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.
