The role of IL-1B in breast cancer bone metastasis.

Interleukin-1B (IL-1B) is a potent pro-inflammatory cytokine that plays multiple, pivotal roles, in the complex interplay between breast cancer cells and the bone microenvironment. IL-1B is involved in the growth of the primary tumours, regulation of inflammation within the tumour microenvironment, promotion of epithelial to mesenchymal transition (EMT), migration and invasion. Moreover, when breast cancer cells arrive in the bone microenvironment there is an upregulation of IL-1B which promotes the creation of a conducive niche for metastatic breast cancer cells as well as stimulating initiation of the vicious cycle of bone metastasis. Pre-clinical studies have demonstrated that inhibition of IL-1 signalling reduces bone metastasis from oestrogen receptor positive/triple-negative breast cancers in various mouse models. However, effects on primary tumours and soft tissue metastasis remain controversial with some studies showing increased tumour growth in these sites, whilst others show no effects. Notably, combining anti-IL-1 therapy with standard-of-care treatments, such as chemotherapy and immunotherapy, has been demonstrated to reduce the growth of primary tumours, bone metastasis, as well as metastatic outgrowth in other organs. This review focuses on the mechanisms by which IL-1B promotes breast cancer bone metastasis.

The CDK4/6 Inhibitor Palbociclib Inhibits Estrogen-Positive and Triple Negative Breast Cancer Bone Metastasis In Vivo.

CDK 4/6 inhibitors have demonstrated significant improved survival for patients with estrogen receptor (ER) positive breast cancer (BC). However, the ability of these promising agents to inhibit bone metastasis from either ER+ve or triple negative BC (TNBC) remains to be established. We therefore investigated the effects of the CDK 4/6 inhibitor, palbociclib, using in vivo models of breast cancer bone metastasis. In an ER+ve T47D model of spontaneous breast cancer metastasis from the mammary fat pad to bone, primary tumour growth and the number of hind limb skeletal tumours were significantly lower in palbociclib treated animals compared to vehicle controls. In the TNBC MDA-MB-231 model of metastatic outgrowth in bone (intracardiac route), continuous palbociclib treatment significantly inhibited tumour growth in bone compared to vehicle. When a 7-day break was introduced after 28 days (mimicking the clinical schedule), tumour growth resumed and was not inhibited by a second cycle of palbociclib, either alone or when combined with the bone-targeted agent, zoledronic acid (Zol), or a CDK7 inhibitor. Downstream phosphoprotein analysis of the MAPK pathway identified a number of phosphoproteins, such as p38, that may contribute to drug-insensitive tumour growth. These data encourage further investigation of targeting alternative pathways in CDK 4/6-insensitive tumour growth.

Novel Methods of Targeting IL-1 Signalling for the Treatment of Breast Cancer Bone Metastasis.

Breast cancer bone metastasis is currently incurable. Evidence suggests that inhibiting IL-1 signalling with the IL1R antagonist, Anakinra, or the IL1ß antibody, Canakinumab, prevents metastasis and almost eliminates breast cancer growth in the bone. However, these drugs increase primary tumour growth. We, therefore, investigated whether targeting other members of the IL-1 pathway (Caspase-1, IL1ß or IRAK1) could reduce bone metastases without increasing tumour growth outside of the bone. Inhibition of IL-1 via MLX01 (IL1ß secretion inhibitor), VRT043198/VX765 (Caspase-1 inhibitor), Pacritinib (IRAK1 inhibitor) or Anakinra (IL1R antagonist) on tumour cell viability, migration and invasion were assessed in mouse mammary E0771 and Py8119 cells in vitro and on primary tumour growth, spontaneous metastasis and metastatic outgrowth in vivo. In vitro, Inhibition of IL-1 signalling by MLX01, VRT043198 and Anakinra reduced migration of E0771 and Py8119 cells and reversed tumour-derived IL1ß induced-increased invasion and migration towards bone cells. In vivo, VX765 and Anakinra significantly reduced spontaneous metastasis and metastatic outgrowth in the bone, whereas MLX01 reduced primary tumour growth and bone metastasis. Pacritinib had no effect on metastasis in vitro or in vivo. Targeting IL-1 signalling with small molecule inhibitors may provide a new therapeutic strategy for breast cancer bone metastasis.

