), FAM207a (nucleolar protein regulating ribosome biogenesis), OPN (osteopontin) and GIT1 (ARF GTPase-activating protein) \u0026mdash; that were differentially expressed in both CIS-exposed versus Ctrl mice, and in denervated versus sham mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Protein levels of COL11a1, COL1a1 and FAM207a were lower in CIS-exposed mice and higher in denervated mice compared to their respective controls. Notably, OPN exhibited an inverse expression pattern, being higher in CIS-exposed mice and lower in denervated mice compared to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eOPN, a glycoprotein involved in ECM-receptor interactions through binding with collagens, integrins and CD44, et al\u003csup\u003e31, 32\u003c/sup\u003e, was confirmed to be significantly higher in the BM of CIS-exposed mice and lower in denervated mice by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I), suggesting a positive correlation between BM NE and OPN. Immunofluorescence (IF) staining was carried out to determine cellular source of OPN and indicated clustered OPN\u003csup\u003e+\u003c/sup\u003e cell populations in the BM, which were significantly increased in CIS-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Flow cytometry corroborated this increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK; Suppl. Figure\u0026nbsp;3E), and identified monocytes (CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e) and macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e) as the primary OPN-expression cells in the BM, and these cells, but not neutrophils (CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e), were significantly increased in the BM of CIS-exposed compared to Ctrl mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN; Suppl. Figure\u0026nbsp;3F).\u003c/p\u003e\u003cp\u003eTo determine whether NE directly stimulates OPN expression in monocytes/macrophages, BM Ly6C\u003csup\u003e+\u003c/sup\u003e monocytes were isolated from Ctrl and CIS-exposed mice and treated with increasing doses of NE \u003cem\u003ein vitro\u003c/em\u003e. NE significantly stimulated \u003cem\u003eOpn\u003c/em\u003e gene transcription in monocytes in a dose-dependent manner, with a stronger response observed in monocytes from CIS-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). These findings demonstrate that CIS-induced elevated NE release enhances OPN expression in BM monocytic cells.\u003c/p\u003e\n\u003ch3\u003eExcessive OPN targets CD44 neutrophils to promote bone metastasis in stress-exposed mice\u003c/h3\u003e\n\u003cp\u003eTo elucidate the role of OPN in bone metastasis, a protein array comparing serum from breast cancer patients with newly diagnosed bone metastasis versus those without bone metastasis was performed. Patients with bone metastasis exhibited increased serum levels of OPN and IL-4, alongside reduced levels of CCL24, OPG and MIP-1b (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Notably, breast cancer patients with higher expression of OPN receptors, including ITGB5, ITGA5 and CD44, had significantly shorter bone metastasis-free survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting a role for environmental OPN-receptor interactions in promoting metastasis. Transwell cell migration assay demonstrated that NE-treated BM cells recruited significantly more E0771 cells compared to vehicle-treated controls, and this enhanced migration was partially abrogated by a CD44-blocking antibody in BM cells, implying a critical role for CD44\u003csup\u003e+\u003c/sup\u003e BM cells in promoting E0771 cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn CIS-exposed mice, CD44\u003csup\u003e+\u003c/sup\u003e BM cells were primarily enriched within the CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cell population. Sorted CD44\u003csup\u003e+\u003c/sup\u003e cells from CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells showed significantly higher transcriptional levels of \u003cem\u003eItgα5\u003c/em\u003e and \u003cem\u003eItgβ5\u003c/em\u003e compared to CD44\u003csup\u003e\u0026minus;\u003c/sup\u003e myeloid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), suggesting preferential co-expression of ITGA5 and ITGB5 with CD44 in these cells. Phenotypic analysis of CD44\u003csup\u003e+\u003c/sup\u003e BM cells revealed that approximately 70% of CD44\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e myeloid BM cells were Ly6C\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e neutrophils in both CIS-exposed and Ctrl mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Furthermore, the percentage of CD44\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e neutrophils in BM was significantly elevated in CIS-exposed mice, and over 90% of total neutrophils expressed CD44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK).\u003c/p\u003e\u003cp\u003eTo investigate the functional role of CD44\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e neutrophils in CIS-induced breast cancer bone metastasis, Ly6G\u003csup\u003eCreERT2\u003c/sup\u003eROSA26\u003csup\u003eDTA\u003c/sup\u003e mice (hereafter referred to as Ly6G-DTA mice), allowing for inducible neutrophil depletion, were utilized. Tamoxifen administration effectively depleted\u0026thinsp;\u0026gt;\u0026thinsp;99% neutrophils in the BM of Ly6G-DTA mice (Suppl. Figure\u0026nbsp;4A-C), in which osteoclastic activity was slightly increased (Suppl. Figure\u0026nbsp;4D). Following 3 weeks of CIS treatment and concurrent tamoxifen-induced neutrophil clearance, E0771 cells were transferred into the left ventricle, and metastasis was monitored for an additional 4 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Notably, bone metastasis was markedly reduced in Ly6G-DTA mice compared to ROSA26\u003csup\u003eDTA\u003c/sup\u003e (wild-type, WT) controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM-N), and Ly6G-DTA mice exhibited significantly longer overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO). Histomorphometry of tibial paraffin sections consistently showed significantly smaller metastatic breast cancer lesions in Ly6G-DTA mice compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP-Q). These findings suggest that neutrophils are a major BM immune cell population targeted by OPN, thereby contributing to the promotion of bone metastasis.\u003c/p\u003e\n\u003ch3\u003eChronic stress stimulates excessive arginine metabolism in neutrophils\u003c/h3\u003e\n\u003cp\u003eTo determine how CIS models the BM microenvironment via neutrophils, Ly6G\u003csup\u003e+\u003c/sup\u003e BM neutrophils were magnetically sorted and subjected to bulk mRNA sequencing (RNA-Seq). KEGG analysis identified \"Arginine and proline metabolism\" as the sole metabolism-related pathway among the top 10 enriched pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistently, transcriptional levels of arginase-2 (\u003cem\u003eArg2\u003c/em\u003e) and inducible nitric oxide synthase (iNOS, \u003cem\u003eNos2\u003c/em\u003e), key enzymes in arginine metabolism, were significantly higher in BM neutrophils from CIS-exposed mice compared to Ctrl mice, regardless of tumor burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMetabolic profiling further validated these findings. Principal component analysis (PCA) revealed strong internal consistency within CIS and Ctrl groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). KEGG analysis of differentially abundant metabolites highlighted \"D-arginine and D-ornithine metabolism\" as the most significantly altered pathway, with \"Arginine biosynthesis\" and \"Arginine and proline metabolism\" also ranking among the top 10 metabolism-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Among 1,135 identified metabolites, 5 were upregulated and 32 were downregulated (FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5 or FC\u0026thinsp;\u0026lt;\u0026thinsp;0.67, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Notably, arginine and DL-arginine levels were significantly lower in neutrophils from CIS-exposed mice compared to Ctrl mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). Additionally, arginine-containing di- and tri-peptides were reduced in CIS-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), suggesting that enhanced arginine metabolism and consumption drive the depletion of arginine in the BM microenvironment.\u003c/p\u003e\u003cp\u003eTo determine whether neutrophils contribute to arginine consumption under CIS, arginine levels were measured in the interstitial fluid of CIS-exposed WT and Ly6G-DTA mice bearing breast tumors. Arginine levels were significantly lower in CIS WT mice compared to Ctrl WT mice, but this reduction was completely reversed in CIS-exposed Ly6G-DTA mice with specific neutrophil depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). To further investigate whether OPN promotes arginine consumption by neutrophils through upregulating \u003cem\u003eArg2\u003c/em\u003e or \u003cem\u003eNos2\u003c/em\u003e expression, BM cells from WT and Ly6G-DTA mice were cultured with vehicle, OPN plus vehicle or arginine. Subsequent qPCR analysis of sorted neutrophils revealed that OPN treatment significantly increased transcriptional levels of \u003cem\u003eNos2\u003c/em\u003e and \u003cem\u003eArg1\u003c/em\u003e (but not \u003cem\u003eTnfα\u003c/em\u003e, \u003cem\u003eTgfβ1\u003c/em\u003e and \u003cem\u003eIfnγ\u003c/em\u003e) in WT neutrophils compared to vehicle-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). Consistently, flow cytometry analysis revealed a significantly higher frequency of iNOS\u003csup\u003e+\u003c/sup\u003e neutrophils in OPN-treated WT BM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ; Suppl. Figure\u0026nbsp;5A-B). Moreover, OPN treatment significantly reduced the frequencies of CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK) and IFNγ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL; Suppl. Figure\u0026nbsp;5C-D), and this reduction was effectively prevented by arginine supplementation or neutrophil depletion in Ly6G-DTA BM cells. These findings indicate that OPN stimulates arginine consumption by neutrophils, establishing a low-arginine BM microenvironment that suppresses CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells.\u003c/p\u003e\n\u003ch3\u003eNE/ADRβ2 axis sustains OPN-expressing monocytes/macrophages and neutrophil arginine metabolism\u003c/h3\u003e\n\u003cp\u003eTo investigate the mechanism by which NE induces OPN expression in monocytes/macrophages, transcription levels of NE receptors (\u003cem\u003eAdrβ1\u003c/em\u003e, \u003cem\u003eAdrβ2\u003c/em\u003e and \u003cem\u003eAdrβ3\u003c/em\u003e) in the BM cells from CIS-exposed and Ctrl mice were compared. \u003cem\u003eAdrβ2\u003c/em\u003e mRNA expression were significantly more abundant than other isoforms in both of CIS-exposed and Ctrl mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), suggesting ADRβ2 as the dominant NE receptor in BM. In global ADRβ2 knockout (Adrβ2-KO) mice, the percentages of myeloid lineage cells, such as neutrophils, monocytes and macrophages, were comparable to those in WT mice (Suppl. Figure\u0026nbsp;6A-D). However, the percentage of OPN\u003csup\u003e+\u003c/sup\u003e BM cells in Adrβ2-KO mice was significantly lower than in WT littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), with marked reductions observed particularly in OPN\u003csup\u003e+\u003c/sup\u003e monocytes (Ly6C\u003csup\u003e+\u003c/sup\u003e) and macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e), the two major OPN-expressing cell populations in BM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In addition, the percentages of total neutrophils and CD44\u003csup\u003e+\u003c/sup\u003e neutrophils in the BM of Adrβ2-KO mice were comparable to WT mice, while the percentage of iNOS\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e neutrophils was significantly lower in Adrβ2-KO mice than WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Consistently, BM arginine levels were higher in Adrβ2-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), reflecting reduced neutrophil-mediated arginine consumption.