Ovarian Cancer Ascites Inhibits Transcriptional Activation of NK Cells Partly through CA125.

Malignant ascites is a common clinical problem in ovarian cancer. NK cells are present in the ascites, but their antitumor activity is inhibited. The underlying mechanisms of the inhibition have yet to be fully elucidated. Using an Fcγ receptor-mediated NK cell activation assay, we show that ascites from ovarian cancer patients potently inhibits NK cell activation. Part of the inhibitory activity is mediated by CA125, a mucin 16 fragment shed from ovarian cancer tumors. Moreover, transcriptional analyses by RNA sequencing reveal upregulation of genes involved in multiple metabolic pathways but downregulation of genes involved in cytotoxicity and signaling pathways in NK cells purified from ovarian cancer patient ascites. Transcription of genes involved in cytotoxicity pathways are also downregulated in NK cells from healthy donors after in vitro treatment with ascites or with a CA125-enriched protein fraction. These results show that ascites and CA125 inhibit antitumor activity of NK cells at transcriptional levels by suppressing expression of genes involved in NK cell activation and cytotoxicity. Our findings shed light on the molecular mechanisms by which ascites inhibits the activity of NK cells and suggest possible approaches to reactivate NK cells for ovarian cancer immunotherapy.

High-Throughput Quantitative Glycomics Enabled by 12-plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags.

Glycans, which are widely distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as immune response and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probes greatly facilitates routine analysis of glycans. However, the requirement of high-throughput analysis still calls for advanced tools to be developed. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the subtle mass differences among different isotopologues and expanded the multiplexing capacity to 12 channels, a 3-fold throughput improvement for the original SUGAR tag design and achieved high-throughput N-glycan analysis in a single LC-MS/MS injection. We then applied 12-plex SUGAR tags to profile the N-glycans in four subtypes of human Immunoglobulin G (IgG) and to investigate the N-glycan changes in the endometrial cancer cells (ECC1) treated with Atovaquone, a quinone antimicrobial medication, and a dihydroorotate dehydrogenase (DHODH) inhibitor. Data are available via ProteomeXchange with the identifier PXD038501.

6-Plex mdSUGAR Isobaric-Labeling Guide Fingerprint Embedding for Glycomics Analysis.

Glycans are vital biomolecules with diverse functions in biological processes. Mass spectrometry (MS) has become the most widely employed technology for glycomics studies. However, in the traditional data-dependent acquisition mode, only a subset of the abundant ions during MS1 scans are isolated and fragmented in subsequent MS2 events, which reduces reproducibility and prevents the measurement of low-abundance glycan species. Here, we reported a new method termed 6-plex mdSUGAR isobaric-labeling guide fingerprint embedding (MAGNI), to achieve multiplexed, quantitative, and targeted glycan analysis. The glycan peak signature was embedded by a triplicate-labeling strategy with a 6-plex mdSUGAR tag, and using ultrahigh-resolution mass spectrometers, the low-abundance glycans that carry the mass fingerprints can be recognized on the MS1 spectra through an in-house developed software tool, MAGNIFinder. These embedded unique fingerprints can guide the selection and fragmentation of targeted precursor ions and further provide rich information on glycan structures. Quantitative analysis of two standard glycoproteins demonstrated the accuracy and precision of MAGNI. Using this approach, we identified 304 N-glycans in two ovarian cancer cell lines. Among them, 65 unique N-glycans were found differentially expressed, which indicates a distinct glycosylation pattern for each cell line. Remarkably, 31 N-glycans can be quantified in only 1 × 103 cells, demonstrating the high sensitivity of our method. Taken together, our MAGNI method offers a useful tool for low-abundance N-glycan characterization and is capable of determining small quantitative differences in N-glycan profiling. Therefore, it will be beneficial to the field of glycobiology and will expand our understanding of glycosylation.

Oxidative phosphorylation inhibitors inhibit proliferation of endometriosis cells.

