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New study investigates how growth factors in our gut could initiate cancer

The cells that make up our tissues are strictly organised, and various differentiated cell types do different jobs in specific locations. The cell composition of tissues and the way the cells are organised is often different in pre-cancerous conditions, or even severely disrupted when they progress to tumours.

Understanding the molecular signals that cause cell differentiation and prompt the cells to find their location within the tissue, may explain the morphological changes observed in patients with pre-cancerous conditions. Ultimately, the alteration of these signals might be a driving force in tumour development and progression.

A recent paper from the Boccellato Lab at the Ludwig Institute for Cancer Research, University of Oxford, has investigated how the epithelial cell lining of our gastrointestinal tract differentiates based on different growth factors, and how this could ultimately determine how a patient progresses to precancerous conditions that could lead to stomach cancer.

Image: A picture from the published paper showing how normal gastric pits can change shape and functionality if EGF levels are altered, and eventually lead to the pre-cancerous condition Atrophic Gastritis

The team exposed healthy human gut tissue (the mucosoid cultures, patented) to a variety of growth factors, including EGF, BMP and NOGGIN. What they found is that different combinations of these factors help to determine which cells differentiate to form the gastric glands. These glands line the stomach, and contain a variety of different cells that produce digestive enzymes and gastric acid to help to digest our food, or mucus to protect the stomach lining.

For example, exposure to growth factors including EGF and BMP formed the foveolar cells that produce the mucus to line our gut, whereas inhibition of EGF induces the differentiation of cells producing gastric acid and digestive enzymes.

Patient with the pre-cancerous condition called Atrophic Gastritis have a problem with digestion due to the lack of digestive enzymes and gastric acid producing cells. In the biopsies of this pre-cancerous condition, the team have found elevated levels of EGF, which correlated with the lack of those gastric acid producing cells and with a flattened shape of the stomach tissue.

What this study has shown is that specific localisation of growth factors in the tissue microenvironment may be responsible for the differentiation process. So changing the relative quantities or localisation of these growth factors could trigger a change in the epithelium structure and cellular composition over time, potentially leading to cancer.

Building a high-resolution, dynamic map of the growth factors during cancer progression is the next step in this research. The team will also be investigating causes for these growth factor level changes. For example, long-term infection with  Helicobacter pylori bacteria is associated with increased risk of gastric cancer. Investigating how infection alters the growth factor microenvironment is essential to understand the response of the tissue and its potential aberration leading to cancer.

Dr Francesco Boccellato says:

“By better understanding the role of growth factors underlying the epithelial structures in pre-cancerous conditions, we can detect when cancers may appear and thus treat them earlier.

“This study has allowed us to draw up a new, detailed map of the signalling microenvironment in the healthy human gastric glands, which we can now draw upon in future studies as we investigate how growth factors influence cancer occurrence.”

About the Boccellato lab

The Boccellato lab is investigating oncogenic pathogens and how they contribute to cancer.  Patients infected with those pathogens have a higher chance of developing cancer, but the malignancy arises many years after the initial infection event. Cancer may develop as a result of a long battle between the pathogen that persists, hides and damages the tissue, and the host that attacks the pathogen and continuously repairs the damage caused by the infection.

DeLIVER clinical research study underway as recruitment opens

DeLIVER is a five-year Cancer Research UK-funded research programme led by Professor Ellie Barnes (Nuffield Department of Medicine) that aims to detect liver cancer earlier. Liver cancer is the fastest rising cause of cancer death in the UK, with more than 5,000 deaths per year. To improve survival, it is crucial to diagnose liver cancer earlier, when current treatments are more likely to be successful. However, this is challenging because symptoms are vague and late-presenting, and are frequently masked by co-occurring liver disease, such as cirrhosis.

