Development of human ex vivo bone-tumour niche for the study of cancer bone disease
Lead Research Organisation:
University of Oxford
Department Name: NDORMS
Abstract
The skeleton is the preferential site for tumour metastases and the incidence and management of primary bone tumours (e.g. osteosarcoma) typically affecting young adults, have remained unchanged for over a decade. Moreover, the spread of tumours to the skeleton often represents the final stages of disease. Consequently, there is a large field of research aimed at reducing the spread and development of tumours within the skeleton, and which involve a wide range of animal models. These include models where tumours are injected into distant sits (e.g. heart) and allowed to spread to bone, or where tumour cells are implanted directly into skeletal sites (e.g. tibia). In addition, human tumour cells are often used in such mouse models, and where animals lacking an efficient immune system are used, and which do not fully capitulate the human bone-human tumour microenvironment seen in patients with cancer-bone disease.
This proposal aims to address this issue and reduce/replace the use of animals as a first line tool in such studies, through the development of a new ex vivo model where human bone is isolated under sterile, ethical- and human tissue act-approved procedures, and colonised in culture with human tumour cells. The extent of bone destruction and tumour growth will be assessed as is used for bones dissected from mouse models of cancer-bone disease. This will include using computer-generated 3D images of the bone and histology, with tissue culture media stored for future analysis or bone- and tumour-derived factors. In addition, this proposal will re-use data from previous in vivo studies conducted within our group to compare directly against data generated using this ex vivo assay, and which will include the testing of anti-cancer therapies.
This proposal aims to address this issue and reduce/replace the use of animals as a first line tool in such studies, through the development of a new ex vivo model where human bone is isolated under sterile, ethical- and human tissue act-approved procedures, and colonised in culture with human tumour cells. The extent of bone destruction and tumour growth will be assessed as is used for bones dissected from mouse models of cancer-bone disease. This will include using computer-generated 3D images of the bone and histology, with tissue culture media stored for future analysis or bone- and tumour-derived factors. In addition, this proposal will re-use data from previous in vivo studies conducted within our group to compare directly against data generated using this ex vivo assay, and which will include the testing of anti-cancer therapies.
Technical Summary
This proposal will standardise and validate the use of a human ex vivo assay for the study of cancer-bone disease, thereby replacing mice as the first stage model for the study of cancer cell interactions within the skeleton, and providing a new system to assess novel drugs aimed at reducing cancer metastasis to and proliferation within bone, in addition to blocking tumour-induced osteolysis.
Uniform slices (0.5cm thick) of human bone from cores (0.5cm diameter, approx. 5 cm length) isolated from femoral heads removed during surgery will be cultured ex vivo. Human tumour cells (breast cancer, myeloma) will be seeded within each bone slice at a known density using either A) direct injection over 30 mins, followed by an overnight colonisation and transfer to experimental well plate; B) implantation as above followed by 1hr gentle agitation prior to overnight colonisation; C) immersion of bone slice in matrigel and implantation of tumour cells within bone/gel environment, followed by culture as in A. All cultures will be maintained for either 3, 7 or 10 days in aMEM (+1%BSA) as used previously and replenished every 3 days. Anti-cancer therapies previously tested within our group (bortezomib, zoledronate) will also be added to culture media in this assay and results compared to previous in vivo studies.
As with murine skeletal samples from tumour-bone disease models, bone from this assay will be assessed by microCT scanning and histomorphometric analysis to quantify bone volume and architecture, bone mineral density, tumour and bone cell distribution. In addition, media will be collected to assess the release of bone- (alkaline- and acid phosphatase) and tumour-derived factors (PTHrP, TGF-B, myeloma-specific IgG2bk) and correlated with measurements obtained using stored serum from mice from past experiments. Representative STANDARD slices from throughout the bone core will generate baseline data with SAMPLE slices cultured under experimental conditions.
Uniform slices (0.5cm thick) of human bone from cores (0.5cm diameter, approx. 5 cm length) isolated from femoral heads removed during surgery will be cultured ex vivo. Human tumour cells (breast cancer, myeloma) will be seeded within each bone slice at a known density using either A) direct injection over 30 mins, followed by an overnight colonisation and transfer to experimental well plate; B) implantation as above followed by 1hr gentle agitation prior to overnight colonisation; C) immersion of bone slice in matrigel and implantation of tumour cells within bone/gel environment, followed by culture as in A. All cultures will be maintained for either 3, 7 or 10 days in aMEM (+1%BSA) as used previously and replenished every 3 days. Anti-cancer therapies previously tested within our group (bortezomib, zoledronate) will also be added to culture media in this assay and results compared to previous in vivo studies.
As with murine skeletal samples from tumour-bone disease models, bone from this assay will be assessed by microCT scanning and histomorphometric analysis to quantify bone volume and architecture, bone mineral density, tumour and bone cell distribution. In addition, media will be collected to assess the release of bone- (alkaline- and acid phosphatase) and tumour-derived factors (PTHrP, TGF-B, myeloma-specific IgG2bk) and correlated with measurements obtained using stored serum from mice from past experiments. Representative STANDARD slices from throughout the bone core will generate baseline data with SAMPLE slices cultured under experimental conditions.
Planned Impact
Academic and Industry-based researchers will benefit from this research, in addition to patients suffering from cancer-bone disease who (in the long term) may benefit from a more rapid experimental approach to the testing of anti-cancer therapies, which this assay will provide.
These groups will benefit from this research through:
i) increased volumes of data can be generated in a mid/high-throughput manner (when compared to animal studies).
ii) decreased cost to PI researchers leading and managing the work (including animals purchase and housing, consumables, analysis).
iii) decreased time and effort necessary to run the ex vivo assay and generate equally valid data through the use of this system.
With regard to the 3Rs (detailed in Pathways to Impact & Case for Support), it is estimated based on our current animal usage, that over 400 animals may be replaced by the use of this assay in our dept., and based upon our knowledge of researchers in the field we estimate that 2095 animals are currently used annually to study cancer-bone disease in the UK, (or 8380 in the US and UK combined).
These groups will benefit from this research through:
i) increased volumes of data can be generated in a mid/high-throughput manner (when compared to animal studies).
ii) decreased cost to PI researchers leading and managing the work (including animals purchase and housing, consumables, analysis).
iii) decreased time and effort necessary to run the ex vivo assay and generate equally valid data through the use of this system.
With regard to the 3Rs (detailed in Pathways to Impact & Case for Support), it is estimated based on our current animal usage, that over 400 animals may be replaced by the use of this assay in our dept., and based upon our knowledge of researchers in the field we estimate that 2095 animals are currently used annually to study cancer-bone disease in the UK, (or 8380 in the US and UK combined).