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmune cell profiling revealed that Adrβ2-KO mice exhibited significantly higher percentage of CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic and CD4\u003csup\u003e+\u003c/sup\u003e T cells in BM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK-L), with no significant differences observed in CD11b\u003csup\u003e+\u003c/sup\u003e leukocytes or B220\u003csup\u003e+\u003c/sup\u003e B cells, compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-J). Transwell cell migration assay confirmed that ADRβ2 mediates NE-dependent tumor cell attraction: NE-treated WT BM cells attracted more E0771 cells than vehicle-treated controls, but this effect was abolished in NE-treated Adrβ2-KO BM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM-N). Furthermore, NE-induced \u003cem\u003eOpn\u003c/em\u003e transcription in WT BM cells was largely blocked by propranolol, an FDA-approved non-selective adrenergic β receptor antagonist (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). These findings collectively indicate that ADRβ2 knockout or blockade inhibits OPN expression in BM cells, thereby disrupting the NE-driven cascade that promotes breast cancer cell migration.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBone-targeted delivery of propranolol inhibits chronic stress-induced bone metastasis\u003c/h2\u003e\u003cp\u003eAbove results suggest that blocking ADRβ2 in bone microenvironment represents a potential therapeutic strategy for preventing CIS-induced metastasis. To investigate this, we endeavored to synthesize a bone-targeting propranolol through generating an \u003cb\u003ea\u003c/b\u003elendronate-\u003cb\u003ep\u003c/b\u003eropranolol (AP) conjugate, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. Assessment of the toxicity and bone-targeting efficiency of AP confirmed that it exhibited high bone-specific accumulation (Suppl. Figure\u0026nbsp;7A-D) and no overt liver and kidney toxicity (Suppl. Figure\u0026nbsp;7E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the therapeutic efficiency of AP, CIS-exposed, tumor-bearing mice with intratibial injection of E0771 cells were treated with vehicle and AP twice weekly throughout the entire CIS-exposure period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Notably, the percentage of OPN\u003csup\u003e+\u003c/sup\u003e cells in the BM from AP-treated CIS-exposed mice was decreased in a dose-dependent pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). To compare the therapeutic potential of AP with propranolol and alendronate, CIS-exposed, tumor-bearing mice were treated with AP at an optimized dose of 9.05 mg/kg body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; Suppl. Figure\u0026nbsp;7E), or with equimolar concentrations of β-cyclodextrin (β-CD), propranolol, or alendronate as controls. Mice treated with alendronate or AP exhibited increased trabecular bone mass and a reduction in osteoclast number and surface compared to other two groups (Suppl. Figure\u0026nbsp;7F-G). In addition, the survival rate of AP-treated mice was significantly higher than that of CIS-exposed mice treated with vehicle, propranolol alone, or alendronate alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Luciferin flux, a quantifiable indicator of metastasis burden, was significantly lower in the tibiae of AP-treated mice compared to vehicle- or propranolol-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F). AP treatment also effectively suppressed tumor growth for up to 5 weeks post-intratibial injection of E0771 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Histomorphometry analysis revealed markedly smaller metastatic breast cancer lesions in the tibiae of AP-treated mice compared to vehicle- and propranolol-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-I).\u003c/p\u003e\u003cp\u003eImmunophenotyping of the BM revealed that AP-treated CIS-exposed, tumor-bearing mice exhibited significantly lower frequencies of OPN\u003csup\u003e+\u003c/sup\u003e monocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ), OPN\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK) and iNOS\u003csup\u003e+\u003c/sup\u003e neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM) in the BM compared to vehicle- and propranolol-treated mice. No significant differences were observed in the frequencies of total monocytes, macrophages, neutrophils or CD44\u003csup\u003e+\u003c/sup\u003e neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL; Suppl. Figure\u0026nbsp;8A-D). Consistent with these findings, BM arginine levels were elevated in AP-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN), in which the frequency of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the BM was significantly increased compared to vehicle-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO). Dendritic cells and B lymphocyte populations did not show a similar increase (Suppl. Figure\u0026nbsp;8F).\u003c/p\u003e\u003cp\u003eIn summary, the bone-targeted AP delivery effectively inhibited CIS-induced breast cancer bone metastasis. This effect was associated with reduced frequencies of OPN\u003csup\u003e+\u003c/sup\u003e monotypes/macrophages and iNOS\u003csup\u003e+\u003c/sup\u003e neutrophils, decreased arginine consumption, and a restored cytotoxic CD8\u0026thinsp;+\u0026thinsp;T cell population.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChronic stress, a pervasive factor in the lives of cancer patients, is increasingly recognized as a significant driver of tumor progression and distant metastasis, notably in bone-metastasis-prone cancers like breast cancer. The BM microenvironment, in particular, may offer a protective niche for disseminated cancer cells, potentially contributing to increased resistance to immune checkpoint inhibitors. Therefore, a comprehensive understanding of the mechanisms by which chronic stress promotes cancer bone metastasis and the development of targeted therapeutic strategies to counteract this process are imperative. This study unveils a novel neuro-immune-metabolic axis, by which chronic immobilization stress reshapes the BM niche, creating a low-arginine, pro-metastatic environment that impairs CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells and ultimately promotes breast cancer bone metastasis. Mechanistically, OPN emerges as a central mediator in this process. Elevated environmental NE, a consequence of chronic stress, stimulates OPN expression in BM monocytes/macrophages through ADRβ2 signaling. Subsequently, this increased OPN binds to its receptor, CD44, on neutrophils, which in turn upregulates NOS2 expression and accelerates arginine metabolism within these cells. This excessive arginine consumption by neutrophils and an exhaustion of CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells consequently establishes a low-arginine, pro-metastatic BM microenvironment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This novel neuro-immune-metabolic axis was further validated using neutrophil-specific depletion or global \u003cem\u003eAdrβ2\u003c/em\u003e knockout mouse models. To therapeutically prevent this NE-ADRβ-initiated cascade, we developed alendronate-conjugated propranolol (AP), a bone-targeting ADRβ antagonist. AP administration effectively restored BM arginine levels and prevented CIS-induced breast cancer bone metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Taken together, our findings not only offer critical insights into how chronic stress reshapes the BM microenvironment through neural-immune interactions but also propose a promising therapeutic strategy for mitigating this cascade in bone metastasis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCancer patients frequently experience profound psychological stress and sympathetic nerve system (SNS) activation, a response exacerbated by factors such as diagnosis, treatment implications, and disease progression. Tumors themselves can also initiate stress responses, activating anxiety-related circuits and further stimulating SNS\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Elevated SNS activity, mediated by catecholamines like NE and epinephrine (EPI), profoundly alters the tumor microenvironment, potentially enhancing primary tumor cell proliferation, survival, migration, invasion\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and neoangiogenesis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Moreover, SNS activation significantly modulates the tumor immune microenvironment, notably by suppressing anti-tumor immune responses through catecholamine release\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The bidirectional interaction between the tumor and SNS establishes a positive-feedback loop that accelerates cancer progression and metastasis. Intervening this loop could involve either suppressing the intrinsic invasive capacity of primary tumor cells or preventing the formation of pro-metastatic niche in distant organs. However, the relative contribution of these mechanisms to distant metastasis remains a critical area of investigation. Our orthotopic breast cancer model revealed that CIS significantly promoted primary tumor growth and enhanced breast cancer cell metastasis to bone, liver and lung. Crucially, \u003cem\u003ein vitro\u003c/em\u003e analyses demonstrated that cancer cells isolated from primary tumors in CIS-exposed mice exhibited comparable metastatic potential to those from Ctrl mice upon secondary transplantation. These findings collectively indicate that CIS primarily drives remote metastasis by remodeling pro-metastatic niches in distant organs, such as bone, via peripheral SNS activation or other stress-related factors, rather than by increasing the intrinsic invasive capacity of tumor cells.\u003c/p\u003e\u003cp\u003ePersistent elevation of catecholamines, such as NE and EPI, due to chronic stress exerts several detrimental effects on the immune system. This includes impairing the proliferation and function of T and B lymphocytes, which compromises T-cell-mediated immunity and antibody responses\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Elevated catecholamine disrupts the normal function of hematopoietic stem and progenitor cells (HSPCs) and natural killer (NK) cells, also drives the production of pro-inflammatory cytokines like IL-6 and IL-1β, which contributes to the pathogenesis of neurodegenerative and neuroinflammatory diseases\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In our study, we observed that CIS-exposed mice exhibited a significantly higher proportion of myeloid lineage cells (including monocytes and neutrophils) and a significantly lower proportion of lymphoid lineage cells (including CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells and B cells) in the BM compared to Ctrl mice. These findings indicate that CIS exposure increases the neutrophil-to-lymphocyte ratio in the BM, which may contribute to chronic stress-induced bone metastasis.\u003c/p\u003e\u003cp\u003eOur study identified OPN as a key mediator of bone metastasis associated with alterations in the BM immune system. OPN is a multifunctional glycoprotein known to promote cancer progression and therapy resistance via multiple mechanisms\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. These include enhanced angiogenesis, increased cancer cell survival, proliferation, invasion, and migration, recruitment of tumor-associated macrophages (TAMs), and PD-L1 upregulation to suppress anti-tumor T cell activation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. OPN has been reported to stimulate osteoclast differentiation, and it is also produced by osteoclasts to reprogram the extraosseous tumor microenvironment\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. While the role of OPN in primary tumor progression and invasion is well-documented, its function in forming distant pro-metastatic niches is less understood. In contrast to prior research, our results identified BM monocytes and macrophages, not cancer cells, as the primary source of OPN in the BM of metastatic bones. Given the scarcity of breast cancer cells anchored in the BM during early metastasis, cancer cells are unlikely to be the major source of OPN for establishing a pro-metastatic niche. The limited presence of TRAP\u003csup\u003e+\u003c/sup\u003e osteoclasts around breast cancer lesions in metastatic bones, suggesting osteoclasts might play a limited role in mediating CIS-induced bone metastasis. We demonstrate that chronic stress induces OPN upregulation specifically in BM monocytes and macrophages. This upregulation is mediated by NE activation of Adrβ2 isoform, as deletion of \u003cem\u003eAdrβ2\u003c/em\u003e resulted in significantly fewer OPN\u003csup\u003e+\u003c/sup\u003e monocytes/macrophages, and BM cells from \u003cem\u003eAdrβ2\u003c/em\u003e-KO mice failed to mediate tumor cell attraction in response to NE.