A major goal of the DeLIVER programme is to learn more about the biology of liver cancer development and to use this information to design more sensitive detection tests. Because many people being tested for liver cancer have the high-risk condition cirrhosis, these tests need to be specific enough to detect liver cancer on top of other changes in the liver caused by cirrhosis. In order to identify the defining characteristics of early liver cancer, researchers need to perform a detailed molecular analysis of tissue from tumours and the background liver in people with liver cancer and cirrhosis and compare this to liver tissue from people with cirrhosis alone.

The DELPHI (Deep Liver Phenotyping and Immunology) study will recruit 100 participants at Oxford University Hospitals NHS Trust. 80 of these recruited participants will have cirrhosis (caused by hepatitis virus B or C, fatty liver disease or alcohol) and 20-30 participants will have liver cancer in addition to cirrhosis. After giving consent, the participants will undergo fine-needle aspiration to collect tissue from the liver. This is a safe technique established in Oxford as one of only a few centres in the UK. Blood samples will also be taken.

Cancer Research UK Clinical Research Fellow Dr Rory Peters is leading the study. He said,

“We are very pleased to have started the recruitment for the DELPHI study. The in-depth analysis of samples from the DELPHI participants will be critical for increasing our understanding of how liver cancer develops and will give insights into how this cancer can be detected earlier.”

The researchers will look at individual cells to understand the cellular make-up of the tumour and surrounding tissue, including infiltrating immune cells, and how this may influence cancer development. By comparing the tissue from participants with and without cancer, they will also look for changes in protein or metabolite levels and alterations in the levels of chemical modification of DNA by methylation using the TAPS assay developed in Oxford by Dr Chunxiao Song. They will investigate whether the changes that they observe from the tissue analysis can also be detected in the blood, which would provide evidence that a blood-based assay could be developed as a less invasive diagnostic test.

Professor Ellie Barnes, Chief Investigator for DeLIVER said,

“The DELPHI study is one of three clinical projects within the DeLIVER programme. Together, these studies will inform us which of our diagnostic technologies perform best at detecting liver cancer at the earliest stages. We hope this work will lead to a step-change in earlier liver cancer diagnosis and improved patient survival.”

 

Read more about the DeLIVER programme in the OxCODE liver cancer early detection research showcase.

Finding extracellular vesicle biomarkers for oesophageal cancer early detection

Extracellular vesicles (EVs) are entities secreted by cells that can be involved in cell-to-cell communication. They contain messenger proteins and other molecules, which act like ‘instructions’ to recipient cells.  EVs contain proteins both on their inside and outside.

All cells, including cancer cells, release EVs.  EVs from different cell types have slightly different compositions of proteins, which give them the ‘characteristics’ of their parent cell.

Members of the Goberdhan’s lab have previously shown that EVs released by colorectal cancer cells contain different protein when they are subjected to certain types of stress, such as certain nutrient deficiencies.  These ‘switched’ EVs change recipient cell behaviour, for example, increasing cancer cell growth. Researchers can potentially exploit these differences in EV protein composition to define distinct EV sub-populations: a helpful step towards their use as multi-protein biomarkers.

Dr Jennifer Allen and Ms Karen Billington from the Goberdhan lab are now applying this concept to the early detection of oesophageal cancer. Barrett’s Oesophagus is a pre-cancerous condition whereby oesophageal cells become damaged. Over time the damage can increase and cancer can develop.

Monitoring patients with Barrett’s Oesophagus is in place to try and identify when cancer has developed, however this is done through invasive and costly endoscopy, which may miss cancer in the very early stages. There is a need to identify when Barrett’s Oesophagus has progressed using less-invasive methods that can be used more regularly, so that cancer can be caught earlier.

Jen and Karen are investigating the potential of using EV proteins as biomarkers, which could be identified though simple blood tests. They are using different types of cells – such as normal oesophagus cells, Barrett’s Oesophagus cells and Oesophageal cancer cells – to compare the proteins found on the EVs released by each of these cell types.

