To investigate the role of neutrophils in breast cancer progression

Breast cancer is the most common cancer in the UK, affecting 1 in 8 women during the course of their lifetime. Despite advances in treatment, around 12,000 women die from breast cancer in the UK annually, highlighting the urgent need for a deeper understanding of breast cancer biology to develop more effective treatment options.

Over the last decade, the body's immune system has been increasingly found to play a crucial role in cancer. The immune system comprises of the innate immune system (present from birth) and the adaptive immune system (which develops after exposure to infection). Neutrophils (a type of white blood cell) play a key role of the innate immune system whose main function is to fight against infection and help with wound healing. However, there is rapidly growing evidence that neutrophils are much more complex, and have more functions than previously thought, such as encouraging tumour growth and development. Neutrophils can promote the spread of tumour cells to the rest of the body (metastasis) and patients across a range of tumour types, including breast cancer, often have higher levels of neutrophils in their blood. Evidence from mice models of breast cancer shows that neutrophils can either support tumour growth (tumour-promoting neutrophils) or help to suppress tumour growth (tumour-inhibitory neutrophils) (Wculek et al, Nature 2015), (Comen et al, NPJ Breast Cancer 2016). This suggests that there may be different subtypes of neutrophils which account for the different functions.

Gaining a deeper understanding of the characteristic features and functions of the different neutrophil subtypes and how they change in the presence of breast cancer can open up new potential avenues for treatment. This research proposal seeks to address this by analysing patient blood samples undergoing chemotherapy for early, potentially curative breast cancer and from patients with incurable metastatic breast cancer.

A common side effect of chemotherapy is lowering of the neutrophil count which reduces the body's ability to fight infection and can be potentially life threatening (also known as neutropaenic sepsis). To avoid this, patients receiving chemotherapy treatment for early breast cancer are given Granulocyte-Colony Stimulating Factor (G-CSF) as a precautionary measure to boost the neutrophil count and reduce the risk of neutropaenic sepsis. However, it is not known what effect G-CSF has on the types of neutrophils produced in patients with breast cancer. This research seeks to address this through analysing neutrophils in mice models of breast cancer and neutrophils in blood samples of patients with early breast cancer receiving G-CSF treatment during their chemotherapy regimen. Different types of neutrophils will be identified using a process called Flow Cytometry, and then cultured with breast cancer cell lines to further investigate their tumour-promoting and tumour-inhibiting effects. The results of the study have important implications in terms of current treatment protocols, and will help clinicians make more informed decisions involving the use of G-CSF in chemotherapy regimens.

The research will be undertaken at Imperial College London under the primary supervision of Professor Charles Coombes in collaboration with Dr Ilaria Malanchi at the Crick Institute, whose research interest focuses on the role of neutrophils in mice models of breast cancer. Fostering collaboration between two world class institutions will enhance research productivity, promote knowledge sharing, increase networking and improve the UK's international standing in pushing the boundaries of scientific research.

The work will be performed by Dr Anisha Ramessur, a specialist registrar in Medical Oncology who is committed to undertaking research that gives a greater understanding of breast cancer biology that can be translated into novel therapeutic targets in order to improve the outcomes for patients with breast cancer.

Pre-clinical evidence suggests that neutrophils are mobilised from the bone marrow in presence of primary breast cancer and act to promote distant metastatic growth (Wculek et al, 2015). Clinical evidence suggests that the neutrophil:lymphocyte ratio has prognostic value, and that circulatory neutrophils in breast cancer patients are different compared to heathy individuals (Wei et al, 2016), (Sagiv et al, 2015). Our collaboration lab has found specific phenotypical changes within neutrophil pools in mice with metastatic breast tumours (unpublished data). We hypothesise that those phenotypic changes are functionally linked to the pro-metastatic activity of neutrophils and that similar changes could be present in human patients. G-CSF is given to patients with early breast cancer to reduce the risk of chemotherapy-related neutropenic sepsis but its effect on influencing neutrophil subsets and their tumourigenic function is unknown.

