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Single-cell RNA-seq data from 3 HGSOC patient-derived organoids and corresponding tumor or ascites

ABSTRACT: The broad research use of organoids from high-grade serous ovarian carcinoma (HGSOC) has been hampered by low success rates of culture establishment and scarcity of fresh tumor material. Here we describe a novel method that enabled generation and long-term expansion of a HGSOC organoid biobank with efficacy markedly improved over previous reports (55% vs. 23-38%). We established organoids from cryopreserved material, demonstrating the feasibility of using frozen tissue biobanks for HGSOC organoid derivation. Organoids recapitulated patient-specific genomic landscapes and retained the phenotypic heterogeneity of original samples. Organoid drug responses correlated with clinical treatment outcomes, but only for organoids maintained in human plasma-like medium (HPLM), highlighting the importance of relevant culture conditions in organoid-based functional assays. Taken together, we introduce a resource for efficient development and use of HGSOC organoids from cryopreserved material in ovarian cancer research.

PROVIDER: EGAS00001006246 | EGA |

REPOSITORIES: EGA

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Project description:High-grade serous ovarian cancer (HGSOC) creates a unique clinical challenge due to late detection, limited therapeutic options and wide-spread resistance to chemotherapy, all of which lead to very low survival rates. It is now widely recognized that this cancer emerges as metastasis from the fallopian tube and the lack of suitable in vitro disease models poses a major obstacle for efforts to improve our understanding of this deadly disease. We used organoids of healthy human FT epithelium as a model for stepwise genetic modification towards malignancy, using shRNA-mediated knockdown (KD) of three major HGSOC driver genes: P53, PTEN and Retinoblastoma (RB). Moreover, we successfully established patient-derived HGSOC organoids from solid tumor deposits. After combinatorial screening approach of different media compositions we have established 15 organoid lines which depend on the absence of exogenous Wnt3a in the medium. We could show that those cultures closely resemble the parental tumor tissue and express hallmarks of HGSOC phenotype as revealed by histology and global gene expression profiles of the matching organoid and tissue samples. Expression and sequencing analysis revealed that all samples were characterized by p53 mutation and/or aberrant p53 expression. Interestingly, the addition of Wnt agonists to cancer organoids leads to rapid growth arrest and a sharp drop in stemness, as proven by cell viability assay and the number of CD133 positive cells, respectively. Complementary to this finding, we can show that the medium optimized for ovarian cancer organoids also improves the growth of the modified organoid lines, showing that changes within the niche environment are required to promote stemness upon depletion of P53/PTEN/RB. Taken together, our data reveal that critical changes in the paracrine environment are likely to represent early events during HGSOC development and provide a starting point to further analyze how specific mutations and paracrine signals influence pathways of stemness regulation and thus drive malignant transformation in ovarian carcinogenesis.

Project description:Background & Aims Patient-derived gastrointestinal organoids are powerful tools for studying human physiology and disease. However, generating organoids requires prompt isolation and culture of fresh tissue, which is labor and time intensive, placing restraints on basic and clinical research. For example organoid biobanking efforts, or the ability to obtain specimens from distant locations that lack the expertise to culture organoids, is limited by current approaches. We sought to develop a method by which tissue biopsies from patients could be cryo-preserved, stored, or shipped, and later thawed in order to establish live organoid cultures. Methods A method for freezing and recovery, along with straightforward isolation methods using readily available materials were developed. Epithelial isolation and culture conditions were optimized to promote cell survival and the establishment of long-term culture post-thawing. Results Patient biopsies from stomach, duodenum, ileum, colon and from adenomateous colonic polyps were frozen, subsequently thawed and used to successfully establish patient-specific epithelium-only organoid cultures with 100% efficiency (n=31 independent patient biopsies from different regions of the GI tract). Frozen tissue could be shipped internationally and across the United States and used to establish successful organoid cultures. Organoid cultures could be expanded, passaged and frozen down for long-term storage. RNA-sequencing demonstrates that organoids derived from fresh tissue or from frozen biopsies are >99% transcriptionally identical. Conclusions Cryo-preservation of human tissue biopsies followed by the establishment of long-term, expandable cultures has negligible effect on transcription when compared to freshly prepared organoids, and will be of immense benefit to research and for clinical applications. This technique will allow for the establishment of biobanks of human tissues that can be revived and cultured in the future as well as transported across the globe for research, therapeutic or diagnostic use in remote labs and/or clinics.

Project description:High-grade serous ovarian cancer (HGSOC) creates a unique clinical challenge due to late detection, limited therapeutic options and wide-spread resistance to chemotherapy, all of which lead to very low survival rates. It is now widely recognized that this cancer emerges as metastasis from the fallopian tube and the lack of suitable in vitro disease models poses a major obstacle for efforts to improve our understanding of this deadly disease. We used organoids of healthy human FT epithelium as a model for stepwise genetic modification towards malignancy, using shRNA-mediated knockdown (KD) of three major HGSOC driver genes: p53, PTEN and Retinoblastoma (Rb). These pathways are known to be disrupted in the vast majority of HGSOC patients (>95%, 45% and 67% of cases, respectively as reported by TCGA consortium). Unexpectedly, combined loss of function of the three tumor suppressor proteins, p53, PTEN and Rb, led to a growth arrest and loss of stemness if organoids are cultured in medium optimized for healthy FT epithelium, which maintains high Wnt signaling and BMP pathway inhibition. This designates a requirement for alterations in the exogenous signaling cues to facilitate transformation. Indeed, we show that successful expansion of patient-derived HGSOC organoids from solid tumor deposits depends on the absence of exogenous Wnt3A in the medium. Using a combinatorial screening approach of different medium compositions, we have established 15 organoid lines, which closely resemble the parental tumor tissue and express hallmarks of HGSOC phenotype as revealed by histology and global gene expression profiles. Strikingly, the medium optimized for ovarian cancer organoids also rescued the growth of the modified organoid lines, showing that changes within the niche environment are required to promote stemness upon depletion of p53/PTEN/RB. Likewise, surface expression of the stemness marker CD133 was strongly increased in triple KD organoids in the absence of Wnt3A/Rspo1. Complementary to this finding, the addition of Wnt agonists to cancer organoids led to rapid growth arrest and a sharp drop in stemness, as evidenced by cell viability assay and the number of CD133 positive cells, respectively. Taken together, our data reveal that critical changes in the paracrine environment constitute early events during HGSOC development.

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Single-cell RNA-seq data from 3 HGSOC patient-derived organoids and corresponding tumor or ascites

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