The inflammatory microenvironment of the post-pregnancy mammary gland and its role in tumourigenesis

The fundamental role of the mammary gland (MG) is to produce milk to feed young, thus during lactation the gland is packed with milk-producing mammary cells. Upon weaning, when milk is no longer needed, these milk-producing cells die and are replaced by fat cells. This process is called involution and is thought to be important in the development of pregnancy-associated breast cancer. In addition, the probability that involution will lead to the development of cancer is thought to increase with age. This is important because in today's society, where women often delay starting a family until later in life, it is likely that we will see increasing numbers of pregnancy-associated breast cancer cases.

This research proposal outlines a number of aims and objectives which will help us identify exactly how involution in the MG leads to breast cancer. To understand this, we use mouse models where we can look inside the MG tissue at specific time-points following pregnancy, and we can study the cells which are there and what they are doing. We do this by analysing different markers which the cells express on their surface. We know that during involution, immune cells called macrophages accumulate in the MG to help clear the extensive debris. These cells help to create an distinct type of inflammatory environment, where they signal to other immune cells to restrict the number of cells coming into the gland. This is important for preventing tissue damage in the gland by an unwanted immune response. However, we think that these signals generate an environment which is prone to developing cancer. To test this, we will use genetically modified mice which lack these macrophage cells or the molecules they produce, to see if tumours develop in the same way. To understand how age contributes to pregnancy-associated breast cancer, we will use aged mice, which are the equivalent of 35-40 years old in human years.

After pregnancy, the mammary gland undergoes extensive remodelling whereby milk-producing epithelial cells are replaced by adipocytes to return the gland to its pre-pregnant state. This regenerative process (involution) is associated with tumourigenesis and is thought to contribute significantly to pregnancy-associated breast cancer. Whilst the physiological processes of involution are well defined, the immunological implications are less well understood and are likely to be of great importance. Furthermore, the tumourigenic potential of involution is thought to increase with age and thus is likely to have an increasing impact in a society where women delay child birth until later in life. We want to investigate the role of the immune system in involution to determine how the mammary microenvironment becomes tumourigenic and how this is exacerbated in older women.

To fulfil these aims, we have an armoury of mechanistic in vivo models which have been established during our current MRC funding. We have already identified many of the leukocyte populations present in the gland during involution. To fully characterise these cells we will interrogate their molecular function, by ex vivo stimulation of isolated cells and subsequent cytokine analysis. Our previous microarray highlighted key players in this respect. To identify the role of these mediators in involution we will use genetically altered mice which lack these components in conjunction with depletion of specific cell types, namely macrophages. We will then utilise our established in vivo syngeneic tumour model to determine firstly how involution contributes to tumourigenesis, and secondly how the molecular mediators outlined above contribute to tumourigenesis. Finally, using cohorts of aged mice we will dissect how age affects involution-mediated tumour development.

The outlined research has the potential to impact a number of scientific disciplines. It is highly relevant to those in the fields of immunology, cancer biology, mammary gland, molecular genetics of cancer, inflammation, microenvironment, ageing and epidemiology; it will advance knowledge in all these disciplines. This proposal builds on previous MRC funding which is now in its final stages. During this funding, methodologies have been developed, for example the isolation of leukocyte populations from the involuting MG. This has enabled our understanding of MG immunology to progress, the outcomes of which were recently presented at the Cell Symposia: Cancer, Inflammation & Immunity in Sitges, Spain and are currently in preparation for publication in Breast Cancer Research. We hope to fully implement these findings and develop ideas further in the proposed project.

The aging aspect of the proposed work is of particular significance because it is likely to generate novel and high impact data. The tumourigenic potential of involution is thought to increase with age and thus is likely to have an increasing incidence in a society where women delay child birth until later in life. To our knowledge, these vital experiments utilising aged mice in the context of immune regulation of involution have not been undertaken in the past. Understanding the epidemiological phenomenon of increased risk following a late pregnancy would facilitate wider dissemination of our findings. This knowledge will be useful to breast oncologists as it may highlight the mechanism by which the breast ages. The relationship between ageing and tumourigenesis may provide potential avenues for the development of future breast cancer therapies.

This project will provide an opportunity to engage the public in mammary gland biology, breast cancer research and basic research methods. The Watson laboratory has taken part in the Cambridge Science Festival for many years. The proposed project can be incorporated into this exhibit and will have an impact on public understanding of science and breast cancer research. Finally, in addition to publication of research in well cited scientific journals, members of the laboratory regularly give talks and seminars at universities and research institutions throughout the UK and the world and present their work at international conferences. Professor Watson is regularly invited to give seminars both in the UK and internationally and thus our work reaches international audiences on a regular basis, providing impact on an international level.

The University of Cambridge as a teaching institution, affords opportunities for fellows to engage in teaching at various levels. In addition to lecturing to undergraduates, supervision of Part II project students is possible. This is mutually beneficial for all concerned as the fellows gain teaching experience and associated skills and the students are able to work with scientists and clinicians doing research at the cutting edge.