IL-1ß in breast cancer bone metastasis.

Bone is the most common site for advanced breast cancer to metastasise. The proinflammatory cytokine, interleukin-1ß (IL-1ß) plays a complex and contradictory role in this process. Recent studies have demonstrated that breast cancer patients whose primary tumours express IL-1ß are more likely to experience relapse in bone or other organs. Importantly, IL-1ß affects different stages of the metastatic process including growth of the primary tumour, epithelial to mesenchymal transition (EMT), dissemination of tumour cells into the blood stream, tumour cell homing to the bone microenvironment and, once in bone, this cytokine participates in the interaction between cancer cells and bone cells, promoting metastatic outgrowth at this site. Interestingly, although inhibition of IL-1ß signalling has been shown to have potent anti-metastatic effects, inhibition of the activity of this cytokine has contradictory effects on primary tumours, sometimes reducing but often promoting their growth. In this review, we focus on the complex roles of IL-1ß on breast cancer bone metastasis: specifically, we discuss the distinct effects of IL-1ß derived from tumour cells and/or microenvironment on inhibition/induction of primary breast tumour growth, induction of the metastatic process through the EMT, promotion of tumour cell dissemination into the bone metastatic niche and formation of overt metastases.

Oestradiol Contributes to Differential Antitumour Effects of Adjuvant Zoledronic Acid Observed Between Pre- and Post-Menopausal Women.

Clinical trials have demonstrated that adding zoledronic acid (Zol) to (neo)adjuvant standard of care has differential antitumour effects in pre- and post-menopausal women: Both benefit from reduced recurrence in bone; however, while postmenopausal women also incur survival benefit, none is seen in premenopausal women treated with adjuvant bisphosphonates. In the current study, we have used mouse models to investigate the role of oestradiol in modulating potential antitumour effects of Zol. Pre-, peri-, and post-menopausal concentrations of oestradiol were modelled in BALB/c wild-type, BALB/c nude, and C57BL/6 mice by ovariectomy followed by supplementation with oestradiol. Mice also received 40 mg/kg/day goserelin to prevent ovariectomy-induced increases in follicle-stimulating hormone (FSH). Metastasis was modelled following injection of MDA-MB-231, 4T1, or E0771 cells after ovariectomy and saline or 100 µg/kg Zol administered weekly. Supplementing ovariectomised mice with 12.5 mg/ml, 1.38 mg/ml, and 0 ng/ml oestradiol, in the presence of goserelin, resulted in serum concentrations of 153.16 ± 18.10 pg/ml, 48.64 ± 18.44 pg/ml, and 1.00 ± 0.27 pg/ml oestradiol, which are equivalent to concentrations found in pre-, peri-, and post-menopausal humans. Osteoclast activity was increased 1.5-1.8-fold with peri- and post-menopausal compared with premenopausal oestradiol, resulting in a 1.34-1.69-fold reduction in trabecular bone. Zol increased trabecular bone in all groups but did not restore bone to volumes observed under premenopausal conditions. In tumour-bearing mice, Zol reduced bone metastases in BALB/c (wild-type and nude), with greatest effects seen under pre- and post-menopausal concentrations of oestradiol. Zol did not affect soft tissue metastases in immunocompetent BALB/c mice but increased metastases 3.95-fold in C57BL/6 mice under premenopausal concentrations of oestradiol. In contrast, Zol significantly reduced soft tissue metastases 2.07 and 4.69-fold in immunocompetent BALB/c and C57BL/6 mice under postmenopausal oestradiol, mirroring the results of the clinical trials of (neo)adjuvant bisphosphonates. No effects on soft tissue metastases were observed in immunocompromised mice, and differences in antitumour response did not correlate with musculoaponeurotic fibrosarcoma (MAF), macrophage capping protein (CAPG), or PDZ domain containing protein GIPC1 (GIPC1) expression. In conclusion, oestradiol contributes to altered antitumour effects of Zol observed between pre- and post-menopausal women. However, other immunological/microenvironmental factors are also likely to contribute to this phenomenon.

