Joanna E. Burdette

Defining the Ovarian Cancer Precancerous Landscape through Modeling Fallopian Tube Epithelium Reprogramming Driven by Extracellular Vesicles

Abstract Serous tubal intraepithelial carcinomas (lesions) in the human fallopian tube epithelium (hFTE) are theorized to give rise to high-grade serous ovarian cancers. Small extracellular vesicles (sEV) are known to mediate key signaling in both normal and cancerous tissues, but few ex vivo systems exist for studying the impact of sEV on hFTE tissue. In this study, we present a microfluidic tissue culture platform with combined spatial transcriptomic and proteomic readouts that allows us to profile dual responses in tissue exposed to sEV “messages”—capturing both short-term transcriptomic shifts in the tissue and long-term changes in protein cargo of secreted EVs (the “reply”). Using spatial transcriptomics, we show that the short-term 1-day exposure to ovarian cancer–derived sEVs alters expression of 68 transcripts in secretory cells, the progenitor of high-grade serous ovarian cancer, notably upregulating immune-related mRNA, including CXCL family chemokines, VCAM1, and pro-inflammatory mediators (NFKB1, IL1B, and IFNA7/17). Additionally, we observed that the long-term 14-day exposure to sEVs alters the expression of seven transcripts and 25 EV cargo proteins of fallopian tube–derived EVs (“secondary release EVs”) following stimulus from cancer EVs. Together, tissue transcriptomics and tissue-derived EV proteomics indicate that ovarian cancer–derived sEVs rewire target cell signaling to modify the tubal immune landscape. This study provides insights into the early molecular changes associated with the pathogenesis of ovarian cancer in its tissue of origin, providing a platform to study EV–tissue interactions and identify how sEVs drive cell signaling reprogramming in hFTE. Significance: We model the fallopian tube preneoplastic landscape using a microfluidic platform to study EV-induced stress and show that cancer EVs promote immune signaling changes representing the earliest stages of ovarian cancer pathogenesis.

Branched-Chain Amino Acid Catabolism Promotes Ovarian Cancer Cell Proliferation via Phosphorylation of mTOR

Abstract Ovarian cancer is the sixth leading cause of cancer-related mortality among individuals with ovaries, and high-grade serous ovarian cancer (HGSOC) is the most common and lethal subtype. Characterized by a distinct and aggressive metastatic pattern, HGSOC can originate in the fallopian tube with the transformation of fallopian tube epithelial (FTE) cells, which metastasize to the ovary and subsequently to the omentum and peritoneal cavity. The omentum is a privileged metastatic site, and the metabolic exchange underlying omental metastasis could provide enzyme or receptor targets to block spread. In this study, we adapted a mass spectrometry imaging (MSI) protocol to investigate spatial location of three-dimensional cocultures of tumorigenic FTE cells when grown in proximity to murine omental explants as a model of early metastatic colonization. Our analysis revealed several altered metabolites in tumorigenic FTE/omentum cocultures, namely changes in branched-chain amino acids (BCAA), including valine. We quantified the heightened consumption of valine, other BCAAs, and other amino acid–derived metabolites in omental cocultures using LC/MS assays. Our analysis revealed that metabolite concentrations when monitored with MSI from cell culture media in living culture systems have notable considerations for production of signatures by MSI data that induce ionization suppression. Supplementation with valine enhanced proliferation and mTOR signaling in tumorigenic FTE cells, suggesting the potential of BCAAs as a nutrient utilized by tumor cells during omental colonization and a possible target for metastasis. Significance: This study uncovers altered amino acid metabolism, specifically increased BCAA catabolism, at the interface of ovarian cancer cells and omental tissue in a coculture model of HGSOC secondary metastasis. Enhanced BCAA catabolism promotes cancer cell proliferation through mTOR signaling, presenting potential therapeutic value. These findings deepen our understanding of HGSOC pathogenesis and the metastatic tumor microenvironment, offering insights for developing new treatment strategies.

Acetogenins from the Stem of Uvaria rufa and Their Cytotoxic Activity

Four new adjacent bis-tetrahydrofuran acetogenins, bullacin C (

Scaffold-Free Endometrial Organoids Respond to Excess Androgens Associated With Polycystic Ovarian Syndrome

AbstractContextPolycystic ovary syndrome (PCOS) is a prevalent disorder in reproductive aged women associated with a number of endocrine and metabolic complications, including increased risk of endometrial cancer.ObjectiveTo study the effect of the characteristic increased androgen levels in PCOS on the endometrium, a novel scaffold-free multicellular endometrial organoid was established.DesignHuman endometrial organoids were constructed using primary endometrial epithelial and stromal cells from endometrial tissues. Organoids were treated for 14 days with physiologic levels of estradiol and testosterone to mimic a normal follicular phase or PCOS hormone profiles. Organoids were harvested for immunostaining and ribonucleic acid sequencing.SettingAcademic institution.PatientsEndometrial tissues from 10 premenopausal women undergoing hysterectomy for benign pathologies were obtained following written consent.Main Outcome MeasuresOrganoid architecture, cell specific markers, functional markers, proliferation, and gene expression were measured.ResultsA method to generate scaffold-free endometrial organoids containing epithelial and stromal cells was established. These organoids exhibited distinct organization with epithelial cells lining the outer surface and stromal cells in the center of the organoids. Epithelial cells were polarized, organoids expressed cell type specific and functional markers, as well as androgen, estrogen, and progesterone receptors. Treatment with PCOS hormones increased cell proliferation and dysregulated genes in endometrial organoids.ConclusionsA new multicellular, scaffold-free endometrial organoid system was established that resembled physiology of the native endometrium. Excess androgens in PCOS promoted cell proliferation in endometrial organoids, revealing new mechanisms of PCOS-associated with risk of endometrial neoplasia.