The team is working with Dr Elizabeth Bird-Lieberman, a Gastroenterology Consultant at the JR Hospital, to collect blood samples from patients with Barrett’s Oesophagus, to see if EV information could be extracted and tested through simple blood tests – such as that being developed by Prof Jason Davis.

The aim is to identify a handful of proteins via proteomic analysis, that allows them to differentiate EVs from oesophageal cancer cells. If the protein biomarkers associated with the more cancerous cell lines can be detected in patient blood samples, Barrett’s Oesophagus patients could then be routinely tested for specific EV proteins that indicate the presence of parent cancer cells. This simple test could be carried out much more regularly than endoscopy surveillance and would enable earlier detection and treatment of oesophageal cancer in these patients.

Colorectal cancer cell extracellular vesicles. These two vesicles have become deflated and have the characteristic cup-shaped morphology caused by preparation for electron microscopy. Images generated by Dr John Mason (DPAG) and Dr Errin Johnson (EM Facility Manager, Dunn School).

About the study

The Goberdhan lab members are interested in intracellular signalling and cell communication. Their major focus is on how this goes wrong in cancer and other major human diseases. Specifically, they investigate:

  1. The role of amino acid sensing and stress-induced signalling in regulating cellular growth and intercellular communication involving exosomes.
  2. The regulation of exosome formation and heterogeneity by intracellular signalling pathways and membrane trafficking.
  3. The effect of exosome signalling on recipient cell behaviour and cancer progression, particularly in response to microenvironmental stresses applied to exosome-secreting cells.

 

This study is funded by the CRUK Early Detection Primer Award.

Understanding how cancer arises from infected tissue

Whilst rates in the UK are relatively low, stomach cancer is still the third highest cause of cancer mortalities worldwide. The largest risk factor for stomach cancer is a chronic infection of the H. pylori bacteria. The contributions of other factors like diets high in salt, smoked foods, smoking and obesity are also important.

H. pylori can be found in the gut, and some strains cause gastritis & stomach ulcers. Long term colonisation can result in persistent cellular and tissue damage. Over time, the damaged gut lining can lose its structure and eventually become so undefined that the patient develops atrophic gastritis – a precancerous condition that could eventually lead to cancer.

A to F shows the increasing change of structure to existing gastric epithelium, as a result of prolonged H. pylori infections. (A) The normal gastric epithelium is organised in invaginations called glands. (B) A remarkable increase in size is observed in the inflamed stomach after H.pylori infection, a condition called chronic gastritis. (C) Atrophic gastritis, a precancerous condition with a higher chance of leading to cancer: the glandular structure is lost. (D) The emergence of a new type of gland with different features: a condition known as intestinal metaplasia to cancer. (E-F) The progression from dysplasia to cancer. Credit: Correa & Piazuelo, 2013

 

Understanding how persistent infection can result in increased risk of cancer is the focus of Dr Francesco Boccellato, Ludwig Institute, and his lab. Improving the knowledge of underlying mechanisms in early cancer biology may help us to understand how cancers originate in various parts of the body, and thus giving doctors more insight to detect cancer earlier in patients with precancerous conditions.

Francesco’s most recent project is investigating the role of growth factors in the determination of gut epithelial cells. The cellular lining of the gut, known as the epithelium, is where most stomach cancers originate. The epithelium is made up of a variety of different types of cells, responsible for different things such as mucus secretion, production of gastric acid and digestive enzymes.

Cross section of the stomach lining showing a gastric gland with different cell types that make up the epithelium. What causes stem cells to differentiate into these different cells is the focus of the Boccellato lab. Credit: Boccellato lab

The team are investigating what it is that activates stem cells to differentiate into different epithelial cells, in the hope of identifying new ways that the cells can become cancerous.

It is Francesco’s hypothesis that the specific localisation of growth factors in the tissue microenvironment may be responsible for the differentiation process. If this is the case, then it may be that a change in the relative quantities or localisation of these growth factors triggers a change in the epithelium structure and cellular composition over time.

