Karaciğer kanserinin modellenmesinde indüklenebilir pluripotent kök hücre (IPSCs) kaynaklı hepatik organoidler

Liver cancer is a heterogeneous disease with very limited treatment options, which ranks 4th in the world in terms of death. Hepatocellular Carcinoma (HCC), the most common type, is cancer that usually develops on the basis of cirrhosis and is characterized by late diagnosis and drug resistance. The most important limitation in understanding the early molecular pathophysiological changes and identifying new treatment targets in HCC is lack of the in vitro and in vivo models mimicking human tumorigenesis. In this context, in this study, the interactions of the microenvironment with stemness and senescence morphology in hepatocytes produced from IPSCs were investigated (Karabicici, 2021) and it was aimed to develop a long-term and large-scale 3D cell culture model that models the formation and development of pre-malignant stages of liver cancer such as fibrosis and cirrhosis. Here we utilize CRISPR/Cas9 technology for targeted gene modification of three of the most commonly mutated liver cancer genes (p53, APC, and NF1) in Induced Pluripotent Stem Cell derived healthy organoids (eHEPO). Although the CRISPR/Cas9 strategy to make these driver genes knockout has been worked successfully in HEK293 cells, we only continued with p53 knockout on eHEPOs. For this, we firstly transfected px458 plasmids containing p53 specific gRNA to the eHEPOs via optimized Nucleofector conditions. After enrichment of the GFP+ cells by FACS, we manually selected p53 KO-eHEPO clones under Nutlin-3a treatment. To mimic pro-carcinogenic TME activated LX2 and M2 polarized cell secretoms were added to growing medium of p53 KO-eHEPOs as well as WT controls. P53-KO highly proliferative (Ki67) and all displayed features of pro-carcinogenic pathological marks of pre-malign stages including increased nuclear–cytoplasmic ratio, pleiomorphic and hyperchromatic nuclei, pseudo glandular and tubular rosettes and inflammatory-like cell morphology. In addition, mason trichome staining area and fibrotic gene expressions have been shown to increase significantly. As a result, it has been shown that a fibrotic organoid model can be created that will make significant contributions to the understanding of tumor formation mechanisms in a carcinogenic microenvironment by using p53 KO-eHEPOs, which is one of the driver genes at the onset of HCC.