Endometrial carcinoma, the most common gynaecological cancer, develops from endometrial epithelium which is composed of secretory and ciliated cells. Pathologic classification is unreliable and there is a need for prognostic tools. We used single cell sequencing to study organoid model systems derived from normal endometrial endometrium to discover novel markers specific for endometrial ciliated or secretory cells. A marker of secretory cells (MPST) and several markers of ciliated cells (FAM92B, WDR16, and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. We performed single cell sequencing on endometrial and ovarian tumours and found both secretory‐like and ciliated‐like tumour cells. We found that ciliated cell markers (DYDC2, CTH, FOXJ1, and p73) and the secretory cell marker MPST were expressed in endometrial tumours and positively correlated with disease‐specific and overall survival of endometrial cancer patients. These findings suggest that expression of differentiation markers in tumours correlates with less aggressive disease, as would be expected for tumours that retain differentiation capacity, albeit cryptic in the case of ciliated cells. These markers could be used to improve the risk stratification of endometrial cancer patients, thereby improving their management. We further assessed whether consideration of MPST expression could refine the ProMiSE molecular classification system for endometrial tumours. We found that higher expression levels of MPST could be used to refine stratification of three of the four ProMiSE molecular subgroups, and that any level of MPST expression was able to significantly refine risk stratification of the copy number high subgroup which has the worst prognosis. Taken together, this shows that single cell sequencing of putative cells of origin has the potential to uncover novel biomarkers that could be used to guide management of cancers. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Purpose: Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum-and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.Experimental Design: We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT.Results: Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo.Conclusions: Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell–cell adhesion molecule E‐cadherin. Loss of E‐cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single‐cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage‐specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation‐dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The human inner ear compared with that of other mammalian species is very complex. Although the mouse’s cochlea is frequently studied the mouse’s inner ear continues to develop postnatally whilst the human inner ear is fully developed by the third month of gestation which leads one to question the applicability of findings based on research on mice to human regenerative therapies. Here, we report a novel in vitro culture of adult porcine (Sus scrofa) inner ear cells developed from post-mortem labyrinth specimens. Anatomical findings based on maximal transverse and vertical axial diameters and the length of the cochlear duct suggest that the pig’s cochlea is similar to the human cochlea. In vitro cultures of porcine cochlear and vestibular cells showed the persistence of both inner ear hair cell (HC), supporting cell (SC) and stem/progenitor cell characteristics across passages up to 6 based on scanning electron microscopy, fluorescence immunocytochemistry and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings showed that porcine cochlear and vestibular epithelia maintained multipotent stem/progenitor cell populations into adulthood although their regenerative capacities differed across the passages. The development of a viable and reproducible method to culture porcine inner ear cells provides an important investigative tool that can be utilized to study and evaluate the pathophysiological causes and cellular consequences of human inner ear disorders.

Endometrial epithelium gives rise to both endometrial and ovarian cancers (of clear-cell and endometrioid subtypes), the latter arising from ectopic endometrium (endometriosis). Endometrial epithelium comprises mainly secretory cells, with a minor ciliated cell population. Due to their scarcity, little is known about the biology or function of endometrial ciliated cells. To understand the biology of endometrial epithelium, and by extension the cancers that arise from it, organoids derived from normal endometrial tissue were cultured. Notch signaling inhibitors were used to induce ciliated cell differentiation. Through single-cell RNA sequencing, distinct secretory and ciliated cell populations were observed, with the ciliated cell population increasing with Notch signaling inhibition. Many novel markers of ciliated cells were observed, but no highly specific markers of secretory cell differentiation. A marker of secretory cells (MST) and several markers of ciliated cells (FAM92B, WDR16 and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. In endometrial tumors, both MST and FAM92B exhibited diffuse staining and were markers of better prognosis. This suggests that tumors expressing differentiation markers have better prognosis, whether it is a marker of secretory or ciliated cells. Interestingly, a small number of endometrial tumors stained positive for DYDC2; however, these tumors exhibited a variable staining pattern with 25-50% tumor cells staining intensely, and the remaining tumor cells not staining at all. A similar variable staining pattern had been observed previously with CTH, another ciliated cell marker. Endometrial and ovarian tumor tissue microarrays were stained with DYDC2, CTH and two ciliated cell markers, FOXJ1 and p73. For all these markers, a subset of tumors displayed a variable staining pattern and for endometrial cancers, the variable staining was a good prognostic indicator. Single-cell sequencing of endometrial tumors has been able to capture these two populations of tumor cells. In ovarian tumors, only variable CTH staining was a significant prognostic indicator. Normal endometrial secretory cells are able to differentiate into ciliated cells, and the variable staining pattern suggests that a subset of tumors retains this ability, and these are clinically less aggressive. Using single-cell sequencing technology on normal tissues to guide development of prognostic markers and provide insight into the biology of the tumors arising from these tissues may be useful for many other tumor types. Citation Format: Dawn R. Cochrane, Kieran R. Campbell, Kendall Greening, Germain C. Ho, James Hopkins, Minh Bui, Vassilena Sharlandjieva, Daniel Lai, Maya DeGrood, Evan W. Gibbard, Samuel Leung, Angela S. Cheng, Jamie L.P. Lim, Samantha Neilson, David Farnell, Friedrich Kommoss, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. Single-cell RNA sequencing of normal endometrial organoids uncovers novel cell-type markers for prognostication of primary tumor samples [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B09.

