DR MARK WILLIAMS AND DR ALYSON PARRIS
PROJECT TITLE: Dependancy of human intestinal stem cells and colon cancer stem cells on the ‘neurotransmitter’ acetylcholine: implications for colon cancer progression
PROJECT TIMESCALE: 31 August 2014 – extended to 30 April 2017
What were the most significant achievements from this grant?
(i) We developed 3D culture systems for the routine cultivation of the healthy and cancerous intestinal epithelium. Human intestinal crypts derived from the healthy mucosa propagate as enclosed spheres that manifest an increasing number of buds (i.e. crypt domains) over time and have been termed ‘organoids’ or ‘mini-guts’. Tumour fragments (aka tumouroids, on the other hand, propagate as expanding spheroids and are not dependent on key growth factors that are essential for the human intestinal organoid culture.
(ii) The expression of cholinergic signalling toolkit components in healthy gut epithelium and tumour tissue was analysed at the transcript and protein level. This included expression of M1-M5 muscarinic receptor subtypes and the enzymes that regulate acetylcholine synthesis, (choline acetyltransferase) and acetylcholine catalysis (acetylcholine esterase).
(iii) Development of a liquid chromatography-tandem mass spectroscopy assay to quantify levels of acetylcholine secretion from intestinal organoid and tumouroid cultures.
(iv) Demonstration of biological activity of secreted acetylcholine at the human intestinal stem cell niche.
(v) Discovery of muscarinic acetylcholine receptor coupling to mobilisation of calcium from endolysosomal calcium stores via two pore calcium channels in stem cells contained within organoids and tumouroids; this is novel because mobilisation from the endoplasmic reticulum calcium store is the more common occurrence for most other receptor subtypes.
(vi) Forged strong links between regulation of organoid and tumouroid growth with activation of muscarinic acetylcholine receptor coupled calcium signals.
Indicate whether or not the main objectives of this grant were met and, for any which were not met, provide a brief explanation:
(i) Characterise expression of cholinergic signalling system in human colonic mucosa (0-9 months)
Yes, we completed a comprehensive characterisation of the cholinergic signalling toolkit at the transcript and protein level.
(ii) Determine the relative contribution of neuronal (exogenous) and non-neuronal (endogenous/crypt autonomous) acetylcholine to intestinal stem cell-driven tissue renewal (10-18 months)
Yes, we demonstrated that cholinergic neurons innervate the intestinal stem cell niche and that the tuft cells, which synthesise and secrete acetylcholine, congregate near to the stem cell niche. Endogenous and exogenous acetylcholine stimulated calcium signals in the stem cell niche and promoted crypt cell proliferation.
(iii) Determine the relative contribution of neuronal (exogenous) and non-neuronal (endogenous/crypt autonomous) acetylcholine to cancer stem cell-driven tumour growth (19-24 months)
Yes, in part. Colorectal tumours and cultured tumouroids expressed cholinergic signalling toolkit components, many of which appeared to be over expressed. However, using a nested RT-PCR approach, it turned out that tumouroids exhibited heterogeneous expression choline acetyltransferase which explained why we could not detect secreted acetylcholine in the conditioned media of some tumouroid cultures. Exogenous acetylcholine stimulated robust calcium signals and appeared to promote tumouroid growth. This work is ongoing.
Europhysiology (2018, to be submitted): Oral presentation
Non-neuronal acetylcholine promotes stem cell-driven renewal of the human colonic epithelium by triggering propagating calcium signals in the stem cell niche. Mark Williams, Alyson Parris, Nicolas Pelaez-Llaneza, Victoria Jeffery, Christy Kam, Alvin Lee, Michael Lewis, Richard Wharton and James Hernon.
Europhysiology (2018, to be submitted): Oral presentation
Guardian goblet cells protect the human intestinal stem cell niche via mucus secretion stimulated by CD38-TPC calcium mobilisation from endolysosomes. Nicolas Pelaez-Llaneza, Alyson Parris, Victoria Jeffery, Christy Kam, Alvin Lee, Martin Loader, George Russam, Nathalie Juge, Michael Lewis, Richard Wharton, James Hernon and Mark Williams.
