Organoid and single cell models of bladder cancer
Lead Research Organisation:
University of Leeds
Department Name: School of Medicine
Abstract
The project aims to develop a range of in vitro organoid models from fresh bladder tumour tissues and use these to develop drug screening assays.
Key objectives:
1. Optimization of conditions for tumour cell isolation, organoid culture and long-term maintenance.
Fresh bladder tumour tissues will be used. More than 250 new bladder cancers are diagnosed in Leeds each year and we collect 4-5 fresh tumour samples each week with patient consent. In addition to material for culture, a portion of each tissue is snap-frozen for molecular characterisation and a blood sample collected to provide germline DNA. A large panel of extensively-characterised bladder cancer cell lines is also available for initial optimisation experiments.
2. Establishment of a frozen bank of organoids representing the range of bladder cancer molecular subtypes. Parallel stored tissues from successful cultures will be characterised as part of other work within the group. This will include targeted mutation screening using a next generation sequencing assay and genome-wide expression profiling. This will assign samples to previously defined molecular subtypes and will identify targetable mutations.
3. Development of microfluidics platform-based drug sensitivity analysis for rapid evaluation of patient samples.
A microfluidics platform (already available) that allows trapping of individual cells and exposure to drug profiles will be used to determine drug sensitivity to cytotoxic and cytostatic agents. Initial experiments will use a tumour cell line with known sensitivity to FGFR inhibitors and to cisplatin. Surface Acoustic Wave Dielectrophoresis (SAW-DEP) will be evaluated for separation of viable from non-viable cells, prior to the sensitivity testing. Both flow-sorted single cell populations from tumours and cells derived from organoids will be assessed. Recent advances in the integration of spheroid / organoid formation with microfluidic devices offer potential advantages in terms of improved screening capability and will be tested.
The project will use a wide range of methods, including tissue dissociation methods, organoid culture from tissue fragments and single cells, flow sorting, cell staining (immunohistochemistry and immunofluorescence), light and fluorescence microscopy (including confocal) and drug sensitivity testing in 2D and 3D cultures and on the microfluidics platform.
Key objectives:
1. Optimization of conditions for tumour cell isolation, organoid culture and long-term maintenance.
Fresh bladder tumour tissues will be used. More than 250 new bladder cancers are diagnosed in Leeds each year and we collect 4-5 fresh tumour samples each week with patient consent. In addition to material for culture, a portion of each tissue is snap-frozen for molecular characterisation and a blood sample collected to provide germline DNA. A large panel of extensively-characterised bladder cancer cell lines is also available for initial optimisation experiments.
2. Establishment of a frozen bank of organoids representing the range of bladder cancer molecular subtypes. Parallel stored tissues from successful cultures will be characterised as part of other work within the group. This will include targeted mutation screening using a next generation sequencing assay and genome-wide expression profiling. This will assign samples to previously defined molecular subtypes and will identify targetable mutations.
3. Development of microfluidics platform-based drug sensitivity analysis for rapid evaluation of patient samples.
A microfluidics platform (already available) that allows trapping of individual cells and exposure to drug profiles will be used to determine drug sensitivity to cytotoxic and cytostatic agents. Initial experiments will use a tumour cell line with known sensitivity to FGFR inhibitors and to cisplatin. Surface Acoustic Wave Dielectrophoresis (SAW-DEP) will be evaluated for separation of viable from non-viable cells, prior to the sensitivity testing. Both flow-sorted single cell populations from tumours and cells derived from organoids will be assessed. Recent advances in the integration of spheroid / organoid formation with microfluidic devices offer potential advantages in terms of improved screening capability and will be tested.
The project will use a wide range of methods, including tissue dissociation methods, organoid culture from tissue fragments and single cells, flow sorting, cell staining (immunohistochemistry and immunofluorescence), light and fluorescence microscopy (including confocal) and drug sensitivity testing in 2D and 3D cultures and on the microfluidics platform.
Organisations
People |
ORCID iD |
| Margaret Knowles (Primary Supervisor) | |
| Daniel Griffiths (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| NC/P001777/1 | 01/10/2017 | 09/12/2019 | |||
| 2302487 | Studentship | NC/P001777/1 | 01/10/2017 | 31/07/2019 | Daniel Griffiths |