Periodic Reporting for period 2 - PRECODE (PancREatic Cancer OrganoiDs rEsearch Network)
Reporting period: 2021-10-01 to 2024-03-31
The PRECODE Network (PancREatic Cancer OrganoiDs rEsearch) central mission is to establish pancreatic organoid research firmly in the European Union, by training the next generation of creative and innovative researchers in pancreatic cancer. This was done through a shared doctorate program of excellence that fulfils the three “I”: international, interdisciplinary and intersectorial. Organoids can be viewed as small micro-organs and as such can be used to reduce animal experiments and help to reach 3Rs goals. Organoids from pancreatic cancer will help us to learn which drugs might help in the therapy of this terrible disease. They can be isolated from a variety of clinical sources with minimal material needed. This makes them a perfect tool for precision medicine. Sequencing, metabolomics and high throughput in vitro experiments to deduct the best possible drug combination fit hand in glove with this approach. Training of ESRs was achieved by e-learning, workshops and secondments. ESRs learnt to cultivate and manipulate organoids as well as cutting edge molecular biology techniques. Therefore, our work group consisted of 15 beneficiaries and 7 partner organizations (including three patient advocate groups from Sweden, Italy and Germany) to develop those technologies and to train ESRs in their application. PRECODE therefore connected the concepts of investigation, education and innovation establishing a capacitation for the ESRs that goes beyond the state of the art and that allowed them to improve their competences and their professional career at the same time that we advanced towards innovation in the field of pancreatic cancer research.
PRECODE has focused on the production of a robust and reliable SOP for the derivation of organoid cultures. The protocol has been produced in the form of Standard Operating Procedures and includes the description of methods for preparation of nucleic acids and protein lysates from the models. In addition to the protocol, PRECODE has also generated a flowchart for the qualification of organoids before their distribution which included: (1) expansion of the organoids for a minimum of five consecutive passages; (2) “resuscitation” of the culture following freeze/thaw cycle; (3) genetic evidence of cancer based on the detection of KRAS mutation and/or alteration of other common PaCa genes.
In total PRECODE can provide 60 live models, the sequence information of about 80 and the proteomics data of about 40 models. We can provide 6 live organoid models which express Cas9 for future CRISPR/Cas9 experiments. We have also defined a minimum medium condition that enables long-term organoid propagation. Common Data Elements to be recorded from the source patients by the clinical collaborators were also defined. Tissues and models related information are anonymized and collected at each contributing site before submission into a RedCap database that has been established at Umea University. We have also successfully developed a protocol for isolation of cancer-associated fibroblasts, and have established normal fibroblasts and cancer-associated fibroblast cultures. To identify metastases associated genes we have established genome wide gene editing in organoids from different sources. Moreover, we have established about eight Zeb1 knockout organoid lines from the KPC PaCa GEMM in order to block the EMT program. We have optimized media conditions for high throughput drug screening of PDAC organoid monocultures and initial drug screens with standard of care drugs have been performed including treatment schedules mimicking clinical applications. Co-cultures of PDAC organoids and platelets have been established and initial drug treatments with standard of care and nanodrugs were performed. In addition, organ-on-a-chip models of PDAC organoids and PDAC organoids-PSCs co-cultures have been established, and applied in drug exposure and immune migration studies. PRECODE has trained the ESR via eLearning and in in-person meetings in the area of organoid production, bioinformatics, entrepreneurship and CRISPR/Cas9 based genome editing. ESR training also included several courses on transferable skills.
Within the reporting period we achieved several results. First, we introduced a standard operating procedure (SOP) for the establishment of organoid lines and the extraction of RNA, DNA and proteins, which is currently also used in the planned clinical trials of ESPAC-6 and ESPAC-7. The participation in PRECODE permitted the beneficiary Peter Bailey from UG to become the head of translational research for the two clinical trials mentioned above. Second, we were able to publish a collection of peer reviewed reviews in the journal “Frontiers in cell and developmental biology” (IF:6.514) in which we presented the anticipated work of PRECODE. Third, we have agreed on a SOP for high throughput screening for the produced organoid lines, so that we achieve comparable results between different beneficiaries. Our efforts in dissemination of PRECODE has enabled us to include organoid lines from three other European groups (Hanna Seppänen from Helsinki, Carles Barceló from IdISBA in Palma and Michael Linnebacher from Rostock) into the PRECODE pipeline indicating the amplifying effect of the MSCA action. The web site of PRECODE will be funded for the next years and includes now all the eLearning modules for public usage. No unforeseen risks occurred in period 1. An unforeseen risk in period 2 was the withdrawal of one beneficiary from integrated activities regarding our sequencing efforts. We have compensated this by engaging other groups versed in bioinformatics.