In brief: Developing novel therapies to cure and manage endometriosis is a major unmet need that will benefit over 180 million women worldwide. Results from the current study suggest that inhibitors of oxidative phosphorylation may serve as novel agents for the treatment of endometriosis. Abstract: Current therapeutic strategies for endometriosis focus on symptom management and are not curative. Here, we provide evidence supporting the inhibition of oxidative phosphorylation (OXPHOS) as a novel treatment strategy for endometriosis. Additionally, we report an organotypic organ-on-a-chip luminal model for endometriosis. The OXPHOS inhibitors, curcumin, plumbagin, and the FDA-approved anti-malarial agent, atovaquone, were tested against the endometriosis cell line, 12Z, in conventional as well as the new organotypic model. The results suggest that all three compounds inhibit proliferation and cause cell death of the endometriotic cells by inhibiting OXPHOS and causing an increase in intracellular oxygen radicals. The oxidative stress mediated by curcumin, plumbagin, and atovaquone causes DNA double-strand breaks as indicated by the elevation of phospho-γH2Ax. Mitochondrial energetics shows a significant decrease in oxygen consumption in 12Z cells. These experiments also highlight differences in the mechanism of action as curcumin and plumbagin inhibit complex I whereas atovaquone blocks complexes I, II, and III. Real-time assessment of cells in the lumen model showed inhibition of migration in response to the test compounds. Additionally, using two-photon lifetime imaging, we demonstrate that the 12Z cells in the lumen show decreased redox ratio (NAD(P)H/FAD) and lower fluorescence lifetime of NAD(P)H in the treated cells confirming major metabolic changes in response to inhibition of mitochondrial electron transport. The robust chemotoxic responses observed with atovaquone suggest that this anti-malarial agent may be repurposed for the effective treatment of endometriosis.

Affinity-free enrichment and mass spectrometry analysis of the ovarian cancer biomarker CA125 (MUC16) from patient-derived ascites.

Developing a mass spectrometry-based assay for the ovarian cancer biomarker CA125 (MUC16) is a desirable goal, because it may enable detection of molecular regions that are not recognized by antibodies and are therefore analytically silent in the current immunoassay. Additionally, the ability to characterize the CA125 proteoforms expressed by individuals may offer clinical insight. Enrichment of CA125 from malignant ascites may provide a high-quality source of this important ovarian cancer biomarker, but a reliable strategy for such enrichment is currently lacking. Beginning with crude ascites isolated from three individual patients with high grade serous ovarian cancer, we enriched for MUC16 using filtration, ion exchange, and size exclusion chromatography and then performed bottom-up proteomics on the isolated proteins. This approach of enrichment and analysis reveals that the peptides detected via mass spectrometry map to the SEA domain and C-loop regions within the tandem repeat domains of CA125 and that peptide abundance correlates with clinical CA125 counts.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Mass Spectrometry Imaging of N-Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using a Novel Subatmospheric Pressure Ionization Source.

N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.

MUC16 suppresses human and murine innate immune responses.

OBJECTIVE: MUC16, the mucin that contains the CA125 epitopes, suppresses the cytolytic responses of human NK cells and inhibits the efficacy of therapeutic antibodies. Here, we provide further evidence of the regulatory role of MUC16 on human and murine NK cells and macrophages. METHODS: Target cell cytolysis and doublet formation assays were performed to assess effects of MUC16 on human NK cells. The effect of MUC16 on ovarian tumor growth was determined in a mouse model by monitoring survival and ascites formation. Innate immune cells from spleens and peritoneal cavities of mice were isolated and stimulated in vitro with anti-CD40 antibody, lipopolysaccharide and IFN-γ and their ability to cytolyse MUC16 expressing and non-expressing cells was determined. RESULTS: We confirm that MUC16 inhibits cytolysis by human NK cells as well as the formation of NK-tumor conjugates. Mice implanted with MUC16-knockdown OVCAR-3 show >2-fold increase in survival compared to controls. Murine NK cells and macrophages are more efficient at lysing MUC16-knockdown cells. In vitro cytotoxicity assays with NK cells and macrophages isolated from mice stimulated with anti-CD40 antibody showed 2-3-fold increased activity against the MUC16-knockdown cells as compared to matching target cells expressing this mucin. Finally, knockdown of MUC16 increased the susceptibility of cancer cells to ADCC by murine splenocytes. CONCLUSIONS: For the first time, we demonstrate the immunoregulatory effects of MUC16 on murine NK cells and macrophages. Our study implies that the immunoregulatory role of MUC16 on murine NK cells and macrophages should be considered when examining the biology of MUC16 in mouse models.