There is a need for an animal model that closely parallels human cochlea gestational development. This study aims to document porcine inner ear anatomy, and in vitro porcine derived inner ear cell culture characteristics. Twenty‐four temporal bone were harvested from 12 adult pigs (Sus scrofa). Six were formalin fixed and their maximal diameters were measured. The cochlea duct length was determined by the insertion length of a Nucleus 22 cochlear implant in two bones. Four formalin fixed bones were sectioned for histology. Cochlear and vestibular tissues were harvested from non‐fixed bones, cultured and characterized at different passages (P). Gene and protein expression of multipotent stem/progenitor (Nestin and Sox2), inner ear hair (Myosin VIIa, Prestin) and supporting (Cytokeratin 18 and Vimentin) cell markers were determined. The porcine cochlea was a 3.5 turn spiral. There was a separate vestibular compartment. The cochlear mean maximal diameter and height was 7.99 and 3.77 mm, respectively. Sphere forming cells were identified on phase‐contrast microscopy. The relative mRNA expression levels of KRT18, MYO7A and SLC26A5 were significantly positively correlated in cochlear cultures; and MYO7A and SLC26A5; SOX2 and KRT18; NES and SLC26A5 genes were positively correlated in vestibular cultures (p = .037, Spearman correlation [τ] = .900). Inner ear sensory and stem cell characteristics persist in passaged porcine inner ear cells. Further work is required to establish the usefulness of porcine inner ear cell cultures to the study of human inner ear disorders.

INTRODUCTION: Ovarian cancer is the 5th deadliest cancer found in women and is the deadliest involving the gynecological tract. Most epithelial ovarian cancers have extra-ovarian origins and can be stratified into various histotypes: high and low-grade serous (HGS and LGS), endometrioid (ENOC), clear cell (CCOC), and mucinous – each of which are proposed to have distinct precursor lesions. We present organoids as a useful model to study precursor lesions and the process of tumorigenesis in epithelial ovarian carcinomas. Organoids recapitulate the in vivo growth microenvironment and are useful to study the mechanisms of tumorigenesis from healthy cells. We have previously proposed that ENOC arise from the secretory cell lineage, while CCOC originate from the ciliated cell lineage, and organoids are an ideal model to examine in greater depth the impact of mutation on specific cell populations, such as ciliated cells. METHODS: Surgical fallopian tube and endometrial tissues, removed for non-cancer reasons, were cultured in 2D followed by plating into Matrigel. Matrigel cultures were supplemented with media containing stem/progenitor differentiation factors promoting organoid growth. To study the effect of mutations often found in ovarian cancers on organoid growth and development, gene knockouts were produced using CRISPR lentiviruses on cells prior to Matrigel culture. Lentiviral transductions were optimized for organoid formation and for minimizing invasiveness accrued on cells. CRISPR gRNA constructs were validated by Western Blot and qPCR. Organoids containing knockouts of p53, BRCA1 and BRCA2 were used to model precursor lesions of HGS, whereas ARID1A knockouts and an inducible PIK3CA activating mutations were used to model CCOC. To gain further insight into ciliated cells of the endometrium, organoids were treated with the notch inhibitor-DBZ to drive differentiation of cells towards a ciliated cell lineage. We analyzed organoids by single-cell RNA sequencing (scRNA-seq), immunohistochemistry (IHC), and immunofluorescence staining (IF). Single cells were derived by purifying the organoids from Matrigel followed by a chemical and physical digestion. scRNA-seq was performed utilizing the 10X Genomics Platform and analyzed by in-house bioinformaticians. Bioinformatic analyses included stringent QC to remove low-quality and dead cells, before applying unsupervised learning algorithms like PCA and Gaussian mixture modeling as well as differential expression analysis to understand both how samples relate to each other and cell types discovered within each sample. RESULTS: We successfully recapitulated the histology observed in tissues by growing endometrial and fallopian tube organoids. The notch inhibitor, DBZ forced ciliated cell differentiation, as observed by IHC, IF and scRNA-seq. scRNA-seq clustering of DBZ-treated organoid cultures revealed a possible intermediary state between progenitor and ciliated cells. Initial IHC and IF analyses of CRISPR-mediated organoids reveal successful gene manipulation. CONCLUSIONS: Organoid cultures present as a powerful method for modelling precursor lesions; they can be readily manipulated genetically and with rapid turnaround compared to conventional mouse models. Organoids are also amenable to sequencing at single-cell resolution. The ability to model ovarian cancers with permanent knockouts in human tissue serves as a necessary link between animal models and human therapy. Citation Format: Germain C. Ho, Dawn R. Cochrane, Evan W. Gibbard, Kieran Campbell, Basile Tessier-Cloutier, Kendall Greening, Forouh Kalantari, Genny Trigo-Gonzalez, Yemin Wang, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. MODELS AND ANALYTIC TECHNIQUES OF MULLERIAN TISSUE-DERIVED ORGANOIDS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-030.

<div>AbstractPurpose:<p>Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.</p>Experimental Design:<p>We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed <i>in vitro</i> using cell lines and patient-derived xenograft mouse models representing SCCOHT.</p>Results:<p>Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype <i>in vitro</i>. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth <i>in vivo</i>.</p>Conclusions:<p>Preclinical <i>in vitro</i> and <i>in vivo</i> studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.</p></div>