ContextLiver cancer is the third leading cause of cancer-related death worldwide.
At present, there is only an elemental understanding of the molecular, cellular and environmental mechanisms that drive liver cancer pathogenesis. Furthermore, the lack reliable human models for liver cancer where to perform drug testing with highly predictive value, delays the advance of the management of the disease.
In fact, the majority of existing animal models for liver cancer, ranging from genetically engineered mouse models (GEEMM) to dogs, do not fully replicate the human liver cancer and are not suitable for large drug-screening testing.
Recently, patient derived xenografts (PDX) have emerged as good models for human cancer and personalized medicine, including liver cancer. However, for most patients, PDXs are not a feasible approach as these have a low intake rate, grow very slow and require long time for a complete study, which, overall, delays their utility for large drug screenings testing.
An alternative to these models (cell lines, GEMM or PDxs) would be to grow in vitro the primary liver cancer cells derived directly from the patient’s tumour. However, growing human liver cells in culture, both normal or tumoural has remained a challenge.
We recently showed that our novel liver organoid culture technology allows, for the first time, the long- term expansion (>6 months) of mouse, canine, feline and human Broutier liver cells directly from the healthy liver. In this culture system, primary liver cells grow into 3D-epithelial structures termed “liver-organoids”. These cultures, derived directly from the adult liver tissue, are able to expand long-term and differentiate towards functional liver cells, while preserving their genetic stability after culture. Furthermore, we have also recently shown that this organoid culture system allows modelling some aspects of 2 monogenic human liver diseases: Alpha 1 Antitrypsin Deficiency (A1AT-D) and Allagile Syndrome directly from biopsies from affected patients (Huch et al., 2015). However, the technology has not yet been adapted for modelling liver cancer
Concept & ApproachDeveloping novel liver cancer organoid protocol further by establishing and characterizing in vitro and in vivo human cancer organoid models.
Aim1: Characterize 3D-Organoid Cancer Models (Months 3-24)
We will aim to increase our tumour culture collection to 12 patients (8 HCC and 4 CCA patients) and 3 donors and use these for in depth characterization and proof-of-principle that these systems could represent an alternative to existing cancer cell lines or PDXs models as detailed below. To establish the different lines we will use our newly refined protocol for liver cancer, which enriches for the tumoural population. Established liver cancer- derived organoids will be characterized in depth.
Aim2: Validate in vivo the Tumorogenicity of the Organoid Models obtained in Aim 1 (Month 6-24)
To determine whether the models obtained retain the histoarchitecture and characteristics of the patient’s tumour tissue, even after having been expanded long-term in culture (>2 months), organoids generated in Aim 1 and expanded at least for 2 months will be engrafted subcutaneously into immunodeficient NSG (NOD /Scid/Gamma) mice. We will use NSG mice as this strain is well know to have the highest engraftment rates for solid tumours. Furthermore, the use of NSG mice will allow us to compare our engraftment rate to that of PDXs, as this is also the strain of choice for PDXs. Tumour growth and volume will be monitored weekly. These experiments will inform not only if our models represent a good in vitro model for liver cancer and retain its histological features and expression patter in vivo, but also if these are a potential good alternative to patient derived xenografts (PDXs).
Aim 3: Determine whether the models obtained are applicable for drug-screening (Month 18-30)
Specifically, we will: 3.a) Perform the drug screening 3.b) Validate the results in vitro 3.c) Validate the results in vivo
OutcomesImpact on liver cancer patients, liver cancer researchers and the 3Rs.
The use of human liver material will have a tremendous impact on the application of the 3Rs in the liver cancer field. As mentioned above, at present the study of liver cancer biology relies purely on animal models or on patient derived xenografts, which both use many animals and resources.
Therefore, this project will be studying human liver cancer tissue directly from the patient, will prompt liver cancer researchers to replace the actual animal models used in liver cancer research (from rodents to cat or dog) for human models. These will allow testing novel hypothesis ranging from drug testing to human liver cancer biology, while, at the same time will have a tremendous impact on the application of the 3Rs in the liver field. Specifically, our plan of work will result in the replacement and reduction of animal use in liver cancer and, consequently, it will improve animal welfare (refinement), as less animals suffering from liver failure and/or liver cancer would be used.
Overall, the project has the potential to replace and reduce the amount of animals used in the liver and cancer fields.
Impact on the 3Rs
Impact for liver cancer patients and other researchers
Deriving organoid cultures that faithfully recapitulate the histoarchitecture, genetic landscape and transcriptomic profiles of the original patient’s tumour will allow modelling the specific disease of each patient, and thus making reality the promise of finding new therapeutic treatments for cancer personalized medicine.
Furthermore, not only patients, but also liver cancer researchers will extremely benefit from this research. Since organoids mainly contain pure populations of epithelial cancer cells, these might facilitate the discovery of novel cancer biomarkers. This is especially relevant for very highly fibrotic tumours, like CCAs, where the major tumour mass is highly contaminated with non-tumoral cells (fibrotic and some necrotic cells). If so, this will represent a step forward on liver cancer treatment and diagnosis, as it could increase the odds to detect this cancer at earlier (and easy to treat) stages.
The outcome of this research could also influence other cancer fields in need of establishing reliable in vitro models (e.g. lung cancer).