Tissue-specific recruitment of regulatory T cells in colorectal cancer and liver metastases

Colorectal cancer is the third most common cancer in the United Kingdom after breast and lung cancer. Colorectal cancer is deadly in one-third of patients due to either spread of the disease to other organs at the time of diagnosis or recurrence of the disease later on. Patients with colorectal cancers containing a large number of lymphocytes (a type of white blood cell) have a better prognosis in terms of improved survival than patients with cancers containing fewer lymphocytes. It is proposed that these lymphocytes represent a favourable response to the cancer by the body's natural immune system.
A specific group of lymphocytes known as regulatory T cells can suppress the body's immune response and these are also found in abundance in colorectal cancers. Regulatory T cells in tumours can inhibit other lymphocytes from attacking the tumour. Colorectal cancers would therefore ideally contain lymphocytes attacking the cancer and a minimal number of suppressive regulatory T cells.
It is known that lymphocytes, including regulatory T cells, are recruited to different tissues in the body via a network of different molecules known as chemokines and adhesion molecules. Regulatory T cells can also be formed from other lymphocytes, a process known as induction.
We propose to focus our research on identifying which homing molecules are present on regulatory T cells and which are important in trafficking these suppressive cells into colorectal cancers. The intention is to be able to block recruitment of regulatory T cells into tumours using inhibitors of homing molecules while at the same time allowing effector (non-suppressive) lymphocytes into the cancer unabated. This provides a means to support the body's natural anti-tumour immune response and would be a new means of treating colorectal cancer. We will also identify other immune cells present in colorectal cancer which may be responsible for inducing regulatory T cells within the cancer tissue.
The research will be conducted at the University of Birmingham and Queen Elizabeth hospital and will use tissue obtained from patients operated on for colorectal cancer. Lymphocytes will be extracted from the tissue and the homing molecules present will be analysed. Identified important molecules will be studied in further detail including how the lymphocytes respond differently if the homing molecules are blocked by using drugs.

Aims:
1. Determine the expression and function of chemokine receptors and adhesion molecules on tumour-infiltrating Tregs compared to matched colonic tissue.
2. Define signals within the tumour stroma and parenchyma that results in localisation to specific compartments
3. Determine the frequency, distribution and function of dendritic cells (DC) in colorectal tumours and show how they maintain Treg survival and function at the tumour site.

Methodology:
* Tissue from colorectal primary and metastatic liver resections will be obtained from patients at the Queen Elizabeth Hospital, Birmingham.
* Flow cytometry and intracellular cytokine staining will be used to phenotype lymphocyte subsets and determine expression of chemokine receptors and adhesion molecules. Real-time PCR will be used to quantify mRNA expression of adhesion molecules and chemokines. Positive findings will be confirmed by western blotting and immunohistochemistry.
* Receptor function will be assessed using standard chemotaxis and adhesion assays. A flow-based adhesion assay will be used to prove function under conditions of shear stress. Real-time tracking of lymphocytes migrating into explanted CRC tissue slices will be performed using multi-colour confocal microscopy.
* The phenotype and function of dendritic cells (DC) isolated from tumour and normal tissue will be assessed as well as their ability to induce and maintain a Treg phenotype by co-culture experiments.
* Our laboratory data will be correlated with clinical outcome data to determine if expression of chemokine/adhesion molecules relates to outcome and survival.

The information I generate will define the role of Treg recruitment in colorectal cancer, potentially leading to a novel therapeutic target.

My project will feedback the importance of laboratory-based research using human tissue. The knowledge that surgeons can contribute to valuable research by enabling access to human tissue will encourage them to facilitate this process. Compliance with consent procedures and the consideration at the time of operation that fresh specimens may be useful for research purposes is a barrier to obtaining fresh human tissue.
Doctors properly informed of research projects are more likely to help with the above and may be stimulated to undertake a period of research themselves. These impacts are occurring currently and will persist in the short-term.
If my project were to unveil a successful therapeutic strategy for colorectal cancer, there may be an impact on the requirement for surgery in all cases of colorectal cancer. This would then have a subsequent impact on career choices and workforce planning in the surgical sector. This would only be evident in the long-term.
Sampling cannot be performed close to the invasive margin as it may impact on the assessment of disease staging. Therefore, pathologists informed of the aims and methods of my project are more likely to assist me in obtaining tissue for my research and for future research projects.
Pathologists are well-aware that some colorectal cancers exhibit a marked lymphocytic infiltration. The results of my research will provide academic interest as to the role of these lymphocytes in tumour progression. My project may identify important prognostic markers which in the long-term could lead to the requirement of routine immunohistochemical staining of sections, impacting on department funding and need for pathology technicians.
The results of my research project may provide a novel therapy which would require further research in the form of a clinical trial. In the medium-term, oncologists would need to be aware of such research to enrol patients in trials.
My research may reveal important prognostic markers which will help guide neoadjuvant and adjuvant treatment. A new prognostic marker may provide the solution to identify those Dukes B patients who would benefit from adjuvant therapy.
Despite colorectal cancer surgery being curative in many cases, there are operative risks and the potential for a stoma. My research aims to identify novel treatments which may improve patient survival and avoid disease recurrence. It is possible that manipulation of the immune system may provide a treatment for early cancer and even obviate the need for surgery. On the other hand, a novel immunotherapy might be used, as currently, as an adjunct to existing therapy for advanced disease.
The successful treatment of colorectal cancer entails not only improving survival but also improving quality of life. The downstaging of an advanced rectal cancer to enable primary anastomosis instead of an end-colostomy may be made possible by a novel therapy. The availability of a treatment designed to allow a patient's own immune system to destroy cancer cells may be more palatable to patients and may indeed have less side-effects than existing therapies such as chemotherapy. This may improve compliance with therapy.
All of the above impacts would only be evident in the long-term.
A novel therapy identified from my research would have to be rigorously tested in a clinical trial, showing benefit over best existing therapy and involving a large number of patients.
The costs of a new treatment are offset by the healthcare, social care and society costs of disease recurrence. These costs could be huge given the incidence of colorectal cancer.
A prognostic marker which identifies patients who will benefit from adjuvant treatment will avoid the costs of over-treating patients, consisting of the costs of therapy and the wider costs of treatment morbidity and time off work.