2023 Fiscal Year Research-status Report
Pathogenesis roles and therapeutic targets of PBRM1 mutation in intrahepatic cholangiocarcinoma
| Project/Area Number |
22K15336
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| Research Institution | Kanazawa University |
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Principal Investigator |
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Keywords | Cholangiocarcinoma / PBRM1 / Organoid / CRISPR/Cas9 / TP53 / IDH1 / KRAS |
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| Outline of Annual Research Achievements |
We created four more lines of intrahepatic cholangiocyte organoids (iCO) from non-tumor livers, totaling 16 primary iCO lines, and one intrahepatic cholangiocarcinoma organoid (iCCAO). We constructed different vectors for PBRM1, IDH1, KRAS knockout, and optimized conditions for gene-edited organoids and cell lines. We collected 150 human intrahepatic cholangiocarcinomas (iCCA) and analyzed gene mutations, including PBRM1, P53, KRAS, IDH1/2, ARID1A. A panel of markers to support the classification of iCCA tumor microenvironment was established: SMA, TAGLN, CD276 were prominently found in activated stroma; CD163, CD66b, and VISTA for macrophages and myeloid-derived suppressor cells (MDSCs), prominently in early stroma; CD3, CD8, and PD1 for T cells in inflammatory stroma.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Gene editing efficiency and the survival of gene-modified organoids from normal cholangiocytes were varied. Some lines of PBRM1-KO organoids were difficult to maintained.
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| Strategy for Future Research Activity |
We will perform CRISPR/Cas9 experiments on four human iCCA cell lines available in our lab, in addition to gene-modified iCO, and analyze how cell lines and iCO, iCCAO with PBRM1 loss respond to different treatments in the next experiments. We will also analyze human iCCAs, comparing tumor cell histological phenotypes and tumor microenvironment (TME) features, and TME class between PBRM1-mutant and PBRM1-wild type iCCAs.
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| Causes of Carryover |
To buy reagents and equipment for analyzing effects of different treatments on PBRM1-KO organoids and cell lines; ,performed more immunohistochemistry staining and spatial transcriptomic analysis on human iCCAs with different PBRM1 mutation status.
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