Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids

High-grade serous ovarian cancer (HG-SOC)—often referred to as a “silent killer”—is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53, Brca1, Nf1 and Pten. Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients.

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Hans Clevers Apr 1, 2020 Leica LAS X Version 1.1, Bio-Rad CFX Manager Version 3.1 Libraries were sequenced on an Illumina NextSeq500 by using 75-bp paired-end sequencing. Paired-end reads from Illumina sequencing were aligned to the mouse genome (GRCm38 assembly) with BWA (Li and Durbin 2009). The raw datafile consists of a total number of reads for each gene (without UMI correction) that were uniquely mapped to the transcriptome (with a mapping quality above 60), and that had the appropriate transcription direction. DESeq2 (v1.18.0) package was used to normalize count data and for differential gene expression analysis in program R (R version 3.5.1, Bioconductor version 3.8 (BiocManager 1.30.4)). Gene set enrichment analysis (GSEA) was performed using GSEA software v3.0 beta2. qPCR data was analyzed in Microsoft Excel 2019 using delta-delta Ct method.
Immunohistochemistry samples were imaged on DM4000 light microschope using LAS X software (Version 1.1) and processed using ImageJ (Version 1.51p). Immunofluorescent stainings for yH2A.X were imaged on a Leica SP8 confocal microscope, and positive cells quantified by manual counting.
Drug screening kill curves were produced using GraphPad Prism software (version 7.04) and lines were fitted using the option 'log (inhibitor) vs normalized response -variable slope'.

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.  Supplementary Figures 1-4, 6 are provided as a Source Data file. All the other data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request. A reporting summary for this article is available as a Supplementary Information file.
Ovaries and oviducts from at least 6 mice were used to establish a single oviductal and OSE organoid line. The number of mice (n=6) for one experiment was chosen due to small amount of epithelial cells that could be derived from the oviducts and ovaries of a single mice. At least 4 independent organoid lines were established per origin.
qPCR experiment was performed on n=3 independent biological replicates with two technical replicates per each sample. The experiment was repeated three times. This sample size was chosen to confirm that our results are line-independent and reproducible.
RNA-seq analysis was performed on 3 independent tissue samples or organoid lines, except for OSE organoid lines where 1 of the 3 lines was exluded from analysis due to evident contamination. This sample size was chosen to confirm that our results are line-independent and reproducible. Additionally, RNA-sequencing was performed on 6 independent tumor tissues derived from subcutaneously grown tumors. This sample size was chosen to reliably characterize the tumors with statistically significant power.
Two independent organoid clones were analyzed per each mutation or their combination per tissue of origin to guarantee representative results. Each clone was transplanted into at least 3 immunodeficient mice, the injections were done to both sides of each mice (left/right flank or left/right ovarian bursa in subcutaneous and orthotopic transplantations, respectively). At least 2 mice were used for orthotopic transplantations per clone (2 injected ovaries/mouse) and at least 1 mouse was used for subcutaneous transplantations per clone (2 flanks/ mouse). This strategy was favored due to the previous knowledge that orthotopic transplantations will better mimic the original microenvironment of the tumor and orthotopic tumors will therefore recapitulate the disease better. However, as the subcutaneous transplantations are easier to perform and have a higher success rate compared to orthotopic injections, one mouse per clone was assigned for receiving a subcutaneous injection to ensure the success of the overall experiment. Altogether, each clone was injected to in total of 6 locations (4 orthotopic + 2 subcutaneous) and two independent clones were transplanted per mutation, therefore, the tumorigenic effect of each mutation type was evaluated in in total of 12 locations/6 mice. This set-up guaranteed the success of the experiment and provided statistically significant data on the effect of the mutation type and tissue of origin to the tumor development.
Organoid sizes were calculated based on the measurements taken from 12 organoids per line.
In drug screening assays the drug exposure was performed in quadruplicates (n=4) for each concentration over 2 independent experiments.
One OSE organoid line was excluded from analysis since there was evident contamination.
Organoid establishment: at least 4 independent organoid lines were successfully established per origin. qPCR data: The organoid differenatiation assay was confirmed over 3 independent experiments. Growth assay: the diameter of 12 organoids/clone were measured. Assay was replicated twice. Karyotyping: at least 15 spreads were counted per line and in many cases more. yH2A.X quantification: at least 10 organoids were quantified per line for positive staining. Two independent experiments were performed. KI67-and cleaved Caspase-3 quantification: marker-positive cells per 20x magnification field were quantified (5 fields/tumor, 2 tumors/origin) Drug screens: drug screening results were confirmed in quadruplicates (n=4) over two independent experiments. FACS assay: two independent experiment were performed to confirm the apoptotic characteristics of the lines.
Reporting for specific materials, systems and methods For RNA-seq analysis, bulk RNA was extracted from 2-3 drops of BME with organoids (per line) in order to provide sufficient material for library preparation.
Blinded evaluation of tumors was performed by expert pathologist. RNA-seq initial analysis was carried out as a blinded experiment to visualize the overall sample characteristics without allocating data to any distinct groups. During the further analysis investigators were not blinded as the interest was to find out clear differences between distinct groups.
No blinding was performed for other experiments as standard protocols were equally applied on all samples.