Stromal alterations in ovarian cancers via wavelength dependent Second Harmonic Generation microscopy and optical scattering.

BACKGROUND: Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate; however, the specific rates are highly dependent on the stage of the disease upon diagnosis. Current screening and imaging tools are insufficient to detect early lesions and are not capable of differentiating the subtypes of ovarian cancer that may benefit from specific treatments. METHOD: As an alternative to current screening and imaging tools, we utilized wavelength dependent collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe the structural differences in the extracellular matrix (ECM) of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous tumors. RESULTS: The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size scale and encode structural information. The wavelength dependence of these readouts adds additional characterization of the size and distribution of collagen fibrils/fibers relative to the interrogating wavelengths. We found a strong wavelength dependence of these metrics that are related to significant structural differences in the collagen organization and are consistent with the dualistic classification of type I and II serous tumors. Moreover, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. The SHG metrics and optical scattering measurements were used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (>90%) between high-grade serous tumors from the other tissue types. High-grade serous tumors account for ~70% of ovarian cancers, and this delineation has potential clinical applications in terms of supplementing histological analysis, understanding the etiology, as well as development of an in vivo screening tool. CONCLUSIONS: SHG and optical scattering measurements provide sub-resolution information and when combined provide superior diagnostic power over clinical imaging modalities. Additionally the measurements are able to delineate the different subtypes of ovarian cancer and may potentially assist in treatment protocols. Understanding the altered collagen assembly can supplement histological analysis and provide new insight into the etiology. These methods could become an in vivo screening tool for earlier detection which is important since ovarian malignancies can metastasize while undetectable by current clinical imaging resolution.

Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model.

PURPOSE: Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. METHODS: Geranial and neral were synthesized. Commercially-purchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10,000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 and 80 mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. RESULTS: Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 h), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4 to 9.9 µM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. CONCLUSIONS: Geranial is significantly more potent than neral and citral at 80 mg/kg (p < 0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells.

MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress.

Over three decades have passed since the first report on the expression of CA125 by ovarian tumors. Since that time our understanding of ovarian cancer biology has changed significantly to the point that these tumors are now classified based on molecular phenotype and not purely on histological attributes. However, CA125 continues to be, with the recent exception of HE4, the only clinically reliable diagnostic marker for ovarian cancer. Many large-scale clinical trials have been conducted or are underway to determine potential use of serum CA125 levels as a screening modality or to distinguish between benign and malignant pelvic masses. CA125 is a peptide epitope of a 3-5 million Da mucin, MUC16. Here we provide an in-depth review of the literature to highlight the importance of CA125 as a prognostic and diagnostic marker for ovarian cancer. We focus on the increasing body of literature describing the biological role of MUC16 in the progression and metastasis of ovarian tumors. Finally, we consider previous and on-going efforts to develop therapeutic approaches to eradicate ovarian tumors by targeting MUC16. Even though CA125 is a crucial marker for ovarian cancer, the exact structural definition of this antigen continues to be elusive. The importance of MUC16/CA125 in the diagnosis, progression and therapy of ovarian cancer warrants the need for in-depth research on the biochemistry and biology of this mucin. A renewed focus on MUC16 is likely to culminate in novel and more efficient strategies for the detection and treatment of ovarian cancer.

A Perspective Review: Analyzing Collagen Alterations in Ovarian Cancer by High-Resolution Optical Microscopy.

