Methods in Molecular Biology

DBD-FISH Using Specific Chromosomal Region Probes for the Study of Cervical Carcinoma Progression

Genomic instability is an important biomarker in the progression of cervical carcinoma. DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) is a sensitive method that detects strand breaks, alkali-labile sites, and incomplete DNA excision repair in cells of the cervical epithelium. This technique integrates the microgel immersion of cells from a vaginal lesion scraping and the DNA unwinding treatment with the capacity of FISH integrated into digital image analysis. Cells captured within an agarose matrix are lysed and submerged in an alkaline unwinding solution that generates single-stranded DNA motifs at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with DNA-labeled probes. The quantity of a hybridized probe at a target sequence corresponds to the measure of the single-stranded DNA produced during the unwinding step, which is equivalent to the degree of local DNA breakage. DNA damage does not show uniformly throughout the entire DNA of a cell; rather, it is confined to specific chromosomal sites. In this chapter, an overview of the technique is supplied, focusing on its ability for assessing the association between DNA damage in specific sequences and in the progressive stages of cervical carcinoma.

Multiparameter Single-Cell Characterization of Ovarian Intratumor Heterogeneity

Cancer is a complex disease rooted in heterogeneity, which is the phenomenon of individual cells, tissues, or patients having distinct phenotypic and/or genetic characteristics. Observed divergent disease etiology is likely rooted, at least in part, in tumor heterogeneity and the classification of distinct and important subpopulations of cells within the tumor and its associated microenvironment has remained a technical challenge. Standard next-generation sequencing of bulk tumor tissue provides an overall average genetic profile of the sample, and masks contributions from individual cells and minor populations of cells, particularly in heterogeneous samples. Only with the advent of single-cell analysis and sequencing technologies has it become possible to characterize key contributions of cellular subpopulations in order to more comprehensively characterize disease. This chapter describes a method to generate linked phenotypic and genotypic data at single-cell resolution using a real-time single-cell resolved platform. Specifically, the example method provided here is used to link cellular growth kinetics and expression of a prognostic marker protein, CA-125, in cells derived from ovarian cancer patients with their single-cell genomic profiles, but the method is translatable to other cell types and phenotypes of interest.

Multispectral Staining and Analysis of Extracellular Matrix

Multiplexed immunofluorescent (IF) techniques enable the detection of multiple antigens within the same sample and are therefore useful in situations where samples are rare or small in size. Similar to standard IF, multiplexed IF yields information on both the location and relative amount of detected antigens. While this method has been used primarily to detail cell phenotypes, we have recently adapted it to profile the extracellular matrix (ECM), which provides technical challenges due to autofluorescence and spatial overlap. This chapter details the planning, execution, optimization, and troubleshooting to use multiplexed IF to profile the ECM of human fallopian tube tissue.

Protocol for the Detection of Organoid-Initiating Cell Activity in Patient-Derived Single Fallopian Tube Epithelial Cells

Identification of serous tubal intraepithelial carcinomas (STIC) in the fallopian tubes of women who are carriers of germ line pathogenic variants in tubo-ovarian cancer predisposition genes (i.e., BRCA1 and BRCA2) has led to the hypothesis that many high-grade serous carcinomas (HGSC) arise from the fimbria of the fallopian tube. However, the primitive (stem and progenitor) tubal epithelial cells that give rise to STIC and HGSC have not been defined. Further, as putative HGSC precursors are discovered at salpingectomy, the natural history of such lesions is truncated at diagnosis. Thus, living cultures of human fallopian tubes suitable for experimental studies are needed to define and characterize the cellular origin of HGSCs and thereby advance the discovery of improved methods to assess risk, develop effective early detection tests and identify novel prevention approaches. Accordingly, patient-derived tissue-organoids and isolated epithelial stem cell derived-organoids generated from average and high-risk patients are vital resources to understand the developmental biology of aging fallopian tubes and pathogenesis of HGSCs. With a vision to boost HGSC prevention research, we have established state-of-the-art protocols for the collection, processing, storage, distribution, and management of fallopian tube tissues. Here we describe the protocol for preparing these organoids, with emphasis on the key steps that require meticulous attention to achieve success.

Ex Vivo Ovarian Culture to Model the Initial Metastasis in Ovarian Cancer

Being able to accurately model metastasis is an important tool in cancer research. Several in vitro and ex vivo models have been developed to model metastasis from the ovary to the omentum, the most frequent metastatic site after leaving the ovary. However, the recent discovery that high-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube and then metastasize to the ovary has necessitated the development of assays that can quantify the adhesion of tumor cells to the ovary. Here we describe a protocol for accessing the adhesion of fluorescent cells to mouse ovaries. This assay can be used to investigate the role of ovarian function, hormones, and adhesion molecules in metastasis of cancer cells originating in the fallopian tube to the ovary, an important step in the progression of HGSOC.

