the roleDefining of IL-1B in breast cancer bone metastasis

Despite advances in detection and treatment, breast cancer still causes over 12,000 deaths per year in the UK. The introduction of screening programmes, increased specialisation of care and the availability of new drugs have undoubtedly improved survival rates for patients whose breast cancer is picked up at an early stage. However survival rates have not improved for patients with breast cancer that is more advanced and has moved to other parts of the body. Once breast cancer has spread it unfortunately becomes incurable and the only treatment that is available is aimed at slowing disease progression and limiting pain.

One of the most common places for breast cancer to spread to is the skeleton. Most people who die from breast cancer have traces of the disease in their bones. Working out why this is and how breast cancer develops and grows in bone could help researchers develop new treatments to slow the progression of advanced breast cancer or stop it spreading to bone altogether.
Scientists at the University of Sheffield are now gathering evidence that a molecule known as IL-1B plays an important role in the spread of breast cancer to bone. This suggests that drugs which can "switch off" IL-1B in early stage breast cancer could stop the disease spreading. Drugs that target IL-1B might also be able to slow, stop or even reverse the further growth of breast cancer that has spread to a patient's bones.

First researchers need to understand how IL-1B from breast cancer cells alters the environment around the main tumour, making the disease more aggressive and likely to spread. We also need to learn how IL-1B alters the way that molecules behave and organise in bones, creating sites that breast cancer cells can live and grow in.

I plan to test this in a series of laboratory experiments using specially engineered breast cancer cells. I will compare the behaviour of breast cancer cells that have been modified to have no IL-1B against breast cancer cells that that have been produced with excessive levels of IL-1B. I will also examine the effects that these cells have on mice that are not able to produce IL-1B and mice that produce IL-1B normally. These experiments will be designed to model the conditions in which human breast cancer develops and grows as accurately as possible.

Tumour cells must progress through many different stages if they are to spread from the breast to the skeleton. These include growing at the primary tumour site in the breast, moving into the bloodstream, homing towards the skeleton, finding a suitable site to live and develop in the bone, and then finally growing and spreading to produce secondary tumours within the skeleton.

Taken together, the different elements of my project will help reveal how IL-1B and its receptor are involved with all of the many different stages of breast cancer spread to the skeleton. Specifically, the experiments in my project should show:

1. How IL-1B from breast cancer cells affects the spontaneous spread of breast cancer cells to bone;

2. The importance of direct interactions between IL-1B and cancer cells to the spread of early stage disease;

3. How IL-1B produced by breast cancer cells and IL-1B produced by bone each alter the environment in the skeleton that cancer cells live and grow in;

4. Whether drugs that "switch off" IL-1B could help prevent early stage breast cancer from spreading to bone, and/or help patients whose disease has already moved into the skeleton.

Drugs that target IL-1B and its receptor have already been made and have an exceptional safety record. This means that if a specific role for IL-1B in the spread of breast cancer can be identified, it should be relatively quick to "repurpose" these existing drugs for breast cancer treatment.

This project aims to determine the specific roles played by IL-1B in the development and progression of breast cancer bone metastasis. Genetically manipulated breast cancer cell lines and in vitro modelling will be used to elucidate effects of IL-1B on parameters associated with early metastasis. Mouse models of human breast cancer metastasis to human bone as well as syngeneic IL-1B/IL1-R1 knockout models of bone metastasis will be employed to assess the specific effects of IL-1B from tumour cells and from the host environment on different stages of metastasis. Dr Ottewell is experienced in all proposed techniques:
Genetic manipulation of cell lines: IL-1B/IL1-R1 knockout and control bone metastatic mammary cancer cell lines will generated using CRISPR. Overexpressing/control cells will be generated by stable transfection with IL-1B, IL-1R1 or scramble sequence. Cells will be transfected with GFP and luciferase to allow in vivo imaging and detection of tumour cells in blood.
In vitro analysis of metastasis: Knockout, overexpressing and control cells will be assessed for: Proliferation - Z2 Coulter counter; migration - scratch assay; invasion towards media or bone cells -transwell assay.
Mouse models of human breast cancer metastasis to human bone: Human sub-chondral bone will be implanted subcutaneously into female NODSCID mice. 4 weeks later wild-type or genetically manipulated MDA-MB-231 or T47D breast cancer cells will be injected into each hind mammary fat pad and mice monitored for bone metastasis. Identification of bone metastasis in syngeneic models will be assessed following intra cardiac or intravenous injection of IL-1B/IL-1R1 knockout, overexpressing or control cells.
Analysis: Primary tumour growth and bone metastases- callipers/luciferase imaging; dissemination of tumour cells in the blood - flow cytomentry; dissemination of tumour cells in bone - two photon microscopy; bone lesions- uCT; apoptosis - caspase; proliferation - Ki67; angiogenesis.

