Interferon-epsilon is a novel regulator of NK cell responses in the uterus.

The uterus is a unique mucosal site where immune responses are balanced to be permissive of a fetus, yet protective against infections. Regulation of natural killer (NK) cell responses in the uterus during infection is critical, yet no studies have identified uterine-specific factors that control NK cell responses in this immune-privileged site. We show that the constitutive expression of IFNε in the uterus plays a crucial role in promoting the accumulation, activation, and IFNγ production of NK cells in uterine tissue during Chlamydia infection. Uterine epithelial IFNε primes NK cell responses indirectly by increasing IL-15 production by local immune cells and directly by promoting the accumulation of a pre-pro-like NK cell progenitor population and activation of NK cells in the uterus. These findings demonstrate the unique features of this uterine-specific type I IFN and the mechanisms that underpin its major role in orchestrating innate immune cell protection against uterine infection.

Prevalence and clinical significance of co-existing mutations in MED12 and FH in uterine fibroids of Australian women.

Uterine fibroids are exceedingly common benign tumours of the female reproductive system and cause severe symptoms, including acute pain, bleeding, and infertility. Fibroids are frequently associated with genetic alterations affecting mediator complex subunit 12 (MED12), fumarate hydratase (FH), high mobility group AT-hook 2 (HMGA2) and collagen, type IV alpha 5 and alpha 6 (COL4A5-COL4A6). Recently, we reported MED12 exon 2 mutations in 39 out of 65 uterine fibroids (60%) from 14 Australian patients. The aim of this study was to evaluate the status of FH mutations in MED12 mutation-positive and mutation-negative uterine fibroids. FH mutation screening of altogether 65 uterine fibroids and corresponding adjacent normal myometrium (n = 14) was carried out by Sanger sequencing. Three out of 14 patients displayed somatic mutations in FH exon 1 in addition to harbouring MED12 mutation in uterine fibroids. This study is the first to report that the mutations in MED12 and FH co-exist in uterine fibroids of Australian women.

Loss of liver kinase B1 in human seminoma.

Testicular cancer is a common malignancy of young males and is believed to be originated from defective embryonic or adult germ cells. Liver kinase B1 (LKB1) is a serine/threonine kinase and a tumor suppressor gene. LKB1 is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, often inactivated in many human cancer types. In this study, we investigated the involvement of LKB1 in the pathogenesis of testicular germ cell cancer. We performed immunodetection of LKB1 protein in human seminoma samples. A 3D culture model of human seminoma was developed from TCam-2 cells, and two mTOR inhibitors were tested for their efficacy against these cancer cells. Western blot and mTOR protein arrays were used to show that these inhibitors specifically target the mTOR pathway. Examination of LKB1 showed reduced expression in germ cell neoplasia in situ lesions and seminoma compared to adjacent normal-appearing seminiferous tubules where the expression of this protein was present in the majority of germ cell types. We developed a 3D culture model of seminoma using TCam-2 cells, which also showed reduced levels of LKB1 protein. Treatment of TCam-2 cells in 3D with two well-known mTOR inhibitors resulted in reduced proliferation and survival of TCam-2 cells. Overall, our results support that downregulation or loss of LKB1 marks the early stages of the pathogenesis of seminoma, and the suppression of downstream signaling to LKB1 might be an effective therapeutic strategy against this cancer type.

Establishment and characterization of oviductal organoids from farm and companion animals†.

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.

Extracellular matrix in high-grade serous ovarian cancer: Advances in understanding of carcinogenesis and cancer biology.

High-grade serous ovarian cancer (HGSOC) is notoriously known as the "silent killer" of post-menopausal women as it has an insidious progression and is the deadliest gynaecological cancer. Although a dual origin of HGSOC is now widely accepted, there is growing evidence that most cases of HGSOC originate from the fallopian tube epithelium. In this review, we will address the fallopian tube origin and involvement of the extracellular matrix (ECM) in HGSOC development. There is limited research on the role of ECM at the earliest stages of HGSOC carcinogenesis. Here we aim to synthesise current understanding of the contribution of ECM to each stage of HGSOC development and progression, beginning at serous tubal intraepithelial carcinoma (STIC) precursor lesions and proceeding across key events including dissemination of tumourigenic fallopian tube epithelial cells to the ovary, survival of these cells in peritoneal fluid as multicellular aggregates, and colonisation of the ovary. Likewise, as part of the metastatic series of events, serous ovarian cancer cells survive travel in peritoneal fluid, attach to, migrate across the mesothelium and invade into the sub-mesothelial matrix of secondary sites in the peritoneal cavity. Halting cancer at the pre-metastatic stage and finding ways to stop the dissemination of ovarian cancer cells from the primary site is critical for improving patient survival. The development of drug resistance also contributes to poor survival statistics in HGSOC. In this review, we provide an update on the involvement of the ECM in metastasis and drug resistance in HGSOC. Interplay between different cell-types, growth factor gradients as well as evolving ECM composition and organisation, creates microenvironment conditions that promote metastatic progression and drug resistance of ovarian cancer cells. By understanding ECM involvement in the carcinogenesis and chemoresistance of HGSOC, this may prompt ideas for further research for developing new early diagnostic tests and therapeutic strategies for HGSOC with the end goal of improving patient health outcomes.