High-grade serous ovarian cancer (HGSOC) is the predominant subtype of ovarian cancer (OC), occurring in more than 80% of patients diagnosed with this malignancy. Histological and genetic analysis have confirmed the secretory epithelial of the fallopian tube (FT) as a major site of origin of HGSOC. Although there have been significant strides in our understanding of this disease, early stage detection and diagnosis are still rare. Current clinical imaging modalities lack the ability to detect early stage pathogenesis in the fallopian tubes and the ovaries. However, there are several microscopic imaging techniques used to analyze the structural modifications in the extracellular matrix (ECM) protein collagen in ex vivo FT and ovarian tissues that potentially can be modified to fit the clinical setting. In this perspective, we evaluate and compare the myriad of optical tools available to visualize these alterations and the invaluable insights these data provide on HGSOC initiation. We also discuss the clinical implications of these findings and how these data may help novel tools for early diagnosis of HGSOC.

A Revised Molecular Model of Ovarian Cancer Biomarker CA125 (MUC16) Enabled by Long-read Sequencing.

The biomarker CA125, a peptide epitope located in several tandem repeats of the mucin MUC16, is the gold standard for monitoring regression and recurrence of high-grade serous ovarian cancer in response to therapy. However, the CA125 epitope along with several structural features of the MUC16 molecule are ill defined. One central aspect still unresolved is the number of tandem repeats in MUC16 and how many of these repeats contain the CA125 epitope. Studies from the early 2000s assembled short DNA reads to estimate that MUC16 contained 63 repeats.Here, we conduct Nanopore long-read sequencing of MUC16 transcripts from three primary ovarian tumors and established cell lines (OVCAR3, OVCAR5, and Kuramochi) for a more exhaustive and accurate estimation and sequencing of the MUC16 tandem repeats.The consensus sequence derived from these six sources was confirmed by proteomics validation and agrees with recent additions to the NCBI database. We propose a model of MUC16 containing 19-not 63-tandem repeats. In addition, we predict the structure of the tandem repeat domain using the deep learning algorithm, AlphaFold.The predicted structure displays an SEA domain and unstructured linker region rich in proline, serine, and threonine residues in all 19 tandem repeats. These studies now pave the way for a detailed characterization of the CA125 epitope. Sequencing and modeling of the MUC16 tandem repeats along with their glycoproteomic characterization, currently underway in our laboratories, will help identify novel epitopes in the MUC16 molecule that improve on the sensitivity and clinical utility of the current CA125 assay. SIGNIFICANCE: Despite its crucial role in clinical management of ovarian cancer, the exact molecular sequence and structure of the biomarker, CA125, are not defined. Here, we combine long-read sequencing, mass spectrometry, and in silico modeling to provide the foundational dataset for a more complete characterization of the CA125 epitope.

Human CD4+ memory phenotype T cells use mitochondrial metabolism to generate sensitive IFN-γ responses.

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however, an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4+ MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity, but MP cells retain less compromised mitochondria compared to effector CD4+ T cells, and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells.

Mononuclear Cells Negatively Regulate Endothelial Ca2+ Signaling.

Endothelial Ca2+ signaling has important roles to play in maintaining pregnancy associated vasodilation in the utero-placenta. Inflammatory cytokines, often elevated in vascular complications of pregnancy, negatively regulate ATP-stimulated endothelial Ca2+ signaling and associated nitric oxide production. However, the role of direct engagement of immune cells on endothelial Ca2+ signaling and therefore endothelial function is unclear. To model immune-endothelial interactions, herein, we evaluate the effects of peripheral blood mononuclear cells (PBMCs) in short-term interaction with human umbilical vein endothelial cells (HUVECs) on agonist-stimulated Ca2+ signaling in HUVECs. We find that mononuclear cells (10:1 and 25:1 mononuclear: HUVEC) cause decreased ATP-stimulated Ca2+ signaling; worsened by activated mononuclear cells possibly due to increased cytokine secretion. Additionally, monocytes, natural killers, and T-cells cause decrease in ATP-stimulated Ca2+ signaling using THP-1 (monocyte), NKL (natural killer cells), and Jurkat (T-cell) cell lines, respectively. PBMCs with Golgi-restricted protein transport prior to interaction with endothelial cells display rescue in Ca2+ signaling, strongly suggesting that secreted proteins from PBMCs mediate changes in HUVEC Ca2+ signaling. We propose that endothelial cells from normal pregnancy interacting with PBMCs may model preeclamptic endothelial-immune interaction and resultant endothelial dysfunction.