In total PRECODE can provide 60 live models, the sequence information of about 80 and the proteomics data of about 40 models. We can provide 6 live organoid models which express Cas9 for future CRISPR/Cas9 experiments. We have also defined a minimum medium condition that enables long-term organoid propagation. Common Data Elements to be recorded from the source patients by the clinical collaborators were also defined. Tissues and models related information are anonymized and collected at each contributing site before submission into a RedCap database that has been established at Umea University. We have also successfully developed a protocol for isolation of cancer-associated fibroblasts, and have established normal fibroblasts and cancer-associated fibroblast cultures. To identify metastases associated genes we have established genome wide gene editing in organoids from different sources. Moreover, we have established about eight Zeb1 knockout organoid lines from the KPC PaCa GEMM in order to block the EMT program. We have optimized media conditions for high throughput drug screening of PDAC organoid monocultures and initial drug screens with standard of care drugs have been performed including treatment schedules mimicking clinical applications. Co-cultures of PDAC organoids and platelets have been established and initial drug treatments with standard of care and nanodrugs were performed. In addition, organ-on-a-chip models of PDAC organoids and PDAC organoids-PSCs co-cultures have been established, and applied in drug exposure and immune migration studies. PRECODE has trained the ESR via eLearning and in in-person meetings in the area of organoid production, bioinformatics, entrepreneurship and CRISPR/Cas9 based genome editing. ESR training also included several courses on transferable skills.
Within the reporting period we achieved several results. First, we introduced a standard operating procedure (SOP) for the establishment of organoid lines and the extraction of RNA, DNA and proteins, which is currently also used in the planned clinical trials of ESPAC-6 and ESPAC-7. The participation in PRECODE permitted the beneficiary Peter Bailey from UG to become the head of translational research for the two clinical trials mentioned above. Second, we were able to publish a collection of peer reviewed reviews in the journal “Frontiers in cell and developmental biology” (IF:6.514) in which we presented the anticipated work of PRECODE. Third, we have agreed on a SOP for high throughput screening for the produced organoid lines, so that we achieve comparable results between different beneficiaries. Our efforts in dissemination of PRECODE has enabled us to include organoid lines from three other European groups (Hanna Seppänen from Helsinki, Carles Barceló from IdISBA in Palma and Michael Linnebacher from Rostock) into the PRECODE pipeline indicating the amplifying effect of the MSCA action. The web site of PRECODE will be funded for the next years and includes now all the eLearning modules for public usage. No unforeseen risks occurred in period 1. An unforeseen risk in period 2 was the withdrawal of one beneficiary from integrated activities regarding our sequencing efforts. We have compensated this by engaging other groups versed in bioinformatics.
We created a European resource of 60 human organoid lines, which are fully characterised and available for other European researchers. By this PRECODE will increase its impact since such models are highly sought after. We were able to confirm, that the published signatures are valuable for subclassifying pancreatic cancer. We also expect to identify genes crucial for the early development of metastasis in pancreatic cancer. In a chemotactic model we identified the loss STK11 as crucial for the metastasis of pancreatic cancer cells. Additional validated target genes will serve as a resource for further investigation and European drug development. Finally, PRECODE will have further impact on the European research by integrating former ESRs with knowledge in state-of-the-art technologies, including organoid production, advanced in vitro modelling, next generation sequencing and genome editing into the European workforce. These researchers of the future will drive the European research landscape by becoming the next generation research leader. To date three ESRs have obtained their PhDs already and we expect that most of the ESRs will obtain their scientific degree in early 2025. All of these three former ESRs have found positions in the Life Science industry in Europe. To exploit our research to which PRECODE contributed, more than 45 manuscripts with 758 citations have been published to date and we expect several key publications with PRECODE participation in the next years.
PRECODE has also developed model agreements for the material transfer and data protection and management, which might serve as a blueprint for other European cooperation’s.
PRECODE has also developed model agreements for the material transfer and data protection and management, which might serve as a blueprint for other European cooperation’s.