Mass Spectrometry Proteomic Analysis of Four p53 Patterns in Differentiated Vulvar Intraepithelial Neoplasia.

OBJECTIVE: The histopathologic diagnostic criteria of differentiated vulvar intraepithelial neoplasia (dVIN), the precursor of human papillomavirus-independent squamous cell carcinoma, are basal atypia, a negative or non-block-positive p16, and a supportive p53 immunohistochemistry (IHC). Several different patterns of supportive p53 IHC have been described. This study aims to determine the relationship between p53 IHC patterns and mass spectrometry analysis of cellular proteins in dVIN. METHODS: Four patterns of p53 IHC were studied: overexpression, cytoplasmic, wild type, and intermediate expression between wild type and overexpression. For each pattern, tissue samples of 4 examples were subjected to mass spectrometry. RESULTS: The protein profile within each p53 IHC pattern shared common features. Each of the 4 p53 patterns had a distinguishable protein profile when compared with the other 3 patterns. CONCLUSIONS: The distinguishable protein profiles in different p53 IHC patterns suggest diverse mechanisms of TP53 dysfunction. Subtyping dVIN by p53 IHC is worthy of further study because varied protein expression profiles may translate into different clinical behavior.

Bovine and human endometrium-derived hydrogels support organoid culture from healthy and cancerous tissues.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth-Holm-Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting high-throughput, cell culture-based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.

Proteomic and functional characterization of intra-tumor heterogeneity in human endometrial cancer.

Endometrial cancer is one of the most frequently diagnosed gynecological cancers worldwide, and its prevalence has increased by more than 50% over the last two decades. Despite the understanding of the major signaling pathways driving the growth and metastasis of endometrial cancer, clinical trials targeting these signals have reported poor outcomes. The heterogeneous nature of endometrial cancer is suspected to be one of the key reasons for the failure of targeted therapies. In this study, we perform a sequential window acquisition of all theoretical fragment ion spectra (SWATH)-based comparative proteomic analysis of 63 tumor biopsies collected from 20 patients and define differences in protein signature in multiple regions of the same tumor. We develop organoids from multiple biopsies collected from the same tumor and show that organoids capture heterogeneity in endometrial cancer growth. Overall, using quantitative proteomics and patient-derived organoids, we define the heterogeneous nature of endometrial cancer within a patient's tumor.

Development and characterization of human fetal female reproductive tract organoids to understand Müllerian duct anomalies.

Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.

Protocol for In Vitro Establishment and Long-Term Culture of Mouse Vaginal Organoids.

Healthy vaginal epithelium is essential for normal reproductive functions and protects against infectious diseases. Here, we present a protocol for developing mouse vaginal organoids from single epithelial cells. These organoids recapitulate both functional and structural characteristics of vagina in situ. This model is a powerful tool for investigating how vaginal microbiome or chemicals in contraceptives and personal hygiene products interact with stem cells and alter the epithelial dynamics, which will lead to new insights into the pathogenesis of vaginal diseases. For complete details on the use and execution of this protocol, please refer to Ali et al. (2020).

Schwann Cell Stimulation of Pancreatic Cancer Cells: A Proteomic Analysis.

Schwann cells (SCs), the glial component of peripheral nerves, have been identified as promoters of pancreatic cancer (PC) progression, but the molecular mechanisms are unclear. In the present study, we aimed to identify proteins released by SCs that could stimulate PC growth and invasion. Proteomic analysis of human primary SC secretome was performed using liquid chromatography-tandem mass spectrometry, and a total of 13,796 unique peptides corresponding to 1,470 individual proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were conducted using the Database for Annotation, Visualization, and Integrated Discovery. Metabolic and cell-cell adhesion pathways showed the highest levels of enrichment, a finding in line with the supportive role of SCs in peripheral nerves. We identified seven SC-secreted proteins that were validated by western blot. The involvement of these SC-secreted proteins was further demonstrated by using blocking antibodies. PC cell proliferation and invasion induced by SC-conditioned media were decreased using blocking antibodies against the matrix metalloproteinase-2, cathepsin D, plasminogen activator inhibitor-1, and galectin-1. Blocking antibodies against the proteoglycan biglycan, galectin-3 binding protein, and tissue inhibitor of metalloproteinases-2 decreased only the proliferation but not the invasion of PC cells. Together, this study delineates the secretome of human SCs and identifies proteins that can stimulate PC cell growth and invasion and therefore constitute potential therapeutic targets.