\u003c/p\u003e\u003cp\u003eInnate and adaptive immunity are both essential for the body\u0026rsquo;s defense against cancer progression, with their interplay being crucial for effective anti-tumor response. For example, tumor-activated dendritic cells present tumor antigens to T cells\u0026mdash; a process vital for initiating anti-tumor immunity. A key question is how chronic stress disrupts this interplay between innate and adaptive immunity. Neutrophils, a distinctive type of innate immune cells, possess several defining features: they are the most abundant type of leukocytes, they rapidly response to physiological perturbations like infection, they have a short half-life ranging from hours to days, and they defend against pathogens through mechanisms such as neutrophil extracellular trap (NET) formation. Neutrophils also play a complex role in cancer. They can also release NETs to trap circulating cancer cells and promote their adhesion to distant organs\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Conversely, neutrophils may generate reactive oxidative species (ROS) and granule proteins like elastase and MPO to kill cancer cells\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. While the role of neutrophils in stress-induced metastasis remained largely unknown until recently, when it was reported that glucocorticoids released during chronic stress induce NET formation, establishing a lung metastasis-promoting microenvironment in breast cancer-bearing mouse models\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In our study, we first demonstrated that inducible depletion of Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophils effectively prevented bone metastasis in breast cancer-bearing mice. Through metabolomics analysis and bulk mRNA sequencing, we further found that chronic stress significantly enhanced arginine metabolism in neutrophils. Notably, over 90% neutrophils express CD44 on their surface, suggesting an intimate interplay between CD44\u003csup\u003e+\u003c/sup\u003e neutrophils and OPN\u003csup\u003e+\u003c/sup\u003e monocytes/macrophages, though the mechanisms by which OPN promotes arginine metabolism in neutrophil requires further investigation.\u003c/p\u003e\u003cp\u003eClinical evidence indicates that lower plasma arginine levels correlate with higher tumor burden and poor prognosis\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. T cells are vulnerable to a low-arginine microenvironment due to their low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In our model, excessive arginine consumption by neutrophils drives a low-arginine BM microenvironment in CIS-exposed mice, and this effect was effectively reversed in Ly6G-DTA mice with a specific depletion of neutrophils. This reduction in BM arginine is associated with a significant decrease in CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells, likely because arginine is essential for T cell activation, survival and proliferation, and its deficiency induces T cell anergy\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. We also observed that the genes \u003cem\u003eArg2\u003c/em\u003e and \u003cem\u003eNos2\u003c/em\u003e (but not \u003cem\u003eArg1\u003c/em\u003e) were significantly upregulated in BM neutrophils from CIS-exposed mice, regardless of tumor burden. Our findings, therefore, address how chronic stress disrupts the interplay between innate and adaptive immunity. Chronic stress activates a cascade involving innate immunity cells\u0026mdash;specifically OPN\u003csup\u003e+\u003c/sup\u003e monocytes/macrophages and CD44\u003csup\u003e+\u003c/sup\u003e neutrophils\u0026mdash;leading to the generation of a low-arginine BM microenvironment. This microenvironment subsequently suppresses CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells, disrupting the effective anti-tumor immune response.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe conjugation of therapeutic agents to bisphosphonates (BPs) is a highly effective strategy for achieving selective drug accumulation in bone\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Due to their stable P-C-P backbone and strong affinity for hydroxyapatite in bone, bisphosphonate-drug conjugates have been extensively explored for applications in chemotherapy, radiotherapy, immune modulation, and nanoparticle delivery for the treatment of bone-related conditions, including osteoporosis and bone cancers (primary and metastatic tumors)\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Propranolol, a β-adrenergic receptor blocker, is widely used to treat various cardiovascular and neurological conditions by blocking β1 and β2 adrenergic receptors. In our study, we observed that \u003cem\u003eAdrβ2\u003c/em\u003e transcription levels were higher than those of the other two isotypes (\u003cem\u003eAdrβ1\u003c/em\u003e and \u003cem\u003eAdrβ3\u003c/em\u003e). Global \u003cem\u003eAdrβ2\u003c/em\u003e knockout mice had fewer OPN-expressing monocytes/macrophages and iNOS-expressing neutrophils, along with higher BM arginine levels, compared to WT mice. Propranolol was found to effectively prevented NE-induced OPN expression, supporting its potential to inhibit the OPN/arginine metabolism axis and CIS-induced bone metastasis. To enable bone-specific delivery of propranolol, we conjugated it to alendronate via a urethane (carbamate) bond to generate the conjugate AP. We hypothesized that this conjugate would be cleaved in the acidic BM microenvironment, achieving bone-specific delivery of propranolol. Consistent with our hypothesis, CIS-exposed mice treated with AP exhibited significantly fewer OPN-expressing monocytes/macrophages and iNOS-expressing neutrophils, higher BM arginine levels, and an increase in CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells, compared to vehicle-treated, CIS-exposed mice. Notably, bone metastatic lesions were significantly smaller in AP-treated, CIS-exposed mice. Basing on these findings, we propose that AP could effectively block the CIS-induced NE/ADRβ axis activation, thereby inhibiting bone metastasis not only in breast cancer but also in other cancers prone to bone metastasis under chronic stress conditions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThis study acknowledges certain limitations. Direct evidence for the causal role of OPN\u003csup\u003e+\u003c/sup\u003e monocytes and macrophages in chronic stress-induced bone metastasis would benefit from a conditional knockout mouse model with inducible \u003cem\u003eOpn\u003c/em\u003e deletion in the monocyte/macrophage lineage (e.g., LyzM\u003csup\u003eCreER\u003c/sup\u003eOpn\u003csup\u003efl/fl\u003c/sup\u003e mice). Additionally, given the systemic distribution of sympathetic nerves and neutrophils, the mechanisms identified here for bone metastasis may also contribute to stress-induced metastasis in other organs or in other bone-metastasis-prone cancers (e.g., lung and prostate cancers), warranting further investigation beyond the scope of the current study.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperimental animals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight- to ten-week-old BALB/c and C57BL/6J female mice were purchased from Beijing Huafukang Biotechnology Co., LTD. and Beijing Charles River Laboratories (CRL), respectively. Mice were kept in an SPF barrier animal center. Except under special experimental conditions, mice had free access to sterilized food and water. Ly6G\u003csup\u003eCreER\u003c/sup\u003eROSA26\u003csup\u003eDTA\u003c/sup\u003e (Ly6G-DTA) mice were generated by crossing Ly6G\u003csup\u003eCreER\u0026nbsp;\u003c/sup\u003emice with ROSA26\u003csup\u003eDTA\u003c/sup\u003e mice. Four-month-old Ly6G-DTA female mice and wildtype (Ly6G\u003csup\u003eCreER\u003c/sup\u003e) control female mice were restricted for 3 weeks and then implanted with tumor cells. To deplete Ly6G-expressing neutrophils, tamoxifen (100 mg/Kg body weight) in corn oil were intraperitoneally injected into Ly6G-DTA mice and, 3 doses per week for another 4 weeks. Subsequently, the mice were sacrificed and the efficiency and specificity of Ly6G-expressing neutrophil depletion was confirmed. During the period of observation, the survival curve was recorded and the growth of metastatic tumors were analyzed. Eight-week-old C57BL/6J female mice were purchased from Charles River Laboratories. Adrenal β2 and β3 receptor knockout (Adrb2- and Adrb3-KO) mice were purchased from Shanghai Model Organisms Center, Inc. Mice were raised in a SPF-level barrier animal center. Except under special experimental conditions, mice had free access to food and water, which were sterilized. The feeding environment conditions: 12-hour light-dark cycle, ambient temperature of 22 °C -26 °C, and relative humidity of 50%-60%. The bedding was changed twice a week, and the cages, water bottles and bedding were sterilized by high temperature and high pressure. All animal experiments were carried out according to the protocol approved by the Experimental Animal Ethics Committee of Hebei Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eReagents\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe breast cancer cell lines used in this experiment were 4T1-Luciferase, derived from BALB/c mice, and E0771-Luciferase, derived from C57BL/6J mice. Both cell lines were purchased from Yuanjing Biotechnology Co., Ltd. (Wuhan, China) and cryopreserved in liquid nitrogen storage upon receipt and initial amplification.\u003c/p\u003e\n\u003cp\u003eFluorophore-conjugated primary antibodies were used to detect cell-surface antigens. The following antibodies were purchased from eBioscience (San Diego, CA, USA): FITC-conjugated anti-mouse CD4 (Catalog #11-0041-82), Violet 421-conjugated anti-mouse B220 (Catalog #62-0452-82), Violet 650-conjugated anti-mouse CD41a (Catalog #64-0411-82), PE-conjugated anti-mouse CD11b (Catalog #12-0012-82). The following antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA): PE/CF594-conjugated anti-mouse IgM (Catalog #562565), PerCP/Cy5.5-conjugated anti-mouse CD44 (Catalog #560570), Violet 510-conjugated anti-mouse CD8 (Catalog #740155), Violet 605-conjugated anti-mouse Ly6C (Catalog #563011), Violet 786-conjugated anti-mouse CD11c (Catalog #568973), APC-conjugated anti-mouse Ly6G (Catalog #560599), APC/R700-conjugated anti-mouse F4/80 (Catalog #565787), APC/Cy7-conjugated anti-mouse Ter119 (Catalog #560509). Ly6G-Biotin antibody (Catalog #127603) was also purchased from eBioscience (San Diego, CA, USA). Streptavidin microbeads (Catalog #130-048-101) for cell sorting were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). For flow cytometry analysis, bone marrow cells were stained using the fluorophore-conjugated antibodies listed above. Antibody solutions were prepared at a 1:100 dilution in FACS buffer (Phosphate-Buffered Saline (PBS) containing 2% fetal bovine serum). The staining was performed on ice and in the dark, with antibody solutions prepared and used immediately.\u003c/p\u003e\n\u003cp\u003eThe primary antibody for Osteopontin (OPN) (Catalog #AF808) was purchased from R\u0026amp;D Systems (Minneapolis, MN, USA), and the FITC-conjugated secondary antibody (Catalog #ab150129) was acquired from Abcam (Cambridge, UK). Mouse L-Arginine (L-Arg) ELISA Kit (Catalog #FT-P9S1390X) for the measurement of arginine levels in bone marrow supernatant was purchased from Fantaibio (Shanghai, China). \u003cstrong\u003e\u003cem\u003eSolutions\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreparation of sugar water preference experiment for mouse behavior experiment: weigh 10 g of sucrose and dissolve it in 1 L of mouse drinking water. Shake it well to fully dissolve it, and get 1% sucrose drinking water. Preparation of matrix gel solution for mouse breast cancer orthotopic tumor implantation: the ABW high-concentration matrix gel used in the construction of mouse orthotopic breast cancer model is stored in a refrigerator at -20 °C. Take it out and put it in an ice box the day before use, and put the ice box in a refrigerator at 4 °C to thaw overnight. The matrix gel is in liquid state on the day of use. Please note that it should be operated on ice during use. When the temperature exceeds 10 °C, the matrix gel will solidify. Preparation of mouse breast cancer tumor tissue digestion solution: prepare 100× tissue digestion solution stock solution: 100 mg/ml collagenase IV, 20 mg/ml DNase I, and then dilute 100 times with RPMI-1640 medium with 5% fetal bovine serum to make the working concentration 1 mg/ml collagenase IV and 200 μg/ml DNase I. Preheat the 1× digestion solution in a 37 °C water bath for use. Preparation of mouse bone tissue decalcification solution: first, dissolve 100 g EDTA powder in 800 ml PBS solution, and use a magnetic stirrer to assist dissolution. At this time, the EDTA powder cannot be completely dissolved. Add sodium hydroxide powder to the solution in small amounts and multiple times, and measure the pH value of the solution at the same time. When the pH value is adjusted to 7.2, the EDTA powder