Advancing therapeutics by exploiting single cell functional analysis

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

The human body contains trillions of cells of many different lineages and functions, yet all are descended from the same fertilised embryo. Understanding their similarities and differences (cell 'heterogeneity') is a huge barrier to the design of all therapies that need to target particular rare cells within the body. There is an increasing need to analyse the basis of how varying groups of cells arise, as well as the set of characteristics (biomarkers) that mark them out as being different. This may be due to stable changes, or dynamic changes in these cells that lead to repeatedly fluctuating cell characteristics. It is important to understand when and whether each of these possible situations applies. This requires new technical advances that can better analyse these cells to show the basis of this variation and to identify, characterise and manipulate important groups of cells that underly disease processes.
We focus on characterising a group of rare cells (called circulating tumour cells, or CTCs) that give rise to drug-resistant cancers such as certain lung cancers and lead to relapse, and specific stem cells (cells with the potential to self-renew or differentiate) that can enable the regeneration of damaged tissues such as muscle, joints, skin and blood vessels.
To achieve a 'step-advance' in our ability to design therapies that can specifically target such specific 'progenitor' cell populations, we will establish a ground-breaking Single Cell Research Centre (SCRC) at the University of Manchester. Exploiting and innovating in the very latest single cell technologies, it will comprise a common integrated pipeline from receipt of clinical samples, to identification and characterisation of single target cells within each sample, to the design of treatments that target these specific cells. In this way, it will expedite progress to cell-based and 'personalised' treatments for some of the most challenging diseases and degenerative conditions of man.
Our vision for SCRC is that, within the first 3-5 years, the cellular 'signatures' will have been obtained and verified that will form the basis for potent new 'precision' therapies. The establishment of SCRC is thus particularly timely, given the rapidly growing numbers of people with degenerative conditions and untreatable cancers.

Technical Summary

We will establish a Single Cell Research Centre (SCRC) to enable targeted cancer and regenerative medicine therapies. It will deliver a unified track from clinical tissue to cell selection, 'omics and imaging analysis, bioinformatics and e-Health.
Lab 1 will handle and store clinical tissues and cells. Laser capture microscopy will enable identification and isolation of single cells from tissues. Innovative microfluidics will enable live single cell selection, delivery and imaging (with Zeiss). Labs 2 and 3 will support multi-parameter flow cytometry. Lab 2 will house an ImageStreamX flow cytometer to image, quantify and select cells in suspension; Lab 3 will house a time of Flight Mass Cytometry (cyTOF) for single cell high speed flow cytometry with targeted mass spectrometry. We will innovate in imaging cyTOF for high spatial resolution of multiple proteins in cells within clinical tissues. Lab 4 will undertake single cell genomic/transcriptomic analysis using a Fluidigm C1 and Fluidigm IFC Controller HX, building on existing platforms. The SCRC - CRUK Manchester Institute oncology lab, fully integrated in 'omics technologies and informatics, is defining circulating tumour cells. it will use NextSeq 500 and Nanostring in on-site GCLP facilities for RNA/DNA analyses of patient CTCs, to enable transition to CTC-based diagnostics. Lab 5 will innovate in imaging single cells from clinical tissues, using confocal microscopy with multiphoton laser, 4D light sheet and luminescence imaging. Fluorescence correlation spectroscopy will enable dynamic quantification of molecules in cells. Lab 6 will integrate bioinformatics with network analysis and co-varying signals, infer regulatory interactions underlying differentiation, model gene expression dynamics, and develop models of cell processes from clinical samples. All data will be integrated with our Health e-Research Centre (HeRC), part of MRC-funded Farr Institute for Health Informatics.

Planned Impact

We will establish a ground-breaking Single Cell Research Centre (SCRC) at the University of Manchester in order to deliver a step-advance in the design of therapies to target specific 'progenitor' cell populations. It will exploit and advance single cell technologies (genomics/transcriptomics, flow cytometry, proteomics, imaging and bioinformatics. It will be a fully integrated SCRC pipeline from receipt of clinical samples, to identification and genomic and phenotypic characterisation of target cells, to technology innovation and design of treatments that target specific cells.
Our vision for SCRC is that, within the first 3-5 years, cellular 'signatures' will have been obtained and verified that will form the basis for potent precision therapies for many challenging diseases and degenerative conditions.

Biomedical scientists
SCRC will deliver essential insights into cell heterogeneity and the very nature of multicellularity, and thus both how tissues form and can be remodelled and how mutations can cause drug-resistant cancers.
Realising the benefits: In Manchester, our research will be translated through Manchester Academic Health Science Centre (MAHSC) Experimental Medicine. Nationally, SCRC is a founding partner of (i) the Northern Single Cell Consortium (with Newcastle and Leeds) which will maximise added value by shared technology innovation and access to clinical material; (ii) the UK cyTOF user group which will work together to advance this new technology. SCRC is strongly linked with national CRUK collaborators and has UK Regenerative Medicine Platform hubs partnerships. We will share SCRC advances with regional colleagues in the Mercia Stem Cell Alliance (http://www.msca.ls.manchester.ac.uk/). Internationally, we will extend existing worldwide collaborations in cancer and regenerative medicine.