Potential Tumor Suppressor Role of Polo-like Kinase 5 in Cancer.

The polo-like kinase (PLK) family of serine/threonine kinases contains five members (PLK1-5). Most PLKs are involved in cell cycle regulation and DNA damage response. However, PLK5 is different as it lacks a functional kinase domain and is not involved in cell cycle control. PLK5 remains the least-studied family member, and its role in oncogenesis remains enigmatic. Here, we identified tissues with high PLK5 expression by leveraging the Protein Atlas and GTEx databases with relevant literature and selected ovarian, lung, testis, endometrium, cervix, and fallopian tube tissues as candidates for further investigation. Subsequently, we performed immunohistochemical staining for PLK5 on multiple tissue microarrays followed by Vectra scanning and quantitative inForm analysis. This revealed consistently downregulated PLK5 expression in these cancers compared to normal tissues. To validate and extend our findings, we performed pan-cancer analysis of PLK5 expression using public RNAseq databases (TCGA and GTEx). We found PLK5 is downregulated in 18 cancer types, including our selected candidates. Interestingly, we also observed PLK5 expression remains consistently low in later stages of cancer, suggesting PLK5 may have a greater role in tumor initiation than cancer progression. Overall, our study demonstrates PLK5 downregulation in multiple cancers, highlighting its role as a tumor suppressor.

Assessing the roles of collagen fiber morphology and matrix stiffness on ovarian cancer cell migration dynamics using multiphoton fabricated orthogonal image-based models.

Ovarian cancer remains the deadliest of the gynecological cancers, where this arises from poor screening and imaging tools that can detect early disease, and also limited understanding of the structural and functional aspects of the tumor microenvironment. To gain insight into the underlying cellular dynamics, we have used multiphoton excited fabrication to create Second Harmonic Generation (SHG) image-based orthogonal models from collagen/GelMA that represent both the collagen matrix morphology and stiffness (∼2-8 kPa) of normal ovarian stroma and high grade serous ovarian cancers (HGSOC). These scaffolds are used to study migration/cytoskeletal dynamics of normal (IOSE) and ovarian cancer (OVCA433) cell lines. We found that the highly aligned fiber morphology of HGSOC promotes aspects of motility (motility coefficient, motility, and focal adhesion expression) through a contact guidance mechanism and that stiffer matrix further promotes these same processes through a mechanosensitive mechanism, where these trends were similar for both normal and cancer cells. However, cell specific differences were found on these orthogonal models relative to those providing only morphology, showing the importance of presenting both morphology and stiffness cues. Moreover, we found increased cadherin expression and decreased cell alignment only for cancer cells on scaffolds of intermediate modulus suggesting different stiffness-dependent mechanotransduction mechanisms are engaged. This overall approach affords decoupling the roles of matrix morphology, stiffness and cell genotype and affords hypothesis testing of the factors giving rise to disease progression and metastasis. Further, more established fabrication techniques cannot simultaneously reproduce both the 3D collagen fiber morphology and stiffness. STATEMENT OF SIGNIFICANCE: Ovarian cancer metastasizes when lesions are small, where cells exfoliate from the surface of the ovary and reattach at distal sites in the peritoneum. The adhesion/migration dynamics are not well understood and there is a need for new 3D in vitro models of the extracellular matrix to study the biology. Here we use multiphoton excited crosslinking to fabricate ECM orthogonal models that represent the collagen morphology and stiffness in human ovarian tissues. These are then used to study ovarian cancer cell migration dynamics and we found that contact guidance and a mechanosensitive response and cell genotype all combine to affect the behavior. These models provide insight into disease etiology and progression not readily possible by other fabrication methods.

