Metabolomics applications to in-cell NMR based processes for monitoring the uptake of drugs and biologics
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
The development portfolio is changing as many therapeutic targets are not "drugable" with conventional approaches using either small molecules or antibody proteins. Nucleic acid based therapeutics offer the possibility to engage with these targets, but only if the major translational challenges can be addressed. Optimal intracellular delivery is the bottleneck for the advancement of nucleic-acid based therapies. However, a central question is posed with the development of many new treatments. Have we got what we want, where we want it?
Foremost amongst the challenges is selecting and developing effective and safe transfection vectors for in-vivo studies. A major gap at present is that our existing in-vitro models are poorly predictive of animal studies. We will use NMR spectroscopy based methods to help bridge this gap in this studentship. Current in-vitro approaches to determine effective cellular uptake typically make extensive use of microscopy based methods using labelled nucleic acids. Here we propose to use High Resolution Magic Angle Spinning (HRMAS) NMR spectroscopy to measure the metabolic response of whole living cells to treatment. The cells would then be available for conventional analysis too. By multivariate statistical analysis it is possible to use this technique to:
Determine the metabolic profile of living cells prior to and following treatment with anti-cancer drugs.
Infer cellular uptake based on metabolic profile.
Propose a mechanism of action based on changes in the cell's metabolic profile.
In this studentship we will use a combination of HRMAS 1H NMR spectroscopy and mass spectrometry based approaches to model the uptake and effects of biologics and drugs in a range of cell lines to develop an in vitro test for drug uptake at the start of the development process. Should this project be successful it will contribute significantly to reducing the number of animals used in drug toxicity studies by providing a simple and robust in vitro test for the uptake of compounds into cells.
Foremost amongst the challenges is selecting and developing effective and safe transfection vectors for in-vivo studies. A major gap at present is that our existing in-vitro models are poorly predictive of animal studies. We will use NMR spectroscopy based methods to help bridge this gap in this studentship. Current in-vitro approaches to determine effective cellular uptake typically make extensive use of microscopy based methods using labelled nucleic acids. Here we propose to use High Resolution Magic Angle Spinning (HRMAS) NMR spectroscopy to measure the metabolic response of whole living cells to treatment. The cells would then be available for conventional analysis too. By multivariate statistical analysis it is possible to use this technique to:
Determine the metabolic profile of living cells prior to and following treatment with anti-cancer drugs.
Infer cellular uptake based on metabolic profile.
Propose a mechanism of action based on changes in the cell's metabolic profile.
In this studentship we will use a combination of HRMAS 1H NMR spectroscopy and mass spectrometry based approaches to model the uptake and effects of biologics and drugs in a range of cell lines to develop an in vitro test for drug uptake at the start of the development process. Should this project be successful it will contribute significantly to reducing the number of animals used in drug toxicity studies by providing a simple and robust in vitro test for the uptake of compounds into cells.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/P510440/1 | 01/10/2016 | 30/09/2022 | |||
| 1789837 | Studentship | EP/P510440/1 | 01/10/2016 | 31/12/2020 | Marta Wylot |
| Description | Apoptosis is a cellular process that maintains an equilibrium between cell proliferation and cell death. Induction of apoptosis is a well-known strategy in developing cancer treatments, therefore non-invasive monitoring of apoptosis in intact cells could contribute towards the characterisation of drug efficacy and hence drug discovery. A known metabolic marker of apoptosis is a notable increase in proton NMR resonances associated with lipids stored in lipid droplets (LDs). MRI-based studies of lipid accumulation have been used to monitor apoptosis-based cancer treatments. However, the cellular processes which leads to the accumulation of lipid droplets remain poorly understood, causing a bottleneck for targeting lipid metabolism in cancer cells. This thesis investigates the application of High-Resolution Magic Angle Spinning (HRMAS) 1H NMR spectroscopy in monitoring metabolic changes in intact cells during apoptosis. The technique is used to analyse metabolic profiles of cells treated with cisplatin and etoposide after 3, 8, 24 and 48 h. The results are compared to the analysis of organic cell extracts by solution-state 1H NMR spectroscopy to demonstrate lipid compartmentalisation and highlight the advantages of HRMAS 1H NMR spectroscopy in monitoring lipid metabolism during apoptosis. The lipid compartmentalisation is also confirmed by differential centrifugation and mass spectrometry-based lipidomics further validating the importance of LDs. Previous work linked NMR-visible lipid resonances to increased LD size. In this thesis, an NMR-based diffusion method is described for differentiating between control, apoptotic and necrotic cells. I demonstrate that as apoptosis-induced LDs become larger, the diffusion coefficient of NMR-visible lipids decreases. Therefore, diffusion measurements in conjunction with HRMAS 1H NMR-derived lipid signals provide a novel means of following apoptosis in intact cells. Mass spectrometry and transcriptomic analysis was used to elucidate the origin of LD during cisplatin and etoposide treatments. This work identifies two treatment-dependent mechanisms of lipid particle organisation, contributing to our understanding of LD formation during apoptosis. It may help validate magnetic resonance spectroscopy as a non-invasive tool for following the efficacy of apoptosis-inducing drugs. |
| Exploitation Route | My results may help to understand lipid metabolism during apoptosis; possible application in drug development process. HRMAS 1H NMR spectroscopy offers a label-free approach for monitoring cellular changes in lipid behaviour during apoptosis. It may also provide new applications of HRMAS NMR for probing intact cells. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Title | Differentiating between intact control and apoptotic cells by high-resolution magic angle spinning 1H NMR spectroscopy. |
