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. 2018 Sep 8;13:1-10.
doi: 10.1016/j.jbo.2018.09.002. eCollection 2018 Nov.

β2ARs Stimulation in Osteoblasts Promotes Breast Cancer Cell Adhesion to Bone Marrow Endothelial Cells in an IL-1β and Selectin-Dependent Manner

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Free PMC article

β2ARs Stimulation in Osteoblasts Promotes Breast Cancer Cell Adhesion to Bone Marrow Endothelial Cells in an IL-1β and Selectin-Dependent Manner

Lise Clément-Demange et al. J Bone Oncol. .
Free PMC article

Abstract

Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to impact the hypothalamic-pituitary axis and the autonomic nervous system. Preclinical and clinical evidence support the involvement of the sympathetic nervous system in the control of bone remodeling and in pathologies of the skeleton, including bone metastasis. In experimental mouse models of skeletal metastasis, administration of the βAR agonist isoproterenol (ISO), used as a surrogate of norepinephrine, the main neurotransmitter of sympathetic neurons, was shown to favor bone colonization of metastatic breast cancer cells via an increase bone marrow vascularity. However, successful extravasation of cancer cells into a distant organ is known to be favored by an activated endothelium, itself stimulated by inflammatory signals. Based on the known association between high sympathetic outflow, the expression of inflammatory cytokines and bone metastasis, we thus asked whether βAR stimulation in osteoblasts may alter the vascular endothelium to favor cancer cell engraftment within the skeleton. To address this question, we used conditioned medium (CM) from PBS or ISO-treated bone marrow stromal cells (BMSCs) in adhesion assays with bone marrow endothelial cells (BMECs) or the endothelial cell line C166. We found that ISO treatment in differentiated BMSCs led to a robust induction of the pro-inflammatory cytokines interleukin-1 beta (IL-1β) and interleukin-6 (IL-6). The CM from ISO-treated BMSCs increased the expression of E- and P-selectin in BMECs and the adhesion of human MDA-MB-231 breast cancer cells to these cells in short-term static and dynamic adhesion assays, and a blocking antibody against IL-1β, but not IL-6, reduced this effect. Direct IL-1β treatment of BMECs had a similar effect, whereas the impact of IL-6 treatment on the expression of adhesion molecules by BMECs and on the adhesion of cancer cells to BMECs was negligible. Collectively, these in vitro results suggest that in the context of the multicellular and dynamic bone marrow environment, sympathetic activation and subsequent βAR stimulation in osteoblasts may profoundly remodel the density but also the activation status of bone marrow vessels to favor the skeletal engraftment of circulating breast cancer cells.

Keywords: Adhesion molecules; Bone metastasis; Endothelial activation; Interleukin-1beta; Selectins; Sympathetic nervous system.

Figures

Fig 1
Fig. 1
Confluent and adherent primary mouse bone marrow endothelial cells (BMECs) were treated for 24 h with the conditioned medium (CM) from vehicle or ISO-treated BMSCs prior to addition of GFP-positive (+ ) MDA-MB-231 (A) or GFP +  4T1 (B) cells. After 3.5 h, wells were washed in PBS and the relative adhesion of cancer cells to the endothelial monolayer was quantified by fluorescence-labeled cell count assay. Results are graphed as fold change relative to the CM of PBS-treated BMECs (n = 4 independent experiments, * = p < 0.05, *** = p < 0.001). A and B, right panel: Representative images of tumor cells attached to BMEC monolayers after treatment (blue: DAPI nuclear staining, green: GFP +  tumor cells). Scale bar  =  100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 2
Fig. 2
(A) DiI-labeled BMECs (red) were grown on flow chamber pre-coated with fibronectin (0.8 μg/cm2) until 100% confluent and were then stimulated with the CM from PBS or ISO-treated bone marrow stromal cells (BMSCs) for 6 h prior to perfusion of GFP +  MDA-MB-231 cancer cells at 1 dyn/cm2 shear stress for 5 min. The number of adherent tumor cells was calculated following fluorescence-labeled cell count of ten observation fields. The results are graphed as fold change relative to CM PBS (n = 3 independent experiments, *** = p < 0.001). Representative images are shown for each condition. Scale bar  =  100 µm. (B) The 24 h CM from WT or Adrb2-deficient BMSCs following treatment with either PBS or ISO was collected and used for BMECs adhesion assay as described in 2A (n = 3 independent experiments, *** = p < 0.001). (C) C166 endothelial cells were labeled with Vybrant DiI prior to injection in the flow chamber. Then, adhesion assay was performed as described in 2A (n = 3 independent experiments, *** = p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 3
Fig. 3
BMECs (A) or C166 cells (B) were treated for 1 h with the CM from PBS or ISO-treated BMSCs. The CM from ISO-treated BMSCs induced a 2-fold increase of Sele and Selp expression compared to the CM from PBS-treated BMSCs, whereas Vcam1 level was increased only by 20% and Icam1 expression did not change significantly in both BMECs and C166 cells (qPCR, n = 3 independent experiments, * = p < 0.05, ** = p < 0.01, *** = p < 0.001).
Fig 4
Fig. 4
ISO (10 µM) increased expression of the pro-inflammatory cytokines Il1β (A) and Il6 (B) in mouse BMSCs within 2 h (qPCR, n = 5 independent experiments, *** = p < 0.001). Il1β (C) and Il6 (D) expression was increased in WT BMSCs but not in BMSCs extracted from Adrb2-deficient mice following 2 h of ISO treatment (qPCR, n = 4 independent experiments, ** = p < 0.01, *** = p < 0.001). One hour treatment with murine rIL-1β (10 ng/mL, E) but neither ISO (10 µM, F) nor murine rIL-6 (50 ng/mL, G) increased expression of Sele, Selp, and Icam1 in BMECs (qPCR, n = 5 independent experiments, * = p < 0.05, ** = p < 0.01).
Fig 5
Fig. 5
(A) C166 endothelial cells were treated for 6 h with rIL-1β (10 ng/mL) before immunofluorescence staining for E-selectin (blue: Hoechst nuclear staining, red: E-selectin/CD62E). Scale bar  =  100 µm. (B–D) Murine rIL-1β (10 ng/mL) increased the adhesion of GFP +  MDA-MB-231 cancer cells to a C166 monolayer 3-fold compared to vehicle-treated endothelial cells in flow conditions (flow chamber assay, B) and 2-fold in static conditions (in a coverslip assay, C and in a gravity assay, D). Quantification of adherent tumor cells was calculated by fluorescence-labeled cell count assay and graphed as fold change relative to PBS (n = 3 independent experiments, ** = p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 6
Fig. 6
C166 cells were treated for 6 h with the CM of PBS (-) or ISO (+ ) treated BMSCs along with an IL-1β neutralizing antibody or an IgG antibody control (A) or cimetidine (B), prior to addition of GFP +  MDA-MB-231 cells in the flow chamber (n = 3 independent experiments, * = p < 0.05, *** = p < 0.001). Number of adherent tumor cells was calculated by fluorescence-labeled cell count assay and graphed as fold change relative to untreated group.

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