Patients
SCRC will allow characterisation of rare circulating tumour cells that give rise to drug-resistant populations, for directed early therapy to block relapse and metastasis, and the selection, expansion and characterisation of stem/progenitor cells to underpin repair and regeneration of muscle, joints, skin and vasculature.
Realising the benefits: Within its first 3-5 years, these cellular signatures will lead to targeted cell-based precision therapies.

Biopharma
Single cell research is urgently needed to inform the design and delivery of cell-based biologics. This need is highlighted by the many industrial partnerships we have established for SCRC. As an example, Zeiss is committing £428K to SCRC in order to work together in partnership to innovate in single cell imaging, and in microfluidics of single cells, on top of ~£1m for our Systems Microscopy Centre. Our SCRC capabilities will also be integrated with N8 which has >240 industrial partners.
Realising the benefits: University of Manchester Intellectual Property (UMIP) and the CRUK Manchester Institute's Drug Discovery Unit will support commercialisation of our single cell advances towards therapies targeting cells that give rise to drug-resistant cancers and stem cells for tissue regeneration. We will work towards a spin-out to market single cell discoveries.

Graduates
Graduate training of future UK leaders is a priority of the University of Manchester. Our EPSRC & MRC Centre for Doctoral Training in Regenerative Medicine (>30 industrial partners) will be linked to SCRC to ensure training in single cell technologies. Other PhD programmes that will greatly benefit from SCRC include CRUK, BBSRC DTP and Wellcome Trust and BHF 4-year programmes.
Realising the benefits: Major impact will arise by graduate training in the latest technologies to analyse and exploit cell heterogeneity.

General Public
Public engagement is a top University priority. .
Realising the benefits: SCRC will be a powerful platform to inform the public about our translational research, through NowGen and Faculty activities (see also Communications)
 
Description Contribution to the Manchester BRC award 2017
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact The single cell facility provides training, supervision, and access to equipment for the new Manchester BRC. The core is an essential part of the BRC allowing investigators to apply appropriate technology to human samples.
 
Description Member and deputy chair of UKRI Future Leaders Fellowship panel (sift and interview)
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
URL https://www.ukri.org/funding/funding-opportunities/future-leaders-fellowships/
 
Description Member of MRC Human Cell Atlas Panel
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
URL https://mrc.ukri.org/funding/browse/hca/human-cell-atlas/
 
Description Member of joint research council tecnology touching life advisory group
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
URL https://www.ukri.org/research/themes-and-programmes/technology-touching-life/
 
Description A "Molecular Imaging (FLIM/FCS) toolbox" to investigate molecular interactions and activation in super-resolution and widefield mode
Amount £255,000 (GBP)
Funding ID 202923/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2016 
End 10/2020
 
Description An upright confocal microscope for multidisciplinary research
Amount £282,781 (GBP)
Funding ID BB/R014361/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2019
 
Description MRC project grant
Amount £675,328 (GBP)
Funding ID MR/P011853/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 02/2020
 
Description NIHR Biomedical research centre
Amount £25,000,000 (GBP)
Funding ID Manchester BRC 2017-2022 
Organisation National Institute for Health Research 
Department NIHR Biomedical Research Centre
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2022
 
Description Quantification of protein dynamics driving the circadian clock
Amount £610,426 (GBP)
Funding ID BB/P017347/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2017 
End 11/2020
 
Description Temporal manipulation of genetic circuits in single cells
Amount £122,019 (GBP)
Funding ID BB/P027040/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 09/2018
 
Description Wellcome Trust 4-year PhD Programme in Quantitative and Biophysical Biology
Amount £2,555,000 (GBP)
Funding ID 108867/B/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 01/2023
 
Description clinical research fellowship
Amount £269,390 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2016 
End 07/2019
 
Description programme grant
Amount £2,000,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 08/2022
 
Description research grant
Amount £844,160 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 02/2020
 
Title Data from: Dynamic NF-?B and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation 
Description  
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
 
Title algorithm for single cell genomics 
Description The primary focus of computational (including use of the MRC fundedcomputation and storage hardware) and bioinformatics (MRC funded Post Doc) aspects of this grant has been the development of novel analysis pipelines that reduce NGS based error and make single cell analysis more reliable. As a first step in this process we have repurposed techniques from transcript assembly to exploit the redundancy that result from sequencing multiple PCR products from each initial starting DNA fragment. This allows us to identify and correct both PCR and imaging errors in Illumina deep sequencing data. Our algorithm reduces background noise in the normal control samples and eliminates false positives from the tumour samples. Together these lead to significant improvement in mutation calling accuracy. The code is highly parallelised and makes use of a MapReduce framework to run efficiently on HPC hardware, such as that provided through the SCRC. The first manuscript from this research is in final stages of drafting. 
Type Of Material Computer model/algorithm 
Year Produced 2017 
Provided To Others? Yes  
Impact We intend to extend these approaches to allow us to align and assemble data from all the CTCs from a patient simultaneously, alongside patient matched normal controls. This exploits the evolutionary hierarchy resulting from all cells in the dataset being ultimately derived from the same ancestor cell. In this way, the algorithm can, for each CTC, 'borrow' information from the other CTCs in the set, improving the overall signal to noise ratio. We expect this to improve mutation-calling accuracy and to be substantially faster than other techniques. When used in a clinical setting, reducing processing time is particularly beneficial. 
 