The team are investigating this through in vitro models known as mucosoid cultures – growing human epithelial cells outside of the body and exposing them to different conditions to see how the cells regenerate and differentiate. Mucosoids are an innovative stem cell based cultivation system developed by the Boccellato lab, which enables an exceptional long term regeneration and maintenance of epithelial cells. The cells form a polarised monolayer producing mucus on the top side similar to the epithelium in a patient.

Top: example of a mucosoid with cells (the plasma membrane is labelled in green) producing protective mucins (MUC5AC) labelled in red (the yellow is where the two labels overlap creating the mucus layer). Bottom: example of a mucosoid with cells (the plasma membrane is labelled in red and the nuclei in blue) showing one cells producing Pepsinogen (in green) the precursor of pepsin, the main digestive enzyme. Source: Boccellato et al., GUT 2019

The results of Francesco’s investigation into the role of growth factors in determining gut cell differentiation and progression into atrophic gastritis are expected in Spring 2021. It is hoped that by better understanding the role of growth factors underlying the epithelial structures in pre-cancerous conditions, we can detect when cancers may appear and thus treat them earlier. Further studies will elucidate the role of bacterial infections (like H.pylori) in this process of re-shaping the tissue.

The H. pylori-cancer relationship is a great model for understanding other infection-based cancers. Colon cancer, gallbladder cancer, cervical cancer, stomach cancer and lymphoma are all examples of cancers that can be caused by bacterial infection. By better understanding how gut tissues work and progress to pre-cancerous conditions, we can apply this to other cancer models to see if the same is true.

A final line of investigation by the team will be into how H. pylori bacteria access gut cells to cause damage. The epithelium is usually protected by a mucus barrier, on which our natural and harmless microflora grow. Healthy gut bacteria cannot perforate this mucus barrier to reach epithelial cells, but H. pylori appears to be able to. Francesco is investigating what makes this possible, so that we may be able to develop drugs that prevent H. pylori infections from reaching the epithelium and causing damage.

About the Boccellato lab

The Boccellato lab is investigating oncogenic pathogens and how they contribute to cancer. Patients infected with those pathogens have a higher chance of developing cancer, but the malignancy arises many years after the initial infection event. Cancer may develop as a result of a long battle between the pathogen that persists, hides and damages the tissue, and the host that attacks the pathogen and continuously repairs the damage caused by the infection.

The team use innovative tissue culture systems of human primary cells to re-build the infection niche in vitro and to understand the long term effect of infection on epithelial cells.  

References

Boccellato F.  GUT. 2019 Mar;68(3):400-413. doi: 10.1136/gutjnl-2017-314540. Epub 2018 Feb 21.

Sepe LP, Hartl., mBio. 2020 Sep 22;11(5):e01911-20.doi: 10.1128/mBio.01911-20.

Boccellato F, Meyer F. Cell Host Microbe. 2015 Jun 10;17(6):728-30.doi: 10.1016/j.chom.2015.05.016.

Piazuelo MB, Correa P. Gastric cáncer: Overview. Colomb Med (Cali). 2013;44(3):192-201. Published 2013 Sep 30.

 

Following the cancer metabolomic breadcrumb trail

By analysing the metabolic molecules that tumour cells leave behind, Dr James Larkin is investigating the applications of metabolomics in the early detection of many cancers.

New form of gift wrap drives male reproductive success

The transfer of complex mixtures of signals and nutrients between individuals is a key step in several biologically important events in our lives, such as breastfeeding and sexual intercourse. However, we know relatively little about the ways in which the molecular gifts involved are packaged to ensure their successful delivery to the recipient.

By studying such events during mating in the fruit fly, researchers at the University of Oxford have identified a new communication mechanism in which nutrients and signals are combined in fatty droplets that stably store their bioactive cargos in males, until they are transferred to females when they dissipate within minutes. These specialised multi-molecular assemblies called microcarriers, are made by the prostate-like accessory gland of the male and contain a central fatty (lipid) core wrapped with multiple proteins, including a molecule called Sex Peptide. When Sex Peptide is released in the mated female, it stimulates her to produce more progeny and reprogrammes her brain so she rejects other male suitors.

