Award details

Use of nanoparticles and macrophages to target two novel therapies to hypoxic areas of tumours: a combined biological and mathematical study

Reference	BB/C506113/1
Principal Investigator / Supervisor	Professor Claire Elizabeth Lewis
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Genomic Medicine
Funding type	Research
Value (£)	166,456
Status	Completed
Type	Research Grant
Start date	01/11/2004
End date	31/10/2007
Duration	36 months

Abstract

(i) Tumour spheroids will be grown from human breast cancer cell lines in vitro. Primary macrophages will be loaded with magnetic particles and co-cultured with spheroids in the presence of a magnetic field. The spatiotemporal pattern of their uptake will be assessed using fluorescent cell markers/confocal microscopy and/or CD68 (a pan macrophage marker) immunostaining of spheroid sections. Theoretical models of this process will be developed, using a multiphase modelling framework. Model simulations will describe the migration of the magnetically loaded macrophages within the spheroid in the presence/absence of magnetic forces and be validated against the experimental data. (ii) Macrophages will be infected with a replication-deficient adenovirus bearing a reporter gene (LacZ) under the control of a trimer of a well-defined, hypoxia-regulated enhancer from a human gene promoter. Activation of the LacZ gene (beta-gal expression) by hypoxia will first be assessed using Western blotting of macrophage extracts, then infected macrophages will be loaded with magnetic particles and added to spheroid cultures in the presence of a magnetic field. The hypoxic cell marker piminadazole will be used to demonstrate hypoxia in sections. Expression of beta-gal by macrophages in the outer, well oxygenated region of the spheroids and the inner, hypoxic regions will be assessed using CD68 and beta-gal immunostaining (IS). The reporter gene will then be replaced by either: (a) the prodrug activating enzyme, P450 (in which case the macrophage-infiltrated spheroids will be incubated with the associated prodrug, cyclophosphamide and cell death assessed using both the TUNEL assay on and a clonogenic cell survival assay) or (b) our new anti-angiogenic drug, Alphastatin - with an in-house antibody being used to demonstrate its expression in spheroid sections. The theoretical models from (i) will be extended on account for macrophage loading with therapeutic genes and their likely therapeutic impact, in terms of tumour shrinkage, will be assessed. (iii) A flow model will be constructed in which macrophages flow over a filter coated with endothelial cells. A magnetic field will be applied to enhance macrophage extravasation and spheroid infiltration. The spatiotemporal pattern of macrophage attachment to, and movement across the endothelial cell layer, and then infiltration into spheroids will be assessed using fluorescent membrane markers. Reporter gene expression by transfected macrophages will be assessed in the spheroids as described in (ii). A mathematical model of macrophage flow will be constructed (including the forces acting on macrophages in flow) and coupled to models of extravasation and infiltration into the tumour. The resulting model will be used to predict changes in macrophage uptake that arise when system parameters (e.g. tumour size, flow rate, membrane properties and magnetic field) vary. (iv) Using experimental and theoretical models from (ii) and (iii) to specify macrophage distribution within the spheroid mathematical models of heat flow will be developed to simulate the application of a radio frequency magnetic field to the magnetically loaded macrophages and the consequent heating. The extent to which cooling caused by fluid flow in the model limits the size of tissue that can be destroyed using magnetic hyperthermic and the efficiency of different heating protocols will be determined and compared with experiment. Model predictions will also be tested against experiments in which 2 spheroids (one composed of non-malignant cells and the other of malignant cells) are placed in the lower compartment of the flow chamber assay in order to assess the level of killing cells in malignant tissues (macrophage density in higher in malignant than in healthy spheroids) while leaving adjacent healthy tissue unaffected. (Joint with BB/C506156/1 and BB/C506172/1).

Summary

unavailable

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/C506156/1 Use of nanoparticles and macrophages to target two novel therapies to hypoxic areas of tumours: a combined biological and mathematical study

BB/C506172/1 Use of nanoparticles and macrophages to target two novel therapies to hypoxic areas of tumours: a combined biological and mathematical study

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