Medical impact
Drugs that target IL-1B and IL-1R1 (Anakinra and Ilaris) have already been approved by the National Institute for Health and Care Excellence (NICE) for use in patients with rheumatoid arthritis and myocardial infarction and these drugs have an excellent safety record. Part of this project includes using in vivo model systems to test the efficacy of these drugs against breast cancer bone metastasis. Sheffield has a large number of clinical academics with outstanding international reputations for performing clinical trials in the field of bone metastasis and I currently have ongoing collaborations with Prof. Robert Coleman and Prof. Janet Brown. Data from this MRC NIRG project will be discussed in regular meetings with clinical colleagues (based at Weston Park Hospital) with the aim of developing pharmacological strategies, translational studies and future clinical trials for IL-1B inhibitors as potential treatments for breast cancer bone metastasis. To facilitate further pre-clinical and clinical studies collaboration with industrial partners will be explored with support from Sheffield Healthcare Gateway. Specifically I will build up collaborations with Amgen to further explore the therapeutic potential of Anakinra. I have already established collaboration with Novartis Pharmaceuticals to allow me to test Ilaris in pre-clinical models (proposed in this application) and with positive pre-clinical data would anticipate extending this collaboration with the aim of initiating a clinical trial.
Economic impact
Data generated in this NIRG have the potential to lead to the first preventative or curative treatment for breast cancer bone metastasis. This debilitating condition leads to severe pain and bone fractures resulting in significant economic drains on individuals and society in terms of patients being unable to work and requiring hospitalisation / specialist care. If we can establish a treatment strategy that prevents or reduces breast cancer growth in bone (see Medical impact) patients will be able to lead productive and pain free lives.
Ethical impact
This research will closely adhere to the principals of the 3R's (reduction, refinement and replacement) for all experimental procedures performed as part of this project. At the end of the project, to facilitate continued reduction in animal use and dissemination of materials, all surplus in vivo samples (tissue, serum, RNA and protein) will be stored and made open access (and free of charge) to other researchers via the SEARCHBreast initiative (https://searchbreast.org). I have costed £100 onto this application to enable long-term storage of material for future use by other researchers. In addition the University of Sheffield is a member of the Concordat on Openness in Animal Research and recognises the need for greater public understanding about animal research and the benefits of this work. Dissemination of key results from in vivo experiments will be facilitated with the support from the University of Sheffield Media Relations team and Public Engagement with Research team.
Social Impact
Many people have either experienced breast cancer first hand or know someone who has been affected by this condition. This project would be of direct interest to this group of people as it aims to identify how a molecules promote breast cancer bone metastasis and new thrapies for this currently incurable condition. My research team has an established programme of outreach designed to explain why research is necessary, what types of research are undertaken and how research benefits society. Through this scheme I currently engage with an active Breast Cancer Support group comprising patients and their families that has formed at Weston Park Hospital for whom I carry out seminars and laboratory tours. I also undertake public lectures through the University of Sheffield "Inspire" lecture series. Information obtained from this NIRG will be shared via these mechanisms.