Although Sex Peptide is only produced by a limited group of fruit fly species, lipids and lipid droplets are secreted by many glands, including the human prostate and breast. Preliminary work in flies suggests that one of the genes that is essential for the release of microcarriers from secreting cells also plays a critical role in human glands, including the breast. In fact, this regulatory gene is highly expressed in some breast cancers. This suggests that the mechanisms controlling microcarrier formation may be evolutionarily conserved and that microcarriers may play much broader roles in physiological and pathological cell-cell communication, which have yet to be recognised.

This new cell communication mechanism could have implications for breast cancer and understanding its pathology because the regulatory gene essential for driving reproductive success is also expressed in some breast cancers, so if we can better understand the gene’s mechanism, it might be possible to understand how breast cancer can become pathological.

Please see the full article on the Department of Physiology, Anatomy & Genetics website.

The study was a collaboration between the groups of Professor Clive Wilson and Associate Professor Deborah Goberdhan from the Department of Physiology, Anatomy and Genetics, at The University of Oxford. This work has been supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC), Cancer Research UK, the Wellcome Trust and the Medical Research Council

Paper to be published in PNAS:

Drosophila Sex Peptide Controls the Assembly of Lipid Microcarriers in Seminal Fluid

New hydrogel technology has promise in breast cancer modelling

In science, a model is used as a representation of something in the real world, so that ideas and concepts may be tested out. Models have a variety uses, but in cancer biology they are often popular as they can help to mimic the complex environment seen in human   disease. Models are used to explore the effects of new drugs, understand genetic or cellular pathways on tumour development or predict the potential response of a patients cancer.

It’s in a researcher’s best interest to create a model that is as faithful to the real world as possible, so that the outcomes are accurate and can translate successfully into humans. However, the go-to models to recapitulate human cells in a lab use, a protein matrix extracted from mouse tumours, which is used to resemble the extracellular environment found human tumours. But the extent to which mouse matrix can be used is limited by its fixed extracellular matrix components, which are often not representative of the human tissue, and the inability to add or remove the individual extracellular components to explore the influence these on tumour growth.

Dr Gillian Farnie, Nuffield Department of Orthopaedics, Rheumatology and Musculorskeletal Sciences, has focused her work on developing new models that allow human breast cancer cells to be grown and researched, whilst overcoming these limitations.

A recent publication in Matrix Biology, funded by the NC3Rs, outlines a new peptide hydrogel developed by the Farnie group in collaboration with Prof Merry (University of Nottingham).  This new peptide hydrogel offers the added benefit of being customisable, by incorporating or removing specific extracellular matrix components that researchers want to test, to better understand their influence on cancer cells. It therefore allows full control over the biochemical and physical properties of the model, providing researchers with the opportunity to more accurately adapt the model to the real-life environment of human breast tumour.

The new technology’s applications are incredibly widespread and promising. For example, certain extracellular matrix proteins, when found in high quantities in a tumour, can often be associated with a poorer prognosis for a patient. Researchers may want to understand if this is a simple correlation, or if the proteins are assisting the cancer in some way, such as promoting treatment resistance. The ability to remove these proteins from a cancer model and test the response, whilst remaining faithful and accurate to human cells, is incredibly useful and can allow us to discover therapeutic targets.

Dr Gillian Farnie is currently working with the breast cancer research community to apply this new technology in multiple breast tumour research projects. The hydrogel’s applications are not limited to just matrix biology, but also in investigating areas such as the biological significance of blood vessel supply to tumours or even other cancer types outside the breast.

This new hydrogel provides an opportunity to better understand the individual influences of the extracellular matrix, mechanical properties and cell-cell interactions on breast cancer and other disease. It is an open and reproducible model that Dr Farnie is currently publishing a detailed methodology in JOVE, so that more cancer researchers can have access to the new technology.