Transcriptomic Profiling Reveals Novel Candidate Genes and Signalling Programs in Breast Cancer Quiescence and Dormancy.

Metastatic recurrence, the major cause of breast cancer mortality, is driven by reactivation of dormant disseminated tumour cells that are defined by mitotic quiescence and chemoresistance. The molecular mechanisms underpinning mitotic quiescence in cancer are poorly understood, severely limiting the development of novel therapies for removal of residual, metastasis-initiating tumour cells. Here, we present a molecular portrait of the quiescent breast cancer cell transcriptome across the four main breast cancer sub-types (luminal, HER2-enriched, basal-like and claudin-low) and identify a novel quiescence-associated 22-gene signature using an established lipophilic-dye (Vybrant® DiD) retention model and whole-transcriptomic profiling (mRNA-Seq). Using functional association network analysis, we elucidate the molecular interactors of these signature genes. We then go on to demonstrate that our novel 22-gene signature strongly correlates with low tumoural proliferative activity, and with dormant disease and late metastatic recurrence (≥5 years after primary tumour diagnosis) in metastatic breast cancer in multiple clinical cohorts. These genes may govern the formation and persistence of disseminated tumour cell populations responsible for breast cancer recurrence, and therefore represent prospective novel candidates to inform future development of therapeutic strategies to target disseminated tumour cells in breast cancer, eliminate minimal residual disease and prevent metastatic recurrence.

IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer.

Breast cancer bone metastasis is currently incurable, ~75% of patients with late-stage breast cancer develop disease recurrence in bone and available treatments are only palliative. We have previously shown that production of the pro-inflammatory cytokine interleukin-1B (IL-1B) by breast cancer cells drives bone metastasis in patients and in preclinical in vivo models. In the current study, we have investigated how IL-1B from tumour cells and the microenvironment interact to affect primary tumour growth and bone metastasis through regulation of the immune system, and whether targeting IL-1 driven changes to the immune response improves standard of care therapy for breast cancer bone metastasis. Using syngeneic IL-1B/IL1R1 knock out mouse models in combination with genetic manipulation of tumour cells to overexpress IL-1B/IL1R1, we found that IL-1B signalling elicited an opposite response in primary tumours compared with bone metastases. In primary tumours, IL-1B inhibited growth, by impairing the infiltration of innate immune cell subsets with potential anti-cancer functions but promoted enhanced tumour cell migration. In bone, IL-1B stimulated the development of osteolytic metastases. In syngeneic models of breast cancer, combining standard of care treatments (Doxorubicin and Zoledronic acid) with the IL-1 receptor antagonist Anakinra inhibited both primary tumour growth and metastasis. Anakinra had opposite effects on the immune response compared to standard of care treatment, and its anti-inflammatory signature was maintained in the combination therapy. These data suggest that targeting IL-1B signalling may provide a useful therapeutic approach to inhibit bone metastasis and improve efficacy of current treatments for breast cancer patients.

Osteoblast-Derived Paracrine and Juxtacrine Signals Protect Disseminated Breast Cancer Cells from Stress.