is completely dissolved. Finally, use PBS solution to make the volume 1 L. Store at room temperature for use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConstruction of mouse restraint model and behavioral tests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePut the mouse into the barrel of a 50 ml syringe and insert the needle core to fix it at the 50 ml mark. Before the experiment, drill about 20 small holes with a diameter of 5 μm on the side wall of the syringe so that the mouse can fully ventilate in the syringe. The mouse can move freely in this space but cannot run or jump. The mice were deprived of food and water during the restraint process. The mice in the control group were also deprived of food and water during the same period, but were not restrained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCIS model: restraint for 2 hours at a fixed time every day, restrain for 6 consecutive days a week, and not restrain on the 7th day. Restrain for 6 consecutive weeks to complete the construction of a complete model. After the entire restraint period, the mice were subjected to behavioral tests. RS model: restraint for 6 hours at a fixed time period every day, and a complete model is constructed for 8 consecutive days. After the restraint period is over, the mice are subjected to behavioral tests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe experimental steps of the behavioral test are briefly described as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSucrose preference test (SPT): The first 24 hours are the experimental adaptation phase. Two water bottles are filled with ordinary drinking water and 1% sucrose drinking water respectively. The initial weight of the two bottles of water is recorded before placement. The positions of the two water bottles are changed at the 12th hour (to avoid differences in drinking sugar water due to horizontal position preference), and the weight of the remaining water in the two bottles is finally recorded after 24 hours. After the adaptation phase, the formal experimental phase of 24 hours begins. The positions of the water bottles are also changed once every 12 hours in the middle. The weight of the initial and 24 hours after the water bottles are recorded respectively, and then the amount of water consumed by the mice is calculated according to the formula. Finally, the data of the formal experimental phase are used as the results for statistics. Sugar water preference rate calculation formula: sugar water intake / (sugar water intake + ordinary drinking water intake) × 100%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTail suspension test (TST): 2 h before the experiment, transfer mice from the animal breeding room to the behavioral testing laboratory to allow them to adapt to the environment in advance. Before starting the experiment, adjust the equipment such as the hanging rod, camera, and recording system software. Use medical tape to fix the mouse tail on the hanging rod so that its head hangs down, and the fixed position is about 1 cm away from the tip of the mouse tail. The tail suspension time of each mouse is recorded for 6 min, and the immobility time of the mouse in the last 4 min is counted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eForced swimming test (FST): Prepare a transparent swimming bucket with a diameter of 10 cm and a height of 25 cm, and inject 22 °C-25 °C sterilized water into the bucket so that the water surface reaches 15 cm. 2 h before the formal start of the experiment, transfer mice from the animal breeding room to the behavioral testing laboratory to allow them to adapt to the environment in advance. During the experiment, gently put the mouse into the water and start recording the image and time. Each mouse swims for a total of 6 min, and the immobility time of the mouse in the water in the last 4 min is counted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eELISA assay for norepinephrine (NE)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter mouse sacrifice, the leg bones of the left hind limb including tibiae and femora from the CIS-exposed mice, the denervation mice and their respective control mice, were harvested and the muscle tissue were cleaned off from the bones. Remove off the knee joint cartilage from the femora and tibiae, and flush the bone marrow cells out from a tibia and a femur from an individual mouse using 300 μl of pre-cooled PBS in 1 ml syringe. Peptide up and down the bone marrow cell clots for several times, and mildly push the cells through a 25G needle to make single-cell suspension. The cells were next centrifuged at 4 °C and 3000 rpm for 5 min. The supernatant obtained was the bone marrow interstitial fluid. According to the instructions of the ELISA kit, the bone marrow interstitial fluid sample was diluted by 2.5 times for final measurement. For example, 40 μl bone marrow interstitial fluid was mixed with 60 μl sample diluent provided by the ELISA kit for further assay. The assay procedure was strictly carried out according to the manufactory instructions of the kit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn brief, we first extracted and acetylated NE in the samples and the standard. Add the standard and diluted samples to a specific well plate, add TE buffer, cover with film, and shake on a shaker (20 °C to 25 °C, 200 rpm) for 60 min. After washing, add 100 μl Acylation buffer and 25 μl Acylation reagent to each well, shake the plate and then add 100 ul HCl and shake the plate for 10 min. To perform the enzyme catalysis, mixed 90 μl acetylated NE solution above with 25 μl enzyme solution, and incubated at 37 °C for 2 hours. After this incubation, we next added 100 μl of this incubated liquid into designed plates and then added 50 μl NE antiserum, incubated at 4 °C overnight. After washing, add 100 μl Enzyme conjugate, cover the plate with a membrane and shake the plate for 30 min. After washing, add 100 μl substrate and shake the plate in the dark for 20-30 min, then add 100 μl stop solution. Within 10 min after the reaction, use a microplate reader to measure the absorbance (optical density, OD) value of each well at 450 nm and 620 nm. Draw a standard curve based on the OD value of the standard, according to which, the concentrations of NE in each sample were calculated. Duplicate wells were set up for the all standards and samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBreast cancer cell culture and in situ model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1- and E0771-Luciferase cells were cultured in RPMI-1640 medium and DMEM high-glucose medium (containing 10% FBS and 1% penicillin-streptomycin), respectively. These cells were seeded in T75 culture flasks and maintained in 37 °C incubator with 5% CO2, and 70% humidity. To confirmed the expression and activity of luciferase in 4T1- and E0771-Luciferase cells, these cells were counted and seeded in a 96-well cell culture plate at 5×10\u003csup\u003e4\u003c/sup\u003e, 2.5×10\u003csup\u003e4\u003c/sup\u003e, 1.2×10\u003csup\u003e4\u003c/sup\u003e, 0.6×10\u003csup\u003e4\u003c/sup\u003e, 0.3×10\u003csup\u003e4\u003c/sup\u003e cells per well, and the wells with no cell seeded as blank control. Four replicates were set up for each cell concentration. After the cells were plated, they were placed in an incubator for 2-3 hours to allow the cells to adhere to the wall. Thereafter, 10 μl of substrate potassium luciferin (15 mg/ml) was added to each well and incubated for 2 minutes before imaging using the IVIS imaging system under the cell shooting mode. The luminescence values of individual cells were calculated. It is generally believed that cells with a luminescence value of \u0026gt;175 photons/s/cell can be used for in vivo transplanted tumor experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4T1- and E0771-Luciferase cell lines were seeded in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin in T75 culture flasks under conditions of 37 °C, 5% CO2, and 70% humidity. 4T1 cells in the exponential growth phase as they reached 70%-80% of coverage, were rinsed with 4 ml PBS once and then digested using 2 ml 0.25% trypsin solution. When 4T1 and E0771 cells are digested in culture incubator for 2-3 minutes in trypsin, 4 ml complete culture medium was added to terminate the digestion, collected 4T1 and E0771 cells in 15 ml centrifuge tube, and span down in a centrifuge at 1500 rpm for 5 min. The collected tumor cells were next counted and resuspended evenly in appropriate amount of liquid ABW high-concentration matrix gel on ice. The procedure of an in-situ breast cancer model construction presented as following: a small animal gas anesthesia machine to inhale isoflurane through the mask of the mouse for gas anesthesia after 2 weeks of chronic restraint treatment. After the mouse enters deep anesthesia, fix it in a supine position on the operating table, and remove the hair and disinfect it with iodine on the skin of the fourth breast area on the left side. Use a micro-syringe (range 50 μl) to draw 20 μl of matrix gel cell suspension containing 5×10\u003csup\u003e5\u003c/sup\u003e cells and slowly inject it into the fat pad of the fourth mammary gland on the left side of the mouse. After the operation, the mice were continually maintained in the SPF mouse feeding room and imaged using in-vivo IVIS system to observe the growth of the in-situ tumor and the metastasis to distant organs every week. At the same time, the tumor volume was measured using a vernier caliper and recorded every week. To the estimate the volume of tumor, the longest diameter of the tumor as the long diameter was calculated, and the short diameter perpendicular to the long diameter of the tumor was measured as well, and the tumor volume was calculated according to the formula: tumor volume = 1/2 × long diameter × short diameter\u003csup\u003e2\u003c/sup\u003e. The tumor growth was observed after tumor cell in-situ implantation for 4-5 weeks when the size of in-situ tumors reached 1 cm\u003csup\u003e3\u003c/sup\u003e, and the mice were sacrificed and the required tumor samples were obtained. To observe the tumor cell bone metastasis, 100 μl of cell suspension containing 2×10\u003csup\u003e5\u003c/sup\u003e cells and slowly inject it into the left ventricle of recipient mice. When bright red arterial blood is observed to rush into the syringe in a pulsed manner, it proves that the needle tip has entered the left ventricle, then stop inserting the needle and slowly inject the tumor cell suspension. The recipient mice were continually maintained in the SPF mouse feeding room and imaged using in-vivo IVIS system to observe the metastasis to bones and other distant organs weekly for 3 weeks in total.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAssay of cell migration using Transwell system\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow cell preparation: Five-month-old \u003cem\u003eAdrb2\u003c/em\u003e global knockout (KO) female mice and wildtype female littermates were sacrificed and the leg bons from these mice were moved off. Maximally cleaned the excess skin and muscle tissue and flushed out the bone marrow cells with pre-cooled PBS, lysed the red blood cells, and counted the bone marrow cells. The \u003cem\u003eAdrb2\u003c/em\u003e-KO or WT bone marrow cells were next suspended in three different culture mediums as following: 1) Control medium: RPMI-1640 medium containing 1% penicillin-streptomycin, 1% NEAA and 20 ng/ml M-CSF; 2) NE medium: Control medium containing extra 50 μM NE; 3) NE+anti-CD44 medium: Control medium containing extra 50 μM NE and 10 μg/ml anti-CD44 antibody. 