Modifying the bone metastatic niche by targeting hypoxic response: a novel therapeutic approach to reduce breast cancer metastasis?

Background: 70% of patients with advanced breast or prostate cancer develop incurable bone metastases. These have devastating effects in patients being associated with pain, fractures, spinal cord compression and life threatening hypercalcaemia. The mechanisms driving targeted metastases are complex, requiring changes in both the primary tumour and the metastatic niche. The latter creates a permissive environment for the establishment of metastatic growths. Hypoxia is a physiological condition prevalent in solid tumours that is known to associate with metastases. Hypoxia directly induces pro-metastatic behaviour in tumour cells and upregulates a number of proteins associated with breast to bone metastasis including chemokine receptor-4, lysyl oxidase, receptor activator of NF-kB (RANK) and osteopontin. Hypoxia also plays a key role in the bone metastatic niche influencing both osteoclast differentiation and macrophage behaviour that are prerequisites for breast cancer cell colonisation. Hypoxia is a focus for anti-cancer therapies, either directly or via inhibition of oncogenic signalling pathways which impact on activity of the key hypoxia-associated transcription factor HIF-1. Our studies have previously demonstrated that hypoxia-targeted approaches can ameliorate metastatic phenotype in cancer cells, but we have not yet investigated what is co-incidentally happening within the niche environment.

Our aim is to model the influence of hypoxia, directly or through cross talk from the primary tumour, on the bone metastatic niche and investigate the utility of therapeutic approaches that target hypoxia-mediated cell signalling as a means to modify the propensity for metastatic progression to bone.

Objectives:
1) Investigate using media transfer experiments the impact of hypoxic-priming of breast cancer cells on osteoclast and macrophage differentiation under differing oxygen tensions
2) Profile cytokine changes that could impact response
3) Evaluate the effects of molecular targeted therapies on hypoxia-mediated changes
4) Apply in vivo methodology to investigate impact of primary breast tumours on the bone metastatic niche
5) Investigate in vivo impact of targeted approaches on niche formation and cancer cell colonisation

In vitro methods: Mouse bone marrow cells will be isolated from femurs and adherent cells treated with M-CSF (macrophage colony-stimulating factor) to generate bone marrow-derived monocyte/macrophage lineage cells [BMMs]. Metastatic breast cancer cells (MDA231, BT-549, 4T1; luciferase/EGFP tagged) will be cultured under varying oxygen tensions, media removed and applied to recipient BMMs also at varying O2. Osteoclast differentiation will be tracked via TRAP (tartrate-resistant acid phosphatase) staining with RANK-Ligand used as a positive control. Differentiation will be confirmed via pit formation assay (Osteo assay plates). Morphological changes in TRAP-negative BMMs suggestive of changes in macrophage polarisation/activation would indicate evaluation of Galectin-3. Experiments will be repeated in the presence of therapies targeting phosphoinositide 3-kinase (PI3K) and extracellular-signal-regulated kinase 5 (ERK5), known to converge at HIF-1 signalling. Cytokine arrays will be used to identify putative mechanistic drivers of effects.

In vivo methods: Breast tumours will be established in the mammary fat pad. Co-incidental bone specific changes will be imaged using microCT, and fluorescence imaging of MMP-9 activity. Tumour cell dissemination and changes in bone pathology will be investigated post-mortem. Primary tumours can be surgically removed, and/or treated with radiotherapy to extend experimental timeframe and increase likelihood of visible metastases which will be tracked by luciferase-imaging.