Developing human organotypic perfused bioreactors for physiologically reproducible therapeutic compound screening of a tumour microenvironment
Lead Research Organisation:
University of Oxford
Department Name: Oncology
Abstract
'Developing human organotypic perfused bioreactors for physiologically reproducible therapeutic compound screening of a tumour microenvironment'
Pre-clinical testing of new therapeutics relies on the use of in-vitro and in-vivo models. These, however, have intrinsic weaknesses, one major one being the inadequate recapitulation of physiological conditions in-vivo. In cancer, for example, pre-clinical testing systems do not recapitulate the tumour microenvironment. This is a considerable drawback since the interplay of tumour and stroma cells, the extracellular matrix and the consistent supply of nutrients and oxygen via the perfusion of the microcirculation adds to the spectrum of selective pressure that leads to the heterogeneity and ultimately fate of tumour cells. This inability to reflect crucial in-vivo conditions, together with their associated costs and time constraints, and the ethical controversy of animal testing of in-vivo models, makes the search of better ways for pre-clinical therapeutic testing a matter of paramount urgency.
Tissues and organs artificially grown using bioreactors, engineered devices that support a biologically active environment, are seen as a promising type of advanced in-vitro models to initially reduce and eventually replace current conventional in-vitro and in-vivo models. Biomedical engineers, including myself, have developed bioreactors to grow these artificial tissues in order to test therapeutics, including anti-cancer compounds. Nevertheless, most of these prototypes have so far lacked perfusion, without which these advanced in-vitro models cannot recapitulate key aspects of the tumour microenvironment such as the interstitial fluid, the microcirculation of blood vessels and immune system.
Building on the work I carried out during my DPhil, here I propose to develop a culture platform (bioreactor) capable of recapitulating perfusion in the tumour microenvironment, and fit for screening of anti-cancer compounds. The developed culture platform will be tested using brain and pancreatic cancer tissue, and will focus on following up the process of angiogenesis and immune response during tumour development and responses to therapy. Therapeutic compounds known to regulate these processes will then be tested in order to validate the developed in-vitro model.
Our perfused culture platform has the potential to tackle the disadvantages inherit of conventional in-vitro and in-vivo models, relieving the ethical pressures of animal testing, and being more cost and time effective. Additionally, the small amount of cancer tissue required by this platform would make it particularly suitable for cancer types such as brain and pancreatic cancer which suffer from a lack of clinical samples.
Pre-clinical testing of new therapeutics relies on the use of in-vitro and in-vivo models. These, however, have intrinsic weaknesses, one major one being the inadequate recapitulation of physiological conditions in-vivo. In cancer, for example, pre-clinical testing systems do not recapitulate the tumour microenvironment. This is a considerable drawback since the interplay of tumour and stroma cells, the extracellular matrix and the consistent supply of nutrients and oxygen via the perfusion of the microcirculation adds to the spectrum of selective pressure that leads to the heterogeneity and ultimately fate of tumour cells. This inability to reflect crucial in-vivo conditions, together with their associated costs and time constraints, and the ethical controversy of animal testing of in-vivo models, makes the search of better ways for pre-clinical therapeutic testing a matter of paramount urgency.
Tissues and organs artificially grown using bioreactors, engineered devices that support a biologically active environment, are seen as a promising type of advanced in-vitro models to initially reduce and eventually replace current conventional in-vitro and in-vivo models. Biomedical engineers, including myself, have developed bioreactors to grow these artificial tissues in order to test therapeutics, including anti-cancer compounds. Nevertheless, most of these prototypes have so far lacked perfusion, without which these advanced in-vitro models cannot recapitulate key aspects of the tumour microenvironment such as the interstitial fluid, the microcirculation of blood vessels and immune system.
Building on the work I carried out during my DPhil, here I propose to develop a culture platform (bioreactor) capable of recapitulating perfusion in the tumour microenvironment, and fit for screening of anti-cancer compounds. The developed culture platform will be tested using brain and pancreatic cancer tissue, and will focus on following up the process of angiogenesis and immune response during tumour development and responses to therapy. Therapeutic compounds known to regulate these processes will then be tested in order to validate the developed in-vitro model.