5×10\u003csup\u003e5\u003c/sup\u003e bone marrow cells in 600 μl different culture medium were next evenly seeded in each well of 24-well plate, and duplicate wells were set up for each condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTumor cell preparation: As the growth density of E0771-Luc cells reached about 70%-80%, the cells were digested with 2 ml 0.25% trypsin solution per culture flask at 37 °C incubator for 3 minutes, and then 4 ml complete culture medium (containing 10% FBS) was added to terminate the digestion. The cells were span down, counted, and resuspended in DMEM culture medium. Subsequently, gently put the transwell chambers into the 24-well-plate wells seeded with bone marrow cells, and add 3×10\u003csup\u003e4\u003c/sup\u003e E0771-Luc cells in 200 μl culture medium to each chamber, and put the Transwell culture system into the incubator (37 °C, 5% CO2, and 70% humidity) for 24 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlate staining and observation: As the incubation completed, discarded the culture medium, rinsed the chambers and bottom wells with PBS for two times, fixed the cells with 4% PFA at room temperature for 10 minutes, and discarded the fixation solution and rinsed with PBS for 3 times. Subsequently, add 100 μl 0.1% crystal violet stain into each the upper chamber and incubate at room temperature for 10 minutes, then discarded the staining solution and rinsed the chambers with ddH\u003csub\u003e2\u003c/sub\u003eO for 3 times. The cells on the bottom surface of the chamber were gently wiped clean with cotton swabs, and the E0771-Luc cells that migrated to the outer layer of the chamber were observed under an inverted microscope and photographed for further statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSecondary cardiac transplantation of orthotopic tumor cells\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe orthotopic tumor lesions were aseptically stripped and excised, removed of non-tumor cell tissues such as necrotic sites and fascia, and then approximate 5 mm\u003csup\u003e3\u003c/sup\u003e tumor tissues were removed from the orthotopic tumor lesions of each mouse. The tumor tissues were dipped in 0.5 ml RPMI-1640 culture medium in 1.5 ml centrifuge tube and cut into tissue slurry on ice as much as possible with sterile ophthalmic scissors. The tissue slurry was transferred to the pre-prepared tissue digestion solution in 15 ml tubes and placed in an incubator at 37 °C for enzymatic digestion, and vortexed every 10 minutes for 3 times to sufficient isolation of tumor cells. To terminate enzymatic digestion, the tubes containing tumor cells were added 5 ml RPMI-1640 medium containing 10% FBS, mixed well and stored on ice. The isolated tumor cells were next filtered using a 70 μm cell strainer and washed with pre-cooled RPMI-1640 basic medium for 2 times. The isolated tumor cells were span down in a centrifuge at 4 °C, 1500 rpm for 5 min, counted and taken the required cells and placed on ice for later intracardiac injection. To anesthetize recipient mice, the isoflurane gas was pumped and constantly supplied to an inhale mouse mask using a small animal gas anesthesia machine from RWD. After the mouse enters deep anesthesia, fix it on the operating table in a supine position, and remove the hair and disinfect the skin of the precardiac area with iodine. Use an insulin syringe (range 1 ml) to draw 100 μl of cell suspension containing 2×10\u003csup\u003e5\u003c/sup\u003e cells and slowly inject it into the left ventricle. When bright red arterial blood is observed to rush into the syringe in a pulsed manner, it proves that the needle tip has entered the left ventricle, then stop inserting the needle and slowly inject the tumor cell suspension. The recipient mice were continually maintained in the SPF mouse feeding room and imaged using in-vivo IVIS system to observe the metastasis to bones and other distant organs weekly for 3 weeks in total.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMonitoring tumor growth using in-vivo imaging system (IVIS)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore the IVIS test, 15 mg/ml potassium luciferin solution should be made by dissolving potassium luciferin powder in sterile PBS and filtering with a 0.2 μm filter. This solution could be aliquoted and stored in dark at -80 °C, and thaw it in dark at RT before use. In terms of imaging by IVIS, injected potassium luciferin solution into the mouse intraperitoneally at a dose of 100 μl/10 g body weight, and wait for 15 min until the luminescence value entering the plateau phase, and the mice were imaged using IVIS. During this process, the mice are anesthetized by inhalation of isoflurane in the induction box and then continuously inhaled by mask. After in-vivo imaging, the results are quantitatively analyzed using Living Image software. Briefly, the in-situ lesions or bone metastasis lesions of each mouse were circled first, then a unified quantitative analysis of the luminescence value was performed. The in-situ lesions were located at the fourth mammary gland on the right side of the mouse. The most common bone metastasis lesions were the proximal end of the tibia and the distal end of the femur. Therefore, the fluorescence around the knee joint was determined to be bone metastasis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistomorphometry analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the mice being sacrificed, mouse tissues, such as tibia, femur, lung and liver, were harvested immediately and placed in sufficient 4% paraformaldehyde (4% PFA) fixative solution at 4 °C for 48 hours. After fixation, the tissues were rinsed 3 times with PBS solution to clean off the fixative. The soft tissues including the mouse lung and liver were dehydrated overnight using the soft tissue dehydration program of the dehydrator, and then embedded in paraffin blocks for further section. After fixation, the leg bones were then proceeded to decalcification using 10% EDTA solution at 4 °C for 2-3 weeks, and this solution were changed with fresh decalcification solution weekly. After decalcification, the leg bones were gently rinsed 3 times with PBS solution and dehydrated following a hard tissue dehydration program, and then embedded in paraffin blocks for further section. All paraffin blocks were sectioned with a thickness of 4 μm using Minux S700 slicing machine from RWD. Before staining, the paraffin slices were baked in an oven at 65 °C for 30 minutes to fully melt the paraffin on the glass slices.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor H\u0026amp;E staining, the procedure as following: Briefly, the sections were soaked in xylene for 4 minutes × 3 times, then in 100% ethanol, 95% ethanol, 90% ethanol, 85% ethanol, and 75% ethanol in order for 2 minutes for each, respectively. The sections were next rinsed gently in tap water for 2 min, stained in hematoxylin staining solution for 30-60 s (adjust the staining time according to the degree of staining of the cell nucleus), rinsed in tap water for 5 minutes, then soaked in ammonium hydroxide solution for 15 seconds and washed with deionized water for 2 minutes. The sections were next stained in eosin staining solution for about 30 seconds, decolorize with 95% ethanol for 3 minutes, with 100% ethanol for 1 minute × 2 times, and soaked in xylene for 4 minutes × 3 times, and finally sealed with permount mounting medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunofluorescence staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTibial samples of CIS model and control mice were fixed with 4% PFA for 48 hours, and transferred into 10% EDTA solution at 4 °C for decalcification for 21 days. After decalcification, the samples were dehydrated in a tissue dehydrator, embedded in paraffin and sectioned with a thickness of 4 μm using a rotary paraffin microtome from RWD (Shenzhen, China). Before staining, the paraffin sections were baked at 65 °C for 30 minutes, rehydrated with xylene and gradient alcohol step by step. The sections were slowly rinsed with running water for 5 minutes and washed three times with ddH2O to check whether the dewaxing was complete. Antigen retrieval: Set the water bath to 98 °C, add 200 ml Tris-EDTA retrieval solution (pH=8.0) to a slide staining box, gently place the dewaxed slides into the staining box, and dipped the box in the water bath. As the temperature of retrieval solution in the staining box reached 90 °C, start to count for 10 minutes, thereafter lifted the staining box and dipped into running tap water to cool down gradually to room temperature. Washing the slides with PBS for three times, drew a wax circle on the slice using a wax circle pen, incubated tissue on slides with 0.3% Triton-PBS for 15 minutes to punch the cell membrane. Added 20% donkey serum in 0.3% Triton-PBS to block at room temperature for 1 hour. After 3 washes with PBS, added OPN primary antibody solution (AF808; R\u0026amp;D system, 1:100 diluted), and incubate overnight at 4 °C in a wet box. On the second day, the slides in the wet box were continually incubated at room temperature for 30 minutes, and washed with 0.5% PBST for three times, 5 minutes/time. Subsequently, FITC-labeled donkey anti-Goat secondary antibody (A16000; Novex by life technologies, 1:100 diluted) was added and incubated at room temperature for 1 hour. After three washes with 0.5% PBST, the slides were sealed with mounting medium with DAPI (H1200-10; Vector laboratories) and kept in dark at 4 °C, and scanned them for further histomorphometry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eELISA assay for osteocalcin (OCN) and tartrate-resistant acid phosphatase 5b (TRAP5b)\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare mouse serum, mouse peripheral blood was collected into 1.5 ml EP tubes and stand at room temperature over 4 hours until it coagulated naturally. The serum, light yellow supernatant liquid, was slowly aspirated and moved to a new EP tube and stored in a -80°C refrigerator for long-term storage until it is used for testing. Before ELISA tests, thaw the serum sample on ice and warm up the required reagents in the ELISA kit to room temperature. After sufficient vortex, 70 μl of serum samples were mixed with 140 μl sample diluent, and add 100 μl of this diluted serum samples to each well of antibody pre-coated ELISA plates.\u003c/p\u003e\n\u003cp\u003eStandard preparation: centrifuged the powder in Standard tube at 12000 rpm for 1 minute, added 1 ml of standard diluent, vortexed gently and let it stand for 10 minutes. To draw standard curve, dilute 50 ng/ml standard to 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.13 ng/ml, 1.57 ng/ml, 0.78 ng/ml in order, and add equal volume of standard diluent as a blank control. As the various standard and diluted samples added, the ELISA plate was covered with film firmly, and incubated at 37 °C for 90 minutes. Shake dry the sample solutions in the wells, add 100 μl of biotinylated antibody working solution, cover the plate with film, and incubate at 37 °C for 60 minutes. Drain the biotinylated antibody solution in the wells, pat dry on absorbent paper, then add 350 μl washing solution, soak for 1 min, shake off the washing solution and pat dry, repeat the above washing steps 3 times. Then add 100 ul enzyme conjugate working solution to the each well, cover the plate with film, incubate at 37 °C for 30 min. Drain the liquid and wash 5 times with washing solution. Add 90 μl of substrate solution to each well, cover the plate with film, incubate at 37 °C in the dark for 15 min, then add 50 μl of reaction stop solution to each well, and immediately measure the absorbance value at 450 nm with an enzyme reader. Draw a standard curve based on the OD value of the standard and obtain the calculation formula. Then substitute the OD value of the sample into the formula to calculate the concentration of the samples for statistical analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDenervation mouse model\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe anesthetized mice were placed on the operating table in a prone position, maintained in anesthesia via inhalation of isoflurane, depilated the fur at the junction of the left hind limb and the back and disinfected this site with iodine. About 0.5 cm longitudinal incision was cut at the root of the left hind limb using ophthalmic scissors. The muscles were peeled off along the muscle texture and fascia, and the sciatic nerve was easily found underneath. To sever the sciatic nerve, it was cut off about 0.3 cm using ophthalmic scissors. The muscle position was restored, the incision skin was sutured and disinfected with iodine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mice were then placed in a supine position, and the junction of the left hind limb and the abdomen was depilated and disinfected with iodine. About 0.5 cm oblique incision was cut at the junction of the abdomen and the left hind limb (roughly at the left groin). The muscles were peeled off at the groin to find the femoral nerve underneath, and a 0.3 cm-long section was cut off from the femoral nerve. The muscle position was restored, the incision skin was sutured and disinfected with iodine. After the operation, turn off the gas anesthesia machine, remove the mouse mask, and wait for the mouse to wake up and observe if there is no abnormality before putting them back to the feeding room. In order to reduce the impact of the operation on the mouse's mobility, we only performed nerve severance on the left hind limb to ensure that the mouse's eating and drinking activities were not affected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein mass spectrum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described above, the bone marrow cells of one