In Vivo Cell Fate Tracing Provides No Evidence for Mesenchymal to Epithelial Transition in Adult Fallopian Tube and Uterus.

The mesenchymal to epithelial transition (MET) is thought to be involved in the maintenance, repair, and carcinogenesis of the fallopian tube (oviduct) and uterine epithelium. However, conclusive evidence for the conversion of mesenchymal cells to epithelial cells in these organs is lacking. Using embryonal cell lineage tracing with reporters driven by mesenchymal cell marker genes of the female reproductive tract (AMHR2, CSPG4, and PDGFRß), we show that these reporters are also expressed by some oviductal and uterine epithelial cells at birth. These mesenchymal reporter-positive epithelial cells are maintained in adult mice across multiple pregnancies, respond to ovarian hormones, and form organoids. However, no labeled epithelial cells are present in any oviductal or uterine epithelia when mesenchymal cell labeling was induced in adult mice. Organoids developed from mice labeled in adulthood were also negative for mesenchymal reporters. Collectively, our work found no definitive evidence of MET in the adult fallopian tube and uterine epithelium.

Axin2+ endometrial stem cells: the source of endometrial regeneration and cancer.

Menstruation is one of the basic but poorly understood life processes in primates during which females shed inner uterine lining every month only to be completely regenerated back within a week. The definitive evidence for the existence and/or identity of stem cells responsible for this process has remained elusive for more than six decades now. Recently, we reported Axin2, a classical Wnt reporter gene, as a marker for endometrial stem cells that also serve as the cells of origin for endometrial cancer.

Cell Lineage Tracing Identifies Hormone-Regulated and Wnt-Responsive Vaginal Epithelial Stem Cells.

The intact vaginal epithelium is essential for women's reproductive health and provides protection against HIV and sexually transmitted infections. How this epithelium maintains itself remains poorly understood. Here, we used single-cell RNA sequencing (RNA-seq) to define the diverse cell populations in the vaginal epithelium. We show that vaginal epithelial cell proliferation is limited to the basal compartment without any obvious label-retaining cells. Furthermore, we developed vaginal organoids and show that the basal cells have increased organoid forming efficiency. Importantly, Axin2 marks a self-renewing subpopulation of basal cells that gives rise to differentiated cells over time. These cells are ovariectomy-resistant stem cells as they proliferate even in the absence of hormones. Upon hormone supplementation, these cells expand and reconstitute the entire vaginal epithelium. Wnt/ß-catenin is essential for the proliferation and differentiation of vaginal stem cells. Together, these data define heterogeneity in vaginal epithelium and identify vaginal epithelial stem cells.

Endometrial Axin2+ Cells Drive Epithelial Homeostasis, Regeneration, and Cancer following Oncogenic Transformation.

The remarkable regenerative capacity of the endometrium (the inner lining of the uterus) is essential for the sustenance of mammalian life. Over the years, the role of stem cells in endometrial functions and their pathologies has been suggested; however, the identity and location of such stem cells remain unclear. Here, we used in vivo lineage tracing to show that endometrial epithelium self-renews during development, growth, and regeneration and identified Axin2, a classical Wnt reporter gene, as a marker of long-lived bipotent epithelial progenitors that reside in endometrial glands. Axin2-expressing cells are responsible for epithelial regeneration in vivo and for endometrial organoid development in vitro. Ablation of Axin2+ cells severely impairs endometrial homeostasis and compromises its regeneration. More important, upon oncogenic transformation, these cells can lead to endometrial cancer. These findings provide valuable insights into the cellular basis of endometrial functions and diseases.

Extracellular matrix-mediated regulation of cancer stem cells and chemoresistance.