About this research

Dr Gillian Farnie is based in the Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences.  Her research focuses on the development of patient derived pre-clinical breast cancer models that are used to examine mechanisms of inherent and induced therapy resistance, interrogating both intra-tumour heterogeneity (cancer stem cells) and the tumour microenvironment (ECM, Stroma, Immune cells).

New AI technology to help research into cancer metastasis

Cell migration is the process of cells moving around the body, such as immune cells moving through the body’s tissues to fight off disease, or the cells that move to fill the gap where a tissue has been injured. Whilst cell migration is an important process for regeneration and growth, it is also the process that allows cancer cells to invade and spread across the body.

Therefore understanding the factors that regulate and instruct cells to move is an important part of understanding how we can prevent the metastasis of many cancers. One method of doing this is through scratch assays, which as the title suggests, involves inflicting a wound or ‘scratch’ on cells grown in a petri-dish and analysing how the surrounding cells react and migrate to ‘heal’ the scratch under a microscope.

Although cell migration is intensively studied, we still do not have efficient therapies to target it in the context of cancer metastasis. Observing cancer cell behaviour to artificial wounding and how this can be altered in response to pharmacological drug treatment or gene editing is important to fully understand the factors that drive this process in tumours and provide insights on the processes that drive such behaviours. Whilst current microscopic analysis methods of wound healing data are hindered by the limited image resolution in these assays. Therefore, there is a need to develop new methods that overcome current challenges and help to answer these questions.

Dr Heba Sailem a Research Fellow from the Department of Engineering, has led a study to develop a new deep learning technology known as DeepScratch. DeepScratch can detect cells from heterogenous image data with a limited resolution, allowing researchers to better characterise changes in tissue arrangement in response to wounding and how this affect cell migration.

Tests using the technology have found that DeepScratch can accurately detect cells in both membrane and nuclei images under different treatment conditions that affected cell shape or adhesion, with over 95% accuracy. This out-performs traditional analysis methods, and can also be used when the scratch assays in question are applied to genetically mutated cells or under the influence of pharmaceutical drugs – which makes this technology applicable to cancer cell research too.

Dr Heba Sailem says;

“Scratch assays are prevalent tool in biomedical studies, however only the wound area is typically measured in these assays. The change in wound area does not reflect the cellular mechanisms that are affected by genetic or pharmacological treatments.

“By analysing the patterns formed by single cells during healing process, we can learn much more on the biological mechanisms influenced by certain genetic or drug treatments than what we can learn from the change in wound area alone.”

Using this technology, the team have already observed that cells respond to wounds by changing their spatial organisation, whereby cells that are more distant from the wound have higher local cell density and are less spread out. Such reorganisation is affected differently when perturbing different cellular mechanisms. This approach can be useful for identifying more specific therapeutic targets and advance our understanding of mechanisms driving cancer invasion.

The team predicts that DeepScratch will prove useful in cancer research that studies changes in cell structures during migration and improve the understanding of various disease processes and engineering regenerative medicine therapies. You can read more about DeepScratch and its applications in a recent study published in Computational and Structural Biotechnology.

About Heba

Dr Heba Sailem is a Sir Henry Wellcome Research Fellow at the Big Data Institute and Institute of Biomedical Engineering at the University of Oxford. Her research is focused on developing intelligent systems that help further biological discoveries in the field of cancer.

Therapeutic potential for breast cancer found in the matrix

Work currently underway in the laboratory of Prof Kim Midwood is investigating the therapeutic anti-cancer potential of tenascin-C, a molecule found in the extracellular matrix of breast cancer

Previously unknown ‘genetic vulnerability’ in breast cancer found

New Nature paper reveals discovery of a genetic vulnerability in nearly 10% of breast cancer tumours and how this can be targeted to selectively kill cancer cells.