STEM for Britain conference at Houses of Parliament (2017): Poster presentation
Deciphering Cryptic Calcium Signals: ‘Morse code’ for Colon Cancer Stem Cells. Alvin Lee, Nicolas Pelaez Llaneza, Alyson Parris, Victoria Jeffery, Jack Gilliland, Iain Macaulay & Mark Williams
left to right – Dr Alyson Parris, Alvin Lee, Nicolas Pelaez-Llaneza, Jonathon Tang.
Biochemical Society; Calcium signalling – the next generation (2014): Oral presentation
Cryptic calcium signals maintain the fitness of the human intestinal stem cell niche. Christy Kam, Alyson Parris, Martin Loader, Nathalie Juge, Mike Lewis and Mark Williams.
Physiological Society (2014), Oral Presentation:
Inhibition of CD38-NAADP-TPC-induced calcium signalling blocks cholinergic excitation-mucus secretion coupling in native human colonic crypt goblet cells. Christy Kam, Alyson Parris, Martin Loader, Nathalie Juge, Mike Lewis and Mark Williams
Williams MR and Parris A. (2015) A human colonic crypt culture system to study regulation of stem cell-driven tissue renewal and physiological function. Methods in Molecular Biology
Publications in preparation:
We aim to submit two further research papers relating to the above research outcomes.
Outputs from this grant from The Humane Research Trust has provided preliminary data for grants obtained from the Big C Appeal and Norwich Research Park Translational Fund. We have also been fortunate to obtain funding for a PhD studentship and vacation research studentships from The Humane Research Trust.
During this grant from The Humane Research Trust the Williams Laboratory has set up collaboration with Dr Iain Macaulay (group leader, BBSRC genomics centre, Earlham Institute), Dr Aram Saeed (Tissue Engineering Laboratory, School of Pharmacy, UEA), Jonathon Tang (manager of Bioanalytical Facility, UEA) and Professor Doug Winton (Cancer Research UK, Cambridge Institute).
Mark Williams gave talks on human intestinal tissue research to Howbart High School (Loddon, Norfolk), Science Café (The Cut, Halesworth, Suffolk) and the ‘Pint of Science’ festival (Norwich). Outputs from our research on the human gut also form part of lectures delivered to undergraduate Biomedicine students at UEA.
Research tools and methods:
During the course of this project we have extended our human colonic crypt 3D culture system to incorporate intestinal organoid and tumouroid cultures from healthy and cancerous tissue, respectively.
Epilogue: We have made considerable progress in understanding the role of the neurotransmitter acetylcholine in regulating stem cell driven renewal of the gut epithelium. We have also revealed acetylcholine originates from neuronal and non-neuronal sources. Colon cancer cells also express the cholinergic signalling toolkit which plays a role in regulating tumour growth. Calcium signals/channels downstream of acetylcholine receptors may offer novel therapeutic targets for adjuvant chemotherapy. With our collaborators, we now have the infrastructure to develop a live human intestinal tissue bank that incorporates bespoke intestinal 3D culture systems that have been characterised at the genomic and transcriptomic level, and which would provide an invaluable resource throughout the UK for research into human gut health and disease.
The Williams Laboratory for Gastrointestinal Research is extremely grateful to The Humane Research Trustees, staff and supporters for funding this successful project. Thank you.
Background and work leading up to the project:
Colorectal cancer (CRC) is the second biggest cancer killer in the UK, causing around 16,000 deaths each year. Clearly, there is a grave need for improved treatments and chemoprevention. The consideration of colorectal cancer, and other tumours, as a stem cell disease is providing new insights into the mechanisms of cancer initiation, progression and recurrence. In particular, an increased understanding of the relationship between normal intestinal stem cells (ISCs) that drive tissue renewal throughout life, and colon cancer stem cells, which drive tumour growth and relapse, is paving the way for novel chemotherapeutic strategies and chemoprevention. However, due to the previous lack of ex vivo models of the native human intestinal epithelium and colorectal tumour tissue, much of this understanding has been based on studies of lower organisms such as flies and mice.
A defining characteristic of the inner most lining of the human gut is that it is the most rapidly renewing tissue in the body. It is formed from millions of flask-like invaginations called crypts. A population of intestinal stem cells are located at the bottom of each crypt where they are protected from the hostile environment of the gut contents. Remarkably, each crypt is the functional and self-renewing unit of the entire gut lining. It has long been appreciated that their study holds the potential to understand how the processes of stem cell-driven tissue renewal are co-ordinated to help maintain a life-long healthy gut. However, until recently, it was not possible to maintain intact self-renewing human crypt structures in a viable condition so as to study their biology in culture. To this end, our laboratory has developed a near-native human colonic crypt 3D culture system that preserves the stem cell-driven hierarchy of tissue renewal in culture. Using sophisticated imaging and genetic manipulation techniques we are able to monitor the status and regulation of stem cell biology and renewal of human tissue (Figure 1).
