IntroductionBone metastasis is the most common complication of advanced breast cancer. The associated cancer-induced bone disease is treated with bone-sparing agents like zoledronic acid. Clinical trials have shown that zoledronic acid also reduces breast cancer recurrence in bone; potentially by modifying the bone microenvironment surrounding disseminated tumour cells. We have characterised the early effects of zoledronic acid on key cell types of the metastatic niche in vivo, and investigated how these modify the location of breast tumour cells homing to bone.MethodsFemale mice were treated with a single, clinically achievable dose of zoledronic acid (100 μg/kg) or PBS. Bone integrity, osteoclast and osteoblast activity and number/mm trabecular bone on 1, 3, 5 and 10 days after treatment were assessed using μCT, ELISA (TRAP, PINP) and bone histomorphometry, respectively. The effect of zoledronic acid on osteoblasts was validated in genetically engineered mice with GFP-positive osteoblastic cells. The effects on growth plate cartilage were visualised by toluidine blue staining. For tumour studies, mice were injected i.c. with DID-labelled MDA-MB-231-NW1-luc2 breast cancer cells 5 days after zoledronic acid treatment, followed by assessment of tumour cell homing to bone and soft tissues by multiphoton microscopy, flow cytometry and ex vivo cultures.ResultsAs early as 3 days after treatment, animals receiving zoledronic acid had significantly increased trabecular bone volume vs. control. This rapid bone effect was reflected in a significant reduction in osteoclast and osteoblast number/mm trabecular bone and reduced bone marker serum levels (day 3–5). These results were confirmed in mice expressing GFP in osteoblastic linage cells. Pre-treatment with zoledronic acid caused accumulation of an extra-cellular matrix in the growth plate associated with a trend towards preferential [1] homing of tumour cells to osteoblast-rich areas of bone, but without affecting the total number of tumour cells. The number of circulating tumour cells was reduced in ZOL treated animals.ConclusionA single dose of zoledronic acid caused significant changes in the bone area suggested to contain the metastatic niche. Tumour cells arriving in this modified bone microenvironment appeared to preferentially locate to osteoblast-rich areas, supporting that osteoblasts may be key components of the bone metastasis niche and therefore a potential therapeutic target in breast cancer.

The establishment of bone metastasis remains one of the most frequent complications of patients suffering from advanced breast cancer. Patients with bone metastases experience high morbidity and mortality caused by excessive, tumor-induced and osteoclast-mediated bone resorption. Anti-resorptive treatments, such as bisphosphonates, are available to ease skeletal related events including pain, increased fracture risk, and hypercalcemia. However, the disease remains incurable and 5-year survival rates for these patients are below 25%. Within the bone, disseminated breast cancer cells localize in "metastatic niches," special microenvironments that are thought to regulate cancer cell colonization and dormancy as well as tumor progression and subsequent development into overt metastases. Precise location and composition of this "metastatic niche" remain poorly defined. However, it is thought to include an "endosteal niche" that is composed of key bone cells that are derived from both, hematopoietic stem cells (osteoclasts), and mesenchymal stromal cells (osteoblasts, fibroblasts, adipocytes). Our knowledge of how osteoclasts drive the late stage of the disease is well-established. In contrast, much less is known about the interaction between osteogenic cells and disseminated tumor cells prior to the initiation of the osteolytic phase. Recent studies suggest that mesenchymal-derived cells, including osteoblasts and fibroblasts, play a key role during the early stages of breast cancer bone metastasis such as tumor cell homing, bone marrow colonization, and tumor cell dormancy. Hence, elucidating the interactions between breast cancer cells and mesenchymal-derived cells that drive metastasis progression could provide novel therapeutic approaches and targets to treat breast cancer bone metastasis. In this review we discuss evidences reporting the interaction between tumor cells and endosteal niche cells during the early stages of breast cancer bone metastasis, with a particular focus on mesenchymal-derived osteoblasts and fibroblasts.