Our perfused culture platform has the potential to tackle the disadvantages inherit of conventional in-vitro and in-vivo models, relieving the ethical pressures of animal testing, and being more cost and time effective. Additionally, the small amount of cancer tissue required by this platform would make it particularly suitable for cancer types such as brain and pancreatic cancer which suffer from a lack of clinical samples.
Technical Summary
1. Different scales of bioreactors will be developed to mimic the flow of the interstitial environment of tumours, and bioreactor parameters will be optimised to improve the maintenance of homeostasis. This will allow long-term observation (> 21 days) in a physiologically relevant microenvironment [1]. 2. To realise the spatial control for the development of microtissues, micropatterning engineering tools such as soft lithography and three-dimensional bioprinting will be applied to define the distributions of a range of supporting cell types (such as networks of blood vessels) and tumour cells [2]. 3. Cellular and molecular biology techniques and immunofluorescence microscopy will allow us to elucidate the relation between some biophysical and biochemical parameters which are hard to measure or quantify in traditional in-vivo models, but with prognosis significance [3]. More importantly, parameters such as interstitial fluid dynamics, can determine the differentiation of the cells via creating chemokine gradients or shear stress, which will contribute to the heterogeneity of the tumour tissue [4]. Bioreactors with a fluidic culture environment will allow the observation of interstitial flow, which will then make it possible for testing drugs in a physiomimetic avatar. 4. To develop in-vivo pathomimetic avatars for different differentiated stages of tumour remains a bottleneck, while using tissue engineering techniques differentiation can be induced via well-controlled bioreactor parameters (as explained in #3). The differentiated advanced in-vitro models will then mimic the specific stages of tumour development, and the drug screening results obtained from these models will be more predictable of their clinical behaviours. [1] Gantenbein B et al, Curr Stem Cell Res Ther. 2015;10(4): 339-352; [2] Qian X et al, Cell. 2016; 165(5):1238-1254; [3] Morgan J et al, Nat Protoc. 2013; 8(9):1820-1836; [4] Huang Y et al, Integr Biol (Camb). 2015;7(11): 1402-1411
Planned Impact
1. In our institute, most mouse models were used in a pre-clinical validation core for their drug screening. Last year about 2280 mice were used. Most of the mice experiments were carried out as further validation after the newly developed drug proven to be efficient in traditional two-dimensional cell culture models. However nearly 60% of drugs later on showed compromised effects on those mice and had to be stopped from going further. We have the confidence of replacing traditional cell monolayer models with the developed bioreactor system. If the drugs had no effect on the bioreactor system, which has a better prediction on clinical behaviour of drugs, then the later testing on mice can be saved. This adaption of new testing system will potentially save 1368 mice per year in our institute only.
2. Apart from drug validation, in our institute there are seven labs focusing on the tumour microenvironment basic research also require mice experiments in their final publications. Although the number of the mice they use is lower than the ones used for drug screening, which is about 100 mice under each publication, I have the previous successful case to show the whole mice experiments can be avoided [1]. Thus it will be another 700 mice per year to be saved.
3. For the international influence of the developed bioreactors, we would like to make some fairly conservative estimation. Since we already showed promising data from neuroblastoma cells in the bioreactor, we did a PubMed publication research and found about 80 publications about neuroblastoma in a year. On average 60-100 mice were mentioned to have been used in each publication. Via presenting our developed bioreactors in conferences or workshops, we can potentially persuade those research groups to adapt our models into their research. Even if we just convince 30% of them, to replace 50% mice they are using, that will be 720-1200 mice per year to be saved. Together with impact #1 and #2 above, we expect to save 2788-3268 mice per year, only considering the impact on neuroblastoma study.