leg bone from each mouse were flushed out and centrifuged at 4 °C, 3000 rpm for 3 minutes. During the operation, special attention was paid to prevent the sample contamination from mouse hairs to avoid of the introduction of high-abundance proteins such as keratin in this assay. Briefly, lyse the red blood cells in the bone marrow cells efficiently using 1 ml lysis buffer, on ice for 2 minutes, to avoid of the potential influence of hemoglobin. The cells were next centrifuged at 4 °C and 3000 rpm for 3 minutes, discarded the supernatant, and resuspended in 250 μl of 8M urea solution (containing 1:100 diluted protease inhibitor cocktail). Use an ultrasonic cell crusher with a small probe to break the cells with parameters as following: the power was 100 w, it worked for every 5 s and followed by paused for 5 s, and lasted for 1 minute. During the process of protein extraction, the EP tubes containing cells were placed in an ice-water mixture to continuously cool down to prevent the protein from degradation. After protein extraction, the protein concentration was determined using BCA method. Before mass spectrum assay, SDS-PAGE gel electrophoresis was typically performed. Briefly, 10 μg of each protein sample was loaded and separated in 10% concentration SDS-PAGE gel. As the electrophoresis was done, stain the gel with 1× Coomassie brilliant blue dye, and observe the bands of each sample, to preliminarily determine the molecular weight of high-abundance proteins and the protein degradation, and to confirm the protein quantification. Finally, 30 μg protein solution of each sample was applied in the protein mass spectrum assay. Before detection, the isobaric chemical tag (TMT) of the tandem mass spectrometer of Thermo Fisher was used for protein labeling. After obtaining the results, the data was imported into the Lianchuan Biological Cloud Platform (https://www.omicstudio.cn/) to analyze the different expression of proteins between the experimental and the control groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorrelation analysis on GEO data\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GSE2603 gene expression dataset (sequencing platform: Affymetrix Human Genome U133A Array) was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The GSE2603 dataset contains gene expression information of surgical resection specimens of breast cancer patients and corresponding clinical information of patients, including age, tumor volume, estrogen receptor expression status, progesterone receptor expression status, human epidermal growth factor receptor 2 (HER2) expression status, metastasis status including bone and lung metastasis, metastasis-free survival, et. al. The patients were grouped according to the median value of gene expression. The high and low expression groups were defined as above and below the median value, respectively. The correlation between \u003cem\u003eOpn\u003c/em\u003e, integrin α and β family proteins, such as \u003cem\u003eCd44\u003c/em\u003e, \u003cem\u003eItgb1\u003c/em\u003e, \u003cem\u003eItgb3\u003c/em\u003e, \u003cem\u003eItgb5\u003c/em\u003e, \u003cem\u003eItgb7\u003c/em\u003e and \u003cem\u003eItga5,\u003c/em\u003e and the bone metastasis-free survival of breast cancer patients. R (version: 4.0.3) software and \"Survival\" and \"Survminer\" packages were applied in this analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMetabolomic assay\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bone marrow cells flushed out from a tibia plus a femur with 300 μl pre-cooled PBS. After red blood cell lysis, 5×10\u003csup\u003e6\u003c/sup\u003e cells from each mouse were aliquoted and sent to Shanghai APTBIO Co., Ltd. for non-targeted metabolomic assay. After the frozen cell samples were slowly thawed at 4 °C, appropriate amounts of samples were added to pre-cooled methanol/acetonitrile/water solution (2:2:1, v/v/v), vortexed, sonicated at low temperature for 30 minutes, left at -20 °C for 10 minutes, centrifuged at 14,000 g for 20 minutes at 4 °C, and the supernatant was vacuum dried. For mass spectrometry analysis, 100 μl of acetonitrile aqueous solution (acetonitrile: water = 1:1, v/v) was added to resolute the sample, vortexed, centrifuged at 14,000 g for 15 minutes at 4 °C, and took the supernatant for analysis of the different expression of metabolites from bone marrow cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNeutrophil sorting and RNASeq\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old C57BL/6J female CIS and control mice were sacrificed after 6 weeks of CIS treatment. Bone marrow cells from a femur plus a tibia from these mice were collected and resuspended in FACS buffer (PBS containing 2% FBS) after red blood cell lysis. 4×10\u003csup\u003e7\u003c/sup\u003e cells from each mouse were used for neutrophil magnetic sorting. 2 μl of anti-Ly6G primary antibody was added to every 1×10\u003csup\u003e7\u003c/sup\u003e cells in 100 μl staining solution. Incubated at 4 °C for 30 minutes, centrifuged at 1500 rpm for 5 minutes, and washed twice with FACS buffer. Resuspended the cells with 380 μl FACS buffer + 20 μl beads (5 μl beads/1×10\u003csup\u003e7\u003c/sup\u003e cells), incubated at 4 °C for 20 minutes, washed twice with FACS buffer, and resuspended in 1 ml FACS buffer for sorting, according to the instructions of the commercial Miltenyi LS separation columns. Briefly, install the magnetic separation column on the magnetic pole, rinse it with 3 ml pre-cooled PBS, and add the prepared cell suspension in when the PBS is about to drip off. \u0026nbsp;As the cell suspension is about to drip off, add 3 ml FACS buffer into the column to rinse it, and repeat this rinse twice. During the separation process, be careful to avoid air drying of the column, otherwise bubbles will be generated. After the rinse buffer drips off, take off the separation column away from the magnetic pole, change a new 15 ml tube for cell collection, fill the column with 5 ml FACS buffer, and use the tube core to push out the cells. These collected cells are positively selected neutrophils. Count the Ly6G-positive cells, dissolve 2×10\u003csup\u003e6\u003c/sup\u003e sorted cells in 1 ml Trizol, and store it in a -80 °C refrigerator for further transcriptome sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFlow cytometry\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs the mice being sacrificed, the hind limbs were removed and optimally cleaned off the soft tissue, the ends of the femora and tibiae were cut using ophthalmic scissors to expose the bone marrow cavity. The bone marrow in these bones were flushed out from bone cavity in 300 μl pre-cooled PBS solution using a 1 ml sterile syringe. Pulse-flush the bone cavity for 3 times to ensure maximally harvest the bone marrow cells, spin down the cells at 4 °C, 3000 rpm for 3 minutes, and carefully aspirate the supernatant and transfer it into a new EP tube as bone marrow interstitial fluid and freeze it in a -80 °C refrigerator for later use. Add 1 ml of red blood cell lysis buffer to the cell pellet and lyse it on ice for 2 minutes, then add 5 ml PBS containing 2% FBS (so-called FACS buffer) and spin down these cells at 4 °C, 3000 rpm for 3 minutes, discard the supernatant, and resuspend the cell pellet in 3-5 ml FACS buffer. After counting the cells, take 5×10\u003csup\u003e6\u003c/sup\u003e cells in each 1.5 ml EP tube for further flow antibody staining. Fluorophore-conjugated antibodies were diluted by 1:100 in antibody diluent, and 100ul of the antibody mixed solution were added into each sample and incubated at 4 °C in the dark for 30 minutes. For OPN staining, after the staining for cell surface markers, the stained cells were permeabilized and stained with 0.2 μg/μl primary anti-mouse OPN antibody (AF808; R\u0026amp;D system) at room temperature for 1 hour, and then stained with FITC-conjugated donkey anti-goat IgG (secondary antibody) was diluted by 1:200 and incubated at 37 °C in the dark for 60 minutes. A sample incubated with FITC-conjugated secondary antibody only, but not primary antibody, were applied as a negative control for OPN expression. Another sample with all surface antibodies stained, but not OPN, was applied as FMO (full minus one) control for OPN measurement. After primary and secondary antibody staining, the bone marrow cells were washed 2 times with 1 ml FACS buffer, centrifuge at 4 °C, 3000 rpm for 3 minutes, discard the supernatant, resuspend the cell pellet with 200 μl FACS buffer and stored at 4°C in dark for further analysis using Beckman Cytoflex. Data were analyzed using FlowJo. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePrimary monocyte culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter lysing red blood cells, bone marrow cells were resuspended in FACS buffer, add 2 μl anti-Ly6C antibody in every 1×10\u003csup\u003e7\u003c/sup\u003e cells, incubate at 4 °C for 30 minutes, and wash the cells twice with FACS buffer. Resuspend cells with 250 μl FACS buffer containing 12.5 μl microbeads for every 1×10\u003csup\u003e7\u003c/sup\u003e cells, incubate at 4 °C for 20 minutes, wash the cells twice with FACS buffer and finally resuspend the cells with 1 ml FACS buffer for further magnetic sorting. The procedure of cell sorting was the same with neutrophil sorting mentioned above. Count the sorted Ly6C-positive cells, and seed 1×10\u003csup\u003e6\u003c/sup\u003e cells in each well of 12-well plate. Every 1×10\u003csup\u003e6\u003c/sup\u003e cells resuspended in 2 ml α-MEM medium containing 10% FBS, 1% penicillin-streptomycin, 1% NEAA, and 20 ng/ml M-CSF. Three duplicated wells were set up for each treatment condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003emRNA extraction and real-time qPCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdd 1 ml TRIzol to the bone marrow cells or cultured monocytes and mix them by pipetting and place on ice in a fume hood for 5 minutes. Then add 0.2 ml chloroform to each sample, shake vigorously for 15 seconds, and let it stand at room temperature for 5 minutes. Centrifuge at 13000 rpm for 15 min at 4 °C, take 0.4 ml of the upper layer of the aqueous liquid and transfer it to a new 1.5 ml EP tube. Add 0.4 ml of isopropanol to each sample, gently invert and mix for about 10 times, and place still at room temperature for 10 minutes. Centrifuge at 13000 rpm for 10 min at 4 °C to see white RNA precipitate, discard the supernatant and invert the tubes on filter paper to air dry the RNA precipitates. Add 1 ml of 75% ethanol and gently invert to wash away impurities, centrifuge at 7500 rpm for 5 min at 4 °C, discard the supernatant and invert the tubes on filter paper to air dry the RNA precipitates. Add 20-50 μl of DEPC water to dissolve RNA according to the amount of RNA precipitates, measure the concentration using Nanodrop, and judge the RNA contamination and degradation according to the parameters such as OD260/280 value. After digestion with DNase I, 1 μg extracted mRNA was proceeded to reverse transcription to obtain cDNA using a reverse transcription kit (RR047A; Takara). Subsequently, the expression levels of target genes were tested in the cDNA in 25 μl reaction system using a Takara qPCR kit (820A; Takara). The qPCR primers are listed as following: \u003cem\u003eAdrb1\u003c/em\u003e: forward, 5’-CTCATCGTGGTGGGTAACGTG-3’ and reverse, 5’-ACACACAGCACATCTACCGAA-3’; \u003cem\u003eAdrb2\u003c/em\u003e: forward, 5’-GGGAACGACAGCGACTTCTT-3’ and reverse, 5’-GCCAGGACGATAACCGACAT-3’; \u003cem\u003eAdrb3\u003c/em\u003e: forward, 5’-GGCCCTCTCTAGTTCCCAG-3’ and reverse, 5’-TAGCCATCAAACCTGTTGAGC-3’; \u003cem\u003eOpn\u003c/em\u003e: forward, 5’-CCCGGTGAAAGTGACTGATTC-3’ and reverse, 5’-ATGGCTTTCATTGGAATTGC-3’; and Gapdh: forward,5’-GGTCGGTGTGAACGGATTTG-3’ and reverse, 5’-ATGAGCCCTTCCACAATG-3’. The qPCR assay was performed using Thermal Cycler Dice Real Time System from Bio-Rad. The reaction was amplified according to the instrument standard program, with the denaturation at 95 °C and the annealing at 55 °C for 40 cycles. The 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCT\u003c/sup\u003e method was used to calculate gene expression levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMetastatic model treated with bone-targeted propranolol\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell preparation: E0771-Luc cells in the exponential growth phase were digested with 0.25% trypsin, span down to obtain cell precipitates, resuspended with sterile PBS, counted the cells and made single-cell suspension solution with a concentration of 5×10\u003csup\u003e6\u003c/sup\u003e E0771-Luc cells/ml. The cell solution was placed on ice for immediate use. Eight-week-old female C57BL/6J CIS-exposed mice were anesthetized and fixed in a supine position, and disinfected with iodine on both knee joints. 