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A Multi-omics Approach To Decode Epigenetic Lesions In Pancreatic Cancer Development

Periodic Reporting for period 1 - MultiPan (A Multi-omics Approach To Decode Epigenetic Lesions In Pancreatic Cancer Development)

Reporting period: 2019-06-01 to 2021-05-31

Epigenetic mechanisms enable cells to achieve distinct phenotypes while sharing the same genetic information through precise regulation of genome accessibility and gene expression. Pancreatic Ductal Adenocarcinoma (PDAC), the most common form of pancreatic cancer, represents an abysmal disease with a 5-year survival rate of only ~8%. The mutation signature of the disease it is well known (it includes KRAS, TP53, SMAD4 and CDKN2A) and it has been recently shown that epigenetic mechanisms are key in defining the aggressiveness of PDAC since the early stages of the disease. To elucidate the molecular mechanisms of diseases, it is key to use models that accurately recapitulate the sequence of events leading to the malignancy. 3D cultures (such as spheroids or organoids) emerged as a reliable system for in vitro studies and, in case of PDAC-derived organoids, it was shown that when orthotopically transplanted into mice they gave rise to tumours recapitulating the morphology, metabolism and heterogeneous histology of the parental tissue. For this action we made use of the healthy, PDAC patient-derived and Cas9-engineered organoids to carry out the first multi-omics (single-cell RNA and ATAC sequencing) study in these biological entities at single cell resolution, to provide new insights into the heterogeneity of the epigenetic mechanisms driving cancer evolution.
Therefore, the major goal of the proposed project is to perform a systematic survey and analysis of epigenetic lesions occurring in PDAC, as to generate a novel map of epimutations responsible for the growth of cancer cells and create an resource for future drug development studies.
Work performed during this fellowship was ascribed to three specific work packages. In work package 1, protocols for culture and expansion of PDAC organoids were established in the host laboratory. PDAC organoids used in this work had been previously engineered to carry the different mutations to recapitulate, in vitro, the multistep process of pancreatic cancer development. Since the aim of the action was to generate single-cell RNA (scRNA-seq) and ATAC sequencing (scATAC-seq) from the same cells, the conditions for obtaining single-cell suspension while keeping the dissociation time short were optimised. Furthermore, protocols for the generation of single-cell genomics datasets from frozen human pancreatic biopsies were developed in order to establish a “ground truth” reference for the organoid data and these methods have been published and already adopted by the pancreatic research community.
After obtaining single-cell suspension, scRNA-seq and scATAC-seq data were generated. To this end, the solution provided by 10x Genomics (Single Cell Multiome ATAC + Gene Expression) was chosen as it provided the most effective solution in achieving high throughput and high coverage both at the transcriptomic and at the chromatin accessibility level.
In work package 2, the work focused mainly on the bioinformatics analyses of the scRNA/ATAC-seq datasets in different organoids. In particular, different bioinformatic tools were applied for these explorative analyses including Multi-Omics Factor Analysis (MOFA), Seurat v4 and ArchR and we were able to identify changes in cellular composition among the different organoid lines, as well as distinct gene expression and chromatin accessibility programs active in the different cell states. The analyses have not yet been finalised due to the disruptions caused by the lockdown measures introduced during the COVID19 pandemic, but we are hoping to publish the data and make them available for the community in the next six months.
In work package 3, we optimised different protocols enabling the functional investigations of the targets identified in work package 2. In particular, we optimised protocols for RNA in situ hybridisation using frozen or fixed organoids; we obtained plasmids to perform CRISPR/Cas9 perturbations and streamlined the production of constructs including the desired guide RNAs; we tested different systems for the electroporation of organoids and achieved a transfection efficiency as high as ~10%; lastly we successfully performed live-imaging of PDAC organoids for up to 72 hours, using a confocal microscope available in the host laboratory. After the completion of the analyses proposed in work package 2, the methods developed in work package 3 will be applied to functionally validate the targets identified in the study.

Dissemination: the findings of this project were presented in different online seminars including the 1) Francis Crick Stem Cell Forum and the 2) Sparse2Big consortium. This action also included outreach activities, including 1) an interactive laboratory tour and microscopy session at the “Long Night of Sciences 2019”; 2) a seminar with high-school students about concepts of cancer development and epigenetics; 3) a second tour of the laboratory and interactive microscopy session with international journalists visiting the Digital Health Center at Charitè Universitätsmedizin Berlin during the Berlin Science Week 2019.
In the last few decades, novel therapeutic approaches have highly improved the survival rate of cancer patients but, in the specific case of pancreatic cancer, they conferred only a marginal survival advantage. Using an in vitro 3D model system, we have been able to identify changes in cellular composition, as well as alterations at the gene expression and gene regulatory level, paving the way to future studies that will address the role of these molecular changes in PDAC development. Moreover, this work shed light on the early development of pancreatic cancer, a stage that cannot be investigated in patients and that is usually studied in exclusively in animal models (which however do not fully recapitulate the early step of the human disease). Therefore, these data could help implementing methods and algorithm for the early detection of this tumour, which in turn would increase the possibility of curative intervention.

From a personal perspective, this MSCA has improved my teaching and supervising skills as I have been supervising a full-time PhD student at BIH/Charitè Universitätsmedizin (thesis defence expected at the end of 2022). Moreover, throughout the fellowship, I organised small (in-person and virtual) working groups to promote team-work within the host department. This enhanced the rapid, mutual and effective exchange of expertise between laboratory-based and data scientists, enabling faster analyses and interpretations of the complex datasets generated within the department. To strengthen the potential transition to an independent position, I attended the “Laboratory Leadership for Postdocs” organised by EMBO. Lastly, the host supervisor supported my development towards becoming an independent researcher and specialist in the field of quantitative analysis of cancer epigenetics by promoting the collaboration with international (both from Europe and USA) experts in the field of pancreatic research. Therefore, this action, gave me the opportunity to create an international network of collaborations and develop into a fully independent researcher.
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