Bone metastases are a common and devastating feature of late-stage breast cancer. Metastatic bone disease is a consequence of disturbed bone remodeling due to pathological interactions between cancer cells and the bone microenvironment (BME). In the BME, breast cancer cells severely alter the balanced bone formation and bone resorption driven by osteoblasts and osteoclasts. The complex cellular cross talk in the BME is governed by secreted molecules, signaling pathways and epigenetic cues including non-coding RNAs. MicroRNAs (miRNAs) are small non-coding RNAs that reduce protein abundance and regulate several biological processes, including bone remodeling. Under pathological conditions, abnormal miRNA signaling contributes to the progression of diseases, such as bone metastasis. Recently miRNAs have been demonstrated to regulate several key drivers of bone metastasis. Furthermore, miRNAs are implicated as important regulators of cellular interactions within the metastatic BME. As a consequence, targeting the BME by miRNA delivery or antagonism has been reported to limit disease progression in experimental and preclinical conditions positioning miRNAs as emerging novel therapeutic tools in metastatic bone disease. This review will summarize our current understanding on the composition and function of the metastatic BME and discuss the recent advances how miRNAs can modulate pathological interactions in the bone environment.

Bone is the most common metastatic site in breast cancer. Upon arrival to the bone, disseminated tumor cells can undergo a period of dormancy but often eventually grow and hijack the bone microenvironment. The bone marrow microenvironment consists of multiple cell types including the bone cells, adipocytes, endothelial cells, and nerve cells that all have crucial functions in the maintenance of bone homeostasis. Tumor cells severely disturb the tightly controlled cellular and molecular interactions in the bone marrow fueling their own survival and growth. While the role of bone resorbing osteoclasts in breast cancer bone metastases is well established, the function of other bone cells, as well as adipocytes, endothelial cells, and nerve cells is less understood. In this review, we discuss the composition of the physiological bone microenvironment and how the presence of tumor cells influences the microenvironment, creating a pathological crosstalk between the cells. A better understanding of the cellular and molecular events that occur in the metastatic bone microenvironment could facilitate the identification of novel cellular targets to treat this devastating disease.

BackgroundThe bone-targeting agent zoledronic acid (ZOL) increases breast cancer survival in subsets of patients, but the underlying reasons for this protective effect are unknown. ZOL modulates the activity of osteoclasts and osteoblasts, which form hematopoietic stem cell niches, and therefore may affect hematopoietic cells that play a role in breast cancer progression.MethodImmunocompetent and immunocompromised strains of mice commonly used for breast cancer research were injected with a single, clinically relevant dose of ZOL (100 μg/kg) or vehicle control. The effects of ZOL on the bone marrow microenvironment (bone volume, bone cell number/activity, extracellular matrix composition) were established at various time points following treatment, using micro-computed tomography (μCT) analysis, histomorphometry, ELISA and immunofluorescence. The effects on peripheral blood and bone marrow hematopoietic progenitor populations were assessed using a HEMAVET® hematology analyzer and multicolor flow cytometry, respectively. Tumor support function of bone marrow cells was determined using an in vivo functional assay developed in our laboratory.ResultsUsing multiple mouse strains, we observed transient changes in numbers of hematopoietic stem cells, myeloid-biased progenitor cells, and lymphoid-biased cells concurrent with changes to hematopoietic stem cell niches following ZOL administration. Importantly, bone marrow cells from mice treated with a single, clinically relevant dose of ZOL inhibited breast tumor outgrowth in vivo. The ZOL-induced tumor suppressive function of the bone marrow persisted beyond the time point at which numbers of hematopoietic progenitor cells had returned to baseline.ConclusionsThese findings provide novel evidence that alterations to the bone marrow play a role in the anti-tumor activity of ZOL and suggest possibilities for capitalizing on the beneficial effects of ZOL in reducing breast cancer development and progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0815-8) contains supplementary material, which is available to authorized users.