20 μl prepared E0771-Luc cell solution (containing 1×10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells) was in situ injected into bilateral tibial bone marrow cavity using an insulin syringe. During the injection, kept the tibia and femur perpendicular to each other, and slowly inserted the needle tip of the insulin syringe into the tibia in a direction perpendicular to the tibial plateau. A clear breakthrough feeling was the sign that the needle has successfully entered the bone marrow cavity. After that, slowly injected the cell solution into the bone marrow cavity and slowly withdrawn the needle tip after 2 seconds to prevent tumor cells from overflowing from the needle channel. After tumor cell implantation, the recipient mice were continuously treated with CIS, and intraperitoneally injected with alendronic acid-propranolol (9.05 mg/kg body weight) and vehicle (equal volume of solvent) twice a week [5, 6] (on the 1st and 4th days of each week) for 5 weeks. The tumor growth was monitored via IVIS imaging once a week. At the end of observation, mice were killed and tissue samples were collected for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data were processed using GraphPad Prism 9.0 software, and the results were presented as mean ± standard deviation. The unpaired \u003cem\u003et-test\u003c/em\u003e was used for statistical analysis of the two groups, and One-way ANOVA analysis with Tukey repeated measures were applied for the comparison of one parameter among multiple groups, and the Two-way repeated measures ANOVA was used for repeated detection of multiple time points in the two groups. P\u0026lt;0.05 indicated statistical difference. For the analysis of flow cytometry, the raw data were exported from the instrument, analyzed and exported with FlowJo software, and then statistical analysis was performed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch reported in this publication was supported by the National Natural Science Foundation of China under Award Numbers 81702865 (to X.L.), 82172469, 82472481, 32100916 (to J.L.),\u0026nbsp;82071533 and 82374058\u0026nbsp;(to H.Z.), and by the Natural Science Foundation of Hebei Province under Award Number H2025206343 (to J.L.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX. Li coordinated the studies, performed and analyzed the experiments shown in all Figures except Figs. 3B-D, 4G-L, 5G, 6A-G and 6N, and wrote the paper; X. Liu generated the bone-targeting propranolol, tested the delivery specificity and toxicity, and performed and analyzed the experiments shown in Figures in Figs. 6A-G; X. Zhang performed and analyzed the experiments shown in Figures in Figs. 3B-D, 4G-L, 5G and 6N; J. Fan sectioned all paraffin blocks and blindly analyzed the histological parameters shown in Figs. 1C-E, 1K-L, 3P-Q and 5H-I. J. Fan and Y. Xing generated Ly6G\u003csup\u003eCreER\u003c/sup\u003eR26\u003csup\u003eDTA\u0026nbsp;\u003c/sup\u003emice and ADRβ2 knockout mice, respectively, and analyze the mouse phenotype; H. Jiao analyzed the data generated by flow cytometry; P. Li drew the Fig. 7 and all experimental design sketches; D. Kong carried out the protein mass spectrum assay and instructed the data analysis; J. Qi instructed the process of alendronate-propranolol conjugation; X. Du supervised and coordinated the studies, and revised the paper. J. Li. and H. Zhang supervised, conceived, and coordinated the studies, and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are available from the authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLei, S.; Zheng, R.; Zhang, S.; Wang, S.; Chen, R.; Sun, K.; Zeng, H.; Zhou, J.; Wei, W. Global patterns of breast cancer incidence and mortality: A population-based cancer registry data analysis from 2000 to 2020. \u003cem\u003eCancer Commun (Lond) \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e41\u003c/em\u003e (11), 1183-1194. DOI: 10.1002/cac2.12207.\u003c/li\u003e\n\u003cli\u003eOnkar, S.; Cui, J.; Zou, J.; Cardello, C.; Cillo, A. R.; Uddin, M. R.; Sagan, A.; Joy, M.; Osmanbeyoglu, H. U.; Pogue-Geile, K. L.; et al. Immune landscape in invasive ductal and lobular breast cancer reveals a divergent macrophage-driven microenvironment. \u003cem\u003eNat Cancer \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e (4), 516-534. DOI: 10.1038/s43018-023-00527-w.\u003c/li\u003e\n\u003cli\u003eParkes, A.; Clifton, K.; Al-Awadhi, A.; Oke, O.; Warneke, C. L.; Litton, J. K.; Hortobagyi, G. N. Characterization of bone only metastasis patients with respect to tumor subtypes. \u003cem\u003eNPJ Breast Cancer \u003c/em\u003e\u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e, 2. DOI: 10.1038/s41523-018-0054-x.\u003c/li\u003e\n\u003cli\u003eBuijs, J. T.; van der Pluijm, G. Osteotropic cancers: from primary tumor to bone. \u003cem\u003eCancer Lett \u003c/em\u003e\u003cstrong\u003e2009\u003c/strong\u003e, \u003cem\u003e273\u003c/em\u003e (2), 177-193. DOI: 10.1016/j.canlet.2008.05.044.\u003c/li\u003e\n\u003cli\u003eGkikopoulou, E.; Syrigos, C. C.; Mantogiannakou, I.; Petraki, C. E.; Stathopoulou, M.; Dragolia, M.; Rinotas, V.; Ntafis, V.; Rauner, M.; Douni, E. RANKL Drives Bone Metastasis in Mammary Cancer: Protective Effects of Anti-Resorptive Treatments. \u003cem\u003eInt J Mol Sci \u003c/em\u003e\u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e26\u003c/em\u003e (11). DOI: 10.3390/ijms26114990.\u003c/li\u003e\n\u003cli\u003eWang, R.; Zhu, Y.; Liu, X.; Liao, X.; He, J.; Niu, L. The Clinicopathological features and survival outcomes of patients with different metastatic sites in stage IV breast cancer. \u003cem\u003eBMC Cancer \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e19\u003c/em\u003e (1), 1091. DOI: 10.1186/s12885-019-6311-z.\u003c/li\u003e\n\u003cli\u003eJiao, S.; Subudhi, S. K.; Aparicio, A.; Ge, Z.; Guan, B.; Miura, Y.; Sharma, P. Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e179\u003c/em\u003e (5), 1177-1190 e1113. DOI: 10.1016/j.cell.2019.10.029 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eHuang, J. F.; Shen, J.; Li, X.; Rengan, R.; Silvestris, N.; Wang, M.; Derosa, L.; Zheng, X.; Belli, A.; Zhang, X. L.; et al. Incidence of patients with bone metastases at diagnosis of solid tumors in adults: a large population-based study. \u003cem\u003eAnn Transl Med \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e8\u003c/em\u003e (7), 482. DOI: 10.21037/atm.2020.03.55.\u003c/li\u003e\n\u003cli\u003eChida, Y.; Hamer, M.; Wardle, J.; Steptoe, A. Do stress-related psychosocial factors contribute to cancer incidence and survival? \u003cem\u003eNat Clin Pract Oncol \u003c/em\u003e\u003cstrong\u003e2008\u003c/strong\u003e, \u003cem\u003e5\u003c/em\u003e (8), 466-475. DOI: 10.1038/ncponc1134 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eMoreno-Smith, M.; Lutgendorf, S. K.; Sood, A. K. Impact of stress on cancer metastasis. \u003cem\u003eFuture Oncol \u003c/em\u003e\u003cstrong\u003e2010\u003c/strong\u003e, \u003cem\u003e6\u003c/em\u003e (12), 1863-1881. DOI: 10.2217/fon.10.142 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eDai, S.; Mo, Y.; Wang, Y.; Xiang, B.; Liao, Q.; Zhou, M.; Li, X.; Li, Y.; Xiong, W.; Li, G.; et al. Chronic Stress Promotes Cancer Development. \u003cem\u003eFront Oncol \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e, 1492. DOI: 10.3389/fonc.2020.01492 From NLM PubMed-not-MEDLINE.\u003c/li\u003e\n\u003cli\u003eYaribeygi, H.; Panahi, Y.; Sahraei, H.; Johnston, T. P.; Sahebkar, A. The impact of stress on body function: A review. \u003cem\u003eEXCLI J \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e16\u003c/em\u003e, 1057-1072. DOI: 10.17179/excli2017-480 From NLM PubMed-not-MEDLINE.\u003c/li\u003e\n\u003cli\u003eObradovic, M. M. S.; Hamelin, B.; Manevski, N.; Couto, J. P.; Sethi, A.; Coissieux, M. M.; Munst, S.; Okamoto, R.; Kohler, H.; Schmidt, A.; et al. Glucocorticoids promote breast cancer metastasis. \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e567\u003c/em\u003e (7749), 540-544. DOI: 10.1038/s41586-019-1019-4 From NLM Medline.\u003c/li\u003e\n\u003cli\u003ePeinado, H.; Zhang, H.; Matei, I. R.; Costa-Silva, B.; Hoshino, A.; Rodrigues, G.; Psaila, B.; Kaplan, R. N.; Bromberg, J. F.; Kang, Y.; et al. Pre-metastatic niches: organ-specific homes for metastases. \u003cem\u003eNat Rev Cancer \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e17\u003c/em\u003e (5), 302-317. DOI: 10.1038/nrc.2017.6 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eYirmiya, R.; Goshen, I.; Bajayo, A.; Kreisel, T.; Feldman, S.; Tam, J.; Trembovler, V.; Csernus, V.; Shohami, E.; Bab, I. Depression induces bone loss through stimulation of the sympathetic nervous system. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e\u003cstrong\u003e2006\u003c/strong\u003e, \u003cem\u003e103\u003c/em\u003e (45), 16876-16881. DOI: 10.1073/pnas.0604234103 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eGuo, Q.; Chen, N.; Qian, C.; Qi, C.; Noller, K.; Wan, M.; Liu, X.; Zhang, W.; Cahan, P.; Cao, X. Sympathetic Innervation Regulates Osteocyte-Mediated Cortical Bone Resorption during Lactation. \u003cem\u003eAdv Sci (Weinh) \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e (18), e2207602. DOI: 10.1002/advs.202207602 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eColeman, R.; Finkelstein, D. M.; Barrios, C.; Martin, M.; Iwata, H.; Hegg, R.; Glaspy, J.; Perianez, A. M.; Tonkin, K.; Deleu, I.; et al. Adjuvant denosumab in early breast cancer (D-CARE): an international, multicentre, randomised, controlled, phase 3 trial. \u003cem\u003eLancet Oncol \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e21\u003c/em\u003e (1), 60-72. DOI: 10.1016/S1470-2045(19)30687-4 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eStopeck, A. T.; Lipton, A.; Body, J. J.; Steger, G. G.; Tonkin, K.; de Boer, R. H.; Lichinitser, M.; Fujiwara, Y.; Yardley, D. A.; Viniegra, M.; et al. Denosumab compared with zoledronic acid for the treatment of bone metastases in patients with advanced breast cancer: a randomized, double-blind study. \u003cem\u003eJ Clin Oncol \u003c/em\u003e\u003cstrong\u003e2010\u003c/strong\u003e, \u003cem\u003e28\u003c/em\u003e (35), 5132-5139. DOI: 10.1200/JCO.2010.29.7101 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eTerpos, E.; Confavreux, C. B.; Clezardin, P. Bone antiresorptive agents in the treatment of bone metastases associated with solid tumours or multiple myeloma. \u003cem\u003eBonekey Rep \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e, 744. DOI: 10.1038/bonekey.2015.113 From NLM PubMed-not-MEDLINE.\u003c/li\u003e\n\u003cli\u003eCathomas, F.; Lin, H. Y.; Chan, K. L.; Li, L.; Parise, L. F.; Alvarez, J.; Durand-de Cuttoli, R.; Aubry, A. V.; Muhareb, S.; Desland, F.; et al. Circulating myeloid-derived MMP8 in stress susceptibility and depression. \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e626\u003c/em\u003e (8001), 1108-1115. DOI: 10.1038/s41586-023-07015-2 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eKhan, A.; Song, M.; Dong, Z. Chronic stress: a fourth etiology in tumorigenesis? \u003cem\u003eMol Cancer \u003c/em\u003e\u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e24\u003c/em\u003e (1), 196. DOI: 10.1186/s12943-025-02402-x From NLM In-Process.\u003c/li\u003e\n\u003cli\u003eHeidt, T.; Sager, H. B.; Courties, G.; Dutta, P.; Iwamoto, Y.; Zaltsman, A.; von Zur Muhlen, C.; Bode, C.; Fricchione, G. L.; Denninger, J.; et al. Chronic variable stress activates hematopoietic stem cells. \u003cem\u003eNat Med \u003c/em\u003e\u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e20\u003c/em\u003e (7), 754-758. DOI: 10.1038/nm.3589 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eBarrett, T. J.; Corr, E. M.; van Solingen, C.; Schlamp, F.; Brown, E. J.; Koelwyn, G. J.; Lee, A. H.; Shanley, L. C.; Spruill, T. M.; Bozal, F.; et al. Chronic stress primes innate immune responses in mice and humans. \u003cem\u003eCell Rep \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e36\u003c/em\u003e (10), 109595. DOI: 10.1016/j.celrep.2021.109595 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eHe, X. Y.; Gao, Y.; Ng, D.; Michalopoulou, E.; George, S.; Adrover, J. M.; Sun, L.; Albrengues, J.; Dassler-Plenker, J.; Han, X.; et al. Chronic stress increases metastasis via neutrophil-mediated changes to the microenvironment. \u003cem\u003eCancer Cell \u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e42\u003c/em\u003e (3), 474-486 e412. DOI: 10.1016/j.ccell.2024.01.013 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eClezardin, P.; Coleman, R.; Puppo, M.; Ottewell, P.; Bonnelye, E.; Paycha, F.; Confavreux, C. B.; Holen, I. Bone metastasis: mechanisms, therapies, and biomarkers. \u003cem\u003ePhysiol Rev \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e101\u003c/em\u003e (3), 797-855. DOI: 10.1152/physrev.00012.2019 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eWang, L.; Fang, D.; Xu, J.; Luo, R. Various pathways of zoledronic acid against osteoclasts and bone cancer metastasis: a brief review. \u003cem\u003eBMC Cancer \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e20\u003c/em\u003e (1), 1059. DOI: 10.1186/s12885-020-07568-9 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eChu, B.; Marwaha, K.; Sanvictores, T.; Awosika, A. O.; Ayers, D. Physiology, Stress Reaction. In \u003cem\u003eStatPearls\u003c/em\u003e, 2025.