Periodic Reporting for period 2 - RhabdoEvo (Deciphering the cellular origin and evolution of malignant rhabdoid tumors)
Reporting period: 2021-06-01 to 2022-11-30
Malignant rhabdoid tumours (MRT) are rare, but very aggressive childhood cancers. Although they may arise in any body part, MRT usually form in isolation or synchronously in the kidney and the brain (where they are referred to as atypical teratoid/rhabdoid tumours (AT/RT)). MRT, especially metastatic MRT, remain one of the most lethal childhood cancers, even following intense treatment regimens. Although current therapeutic regimens typically result in an initial reduction of tumour volume, resistance develops in nearly all cases. It remains unknown what is causing these tumours to develop and which biological processes are responsible for their aggressive behavior. Identifying such processes could help developing novel therapeutic opportunities. To do so, adequate experimental models are needed that faithfully capture disease in human patients. Furthermore, tools are required to analyze tumour development and behavior in great detail.
In this ERC-funded project, we aim to study the processes that are involved in MRT initiation as well as key mechanisms driving disease progression and therapy resistance. For this, we make use of unique patient-derived tissues, pre-clinical cancer models (3D organoid technology) and state-of-the-art (single-cell) sequencing technologies. We combine these novel models and technologies to characterize the changes that normal cells have to acquire to become a tumour cell as well as the changes tumour cells undergo when they progress and/or are exposed to different treatments (so-called tumour evolution). In doing so, we hope to identify therapeutic targets that in the future can be used to counteract the aggressive behavior of MRT and thereby provide a much-needed treatment option for patients suffering from this lethal disease. In parallel, the knowledge gained in these studies and the technological advances proposed will be applicable to other tumour types as well and will therefore be of great value for the tumour biology field.
In this ERC-funded project, we aim to study the processes that are involved in MRT initiation as well as key mechanisms driving disease progression and therapy resistance. For this, we make use of unique patient-derived tissues, pre-clinical cancer models (3D organoid technology) and state-of-the-art (single-cell) sequencing technologies. We combine these novel models and technologies to characterize the changes that normal cells have to acquire to become a tumour cell as well as the changes tumour cells undergo when they progress and/or are exposed to different treatments (so-called tumour evolution). In doing so, we hope to identify therapeutic targets that in the future can be used to counteract the aggressive behavior of MRT and thereby provide a much-needed treatment option for patients suffering from this lethal disease. In parallel, the knowledge gained in these studies and the technological advances proposed will be applicable to other tumour types as well and will therefore be of great value for the tumour biology field.
During the first half of our ERC-funded project, we devoted a lot of effort to 1) process MRT tissue in such a way that reliable data can be obtained from individual tumour cells and 2) develop and validate the tumour models to reliably and consistently monitor MRT progression.
We succeeded in developing a protocol that allows for isolation of high-quality single cells from patient-derived tumour specimens. These were subsequently subjected to a comprehensive characterization using a technology that allows for extensive gene expression and chromatin density profiling on the individual cell level. At the time of writing, we successfully applied this technology to 17 patient-derived MRT specimens. Our preliminary analyses show distinct differences between tumour cells of different tumours (intertumoural heterogeneity) as well as between tumour cells within the same tumour (intratumoural heterogeneity). This demonstrates the suitability of our approach to, for the first time, comprehensively characterize rhabdoid tumour cell changes during disease progression.
To further characterize MRT progression, we started with a comprehensive characterization of the transplantation model in which we transplant tumour cells in mice at the location these tumours are normally growing (i.e. orthotopic). We determined the timing and dynamics of primary tumour growth as well as metastasis formation, which is crucial for the experimental design (e.g. number of mice per experimental group). The first tracing experiments revealed extensive clonal selection during in vivo tumour growth. Further analyses and experiments are currently ongoing to study tumour cell behaviour in great detail.
Lastly, we established a genetic tracing strategy to trace back the origin of MRT. This approach allowed us to find the cellular origin of a particular MRT subtype (Custer et al., Nat. Comms. 2021). Additional genome analyses are currently being performed to further pinpoint the cellular origin of other MRT subtypes.
We succeeded in developing a protocol that allows for isolation of high-quality single cells from patient-derived tumour specimens. These were subsequently subjected to a comprehensive characterization using a technology that allows for extensive gene expression and chromatin density profiling on the individual cell level. At the time of writing, we successfully applied this technology to 17 patient-derived MRT specimens. Our preliminary analyses show distinct differences between tumour cells of different tumours (intertumoural heterogeneity) as well as between tumour cells within the same tumour (intratumoural heterogeneity). This demonstrates the suitability of our approach to, for the first time, comprehensively characterize rhabdoid tumour cell changes during disease progression.
To further characterize MRT progression, we started with a comprehensive characterization of the transplantation model in which we transplant tumour cells in mice at the location these tumours are normally growing (i.e. orthotopic). We determined the timing and dynamics of primary tumour growth as well as metastasis formation, which is crucial for the experimental design (e.g. number of mice per experimental group). The first tracing experiments revealed extensive clonal selection during in vivo tumour growth. Further analyses and experiments are currently ongoing to study tumour cell behaviour in great detail.
Lastly, we established a genetic tracing strategy to trace back the origin of MRT. This approach allowed us to find the cellular origin of a particular MRT subtype (Custer et al., Nat. Comms. 2021). Additional genome analyses are currently being performed to further pinpoint the cellular origin of other MRT subtypes.
We successfully developed a lentiviral barcode lineage tracing technology allowing for combined lineage tracing and transcriptomics on the single cell level. In combination with our established orthotopic organoid transplantation methods, this presents a break-through achievement, as it for the first time allows for studying clonal evolution during tumour progression, metastasis, and therapy resistance on the single cell level.Furthermore we successfully implemented cutting-edge phylogenetic lineage tracing strategies allowing for tracing back the cellular origin of cancer (Custer et al., Nat. Comms. 2021).