Human stem cell models of de novo tumorigenesis to replace the use of animals for the study of cancer initiation

Why did we fund this project?

This award aims to develop a 3D breast organoid model using human induced-pluripotent stem cells (iPSCs) replacing the use of some transgenic and xenograft animal models.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is challenging to treat, and no targeted therapies are available. There are multiple molecular and clinical subtypes of TNBC, defined by the specific mutations and altered pathways, adding to the complexity of identifying biomarkers that would enable earlier treatment of TNBC. A range of in vitro and in vivo models are used to study TNBC including transgenic animals, which require extensive breeding programmes to introduce relevant mutations. Xenograft models are also used to study early oncogenic events where patient-derived or cell line cells are transplanted into the animal. These oncogenic in vivo experiments are typically classified as causing moderate severity under the UK’s Animals (Scientific Procedures) Act 1986 due to the level of suffering caused by the growth of the tumour.

The student, with Dr Cinzia Allegrucci, will introduce genetic mutations associated with TNBC development into iPSCs. These cells will then be combined with a hydrogel scaffold, previously developed by Professor Cathy Merry (Co-Supervisor) through an NC3Rs Project grant, and the cells differentiated to develop an in vitro 3D breast organoid model. The student will then use the model to study TNBC initiation and progression and to validate gene expression and pathological characteristics against historic in vivo experiments and patient clinical data. The student will develop skills in cell culture, RNAseq, histopathological staining and confocal microscopy.

This project aims to develop a novel complex human cell model to study breast Triple Negative Breast Cancer (TNBC), whilst reducing the use of animals needed for the research.

TNBC represents the most aggressive breast tumour subtype, typically presenting with high grade, high metastatic potential, and poor prognosis. TNBC remains the most clinical challenging type of breast cancer as no effective targeted therapies are available. Progress is further hindered by TNBC heterogeneity as TNBC is also a not a single disease, but further subdivided into distinct molecular and clinical subtypes based on specific molecular pathway signatures and mutational landscapes that impact on patient outcome. To date, the identification of biomarkers of TNBC initiation that can be used for early detection and treatment of TNBC patients remains an unmet clinical need.

The scientific and clinical importance of these questions has seen a wide use of animals in breast cancer research. Genetically engineered mouse models (GEMM) and xenotransplantation mouse model with genetically modified cells currently represent the gold standard for studying cancer initiation as they allow the analysis of early oncogenic events driven by mutations and closely mimic the histopathological and molecular characteristics of human tumours. Consequently, a large number of breast cancer studies use these models (~1,220 studies/year in the last five years), thus requiring an estimated number of more than 24,000 animals per year.

The approach we intend to use in this research project provides a novel way to investigate the function of specific genetic mutations in the initiation of TNBC, which will replace the use of GEMM and xenograft models. Our system involves introducing genetic changes associated with TNBC into normal induced pluripotent stem cells made for mammary cells to recreate the development of cancer in an in vitro 3D breast organoid model. This technology does not involve using animals to expand the cells and it does not use animal products to create the 3D model. Indeed, the model will use a synthetic hydrogel that will provide a superior matrix not derived from animal tumours to study interaction of tumour cells with their environment. Importantly, this advanced cell model provides advantages over other cancer patient-derived cultured cell systems as it allows modelling cancer initiation which is critical for the development of therapeutic strategies to target early-stage disease.

We estimate that this approach will reduce animal model use by at least ~300 mice/year locally and ~7,000 mice /year globally. The use of the synthetic hydrogel will also reduce the use of animal-derived matrix for differentiation studies using organoids, with an estimated reduction of ~40 mice/year in our laboratory. The project will also provide the foundations to develop similar models for other types of cancer, therefore creating a significant impact and legacy on the overall replacement of animals in cancer research. Importantly, creating this unique model will provide new knowledge of the disease and how it differs in different TNBC patients for the development of precise therapies and increased patients’ survival.