| Description | In order to explore this large difference in diffusion rates we have investigated a wider range of gradient strengths than has been used previously. We use high gradient solution state NMR spectroscopy to monitor diffusion rates of lipid droplets in intact cells. For the first time we have shown the comparison between diffusion rates of lipids in intact cells measured using HRMAS and solution-state NMR equipped with high gradient probe. These measurements show that HRMAS NMR and solution diffusion measurements give similar results regardless of sample packing (rotor vs 3 mm tube) and experimental conditions (spinning vs no spinning). In addition, there are lipids diffusing more slowly in intact cells which cannot be studied with standard NMR probes. Slower moving lipid species may correspond to very large LDs or lipids located in different organelles or even in lipid rafts where the movement of lipids is highly restricted. This approach opens up new opportunities for investigating lipid behaviour in intact cells. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | This will be publish soon. |
| Description | Fluorescence Microscopy experiments |
| Organisation | Durham University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Using HRMAS NMR spectroscopy we reposted that the apparent diffusion coefficients of lipid droplets were smaller in apoptotic cells. We wanted to correlate NMR results with fluorescence microscopy. We used microscopy facility in University of Durham and Cambridge Advanced Imaging Centre. |
| Collaborator Contribution | They provided the facilities and expertise. |
| Impact | The diffusion coefficients from HRMAS NMR correlate well with the droplet sizes determined by light microscopy. This data will be included in my fist paper. |
| Start Year | 2019 |
| Description | Fluorescence Microscopy experiments |
| Organisation | University of Cambridge |
| Department | Cambridge Advanced Imaging Centre |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Using HRMAS NMR spectroscopy we reposted that the apparent diffusion coefficients of lipid droplets were smaller in apoptotic cells. We wanted to correlate NMR results with fluorescence microscopy. We used microscopy facility in University of Durham and Cambridge Advanced Imaging Centre. |
| Collaborator Contribution | They provided the facilities and expertise. |
| Impact | The diffusion coefficients from HRMAS NMR correlate well with the droplet sizes determined by light microscopy. This data will be included in my fist paper. |
| Start Year | 2019 |
| Description | 11th NMR: a tool for biology Conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | A number of conference participants stopped by to see my poster and hear about my projects. A couple of PhD student were interested in applying similar methods for their projects. We exchanged contacts and are in touch. I also had an interesting discussion about ideas how I can extend my current project and the person was happy to get involved. |
| Year(s) Of Engagement Activity | 2019 |
| URL | http://www.nmr2019.conferences-pasteur.org |
| Description | Cambridge Science Festival |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | General public visited our stand to watch our engaging demonstrations and learn interesting facts. Children were keen to participate in our activities and asked a lot of questions. We received many positive comments from parents about the activity. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.sciencefestival.cam.ac.uk |
| Description | EBI-Sanger-Cambridge PhD Symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | The symposium was organized for PhD students to share their research and scientific interests. I had discussions with a number of PhD students about my research and exchanged contact details with two people who were interested in my project. |
| Year(s) Of Engagement Activity | 2019 |
| URL | https://escamps.org/escamps19 |
| Description | King's College Natural Sciences Taster session |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Schools |
| Results and Impact | Gave a mini lecture to sixth formers interested in coming to Cambridge University. Held a question and answers session after the lecture and discussed the teaching at the University. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Metabolomic conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | A number of conference participants stopped by to see my poster and hear about my projects. Three PhD students worked on projects using the same technique and we had interesting discussions and exchanged troubleshooting tips and protocols for analysing samples and data. |
| Year(s) Of Engagement Activity | 2018 |
| URL | http://metabolomics2018.org |
| Description | Pint of Science |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | 30 people attended 3 talks about science in past present and future. After each talk there were a number of questions from audience. We organized a hands on activate for younger audience involving decorating safety secs and we promoted active learning through a pub quiz. After an event participants engaged in scientific discussions. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://pintofscience.co.uk |
| Description | The Naked Scientist Podcast |
| Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | Talked about my research while in a punt going along the river Cam as part of the Naked scientist Podcast. |
| Year(s) Of Engagement Activity | 2018 |
| Description | volunteering in COVID testing centre as an RNA scientist |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Patients, carers and/or patient groups |
| Results and Impact | During the pandemic I put science skills to new use by supporting UK national effort to boost COVID-19 testing. I performed fully automated magnetic bead RNA extraction workflows using Beckman I series robotics and tracked sample through the LIMS system in the Cambridge COVID-19 Testing Centre. |
| Year(s) Of Engagement Activity | 2020 |
| Description | volunteering: responsible for sample logistics for the RECOVERY trial during national lockdown (1 day a week) |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Study participants or study members |
| Results and Impact | During the lockdown I volunteered to handle blood samples from Covid patients in Addenbrooks hospital for RECOVERY trial. I did that one day a week between April and June. This national clinical trial aimed to identify treatments that may be beneficial for adults hospitalised with confirmed COVID-19. Now first results have become available on their website (recoverytrial.net). There has also been a publication titled 'Early immune pathology and persistent dysregulation characterise severe COVID-19' (awaiting publication). |
| Year(s) Of Engagement Activity | 2020 |
| URL | https://www.recoverytrial.net/ |