IntroductionBone metastasis remains incurable with treatment restricted to palliative care. Cabozantinib (CBZ) is targeted against multiple receptor tyrosine kinases involved in tumour pathobiology, including hepatocyte growth factor receptor (MET) and vascular endothelial growth factor receptor 2 (VEGFR-2). CBZ has demonstrated clinical activity in advanced prostate cancer with resolution of lesions visible on bone scans, implicating a potential role of the bone microenvironment as a mediator of CBZ effects. We characterised the effects of short-term administration of CBZ on bone in a range of in vivo models to determine how CBZ affects bone in the absence of tumour.MethodsStudies were performed in a variety of in vivo models including male and female BALB/c nude mice (age 6–17-weeks). Animals received CBZ (30 mg/kg, 5 × weekly) or sterile H2O control for 5 or 10 days. Effects on bone integrity (μCT), bone cell activity (PINP, TRAP ELISA), osteoblast and osteoclast number/mm trabecular bone surface, area of epiphyseal growth plate cartilage, megakaryocyte numbers and bone marrow composition were assessed. Effects of longer-term treatment (15-day & 6-week administration) were assessed in male NOD/SCID and beige SCID mice.ResultsCBZ treatment had significant effects on the bone microenvironment, including reduced osteoclast and increased osteoblast numbers compared to control. Trabecular bone structure was altered after 8 administrations. A significant elongation of the epiphyseal growth plate, in particular the hypertrophic chondrocyte zone, was observed in all CBZ treated animals irrespective of administration schedule. Both male and female BALB/c nude mice had increased megakaryocyte numbers/mm2 tissue after 10-day CBZ treatment, in addition to vascular ectasia, reduced bone marrow cellularity and extravasation of red blood cells into the extra-vascular bone marrow. All CBZ-induced effects were transient and rapidly lost following cessation of treatment.ConclusionShort-term administration of CBZ induces rapid, reversible effects on the bone microenvironment in vivo highlighting a potential role in mediating treatment responses.

Background: Osteoclast activation is a hallmark of breast cancer-induced bone disease while little is known about the role of osteoblasts in this process. Recently, we identified the homeodomain protein TG-interacting factor-1 (Tgif1) as a crucial regulator of osteoblast function. In this study, we demonstrate that lack of Tgif1 also restricts the progression of breast cancer bone metastases. Methods: Transwell migration assays were used to investigate the osteoblast-breast cancer cell interaction in vitro. Molecular analyses included RNA sequencing, immunoblotting, and qRT-PCR. To determine the role of Tgif1 in metastatic bone disease, 4T1 breast cancer cells were injected intracardially into mice with a germ line deletion of Tgif1 (Tgif1 −/−) or control littermates (Tgif1 +/+). Progression of bone metastases and alterations in the bone microenvironment were assessed using bioluminescence imaging, immunofluorescence staining, confocal microscopy, and histomorphometry. Results: Medium conditioned by osteoblasts stimulated breast cancer cell migration, indicating a potential role of osteoblasts during bone metastasis progression. Tgif1 expression was strongly increased in osteoblasts upon stimulation by breast cancer cells, demonstrating the implication of Tgif1 in the osteoblast-breast cancer cell interaction. Indeed, conditioned medium from osteoblasts of Tgif1 −/− mice failed to induce breast cancer cell migration compared to control, suggesting that Tgif1 in osteoblasts augments cancer cell motility. Semaphorin 3E (Sema3E), which is abundantly secreted by Tgif1 −/− osteoblasts, dose-dependently reduced breast cancer cell migration while silencing of Sema3E expression in Tgif1 −/− osteoblasts partially restored the impaired migration. In vivo, we observed a decreased number of breast cancer bone metastases in Tgif1 −/− mice compared to control littermates. Consistently, the presence of single breast cancer cells or micro-metastases in the tibiae was reduced in Tgif1 −/− mice. Breast cancer cells localized in close proximity to Endomucin-positive vascular cells as well as to osteoblasts. Although Tgif1 deficiency did not affect the bone marrow vasculature, the number and activity of osteoblasts were reduced compared to control. This suggests that the protective effect on bone metastases might

The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence. Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. .