Figure 1: Short-term 3D culture of native human colonic crypts 7 days following isolation from a colorectal biopsy tissue sample taken at colonoscopy (Reynolds et al., Gut 2014). Open arrowheads indicate crypt base; filled arrowheads indicate crypt opening; * indicates dead crypt fragments
Another defining characteristic of the human gut is that it has its own ‘brain’ that permits rapid decisions to be made in response to local changes in the immediate environment. The inner lining of the gut is known to be responsive to chemicals released by nerve-endings called neurotransmitters. We discovered that intestinal stem cells possess receptors for a common neurotransmitter, acetylcholine, which signifies that stem cells are under the control of the gut brain. It is clear that to maintain its vital integrity, the rate at which the gut lining renews itself needs to respond dynamically to local changes in the gut environment. Somewhat surprisingly, we have also discovered that cells located in the stem cell niche at the bottom of colonic crypts also express the molecular machinery to synthesise acetylcholine, suggesting that intestinal stem cells are dependent on acetylcholine to signal the constant renewal of the gut lining. Given that colon cancer is defined by uncontrolled cell proliferation, we have developed a native human colorectal tumouroid model system to determine the role of acetylcholine in promoting colon cancer stem cell proliferation. Intriguingly, native colorectal tumouroids may have the potential to synthesise acetylcholine.
Major research question
Does secretion of the neurotransmitter acetylcholine derived from the gut brain or the gut lining itself stimulate an age-related expansion of intestinal stem cell number leading to tumourigenesis and tumour growth?
Work in progress
We have broken our study down into the following key questions:
(i) Is ageing and/or colorectal tumour formation associated with increased secretion of acetylcholine?
(ii) What is the acetylcholine receptor profile in normal versus tumour tissue?
(iii) Are tumouroids more sensitive to acetylcholine than normal colonic crypts?
(iv) Does acetylcholine promote intestinal stem cell/colon cancer stem cell survival and proliferation
(v) Does autonomous stimulation of colon cancer stem cells with acetylcholine offer protection against chemotherapy?
Alyson Parris and the research team in the Williams Gastrointestinal Research Laboratory at UEA have made good progress in addressing the above questions. Strong collaborative links with gastroenterologists and surgeons at the Norfolk and Norwich University Hospital have proved invaluable. We are in the final throes of data analysis and collating the data that will form the basis of a research paper publication to communicate our findings from the study to the research community.
During the course of the project, Alyson Parris developed a variation of the 3D culture conditions developed previously (Reynolds et al., Gut 2014; Sato et al., Gastroenterology 2011) to maintain budding structures called intestinal organoids in long-term culture (Figure 3).
Figure 3: Example of human large intestinal organoid in long-term 3D culture (> 5 months). In our hands, colonic organoids grow as a continuous budding structures where each bud is the functional equivalent of a colonic crypt. Outlinehelps to visualise organoid growth during a 5 day period (D1-D5) according to increase in cross-sectional area.
The secretion of the neurotransmitter Acetylcholineis notoriously difficult to quantify. We set up a collaboration with the Bioanalytical Facility at UEA, which is headed by Professor Bill Fraser and managed by Jonathon Tang. Using a technique called liquid chromatography tandem mass spectroscopy (LC-TMS) we developed the sensitivity to measure acetylcholine secreted into the colonic crypt/organoid culture media (Figure 4).
Figure 4: LC-TMS standard curve used to quantify concentration of acetylcholine secreted by normal crypts/organoids and tumouroids placed in long-term 3D culture.
The cholinergic signaling toolkit (i.e. acetylcholine receptors, cholinergic neurons, acetylcholine-secreting cells etc) has been characterised with respect to healthy ageing (i.e. healthy young and old subjects) and tumourigenesis (i.e. matched ‘normal mucosa’ and tumour tissue derived from colon cancer patients). A combination of RT-PCR and immunocytochemistry (e.g. see Figure 5) has been used to confirm the expression of acetylcholine receptor subtypes in normal colonic crypts/organoids and tumouroids. The relative levels of acetylcholine receptor subtype expression have been quantified by quantitative RT-PCR. We have revealed novel expression patterns for different acetycholine receptor subtypes in different cell types within the normal gut epithelium and in tumour tissue.
