Establishment And Application Of Human Ovarian Cancer Organoid Culture System

Part I: Establishment of human ovarian cancer organoid culture systemObjective: By adjusting the culture conditions,Ovarian cancer 3D organoid and ovarian cancer Air-liquid interface（ALI）organoid were constructed and verified at the histological and genomic features.Methods: From April 2018 to August 2019,we collected ovarian cancer tissues and fresh blood from obstetrics and gynecology department of Tongji hospital（Huazhong University of Science and Technology,Wuhan,China）,explored the conditions of organoid culture and successfully constructed ovarian cancer 3D organoids and ovarian cancer ALI organoids.The cultured conditions were compared and optimized by regulating IGF1、HGF、Heregulinβ-1、Forskolin、Hydrocortisone、β-Estradiol、BMP4 and Noggin.We compared ovarian cancer organoids and matched tumor tissues at the histological level by HE staining and immunohistochemistry.CD3 and Vimentin indexes were measured by immunofluorescence assay between ovarian cancer ALI organoids and matched tumor tissues.Whole genome sequencing（WGS）analysis were performed on ovarian cancer organoids,matched tumor tissues and blood.Copy number variations（CNVs）,single nucleotide variants（SNVs）,structural variants（SVs）,homology analysis,somatic mutations analysis,etc.were used to prove the correlation between ovarian cancer organoids and matched tumor tissues at the genomic features.Results: A total of 34 ovarian cancer samples were collected,and 10 ovarian cancer 3D organoids and 2 ovarian cancer ALI organoids were successfully formed.We optimized the culture conditions and proved that IGF1 and HGF did not improve the proliferation efficiency of ovarian cancer organoids.One or several cytokines in Heregulinβ-1 、 Forskolin 、Hydrocortisone and β-Estradiol can significantly promote the growth of ovarian cancer organoids,while the regulation of BMP signal may have opposite effects in organoids from different sources.Ovarian cancer organoids recapitulate histological and genomic features of the matched tumor tissues.Ovarian cancer ALI organoid could retain the tumor microenvironment（TME）,which containing tumor-infiltrating lymphocytes（TILs）and cancer-associated fibroblasts（CAFs）.Conclusions: We successfully constructed the culture conditions of ovarian cancer 3D organoids and ovarian cancer ALI organoids,providing a new method for exploring the mechanism of ovarian cancer progression and the application of precision medicine.Part Ⅱ: Application of human ovarian cancer organoid culture systemObjective: Through immunotherapy experiment and drug sensitivity test,the potential of ovarian cancer organoid in precision medicine has been proved.The drug sensitivity results were analyzed,personalized treatment was proposed for specific patients,and the related mechanisms were explored.Methods: PD1 antibody was added to ovarian cancer ALI organoid,and the apoptosis rate of tumor cells between different treatment groups was determined by flow cytometry.According to clinical guidelines,drug sensitivity tests were conducted on ovarian cancer 3D organoid to screen out potentially effective treatment schemes.Combined with the drug sensitivity results of PARP inhibitors,CNV analysis and HR-related gene expression data,the pathways or genes related to PARP inhibitor resistance in specific patients were screened.We cloned sh CHK1 sequences into the lentiviral vector and infected organoid.The inhibition effect of sh CHK1 on CHK1 expression was verified by PCR and Western blot,and the effect of sh CHK1 on the therapeutic effect of Olaparib was analyzed by drug sensitivity curve.Flow cytometry was used to measure the cell cycle of Olaparib and Prexasertib monotherapy and combination treatment groups.Immunofluorescence assay was used to measure the formation of RAD51 foci in Olaparib and Prexasertib monotherapy and combination treatment groups.Results: Through immunotherapy experiment and drug sensitivity test,we proved that ovarian cancer organoids could be used for immunotherapy analysis and drug screening.After inhibiting CHK1 expression,only HGS-4_O increased sensitivity to Olaparib,while HGS-2₁O and HGS-3₁O did not change.Ovarian cancer 3D organoids HGS-4_O is not sensitive to Olaparib.However,CHK1 inhibitor Prexasertib promotes the sensitivity of HGS-4_O to Olaparib.Prexasertib eliminated Olaparib-induced G2/M cell cycle arrest by inhibiting CHK1,and promotes unrepaired cells to enter mitosis,resulting in a replication disaster that leads to apoptosis.Prexasertib reduced the formation of Rad51 foci,thereby inhibiting HR and promoting therapeutic efficacy of Olaparib.Conclusions: ALI organoid can be used in the exploration of immunotherapy.The application of precision medicine and personalized therapy can be carried out through organoids drug sensitivity test.Through the analysis of drug sensitivity results and whole-genome sequence（WGS）data,we can propose personalized solutions to clinical problems such as drug resistance and explore the mechanisms.