Metastatic breast cancer in bone is incurable and there is an urgent need to develop new therapeutic approaches to improve survival. Key to this is understanding the mechanisms governing cancer cell survival and growth in bone, which involves interplay between malignant and accessory cell types. Here, we performed a cellular and molecular comparison of the bone microenvironment in mouse models representing either metastatic indolence or growth, to identify mechanisms regulating cancer cell survival and fate. In vivo, we show that regardless of their fate, breast cancer cells in bone occupy niches rich in osteoblastic cells. As the number of osteoblasts in bone declines, so does the ability to sustain large numbers of breast cancer cells and support metastatic outgrowth. In vitro, osteoblasts protected breast cancer cells from death induced by cell stress and signaling via gap junctions was found to provide important juxtacrine protective mechanisms between osteoblasts and both MDA-MB-231 (TNBC) and MCF7 (ER+) breast cancer cells. Combined with mathematical modelling, these findings indicate that the fate of DTCs is not controlled through the association with specific vessel subtypes. Instead, numbers of osteoblasts dictate availability of protective niches which breast cancer cells can colonize prior to stimulation of metastatic outgrowth.

Increased Abundance of Tumour-Associated Neutrophils in HPV-Negative Compared to HPV-Positive Oropharyngeal Squamous Cell Carcinoma Is Mediated by IL-1R Signalling.

The incidence of human papillomavirus (HPV)-associated cancer is increasing and HPV is now implicated in the aetiology of more than 60% of all oropharyngeal squamous cell carcinomas (OPSCC). In OPSCC, innate immune cells such as neutrophils and macrophages generally correlate with poor prognosis, whilst adaptive immune cells, such as lymphocytes, tend to correlate with improved prognosis. This may, in part, be due to differences in the immune response within the tumour microenvironment leading to the recruitment of specific tumour-associated leukocyte sub-populations. In this study, we aimed to examine if differences exist in the levels of infiltrated leukocyte sub-populations, with particular emphasis on tumour-associated neutrophils (TAN), and to determine the mechanism of chemokine-induced leukocyte recruitment in HPV-positive compared to HPV-negative OPSCC. Immunohistochemical analysis showed that HPV-negative OPSCC contained significantly more neutrophils than HPV-positive tumours, whilst levels of CD68+ macrophages and CD3+ lymphocytes were similar. Using a 3D tissue culture model to represent tumour-stromal interactions, we demonstrated that HPV-negative tumour-stromal co-cultures expressed significantly higher levels of CXCL8, leading to increased neutrophil recruitment compared to their HPV-positive counterparts. HPV-negative OPSCC cells have previously been shown to express higher levels of IL-1 than their HPV-positive counterparts, indicating that this cytokine may be responsible for driving increased chemokine production in the HPV-negative 3D model. Inhibition of IL-1R in the tumour-stromal models using the receptor-specific antagonist, anakinra, dramatically reduced chemokine secretion and significantly impaired neutrophil and monocyte recruitment, suggesting that this tumour-stromal response is mediated by the IL-1/IL-1R axis. Here, we identify a mechanism by which HPV-negative OPSCC may recruit more TAN than HPV-positive OPSCC. Since TAN are associated with poor prognosis in OPSCC, our study identifies potential therapeutic targets aimed at redressing the chemokine imbalance to reduce innate immune cell infiltration with the aim of improving patient outcome.

Combined administration of a small-molecule inhibitor of TRAF6 and Docetaxel reduces breast cancer skeletal metastasis and osteolysis.

Tumour necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in breast cancer and osteoclastic bone destruction. Here, we report that 6877002, a verified small-molecule inhibitor of TRAF6, reduced metastasis, osteolysis and osteoclastogenesis in models of osteotropic human and mouse breast cancer. First, we observed that TRAF6 is highly expressed in osteotropic breast cancer cells and its level of expression was higher in patients with bone metastasis. Pre-exposure of osteoclasts and osteoblasts to non-cytotoxic concentrations of 6877002 inhibited cytokine-induced NFκB activation and osteoclastogenesis, and reduced the ability of osteotropic human MDA-MB-231 and mouse 4T1 breast cancer cells to support bone cell activity. 6877002 inhibited human MDA-MB-231-induced osteolysis in the mouse calvaria organ system, and reduced soft tissue and bone metastases in immuno-competent mice following intra-cardiac injection of mouse 4T1-Luc2 cells. Of clinical relevance, combined administration of 6877002 with Docetaxel reduced metastasis and inhibited osteolytic bone damage in mice bearing 4T1-Luc2 cells. Thus, TRAF6 inhibitors such as 6877002 - alone or in combination with conventional chemotherapy - show promise for the treatment of metastatic breast cancer.

