Replacing the need for patient-derived xenografts and matrigel organoid culture as preclinical models for breast cancer

Most cells in your body are surrounded by a matrix of proteins and sugars. Cells interact with these and react to the elasticity and stiffness of the matrix. Matrix plays a key role in disease progression but is difficult to study in the lab. Studies are often carried out in human cells grown on plastic (2D) which poorly supports matrix interactions. To understand 3D matrix-driven behavior, labs rely on highly variable animal-derived artificial matrices or use animal models, including the growth of human cells transplanted into mice. Although these are more realistic, neither models the complex human tissue matrix well. We need to improve the 3D growth of cells within the laboratory and reduce the need for animal models. We have developed a fully synthetic, reproducible gel that can mimic the matrix of human tissues. Breast cancer cells, along with other cell types, can be encapsulated and easily grown in the lab. This project aims to develop tools to share our mimics with research scientists throughout the world, providing cheap, functional and robust environments. This will allow researchers to test theories of cancer development, discover new targets for intervention and provide more realistic environments to screen therapeutics. To demonstrate the adaptability of our method we will also modify the gel formulations to (i) work in a fully animal product-free system (ii) to encapsulate breast cancer stem cells. This project links the group who have developed the hydrogels with one of the world-leading groups researching breast cancer. This will ensure that we can reach and influence the global breast cancer research community and companies developing therapeutics. We will use a combination of on-line training packages and physical workshops to create a reliable way of sharing information. We anticipate significantly reducing the numbers of animals used in xenograft studies whilst also improving the relevance of work carried out in the laboratory.

As part of Cathy Merry and Jennifer Ashworth's current NC3Rs project they have developed a peptide hydrogel suitable for the 3D culture of primary cells and cell lines used to model normal breast development and breast cancer. The purpose of the current project is to ensure uptake of this model by the wider breast community by working closely with Robert Clarke and the Manchester Breast Centre (MBC). Our plan is to embed the technology in the MBC, providing training to Katherine Spence, Senior Scientific Officer. A video training package, supported by written protocols, FAQs and guidance will be assembled and hosted locally on the MBC website and on a website with high visibility to interested researchers; the European Network for Breast Development and Cancer (ENBDC http://www.enbdc.org/). To demonstrate the adaptability of the technology and to create a completely animal-product free system we will additionally combine the peptide hydrogel system with the MBCs current methods for serum-free culture and culture of breast cancer stem cells. These methods will then be added to the training package. A major focus of the project is on working directly with breast cancer research groups, taking advantage of Robert Clarke's involvement in multiple large-scale collaborative groups. A large cohort of interested groups have already been recruited (see letters of support) and these will be encouraged to attend one of a number of workshops which will incorporate group training events. Follow-on support will be via the online training package and in-person using online meeting software. We aim to encourage long-term uptake of the peptide hydrogel technology and a change in practice by the partner labs, reducing or replacing their current use of rodent models or rodent-derived 3D matrices. We will, therefore, integrate online support for the peptide hydrogels into the MBC site and link to the work of the University of Nottingham Corporate Partnerships Team.

Currently, Robert Clarke's (RC) immediate group use up to 300 female NOD/SCID/IL2Rgamma-/- (NSG) mice p/a for PDX tumour implantation and experimental work. This requires a further 100 mice p/a for maintenance/support of colonies. The procedures carried out on these mice are rated as low to moderate severity. Throughout the Manchester Breast Centre and with collaborators nationally, this number increases to 1,000 mice p/a. Breast Cancer is a highly researched area and the use of PDX models is widespread, reflected by an estimated publication of ~500 papers last year involving mammary fat pad and/or subcutaneous xenografts in mice (Source: Pubmed; search term: "breast cancer xenografts 2017"). We estimate this results in ~20,000 animals per year (based on 40 animals/paper). The need for a superior in vitro alternative is more pressing considering the dominance of PDX models as a tool for personalized therapy. The EurOPDX consortium holds a panel of >1,500 PDX subcutaneous and orthotopic cancer models, which illustrates this preclinical research aspiration (www.europdx.eu). Such 'Xeno-patient' cohorts are considered the most clinically relevant animal models in cancer drug discovery (Byrne et al., Nature Reviews Cancer, 2017), and is well funded by trans-national sources (Eg. Euro 5million Horizon 2020 EdiREX grant awarded to EurOPDX Consortium) . Similarly, the use of the animal derived tumour product, Matrigel for 3D culture models is well established in cancer research. In RC's immediate group, use of Matrigel is 30 tubes p/a. Typically, 2 tubes of matrigel can be produced from 1 mice therefore this equates to 15 mice p/a. Throughout the Breast Centre this number increases to 200 tubes p/a (100 mice). Novel preclinical breast cancer models such as the in vitro organoid arrays proposed by Hans Clevers (Sachs et al., Cell, 2018) will only increase the use of Matrigel as this is an essential part of their culture platform. Therefore, a change of practice by an influential group is an essential factor in changing the attitudes of other scientists. Following establishment of the validity of the hydrogel culture platform, existing scientists would need to be convinced that such in vitro technologies partially replace the need for in vivo preclinical models, changing practice in reviewing of grants and papers. RC's role on grants committees and on the Editorial Board of the journals such as Breast Cancer Research and the Journal of Mammary Gland Biology and Neoplasia would ensure that this transition in in vitro usage be encouraged. In the first year of establishing the peptide hydrogel technology in RC's group, we would anticipate that we could reduce the use of Matrigel by 50% rising to 100% in 3 years. Similarly, we would aim to reduce the numbers of PDX models, by carrying out more relevant in vitro assays using the hydrogel by 30% in the first 3 years and by 60% in 5 years as more in vitro models come online. If we expand these numbers to the wider community we would hope to reduce the numbers of animals used by 5-10,000 per annum.