4. There are scientific advantages of the bioreactor systems compared with the most widely used MYCN transgenic mice and xenografted models for neuroblastoma: (i) mice models only mimic a portion of human neuroblastoma, while bioreactor can mimic all neuroblastoma type by using specific human-originated cells; (ii) real-time change of tissue in mice models is difficult to be imaged, while bioreactors are designed platforms with optimised optics properties for real-time imaging; (iii) mice models need long time to prepare with high risk of shrinking size sample due to the high death rate, while bioreactor systems require relatively short time to prepare with reproducible sample sizes; (iv) xenograft model is absent of immunology response, while bioreactors have the potential to mimic the immunology of the real tumour microenvironment by perfusing cells with immunological origins [2, 3]. 5. In the second year of my fellowship I would like to extend my bioreactor systems to other hard-to-treat cancers. The patients will benefit from the results tested using their own tumour cells. Compared with long time (months to even a whole year) to get results from mice models, with limitations as explained in #3, it takes 7-14 days to create tumour tissues to be tested in the bioreactor in a more physiologically relevant microenvironment [4]. This will potentially give the clinicians some time to trim and tailor an individualised strategy to treat a specific patient.
[1] Montenegro RC et al, Oncotarget. 2016; 7(28):43997-44012 [2] Althoff K et al, Oncogene. 2015; 34: 3357-3368; [3] Teitz T et al, PLoS ONE. 2011; 6(4): e19133; [4] Wan X et al, Biotechnol Lett. 2016; 38(8): 1389-1395.
2. Apart from drug validation, in our institute there are seven labs focusing on the tumour microenvironment basic research also require mice experiments in their final publications. Although the number of the mice they use is lower than the ones used for drug screening, which is about 100 mice under each publication, I have the previous successful case to show the whole mice experiments can be avoided [1]. Thus it will be another 700 mice per year to be saved.
3. For the international influence of the developed bioreactors, we would like to make some fairly conservative estimation. Since we already showed promising data from neuroblastoma cells in the bioreactor, we did a PubMed publication research and found about 80 publications about neuroblastoma in a year. On average 60-100 mice were mentioned to have been used in each publication. Via presenting our developed bioreactors in conferences or workshops, we can potentially persuade those research groups to adapt our models into their research. Even if we just convince 30% of them, to replace 50% mice they are using, that will be 720-1200 mice per year to be saved. Together with impact #1 and #2 above, we expect to save 2788-3268 mice per year, only considering the impact on neuroblastoma study.
4. There are scientific advantages of the bioreactor systems compared with the most widely used MYCN transgenic mice and xenografted models for neuroblastoma: (i) mice models only mimic a portion of human neuroblastoma, while bioreactor can mimic all neuroblastoma type by using specific human-originated cells; (ii) real-time change of tissue in mice models is difficult to be imaged, while bioreactors are designed platforms with optimised optics properties for real-time imaging; (iii) mice models need long time to prepare with high risk of shrinking size sample due to the high death rate, while bioreactor systems require relatively short time to prepare with reproducible sample sizes; (iv) xenograft model is absent of immunology response, while bioreactors have the potential to mimic the immunology of the real tumour microenvironment by perfusing cells with immunological origins [2, 3]. 5. In the second year of my fellowship I would like to extend my bioreactor systems to other hard-to-treat cancers. The patients will benefit from the results tested using their own tumour cells. Compared with long time (months to even a whole year) to get results from mice models, with limitations as explained in #3, it takes 7-14 days to create tumour tissues to be tested in the bioreactor in a more physiologically relevant microenvironment [4]. This will potentially give the clinicians some time to trim and tailor an individualised strategy to treat a specific patient.
[1] Montenegro RC et al, Oncotarget. 2016; 7(28):43997-44012 [2] Althoff K et al, Oncogene. 2015; 34: 3357-3368; [3] Teitz T et al, PLoS ONE. 2011; 6(4): e19133; [4] Wan X et al, Biotechnol Lett. 2016; 38(8): 1389-1395.