Microenvironmental IL1ß promotes breast cancer metastatic colonisation in the bone via activation of Wnt signalling.

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1ß stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1ß-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.

The bone metastasis niche in breast cancer-potential overlap with the haematopoietic stem cell niche in vivo.

BACKGROUND: Bone metastasis is one of the most common complications of advanced breast cancer. During dissemination to bone, breast cancer cells locate in a putative 'metastatic niche', a microenvironment that regulates the colonisation, maintenance of tumour cell dormancy and subsequent tumour growth. The precise location and composition of the bone metastatic niche is not clearly defined. We have used in vivo models of early breast cancer dissemination to provide novel evidence that demonstrates overlap between endosteal, perivascular, HSC and the metastatic niche in bone. METHODS: Estrogen Receptor (ER) +ve and -ve breast cancer cells were labelled with membrane dyes Vybrant-DiD and Vybrant-CM-DiI and injected via different routes in BALBc/nude mice of different ages. Two-photon microscopy was used to detect and quantitate tumour cells and map their location within the bone microenvironment as well as their distance to the nearest bone surface compared to the nearest other tumour cell. To investigate whether the metastatic niche overlapped with the HSC niche, animals were pre-treated with the CXCR4 antagonist AMD3100 to mobilise hematopoietic (HSCs) prior to injection of breast cancer cells. RESULTS: Breast cancer cells displayed a characteristic pattern of homing in the long bones, with the majority of tumour cells seeded in the trabecular regions, regardless of the route of injection, cell-line characteristics (ER status) or animal age. Breast cancer cells located in close proximity to the nearest bone surface and the average distance between individual tumour cells was higher than their distance to bone. Mobilisation of HSCs from the niche to the circulation prior to injection of cell lines resulted in increased numbers of tumour cells disseminated in trabecular regions. CONCLUSION: Our data provide evidence that homing of breast cancer cells is independent of their ER status and that the breast cancer bone metastasis niche is located within the trabecular region of bone, an area rich in osteoblasts and microvessels. The increased number of breast cancer cells homing to bone after mobilisation of HSCs suggests that the HSC and the bone metastasis niche overlap.

Pharmacological inhibition of the IKKε/TBK-1 axis potentiates the anti-tumour and anti-metastatic effects of Docetaxel in mouse models of breast cancer.

IκB kinase subunit epsilon (IKKε), a key component of NFκB and interferon signalling, has been identified as a breast cancer oncogene. Here we report that the IKKε/TBK1 axis plays a role in the initiation and progression of breast cancer osteolytic metastasis. Cancer-specific knockdown of IKKε in the human MDA-MB-231-BT cells and treatment with the verified IKKε/TBK1 inhibitor Amlexanox reduced skeletal tumour growth and osteolysis in mice. In addition, combined administration of Amlexanox with Docetaxel reduced mammary tumour growth of syngeneic 4T1 cells, inhibited metastases and improved survival in mice after removal of the primary tumour. Functional and mechanistic studies in breast cancer cells, osteoclasts and osteoblasts revealed that IKKε inhibition reduces the ability of breast cancer cells to grow, move and enhance osteoclastogenesis by engaging both IRF and NFκB signalling pathways. Thus, therapeutic targeting of the IKKε/TBK1 axis may be of value in the treatment of advanced triple negative breast cancer.

Endogenous Production of IL1B by Breast Cancer Cells Drives Metastasis and Colonization of the Bone Microenvironment.