Versican secreted by the ovary links ovulation and migration in fallopian tube derived serous cancer

High grade serous ovarian cancers (HGSOC) predominantly arise in the fallopian tube epithelium (FTE) and colonize the ovary first, before further metastasis to the peritoneum. Ovarian cancer risk is directly related to the number of ovulations, suggesting that the ovary may secrete specific factors that act as chemoattractants for fallopian tube derived tumor cells during ovulation. We found that 3D ovarian organ culture produced a secreted factor that enhanced the migration of FTE non-tumorigenic cells as well as cells harboring specific pathway modifications commonly found in high grade serous cancers. Through size fractionation and a small molecule inhibitors screen, the secreted protein was determined to be 50-100kDa in size and acted through the Epidermal Growth Factor Receptor (EGFR). To correlate the candidates with ovulation, the PREDICT organ-on-chip system was optimized to support ovulation in a perfused microfluidic platform. Versican was found in the correct molecular weight range, contained EGF-like domains, and correlated with ovulation in the PREDICT system. Exogenous versican increased migration, invasion, and enhanced adhesion of both murine and human FTE cells to the ovary in an EGFR-dependent manner. The identification of a protein secreted during ovulation that impacts the ability of FTE cells to colonize the ovary provides new insights into the development of strategies for limiting primary ovarian metastasis.

Models for measuring metabolic chemical changes in the metastasis of high grade serous ovarian cancer: fallopian tube, ovary, and omentum

Abstract Ovarian cancer (OC) is the most lethal gynecologic malignancy and high grade serous ovarian cancer (HGSOC) is the most common and deadly subtype, accounting for 70–80% of OC deaths. HGSOC has a distinct pattern of metastasis as many believe it originates in the fallopian tube and then it metastasizes first to the ovary, and later to the adipose-rich omentum. Metabolomics has been heavily utilized to investigate metabolite changes in HGSOC tumors and metastasis. Generally, metabolomics studies have traditionally been applied to biospecimens from patients or animal models; a number of recent studies have combined metabolomics with innovative cell-culture techniques to model the HGSOC metastatic microenvironment for the investigation of cell-to-cell communication. The purpose of this review is to serve as a tool for researchers aiming to model the metastasis of HGSOC for metabolomics analyses. It will provide a comprehensive overview of current knowledge on the origin and pattern of metastasis of HGSOC and discuss the advantages and limitations of different model systems to help investigators choose the best model for their research goals, with a special emphasis on compatibility with different metabolomics modalities. It will also examine what is presently known about the role of small molecules in the origin and metastasis of HGSOC.

Probing the Cytotoxic Signaling Induced by Eupenifeldin in Ovarian Cancer Models

High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Lack of early detection methods, limited therapeutic agents, and low 5-year survival rate reflect the urgent need to develop new therapies. Eupenifeldin, a bistropolone, originally isolated from

Discovery and development of botanical natural products and their analogues as therapeutics for ovarian cancer

This review highlights botanical natural products with activity against ovarian cancer and key drug development considerations for their advancement.

The Cytotoxic Cardiac Glycoside (−)-Cryptanoside A from the Stems ofCryptolepis dubiaand Its Molecular Targets

A cardiac glycoside epoxide, (-)-cryptanoside A (

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

Embellicines C-E: Macrocyclic Alkaloids with a Cyclopenta[b]fluorene Ring System from the Fungus Sarocladium sp.

Macrocyclic alkaloids with a cyclopenta[b]fluorene ring system are a relatively young structural class of fungal metabolites, with the first members reported in 2013. Bioassay-guided fractionation of a

Detection of Ovarian Cancer Using Samples Sourced from the Vaginal Microenvironment

Mass spectrometry (MS) offers high levels of specificity and sensitivity in clinical applications, and we have previously been able to demonstrate that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS is capable of distinguishing two-component cell mixtures at low limits of detection. Ovarian cancer is notoriously difficult to detect due to the lack of diagnostic techniques available to the medical community. By sampling a local microenvironment, such as the vaginal canal and cervix, a MS based method is presented for monitoring disease progression from proximal samples to the diseased tissue. A murine xenograft model of high grade serous ovarian carcinoma (HGSOC) was used for this study, and vaginal lavages were obtained from mice on a weekly basis throughout disease progression and subjected to our MALDI-TOF MS workflow followed by statistical analyses. Proteins in the 4-20 kDa region of the mass spectrum yielded a fingerprint that we could consistently measure over time that correlated with disease progression. These fingerprints were found to be largely stable across all mice, with the protein fingerprint converging toward the end point of the study. MALDI-TOF MS serves as a unique analytical technique for measuring a sampled vaginal microenvironment in a specific and sensitive manner for the detection of HGSOC in a murine model.