The identity of cancer stem cells (CSCs) remains an enigma, with the question outstanding of whether CSCs are fixed entities or plastic cell states in response to microenvironmental cues. Recent evidence highlights the power of the tumor microenvironment to dictate CSC functionality and spatiotemporal regulation that gives rise to tumor heterogeneity. This microenvironmental regulation of CSCs parallels that of normal tissues, whereby resident stem cells reside within specialized microenvironments or 'niches', which provide the cellular and molecular signals that wire every aspect of stem cell behavior and fate. The extracellular matrix (ECM), along with its sequestered growth factors, is a fundamental component of the stem cell niche. Pathological ECM remodeling is an established hallmark of cancer, with the ECM a key mediator of metastasis and drug resistance. In this review, we discuss the controversial identity of CSCs and new understanding of the impact of tumor microenvironment on CSC function and phenotype. We outline parallels between development, wound repair and cancer to discuss how changes in ECM dynamics influence stem cell function during normal physiological processes and pathological states, as well as the transition between the two in the form of precancerous lesions. We then explore examples illustrating the molecular circuits partnering cancer cells with stromal cells and how this communication involving ECM imparts a CSC phenotype and promotes chemoresistance. Understanding the mechanisms underlying CSC functionality and chemoresistance, along with mathematical modeling approaches and advancing technologies for targeting the undruggable proteome, should open opportunities for cancer breakthroughs in the future.

Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids.

Uterine leiomyomas (fibroids) are the most common gynecological tumors, which are enriched in the extracellular matrix (ECM). Fibroids are leading cause of abnormal uterine bleeding and hysterectomy. One of the major questions yet to be answered is the overproduction of specific ECM components in human uterine fibroids, particularly in relation to mutations in the driver gene mediator complex subunit 12 ( MED12). Surgical specimens from 14 patients with uterine leiomyoma having fibroids and corresponding adjacent normal myometrium (ANM) were utilized to analyze genetic and proteomic expression patterns in the tissue samples. MED12 mutations in the fibroids were screened by Sanger sequencing. iTRAQ was used to label the peptides in small-, medium-, and large-sized fibroid samples of annotated MED12 mutation from the same patient. The mixtures of the peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the differential expression proteins. Using isobaric tagged-based quantitative mass spectrometry on 3 selected patients, ECM-related protein tenascin-C (TNC) was observed significantly upregulated (>1.5-fold) with a confidence corresponding to false discovery rate (FDR) <1% in small-, medium-, and large-sized fibroid samples regardless of MED12 mutation status. The TNC was validated on additional patient samples using Western blotting (WB) and immunohistochemistry (IHC) and confirmed significant overexpression of this protein in fibroids compared to matched ANM. Proteomic analyses have identified the increased ECM protein expression, TNC, as a hallmark of uterine fibroids regardless of MED12 mutations. Further functional studies focusing on the upregulated ECM proteins in leiomyogenesis will lead to the identification of novel ECM drug targets for fibroid treatment.

The Emerging Role of the Microenvironment in Endometrial Cancer.

Endometrial cancer (EC) is one of the most frequently diagnosed cancers in women, and despite recent therapeutic advances, in many cases, treatment failure results in cancer recurrence, metastasis, and death. Current research demonstrates that the interactive crosstalk between two discrete cell types (tumor and stroma) promotes tumor growth and investigations have uncovered the dual role of the stromal cells in the normal and cancerous state. In contrast to tumor cells, stromal cells within the tumor microenvironment (TME) are genetically stable. However, tumor cells modify adjacent stromal cells in the TME. The alteration in signaling cascades of TME from anti-tumorigenic to pro-tumorigenic enhances metastatic potential and/or confers therapeutic resistance. Therefore, the TME is a fertile ground for the development of novel therapies. Furthermore, disrupting cancer-promoting signals from the TME or re-educating stromal cells may be an effective strategy to impair metastatic progression. Here, we review the paradoxical role of different non-neoplastic stromal cells during specific stages of EC progression. We also suggest that the inhibition of microenvironment-derived signals may suppress metastatic EC progression and offer novel potential therapeutic interventions.

Oestrogen fuels the growth of endometrial hyperplastic lesions initiated by overactive Wnt/ß-catenin signalling.

Unopposed oestrogen is responsible for approximately 80% of all the endometrial cancers. The relationship between unopposed oestrogen and endometrial cancer was indicated by the increase in the number of endometrial cancer cases due to the widespread use of oestrogen replacement therapy. Approximately 30% of the endometrial cancer patients have mutations in the Wnt signalling pathway. How the unbalanced ratios of ovarian hormones and the mutations in Wnt signalling pathway interact to cause endometrial cancer is currently unclear. To study this, we have developed a uterine epithelial cell-specific inducible cre mouse model and used 3D in vitro culture of human endometrial cancer cell lines. We showed that activating mutations in the Wnt signalling pathway for a prolonged period leads to endometrial hyperplasia but not endometrial cancer. Interestingly, unopposed oestrogen and activating mutations in Wnt signalling together drive the progression of endometrial hyperplasia to endometrial cancer. We have provided evidence that progesterone can be used as a targeted therapy against endometrial cancer cases presented with the activating mutations in Wnt signalling pathway.