PURPOSE: Breast cancer bone metastases are incurable, highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL1B by tumor cells drives metastasis and growth in bone. EXPERIMENTAL DESIGN: Tumor/stromal IL1B and IL1 receptor 1 (IL1R1) expression was assessed in patient samples and effects of the IL1R antagonist, Anakinra, or the IL1B antibody canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell-derived IL1B on bone colonization and parameters associated with metastasis were measured in MDA-MB-231, MCF7, and T47D cells transfected with IL1B/control. RESULTS: In tissue samples from >1,300 patients with stage II/III breast cancer, IL1B in tumor cells correlated with relapse in bone (HR = 1.85; 95% CI, 1.05-3.26; P = 0.02) and other sites (HR = 2.09; 95% CI, 1.26-3.48; P = 0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL1B by tumor cells promoted epithelial-to-mesenchymal transition (altered E-Cadherin, N-Cadherin, and G-Catenin), invasion, migration, and bone colonization. Contact between tumor and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. IL1B alone did not stimulate tumor cell proliferation. Instead, IL1B caused expansion of the bone metastatic niche leading to tumor proliferation. CONCLUSIONS: Pharmacologic inhibition of IL1B has potential as a novel treatment for breast cancer metastasis.

The IL-1/IL-1R axis induces greater fibroblast-derived chemokine release in human papillomavirus-negative compared to positive oropharyngeal cancer.

Human papillomavirus (HPV) is now recognised as a major aetiological agent in the pathogenesis of oropharyngeal carcinoma (OPC). HPV-positive tumours are associated with better outcomes compared to HPV-negative tumours, possibly due to differences in their aetiology and/or the tumour microenvironment. Increased numbers of tumour-associated leukocytes have been observed in many cancers including OPC, with variable influence on prognosis depending on the leukocyte subpopulation investigated. Whether HPV status influences leukocyte recruitment to OPC remains unknown. This in-vitro study examined differences in the chemoattractant capacity of HPV-positive and HPV-negative OPC cell lines. Gene and protein expression analysis demonstrated that whilst both monocultures of HPV-positive and HPV-negative cell lines, along with normal tonsillar fibroblasts (NTF), expressed low chemokine levels, NTF cultured with conditioned medium from HPV-negative OPC cells expressed significantly higher levels of all chemokines tested compared to NTF incubated with the medium from HPV-positive OPC cell lines. HPV-negative OPC lines expressed IL-1ß mRNA whereas HPV-positive cells did not, and NTF constitutively expressed IL-1R1. Pre-treatment with the IL-R antagonist, anakinra or siRNA to IL-1R1 significantly reduced chemokine secretion from NTF stimulated with conditioned medium from HPV-negative tumour cells or recombinant IL-1ß (p < 0.05). These data suggest that secretion of chemokines is driven by the interaction between HPV-negative OPC cells and stromal fibroblasts through an IL-1/IL-1R-mediated mechanism that is less prominent within the HPV-positive tumour microenvironment. These observations may explain differences in leukocyte sub-populations recruited to HPV-positive versus negative OPC and indicate that HPV status is a key determinant in controlling the inflammatory tumour microenvironment.

Chemotherapy resistance and stemness in mitotically quiescent human breast cancer cells identified by fluorescent dye retention.

Metastatic recurrence in breast cancer is a major cause of mortality and often occurs many years after removal of the primary tumour. This process is driven by the reactivation of disseminated tumour cells that are characterised by mitotic quiescence and chemotherapeutic resistance. The ability to reliably isolate and characterise this cancer cell population is critical to enable development of novel therapeutic strategies for prevention of breast cancer recurrence. Here we describe the identification and characterisation of a sub-population of slow-cycling tumour cells in the MCF-7 and MDA-MB-231 human breast cancer cell lines based on their ability to retain the lipophilic fluorescent dye Vybrant® DiD for up to six passages in culture. Vybrant® DiD-retaining (DiD+) cells displayed significantly increased aldehyde dehydrogenase activity and exhibited significantly reduced sensitivity to chemotherapeutic agents compared to their rapidly dividing, Vybrant® DiD-negative (DiD-) counterparts. In addition, DiD+ cells were exclusively capable of initiating population re-growth following withdrawal of chemotherapy. The DiD+ population displayed only partial overlap with the CD44+CD24-/low cell surface protein marker signature widely used to identify breast cancer stem cells, but was enriched for CD44+CD24+ cells. Real-time qPCR profiling revealed differential expression of epithelial-to-mesenchymal transition and stemness genes between DiD+ and DiD- populations. This is the first demonstration that both MCF-7 and MDA-MB-231 human breast cancer lines contain a latent therapy-resistant population of slow-cycling cells capable of initiating population regrowth post-chemotherapy. Our data support that label-retaining cells can serve as a model for identification of molecular mechanisms driving tumour cell quiescence and de novo chemoresistance and that further characterisation of this prospective tumour-reinitiating population could yield novel therapeutic targets for elimination of the cells responsible for breast cancer recurrence.