Multiparameter Flow Cytometry for Detailed Characterization of Peritoneal Immune Cells from Patients with Ovarian Cancer.

Multiparameter flow cytometry is a convenient and efficient method for thorough phenotyping of cells, and especially immune cells from various tissues. We have successfully used multiparameter flow cytometry to characterize immune cells from patients with ovarian cancer and leveraged dimensionality reduction and machine learning for optimized visualization and analysis. Herein, we describe our optimized and established protocols for the labeling of cells with fluorophore-conjugated antibody panels, followed by details on data acquisition. Finally, we describe methods for analysis of the flow cytometry data using both FlowJo as well as R package, Cytofkit, for multidimensional data visualization.

Atovaquone: An Inhibitor of Oxidative Phosphorylation as Studied in Gynecologic Cancers.

Oxidative phosphorylation is an active metabolic pathway in cancer. Atovaquone is an oral medication that inhibits oxidative phosphorylation and is FDA-approved for the treatment of malaria. We investigated its potential anti-cancer properties by measuring cell proliferation in 2D culture. The clinical formulation of atovaquone, Mepron, was given to mice with ovarian cancers to monitor its effects on tumor and ascites. Patient-derived cancer stem-like cells and spheroids implanted in NSG mice were treated with atovaquone. Atovaquone inhibited the proliferation of cancer cells and ovarian cancer growth in vitro and in vivo. The effect of atovaquone on oxygen radicals was determined using flow and imaging cytometry. The oxygen consumption rate (OCR) in adherent cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption and ATP production were inhibited by atovaquone. Imaging cytometry indicated that the majority of the oxygen radical flux triggered by atovaquone occurred in the mitochondria. Atovaquone decreased the viability of patient-derived cancer stem-like cells and spheroids implanted in NSG mice. NMR metabolomics showed shifts in glycolysis, citric acid cycle, electron transport chain, phosphotransfer, and metabolism following atovaquone treatment. Our studies provide the mechanistic understanding and preclinical data to support the further investigation of atovaquone's potential as a gynecologic cancer therapeutic.

Application of a novel microscopic technique for quantifying CA125 binding to circulating mononuclear cells in longitudinal specimens during treatment for ovarian cancer.

BACKGROUND: Measurement of serum CA125, an antigenic fragment of human mucin 16 (MUC16), is used to monitor the clinical progression of epithelial ovarian cancer (EOC). However, rather than simply a passive marker reflecting tumor burden, MUC16 may have a more active role by binding to immune cells and altering their tumor response. We developed a research tool to measure MUC16-binding to the surfaces of peripheral blood mononuclear cell (PBMC) subtypes and tested its research value using specimens collected serially from a woman being treated for high grade serous EOC. METHODS: Cryopreserved PBMCs were mixed with anti-CA125 antibody-labeled plasmonic gold nanoparticles (PNPs) to detect cell surface MUC16-binding along with fluorescent stains to identify B cells, NK cells, NK-T cells, T cells, and monocytes. From 3D darkfield images, a computer algorithm was applied to enumerate PNP-binding and fluorescence microscopy to identify cell lineage. Average MUC16-binding was determined by fitting a Poisson distribution to PNP-counts across similar cell types. MUC16-binding to cell types was correlated with treatment details, CA125 levels, and complete blood count (CBC) data. RESULTS: Over a 21-month period, monocytes had the highest level of MUC16-binding which was positively correlated with serum CA125 and inversely correlated with circulating monocyte and lymphocyte counts. Fluctuations of PNP-binding to NK cells were associated temporally with types of chemotherapy and surgical events. Levels of MUC16 bound to NK cells were positively correlated with levels of MUC16 bound to T and NK-T cells and inversely correlated with circulating platelets. CONCLUSIONS: Assessment of MUC16-binding among cryopreserved PBMC cell types can be accomplished using darkfield and fluorescence microscopy. Correlations observed between level of binding by cell type with serum CA125, CBC data, and treatment details suggest that the new techniques may offer novel insights into EOC's clinical course.