\u003c/li\u003e\n\u003cli\u003eHansen, J. L.; Carroll, J. E.; Seeman, T. E.; Cole, S. W.; Rentscher, K. E. Lifetime chronic stress exposures, stress hormones, and biological aging: Results from the Midlife in the United States (MIDUS) study. \u003cem\u003eBrain Behav Immun \u003c/em\u003e\u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e123\u003c/em\u003e, 1159-1168. DOI: 10.1016/j.bbi.2024.10.022 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eKatayama, Y.; Battista, M.; Kao, W. M.; Hidalgo, A.; Peired, A. J.; Thomas, S. A.; Frenette, P. S. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2006\u003c/strong\u003e, \u003cem\u003e124\u003c/em\u003e (2), 407-421. DOI: 10.1016/j.cell.2005.10.041 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eKamiya, A.; Hayama, Y.; Kato, S.; Shimomura, A.; Shimomura, T.; Irie, K.; Kaneko, R.; Yanagawa, Y.; Kobayashi, K.; Ochiya, T. Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression. \u003cem\u003eNat Neurosci \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e22\u003c/em\u003e (8), 1289-1305. DOI: 10.1038/s41593-019-0430-3 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eSong, Z.; Chen, W.; Athavale, D.; Ge, X.; Desert, R.; Das, S.; Han, H.; Nieto, N. Osteopontin Takes Center Stage in Chronic Liver Disease. \u003cem\u003eHepatology \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e73\u003c/em\u003e (4), 1594-1608. DOI: 10.1002/hep.31582 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eYim, A.; Smith, C.; Brown, A. M. Osteopontin/secreted phosphoprotein-1 harnesses glial-, immune-, and neuronal cell ligand-receptor interactions to sense and regulate acute and chronic neuroinflammation. \u003cem\u003eImmunol Rev \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e311\u003c/em\u003e (1), 224-233. DOI: 10.1111/imr.13081 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eHong, Y.; Zhang, L.; Liu, N.; Xu, X.; Liu, D.; Tu, J. The Central Nervous Mechanism of Stress-Promoting Cancer Progression. \u003cem\u003eInt J Mol Sci \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e23\u003c/em\u003e (20). DOI: 10.3390/ijms232012653.\u003c/li\u003e\n\u003cli\u003eCole, S. W.; Nagaraja, A. S.; Lutgendorf, S. K.; Green, P. A.; Sood, A. K. Sympathetic nervous system regulation of the tumour microenvironment. \u003cem\u003eNat Rev Cancer \u003c/em\u003e\u003cstrong\u003e2015\u003c/strong\u003e, \u003cem\u003e15\u003c/em\u003e (9), 563-572. DOI: 10.1038/nrc3978 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eThaker, P. H.; Han, L. Y.; Kamat, A. A.; Arevalo, J. M.; Takahashi, R.; Lu, C.; Jennings, N. B.; Armaiz-Pena, G.; Bankson, J. A.; Ravoori, M.; et al. Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. \u003cem\u003eNat Med \u003c/em\u003e\u003cstrong\u003e2006\u003c/strong\u003e, \u003cem\u003e12\u003c/em\u003e (8), 939-944. DOI: 10.1038/nm1447 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eLu, Y.; Zhao, H.; Liu, Y.; Zuo, Y.; Xu, Q.; Liu, L.; Li, X.; Zhu, H.; Zhang, Y.; Zhang, S.; et al. Chronic Stress Activates PlexinA1/VEGFR2-JAK2-STAT3 in Vascular Endothelial Cells to Promote Angiogenesis. \u003cem\u003eFront Oncol \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e11\u003c/em\u003e, 709057. DOI: 10.3389/fonc.2021.709057 From NLM PubMed-not-MEDLINE.\u003c/li\u003e\n\u003cli\u003eGlobig, A. M.; Zhao, S.; Roginsky, J.; Maltez, V. I.; Guiza, J.; Avina-Ochoa, N.; Heeg, M.; Araujo Hoffmann, F.; Chaudhary, O.; Wang, J.; et al. The beta(1)-adrenergic receptor links sympathetic nerves to T cell exhaustion. \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e622\u003c/em\u003e (7982), 383-392. DOI: 10.1038/s41586-023-06568-6 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eDelgado-Benito, V.; Berruezo-Llacuna, M.; Altwasser, R.; Winkler, W.; Sundaravinayagam, D.; Balasubramanian, S.; Caganova, M.; Graf, R.; Rahjouei, A.; Henke, M. T.; et al. PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification. \u003cem\u003eJ Exp Med \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e217\u003c/em\u003e (10). DOI: 10.1084/jem.20200137 From NLM Medline.\u003c/li\u003e\n\u003cli\u003ePons-Espinal, M.; Blasco-Agell, L.; Fernandez-Carasa, I.; Andres-Benito, P.; di Domenico, A.; Richaud-Patin, Y.; Baruffi, V.; Marruecos, L.; Espinosa, L.; Garrido, A.; et al. Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson\u0026apos;s disease. \u003cem\u003eJCI Insight \u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e9\u003c/em\u003e (3). DOI: 10.1172/jci.insight.163359 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eLopez-Rodriguez, A. B.; Hennessy, E.; Murray, C. L.; Nazmi, A.; Delaney, H. J.; Healy, D.; Fagan, S. G.; Rooney, M.; Stewart, E.; Lewis, A.; et al. Acute systemic inflammation exacerbates neuroinflammation in Alzheimer\u0026apos;s disease: IL-1beta drives amplified responses in primed astrocytes and neuronal network dysfunction. \u003cem\u003eAlzheimers Dement \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e17\u003c/em\u003e (10), 1735-1755. DOI: 10.1002/alz.12341 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eBill, R.; Wirapati, P.; Messemaker, M.; Roh, W.; Zitti, B.; Duval, F.; Kiss, M.; Park, J. C.; Saal, T. M.; Hoelzl, J.; et al. CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e381\u003c/em\u003e (6657), 515-524. DOI: 10.1126/science.ade2292 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eEun, J. W.; Yoon, J. H.; Ahn, H. R.; Kim, S.; Kim, Y. B.; Lim, S. B.; Park, W.; Kang, T. W.; Baek, G. O.; Yoon, M. G.; et al. Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib. \u003cem\u003eCancer Commun (Lond) \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e43\u003c/em\u003e (4), 455-479. DOI: 10.1002/cac2.12414 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eLi, Y.; Liu, H.; Zhao, Y.; Yue, D.; Chen, C.; Li, C.; Zhang, Z.; Wang, C. Tumor-associated macrophages (TAMs)-derived osteopontin (OPN) upregulates PD-L1 expression and predicts poor prognosis in non-small cell lung cancer (NSCLC). \u003cem\u003eThorac Cancer \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e12\u003c/em\u003e (20), 2698-2709. DOI: 10.1111/1759-7714.14108 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eZhao, L.; Wang, Z.; Tan, Y.; Ma, J.; Huang, W.; Zhang, X.; Jin, C.; Zhang, T.; Liu, W.; Yang, Y. G. IL-17A/CEBPbeta/OPN/LYVE-1 axis inhibits anti-tumor immunity by promoting tumor-associated tissue-resident macrophages. \u003cem\u003eCell Rep \u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e43\u003c/em\u003e (12), 115039. DOI: 10.1016/j.celrep.2024.115039 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eCheng, J. N.; Jin, Z.; Su, C.; Jiang, T.; Zheng, X.; Guo, J.; Li, X.; Chu, H.; Jia, J.; Zhou, Q.; et al. Bone metastases diminish extraosseous response to checkpoint blockade immunotherapy through osteopontin-producing osteoclasts. \u003cem\u003eCancer Cell \u003c/em\u003e\u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e43\u003c/em\u003e (6), 1093-1107 e1099. DOI: 10.1016/j.ccell.2025.03.036 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eCools-Lartigue, J.; Spicer, J.; McDonald, B.; Gowing, S.; Chow, S.; Giannias, B.; Bourdeau, F.; Kubes, P.; Ferri, L. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. \u003cem\u003eJ Clin Invest \u003c/em\u003e\u003cstrong\u003e2013\u003c/strong\u003e, \u003cem\u003e123\u003c/em\u003e (8), 3446-3458. DOI: 10.1172/JCI67484 From NLM Publisher.\u003c/li\u003e\n\u003cli\u003eHedrick, C. C.; Malanchi, I. Neutrophils in cancer: heterogeneous and multifaceted. \u003cem\u003eNat Rev Immunol \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e22\u003c/em\u003e (3), 173-187. DOI: 10.1038/s41577-021-00571-6 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eMa, L. X.; Titmuss, E.; Loree, J. M.; Jonker, D. J.; Kennecke, H. F.; Berry, S.; Couture, F.; Ahmad, C. E.; Goffin, J. R.; Kavan, P.; et al. Plasma arginine as a predictive biomarker for outcomes with immune checkpoint inhibition in metastatic colorectal cancer: a correlative analysis of the CCTG CO.26 trial. \u003cem\u003eJ Immunother Cancer \u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e12\u003c/em\u003e (12). DOI: 10.1136/jitc-2024-010094 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eFultang, L.; Booth, S.; Yogev, O.; Martins da Costa, B.; Tubb, V.; Panetti, S.; Stavrou, V.; Scarpa, U.; Jankevics, A.; Lloyd, G.; et al. Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity. \u003cem\u003eBlood \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e136\u003c/em\u003e (10), 1155-1160. DOI: 10.1182/blood.2019004500 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eGeiger, R.; Rieckmann, J. C.; Wolf, T.; Basso, C.; Feng, Y.; Fuhrer, T.; Kogadeeva, M.; Picotti, P.; Meissner, F.; Mann, M.; et al. L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e167\u003c/em\u003e (3), 829-842 e813. DOI: 10.1016/j.cell.2016.09.031 From NLM Medline.\u003c/li\u003e\n\u003cli\u003eHu, B.; Zhang, Y.; Zhang, G.; Li, Z.; Jing, Y.; Yao, J.; Sun, S. Research progress of bone-targeted drug delivery system on metastatic bone tumors. \u003cem\u003eJ Control Release \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e350\u003c/em\u003e, 377-388. DOI: 10.1016/j.jconrel.2022.08.034 From NLM Medline.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chronic immobilization stress, norepinephrine, ADRβ, osteopontin, monocytes, neutrophils, arginine, breast cancer bone metastasis","lastPublishedDoi":"10.21203/rs.3.rs-7760970/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7760970/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancer bone metastasis is associated with poor prognosis and resistance to immune checkpoint inhibitors. Although chronic stress promotes cancer progression, its role in reshaping the bone marrow (BM) immune microenvironment to facilitate metastasis remains poorly understood. We demonstrate that chronic stress promotes breast cancer bone metastasis primarily by reshaping the BM immune microenvironment. Specifically, chronic stress increases BM neutrophils, and inducible, specific clearance of these neutrophils effectively prevented stress-induced bone metastasis. In mice exposed to chronic stress, abundant BM neutrophils excessively consume arginine by upregulating iNOS, creating a low-arginine, pro-metastatic niche that impairs functional CD8⁺ cytotoxic T cells. Mechanistically, chronic stress stimulates norepinephrine (NE) release in the BM, which boosts osteopontin (OPN) expression by monocytes/macrophages. The elevated OPN then interacts with its receptor CD44 on neutrophils to trigger this excessive arginine consumption. This cascade was shut down in mice with a global knockout of the NE receptor ADRβ2. To therapeutically target this process, we developed a bone-targeting conjugate of propranolol (an ADRβ antagonist) and alendronate (AP). Local accumulation of AP in the bone reduced OPN-positive monocytes/macrophages and iNOS-positive neutrophils, restoring BM arginine levels and CD8⁺ T cell populations, and effectively inhibiting chronic stress-induced breast cancer bone metastasis. Our findings reveal a novel neuro-immune-metabolic axis through which chronic stress promotes breast cancer bone metastasis and highlight AP as a potential therapeutic strategy.\u003c/p\u003e","manuscriptTitle":"Chronic stress reshapes bone marrow microenvironment to facilitate breast cancer bone metastasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 11:29:33","doi":"10.21203/rs.3.rs-7760970/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cbaff257-b0b5-4de3-8614-284653b195fa","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55976534,"name":"Biological sciences/Cancer/Metastasis/Bone metastases"},{"id":55976535,"name":"Biological sciences/Cancer/Breast cancer"}],"tags":[],"updatedAt":"2025-11-25T10:21:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 11:29:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7760970","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7760970","identity":"rs-7760970","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}