Advancements in Microfluidic Systems for the Study of Female Reproductive Biology

AbstractThe female reproductive tract is a highly complex physiological system that consists of the ovaries, fallopian tubes, uterus, cervix, and vagina. An enhanced understanding of the molecular, cellular, and genetic mechanisms of the tract will allow for the development of more effective assisted reproductive technologies, therapeutics, and screening strategies for female specific disorders. Traditional 2-dimensional and 3-dimensional static culture systems may not always reflect the cellular and physical contexts or physicochemical microenvironment necessary to understand the dynamic exchange that is crucial for the functioning of the reproductive system. Microfluidic systems present a unique opportunity to study the female reproductive tract, as these systems recapitulate the multicellular architecture, contacts between different tissues, and microenvironmental cues that largely influence cell structure, function, behavior, and growth. This review discusses examples, challenges, and benefits of using microfluidic systems to model ovaries, fallopian tubes, endometrium, and placenta. Additionally, this review also briefly discusses the use of these systems in studying the effects of endocrine disrupting chemicals and diseases such as ovarian cancer, preeclampsia, and polycystic ovarian syndrome.

The Tumor Immune Profile of Murine Ovarian Cancer Models: An Essential Tool for Ovarian Cancer Immunotherapy Research

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer with an imperative need for new treatments. Immunotherapy has had marked success in some cancer types; however, clinical trials studying the efficacy of immune checkpoint inhibitors for the treatment of EOC benefited less than 15% of patients. Given that EOC develops from multiple tissues in the reproductive system and metastasizes widely throughout the peritoneal cavity, responses to immunotherapy are likely hindered by heterogeneous tumor microenvironments (TME) containing a variety of immune profiles. To fully characterize and compare syngeneic model systems that may reflect this diversity, we determined the immunogenicity of six ovarian tumor models in vivo, the T and myeloid profile of orthotopic tumors and the immune composition and cytokine profile of ascites, by single-cell RNA sequencing, flow cytometry, and IHC. The selected models reflect the different cellular origins of EOC (ovarian and fallopian tube epithelium) and harbor mutations relevant to human disease, including Tp53 mutation, PTEN suppression, and constitutive KRAS activation. ID8-p53−/− and ID8-C3 tumors were most highly infiltrated by T cells, whereas STOSE and MOE-PTEN/KRAS tumors were primarily infiltrated by tumor-associated macrophages and were unique in MHC class I and II expression. MOE-PTEN/KRAS tumors were capable of forming T-cell clusters. This panel of well-defined murine EOC models reflects some of the heterogeneity found in human disease and can serve as a valuable resource for studies that aim to test immunotherapies, explore the mechanisms of immune response to therapy, and guide selection of treatments for patient populations. Significance: This study highlights the main differences in the immunogenicity and immune composition found in six different models of orthotopic ovarian cancer as an essential tool for future preclinical investigations of cancer immunotherapy.

Aulosirazoles B and C from the Cyanobacterium Nostoc sp. UIC 10771: Analogues of an Isothiazolonaphthoquinone Scaffold that Activate Nuclear Transcription Factor FOXO3a in Ovarian Cancer Cells

The known solid-tumor-selective cytotoxin aulosirazole (

PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer

AbstractPHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.

Activity of Didesmethylrocaglamide in High Grade Serous Ovarian Cancer Using Preclinical In Vitro and In Vivo Models

High grade serous ovarian cancer (HGSOC) is the most lethal gynecological cause of death in women and requires new treatments to help tackle chemoresistance. Rocaglamides, a promising class of anticancer natural products, function as protein translation inhibitors and trigger apoptosis in other types of solid tumors. Didesmethylrocaglamide ((±)-DDR), a derivative of rocaglamide with potent antitumor activity, was synthesized, including three additional rocaglamide derivatives, (±)-DDR01, (±)-DDR03, and (±)-DDR04, to evaluate their cytotoxicity in HGSOC. Using in vitro models, it was determined that (±)-DDR induced cytotoxicity in ovarian cancer cell lines as early as 24 h after application and activated caspase-3, indicating pro-apoptotic activity. In addition, (±)-DDR was cytotoxic in the PE04 and MCF7-ADR (OVCAR8-RES) cell lines that are resistant to cisplatin and paclitaxel, respectively. Evaluation of each enantiomer revealed the minus enantiomer to be ∼18-fold more potent compared to the plus enantiomer in the OVCAR8 cell line. (-)-DDR was further evaluated using an OVCAR8 xenograft model in mice, and a reduction in tumor burden was observed. Its effective cytotoxicity in drug-sensitive and -resistant cell models suggests that (±)-DDR and its corresponding minus enantiomer may have potential as a new therapeutic strategy against HGSOC.