Aim: To investigate the role of neutrophils in breast cancer progression

Objectives:
1. To assess how G-CSF affects the function and phenotype of neutrophil subsets in G-CSF knockout mice models of breast cancer
2.To characterize phenotypical changes in circulatory neutrophil subsets in patients with early and advanced stage breast cancer compared to healthy controls
3. To assess how circulatory tumour-educated neutrophil subsets are functionally acting on cancer cells
4. To assess how G-CSF dependent replenishment of circulatory neutrophils can influence neutrophil subsets in patients with breast cancer that have had chemotherapy

Methods:
Neutrophil subsets will be identified using flow cytometry. Functional characteristics will be assessed with techniques including in-vitro culture with breast cancer cell lines, cell viability assays, immunoassays, proteomics and RNA-Seq.

Understanding how neutrophil subsets influence tumour progression has clinical implications for developing new therapies to improve patient outcomes.

The primary aim of the proposal is to significantly enhance our understanding of the role of neutrophils in breast cancer progression. The collaborative lab have published widely on the tumour-promoting effect of neutrophils in pre-clinical mice models of metastatic breast cancer. This project seeks to extend the research to investigate the functional and phenotypical characteristics of neutrophils in human breast cancer and its effect on tumour progression, both in the adjuvant and metastatic setting. This proposal has far reaching potential to impact a wide number of beneficiaries including:

1. Patients
If there are found to be neutrophil subsets which have a tumour promoting or tumour supressing effect on breast cancer progression, as suggested by a review (Coffelt et al, Nat Rev Cancer 2016), this opens avenues to develop novel drugs to promote anti-tumourigenic neutrophil subsets and reduce pro-tumourigenic neutrophil subsets with the aim to reduce the risk of breast cancer recurrence in the adjuvant setting and improve quality of life and overall survival outcomes for patients with metastatic breast cancer. We anticipate that clinical trials with drugs to target neutrophil subsets and associated effector mechanisms to be set up in years following completion of the project.

2. Clinical and academic community
G-CSF is widely used during chemotherapy for early breast cancer to reduce the risk of chemotherapy associated neutropaenic sepsis. If G-CSF treatment is found to influence breast cancer progression through its effect on different neutrophil subsets, this has clinically significant implications for the use of G-CSF in the patients treated with curative intent. This research has the potential to influence breast cancer protocols and guidelines which involve the use of G-CSF for patients both nationally and worldwide. This not only has implications for breast cancer but also for other solid malignancies which use G-CSF as part of the treatment protocol.

The proposed research will enable the applicant to develop lab techniques including flow cytometry, co-culture techniques, method design and data analysis which will be highly useful transferable skills for a future clinical academic career.

Functionally different neutrophil subtypes identified through this project can contribute knowledge to the wider academic community outside the field of oncology where neutrophils are thought to play a key role such as autoimmune diseases.

3. Economic implications
If G-CSF increases the pro-tumourigenic neutrophil subsets in a subgroup of patients, then a more personalised approach such as avoidance of G-CSF can be taken to reduce NHS drug costs. The proposed research has wider implications for the commercial private sector involving new drug development to target particular neutrophil subsets and their associated effector mechanisms either directly or indirectly, either alone or in conjunction with chemotherapy, hormonal therapy or other immune modulating agents. There is the potential for new drugs, job creation, new biotech companies which can generate wealth for the UK and global economy. Fostering collaboration with the host academic institution can expedite the development of new drugs and access to patients for recruitment into clinical trials, enabling identification of benefit earlier.

4. Wider Public and social engagement
This project can promote public awareness of the role of neutrophils in breast cancer which will be communicated through press releases, publications, conferences, and exhibitions during the course of the project. Informing citizens of research knowledge allows them to help shape research agendas and be involved in decision making about how discoveries are used.

This research has vast potential to impact patient outcomes, contribute knowledge to the academic community, promote global health and wealth and inspire the next generation of researchers through public involvement.