Parathyroid Hormone (PTH) Increases Skeletal Tumour Growth and Alters Tumour Distribution in an In Vivo Model of Breast Cancer.

Breast cancer cells colonize the skeleton by homing to specific niches, but the involvement of osteoblasts in tumour cell seeding, colonization, and progression is unknown. We used an in vivo model to determine how increasing the number of cells of the osteoblast lineage with parathyroid hormone (PTH) modified subsequent skeletal colonization by breast cancer cells. BALB/c nude mice were injected for five consecutive days with PBS (control) or PTH and then injected with DiD-labelled breast cancer cells via the intra-cardiac route. Effects of PTH on the bone microenvironment and tumour cell colonization and growth was analyzed using bioluminescence imaging, two-photon microscopy, and histological analysis. PTH treatment caused a significant, transient increase in osteoblast numbers compared to control, whereas bone volume/structure in the tibia was unaffected. There were no differences in the number of tumour cells seeding to the tibias, or in the number of tumours in the hind legs, between the control and PTH group. However, animals pre-treated with PTH had a significantly higher number of tumour colonies distributed throughout skeletal sites outside the hind limbs. This is the first demonstration that PTH-induced stimulation of osteoblastic cells may result in alternative skeletal sites becoming available for breast cancer cell colonization.

Murine models of breast cancer bone metastasis.

Bone metastases cause significant morbidity and mortality in late-stage breast cancer patients and are currently considered incurable. Investigators rely on translational models to better understand the pathogenesis of skeletal complications of malignancy in order to identify therapeutic targets that may ultimately prevent and treat solid tumor metastasis to bone. Many experimental models of breast cancer bone metastases are in use today, each with its own caveats. In this methods review, we characterize the bone phenotype of commonly utilized human- and murine-derived breast cell lines that elicit osteoblastic and/or osteolytic destruction of bone in mice and report methods for optimizing tumor-take in murine models of bone metastasis. We then provide protocols for four of the most common xenograft and syngeneic inoculation routes for modeling breast cancer metastasis to the skeleton in mice, including the intra-cardiac, intra-arterial, orthotopic and intra-tibial methods of tumor cell injection. Recommendations for in vivo and ex vivo assessment of tumor progression and bone destruction are provided, followed by discussion of the strengths and limitations of the available tools and translational models that aid investigators in the study of breast cancer metastasis to bone.

The role of osteoblasts in bone metastasis.

The primary role of osteoblasts is to lay down new bone during skeletal development and remodelling. Throughout this process osteoblasts directly interact with other cell types within bone, including osteocytes and haematopoietic stem cells. Osteoblastic cells also signal indirectly to bone-resorbing osteoclasts via the secretion of RANKL. Through these mechanisms, cells of the osteoblast lineage help retain the homeostatic balance between bone formation and bone resorption. When tumour cells disseminate in the bone microenvironment, they hijack these mechanisms, homing to osteoblasts and disrupting bone homeostasis. This review describes the role of osteoblasts in normal bone physiology, as well as interactions between tumour cells and osteoblasts during the processes of tumour cell homing to bone, colonisation of this metastatic site and development of overt bone metastases.