Description Carl Zeiss 
Organisation Carl Zeiss AG
Country Germany 
Sector Private 
PI Contribution We have advised Zeiss on trends in bioimaging since 1996. We have provided new data and tested prototype equipment. We have spoken at Zeiss organised meetings. We have given them an opportunity to display Zeiss equipment at our training courses. We have organised symposia that have been supported by Zeiss. We have held expert discussion meetings to review microscopy trends that have involved senior Zeiss staff
Collaborator Contribution Zeiss have made a cash contribution to training courses (received) of £16,250. Zeiss estimate of total value of in-kind staff time for collaboration, training courses and other meetings (including visits of teams from Germany) ?25,000. In addition, Zeiss have also committed over £30,000 in cash and ~£80000 in in kind staff for future training meetings and collaborative visits. Zeiss helped to design the new Systems Microscopy Centre in Manchester and made a 45% discount (value ?350k) for the purchase of equipment in 2011. Zeiss are a formal MICA partner on both Liverpool and Manchester awards from the MRC/BBSRC New Microscopy Initiative. In the award to Manchester they have made a contribution of £614,314 in staff time, development costs and equipment contribution. This involves FCS (developed during this project), luminescence fluorescence imaging, light sheet microscopy and SOFI super-resolution imaging. More recently Zeiss have made a further contribution to our new clinical single cell centre. This includes over £400k in equipment discounts and £25k in cash contribution to training and symposia. Over the years the Zeiss contributions have included them helping us with public understanding of science exhibitions where they loaned equipment, provided support for professional poster preparation and used their delivery services to transport our exhibit materials and equipment to the exhibition venues. This included an exhibition in Buckingham Palace in 2006. Zeiss have sponsored 2-3 meetings per year in Manchester. In 2016 this included a session on light sheet imaging and a session on new confocal imaging technologies. In 2017 they have sponsored an image analysis daya and will sponsor a single cell biology workshop.
Impact Annual training courses MICA collaborative MRC grant MICA collaboration on new single cell centre The relationship with Zeiss has been two way. We have been given the opportunity to be early adopters f new technology and to feedback idease for improvement. We receive very favourable deals on microscope purchases and maintenance contracts. Specific areas of successful collaboration lie in improvements to higher throughput live cell imaging using the confocal microscopes; optimisation of truly dark microscopes for quantitative luminescence imaging and the development of FCS. Multiple workshops organised (2-3 per yeat)
 
Description andrew mc donald 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution andrew is developing high resolution analysis of lung immune cells. in human asthma.
Collaborator Contribution he is using the new kit.
Impact none yet
Start Year 2016
 
Description daniel davis 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution daniel is working on The nanoscale organisation of naïve and regulatory T cell surfaces in juvenile idiopathic arthritis patients with wellcome support
Collaborator Contribution he is using single cell sequencing approaches.
Impact none yet
Start Year 2016
 
Description elaine bignell 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution elaine is working on Antifungal potency of the airway epithelium in health and disease: a single cell approach with wellcome support
Collaborator Contribution she is using the kit with wellcome funding the project.
Impact none yet
Start Year 2016
 
Description hannah durrington 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Hannah is working on Applying single cell technology to Steroid Resistant Asthma: identifying the role of circadian mechanisms in Th17 cells. with wellcome support
Collaborator Contribution she is using FACS and single cell genomics
Impact none yet
Start Year 2017
 
Description john blaikley 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution John is working on Does single cell biological noise conceal circadian timing information? with MRC and wellcome support
Collaborator Contribution he is using the kit, with wellcome and MRC salary support.
Impact none yet
Start Year 2016
 
Description sebastian viatte 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Seb is working on Monocyte heterogeneity in health and inflammatory disease with wellcome support
Collaborator Contribution he is using the kit, with funding from wellcome
Impact none yet
Start Year 2016
 
Description School Visit, Altrincham Girls School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dean Jackson - Advice and practice interviews for students
Year(s) Of Engagement Activity 2016
 
Description School visit (Liverpool Life Sciences, University Technical College) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Liverpool Life Sciences UTC is the first school in the UK specialising in Science and Health Care for 14 to 19 year olds. Talk at Liverpool Life Sciences UTC conference on "How Organisms Age"
Year(s) Of Engagement Activity 2016
 
Description School visit, (Manchester Grammar School) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dean Jackson Feb 2017, Science Fair judge for student projects
Year(s) Of Engagement Activity 2017
 
Description school visit (Xaverian College) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dean Jackson - STEM ambassador
Year(s) Of Engagement Activity 2016