Vascular endothelium as the amplifying machinery of oncogene-induced senescence surveillance

The development of cancer occurs when cells of the body accumulate damage to their DNA. This generally occurs slowly, but many patients have an increased risk of cancer development due to long-term inflammation such as liver cirrhosis or inflammatory bowel disease. This inflammation leads to damage to the DNA and activation of cancer-causing genes. At the earliest stages of cancer, prior to our current ability to detect cancer the body has defence mechanisms that suppress these cancer-causing genes and prevent cancer progressing. One such mechanism is called cellular senescence where cells of the body sense this damage and respond by irreversibly preventing that cell from reproducing itself any further. Senescent cells also signal to other cells around themselves preventing them from reproducing and attracting circulating immune cells that kill the senescent cell and the cancer-causing genes that it contains. Therefore, understanding how senescent cells are detected and killed by the immune system is important and could be a way of treating patients to prevent them getting cancer in the first place.

Immune cells, which circulate in our blood, enter different parts of our body by sticking to the walls of the blood vessels when they detect a problem, such as inflammation. Within the liver the blood vessels are lined by sinusoidal endothelial cells, which can attract immune cells into the liver to kill bacteria and viruses. We know that immune cells enter the liver when cancer develops, but we do not know how this happens when cells of the liver become senescent or cancerous. Nor do we understand whether the endothelial cell is important in this process. If we could understand the processes that cause immune cells to enter the liver to kill senescent cells, then we might be able to target this pathway and prevent cancer developing in the first place through boosting the bodies natural defences. Boosting the immune system to fight cancer has already been shown to be effective in other cancers such as melanoma.

In this research proposal, we aim to find out:
1) whether the endothelial cell is activated by senescence in the liver and how the endothelium is altered.
2) which parts of the immune system are attracted by the endothelial cell in response to senescence developing in the liver and whether the endothelial cell activates the immune system as it enters the liver.
3) which molecules and signals are important when sinusoidal endothelial cells respond to senescence and whether boosting these signals improves the ability of the immune system to enter the liver in response to senescence.
4) if this response is prevented by interfering with the normal function of the endothelial cells, whether this allows liver cancer to develop.
5) whether similar changes to the liver sinusoidal endothelial cell occur in the liver of patients with chronic inflammation or other patients with liver cancer.

To achieve this we will study how the endothelial cells from the liver behave when senescent cells are present in the same liver. Much of our research will involve studying cells grown in a laboratory as well as diseased human liver tissue which has been removed after surgery. Our research will also involve studying these cells in mice, as this is the only way to study the complex links between senescent cells, endothelial cells and the immune system.

This proposal will be carried out in two laboratories at the Universities of Birmingham and Cambridge that specialise in looking at immune cells in the liver and looking at senescence respectively.

If we can find out whether it is possible to improve the functioning of the immune system and improve the ability to clear the earliest forms of cancer within the liver we might be able to design treatments that prevent patients in the future from developing cancer.

Oncogene-induced senescence (OIS) is a tumour suppressor mechanism. Critical to this function, senescent hepatocytes, non-autonomously drive their own immune-mediated destruction through the inflammatory senescence-associated secretory phenotype (SASP). Liver sinusoidal endothelial cells (LSEC) are the gatekeepers for immunocyte recruitment into the liver and are known to utilise unique recruitment mechanisms. Our preliminary data suggest that OIS cells could regulate NF-kB activity and inflammatory cytokine expression in LSEC, suggesting a SASP amplification loop.

Currently we do not understand: 1) how senescent cells recruit immunocytes and whether this involves the endothelium; 2) how senescent hepatocytes affect LSEC behaviour and immunocyte recruitment; 3) which immune cells are critical for immune-mediated clearance; 4) can we target LSEC biology to promote the immune-mediated clearance of pre-cancer?

Our research questions are: 1) Do LSECs underpin SASP amplification in the liver, immune surveillance of senescence and prevent tumorigenesis? 2) Are these effects dependent on LSEC NF-kB activity? We plan to: 1) completely describe the LSEC and immunocyte response to in vivo OIS-hepatocytes through transcriptional profiling of senescent hepatocytes and LSEC and deep immunophenotyping of the liver, utilising mass cytometry; 2) model senescence-endothelial cell interactions in vitro to describe and manipulate key senescence-inducible pathways in LSEC, underpinning secretion and immunocyte recruitment; 3) Develop an inducible endothelial-specific NF-kB-knockout mouse in combination with RAS-senescence of hepatocytes to study the dependence of immunocyte recruitment and tumour suppression on LSEC NF-kB activity; we will validate our findings in healthy and diseased human liver tissue. This will allow us to understand the basis of immunity to the earliest forms of cancer, why it fails and how to promote immune-mediated clearance in cancer-prone human tissues.

This project aims to understand the molecular and cellular basis of the immune-mediated clearance of pre-cancer. Our proposal focuses on signalling between oncogene-induced senescent hepatocytes and the liver sinusoidal endothelial cells (LSEC), the physical barrier between the liver and circulating immune cells. The research will have the following impacts:

(A) Academic advancement and collaboration
The primary impact of the project will be to increase knowledge of the role of the immune system in combatting the earliest forms of cancer.
1. Publication of results in Open Access, peer-reviewed, high impact scientific journals and regular presentation of data at high-profile meetings.
2. Fostering collaboration and sharing knowledge between collaborating centres. This project will strengthen active collaborations between the Cambridge research group (non-autonomous signalling in senescence) and Birmingham (LSECs and immune recruitment).

(B) Training highly skilled researchers and inspiring students
1. Professional development and research-specific training will form an integral part of the activity and career development of the researchers named in this proposal.
2. Teaching of undergraduate and postgraduate students attached to the labs in Birmingham and Cambridge.

(C) Innovative methodologies and techniques
1. Publication of methods and technologies in Open Access, peer-reviewed scientific journals or online.

(D) Drug-development and Industry
We will actively seek actionable targets to promote immune-cell clearance of pre-cancerous cells and aim to shape the development and use of new marketable drugs.

(E) Clinical practitioners and patients
1. The investigation of immune-mediated clearance to prevent cancer will be of clear interest to the clinical community in terms of medical research and future practice.

(F) Wider socio-economic impacts
1. Development of effective therapeutic strategies to prevent and treat cancer may ultimately have a significant impact on the health and wellbeing of patients.

(G) Public engagement
We will use well-established routes from CRUK and the Universities of Birmingham and Cambridge to engage with the wider community and reach as wide an audience as possible.

Timeframes:
A. Academic advancement and collaboration: Monthly video-conferences and occasional meet-ups to ensure co-ordination of research across sites from commencement. Attendance at national and international conferences will take place Years 1-4. Scientific publications anticipated from Year 2.
B. Training highly skilled researchers and inspiring students: From commencement of study.
C. Innovative methodologies and techniques: As arises.
D, E & F. Commercialisation and exploitation: When potential commercial avenues are identified.
G. Public engagement: Throughout the study to promote research engagement and receive feedback.

Capacity and Involvement:
The specific resources requested that relate to impact are anticipated to involve all Co-Investigators and named researchers. Support will also be sought from appropriate training and public engagement teams at the Universities of Birmingham and Cambridge.