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Human “mini-liver” stem cell culture systems: from mechanisms behind liver cancer origin and recurrence to new therapeutic treatments

Periodic Reporting for period 1 - mini-liver SCCS (Human “mini-liver” stem cell culture systems: from mechanisms behind liver cancer origin and recurrence to new therapeutic treatments)

Reporting period: 2016-04-20 to 2018-04-19

CONTEXT AND OVERALL OBJECTIVES OF THE PROJECT
Primary liver cancers (PLC) are among the most lethal and prevalent cancers in the human population. Despite their significance, there is only an elemental understanding of PLC pathogenesis, notably due to a lack of models that faithfully recapitulate an original tumor. Indeed, the majority of the existing animal models (from dog to rodent) do not replicate the human disease and existing cell lines do not mimic the complex genetic spectra of these tumors. Therefore, developing a physiologically relevant in vitro cancer model to study the mechanisms behind this disease is of capital importance to facilitate finding new relevant therapeutic treatments for this disease and improve patient care.
Here, we aimed at translating organoid technology to establish and characterize physiologically relevant 3D-liver models and use them to increase our understanding of the mechanisms behind PLC with the final goal to develop new anti-cancer strategies.

OVERVIEW OF THE PROJECT RESULTS
Thanks to the H2020-MSCA-IF fellowship I have pioneered the technique of generating organoids from PLC biopsies (so-called tumoroids) and demonstrated that they faithfully recapitulate and maintain the histopathology and genetic profile of the original tumor, even after long term culture. Therefore, they provide an extremely powerful resource for analysing the biology of PLC and develop new therapeutic strategies. Moreover, I have evaluated tumoroids use as a drug screening platform and provided the proof-of-concept that PLC tumoroids are relevant for in vitro and in vivo drug testing and for identification of actionable therapeutic targets. The outcomes of this research have the potential to significantly impact on the liver cancer research field and might facilitate finding new therapeutic treatments for this disease.
SCIENTIFIC OBJECTIVES:
Generation and characterization of in vitro 3D organoid models for human primary liver cancer:

Aim 1: model liver cancer by directly culturing human primary liver tumors
Current models for PLC either fail to fully recapitulate tumor histology and architecture or are expensive, time consuming and do not allow for personalised drug testing. During this proposal we established a new 3D in vitro model system for PLC. Based on the current knowledge on organoid cultures, we have managed to establish a system to grow human PLC cells long-term in vitro. Interestingly, the tumor-derived organoids (also termed “tumoroids”) recapitulate the original tumor histology and genetic alterations and are also able to generate tumors in an in vivo xenograft mouse model. Furthermore, we have shown that tumoroids can also be successfully used for drug testing, suggesting their use to devise new targeted therapy as well as personalised treatment strategies.

Aim 2: model human liver cancer progression by genetically engineering human liver cancer in vitro using CRISPR/Cas9 technology
Current models to investigate the role of genes in cancer rely mostly on animal studies, which can be very time consuming and cost demanding, especially if resulting in negative outcomes. To overcome this issue, we set up a protocol for introducing mutations in healthy human liver organoids using the CRISPR-Cas9 technology. Interestingly, after mutating TP53, RNF43 and ZNRF3 either alone or in combination, human organoids undergo genetic alterations and phenotypic changes that partially resemble the ones observed in tumoroids. This data suggests that this system could be used as a screening platform to study gene function/dysregulation during tumorigenesis.

EXPLOITATION AND DISSEMINATION:
These results were published in December 2017 in Nature medicine (Broutier et al., Nature medicine 2017). They will open opportunities to study, for the first time, PLC in a physiologically relevant ex-vivo human system. Moreover, PLC-organoids with a short timescale from establishment to drug screening, could be a relevant in vitro liver cancer system for predicting patient-specific drug responses and creating personalized therapies.
I demonstrated the proof-of-concept that organoid technology can recapitulate in vitro human PLC. Therefore, they provide an extremely powerful resource for analysing the biology of PLC and develop new therapeutic strategies. Moreover, I have evaluated PLC tumoroids use as a drug screening platform and provided the proof-of-concept that PLC tumoroids are relevant for in vitro and in vivo drug testing and for identification of actionable therapeutic targets (Broutier et al., Nature medicine 2017).
Recent advances in sequencing technologies have enabled high-throughput quantification of individual cell transcripts. These single-cell assays such as single cell RNA sequencing (scRNAseq) have enormous power to reveal differentiation hierarchies, lineage choice control and cell fate, and are therefore of great importance to study cancer stem cells (CSCs) biology. Then, moving forward I’m currently using my newly established PLC tumoroids and scRNAseq, to study the functional intra-tumoral heterogeneity and CSCs plasticity in human PLC.

Conclusion: These results represent a perfect example of innovative and challenging translational research harnessing the development of new powerful tools to further our knowledge on basic sciences (molecular and cellular mechanisms driving PLC), identify new drugs, and develop new treatment options for patients (precise/personalized medicine).
PROJECT SUMMARY: Primary Liver Cancer-derived organoid for disease modeling and drug screening