Figure 5: Fluorescent immunolabelling of the M3 muscarinic receptor subtype (green) in a culture human colonic crypt. Muc-2 (red) marks mucus-secreting goblet cells. E-cadherin is a marker of epithelial cells. Blue – DAPI, a marker of cell nuclei.
We have also used our culture systems for matched normal colonic crypts and tumouroids to assess the differential effects of acetycholine on supporting intestinal stem cell and cancer cells growth and survival and look forward to updating you of the complete set of results on publication of the associated research paper(s).
The support of the Humane Research Trust has been invaluable to the development and progress of this project. Thank you to the Trustees and for the generous donations made to this most worthy cause.
FIGHTING THE BIG C: Up Close And Personal
(taken from the Winter 2016 Newsletter)
Each year over 41,000 people in the UK are diagnosed with colorectal cancer and only just over half of these individuals will live more than 5 years. There is thus a dire need for more sophisticated chemoprevention and chemotherapy strategies, the development of which has been hindered by the lack of experimental systems that utilise cultured native human tissue from the healthy gut, in comparison with tissue derived from colorectal tumours.
A defining characteristic of the inner most lining of the human gut is that it is the most rapidly renewing tissue in the body. It is formed from millions of flask-like invaginations called crypts. A population of intestinal stem cells are located at the bottom of each crypt where they are protected from the hostile environment of the gut contents. However, colorectal cancer is increasingly considered to be a stem cell disease whereby normal intestinal stem cells are converted into cancer stem cells. The Humane Research Trust is funding a research project in the Gastrointestinal Research Laboratory at the university of East Anglia, (headed by Dr Mark Williams), that has developed 3D culture systems of normal human gut and colorectal tumours, in order to develop chemotherapy agents that specifically target cancer stem cells and not normal, healthy intestinal stem cells. The main focus of the current project is to elucidate the role that the ‘mini-brain’ in our intestine plays in keeping intestinal stem cells healthy, and understand how this ‘control-centre’ is usurped by rogue cancer stem cells. The accompanying images of cultured normal colonic crypts and colorectal ‘tumouroids’ were taken by the senior post-doctoral research scientist, Dr Alyson Parris, who has recently gained an ‘up close and personal’ perspective of the ‘Big C’, on which Alyson reflects:
“I have been fortunate to have had many years of support from The Humane Research Trust, taking me from my first experience of research as an undergraduate Biomedicine student in the summer of 2002 onto funding of my PhD project, and subsequently as an early years’ researcher. There have also been many opportunities to mentor, teach and work alongside the next generation of researchers that the Trust has funded in Dr Williams’ laboratory. In October 2014, I was excited to start a two-year study funded by the Trust entitled ‘Dependency of human intestinal stem cells and colon cancer stem cells on the “neurotransmitter” acetylcholine: implications for colon cancer’. This would utilise the important model system of the human gut that had been developed with THRT funding. Excitingly, there has been a surge in interest in the use of primary tissue organoid cultures across many disciplines in biomedical research, and we have been able to disseminate information about utilising a human gut model in a paper published in April 2015, (‘A human colonic crypt culture system to study regulation of stem-cell driven tissue renewal and physiological function: methods in molecular biology).
Unfortunately, whilst the paper was being published, I was experiencing some rather unwelcome rigours of chemotherapy, surgery and radiotherapy for a breast cancer tumour diagnosed in February 2015. This experience has only increased my drive to continue with basic research, utilising healthy human tissue and tumour material to interrogate normal and subverted renewal pathways and identify possible new targets for disease therapies. I hope for many more years of health to continue researching and mentoring the next generation of researchers. I started back working in Dr Williams’ laboratory in November 2015 and we are making good progress on the project. We have attended two meetings since my return, one in January 2016 on the use of stem cells and organoids, as models of differentiation and disease, and another in June 2016, on the opportunities for collaborations in regenerative medicine, which again emphasised the important questions that can be addressed by our model systems. Currently, we are writing up our next couple of research papers, of which I am very proud and look forward to updating you”.