Discrimination of Multidrug Resistance in Cancer Cells Achieved Using Single-Cell Analysis

The biophysical signatures of single cells, such as multidrug resistance (MDR), may easily change during their various disease states. Therefore, there is an ever-growing need for advanced methods to study and analyze the response of cancer cells to therapeutic intervention. To determine the cancer cells and responses to various cancer therapies, from a cell mortality perspective, we report a label-free and real-time method to monitor the in situ responses of ovarian cancer cells using a single-cell bioanalyzer (SCB). The SCB instrument was used to detect different ovarian cancer cells, such as NCI/ADR-RES cells, which are multidrug resistant (MDR), and non-MDR OVCAR-8 cells. The discrimination of ovarian cells has been achieved at the single-cell level by measuring drug accumulation quantitatively in real time, in which the accumulation is high in non-MDR single cells without drug efflux but is low in MDR single cells which are not efflux-free. The SCB was constructed as an inverted microscope for optical imaging and fluorescent measurement of a single cell that was retained in a microfluidic chip. The single ovarian cancer cell retained in the chip offered sufficient fluorescent signals for the SCB to measure the accumulation of daunorubicin (DNR) in the single cell in the absence of cyclosporine A (CsA). The same cell allows us to detect the enhanced drug accumulation due to MDR modulation in the presence of CsA, which is the MDR inhibitor. The measurement of drug accumulation in a cell was achieved after it was captured in the chip for one hour, with the correction of background interference. The detection of accumulation enhancement due to MDR modulation by CsA was determined in terms of either the accumulation rate or enhanced concentration of DNR in the single cell (same cell, p < 0.01). It showed that with the effectiveness of efflux blocking by CsA, the intracellular DNR concentration in a single cell increased by threefold against its same cell control. This single-cell bioanalyzer instrument has the ability to discriminate MDR in different ovarian cells due to drug efflux in them by eliminating the interference of background fluorescence and by using the same cell control.

Statistical Analysis of Multiplex Immunofluorescence and Immunohistochemistry Imaging Data

Advances in multiplexed single-cell immunofluorescence (mIF) and multiplex immunohistochemistry (mIHC) imaging technologies have enabled the analysis of cell-to-cell spatial relationships that promise to revolutionize our understanding of tissue-based diseases and autoimmune disorders. Multiplex images are collected as multichannel TIFF files; then denoised, segmented to identify cells and nuclei, normalized across slides with protein markers to correct for batch effects, and phenotyped; and then tissue composition and spatial context at the cellular level are analyzed. This chapter discusses methods and software infrastructure for image processing and statistical analysis of mIF/mIHC data.

Culturing Primary Human Mesothelial Cells

Mesothelial cells line the serosal cavities and associated organs. In order to metastasize to distant organs, ovarian tumor cells must first attach and then clear the mesothelial cells. Therefore, human primary mesothelial cells (HPMCs) are necessary to effectively study ovarian cancer metastases. Here, we describe methods to obtain HPMCs from human omentum and to culture in vitro.

Analysis of PD-L1 Transcriptional Activity by Chromatin Immunoprecipitation

Toll-Like Receptor Polymorphisms and the Risk of Cancer: Meta-analysis Study

A systematic review and meta-analysis is a useful method to summarize the different results from primary data, which can then provide an evidence-based outcome. Meta-analysis generates quantitative data by calculating effect sizes, which include odd ratios, relative risks, proportions, correlation coefficients, and so forth. The study of single-nucleotide polymorphisms (SNPs) and the association with the interested outcome is one discipline that has resulted in inconsistent relations. Therefore, the meta-analysis aimed to summarize the relevant data on SNPs associated with the outcome of interest. Herein, we describe a comprehensive meta-analysis on Toll-like receptor-9 polymorphism and the risk of cervical cancer.

Analysis of PD-L1 Transcriptional Regulation in Ovarian Cancer Cells by Chromatin Immunoprecipitation

The immune checkpoint molecule, programmed death ligand 1 (PD-L1; B7-H1, CD274), induces T cell apoptosis and tolerance, thus inhibiting the antitumor immunity. PD-L1 expression is increased in many types of cancer, including ovarian cancer (OC), and correlates with poor prognosis. However, the mechanisms that regulate the PD-L1 expression in cancer cells are incompletely understood. The transcriptional regulation of PD-L1 expression is orchestrated by several transcription factors, including NFκB. The human PD-L1 promoter contains five NFκB-binding sites. Interferon-γ (IFNγ) stimulation of OC cells induces p65, and particularly K314/315 acetylated p65 recruitment to all five NFκB-binding sites in PD-L1 promoter, resulting in increased PD-L1 expression. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of PD-L1 by measuring recruitment of NFκB p65 and K314/315 acetylated p65 to PD-L1 promoter in human OC cells.

Flow Cytometry Analysis of Surface PD-L1 Expression Induced by IFNγ and Romidepsin in Ovarian Cancer Cells

Expression of programmed death ligand-1 (PD-L1, CD274) on cancer cells is regulated by interferon-γ (IFNγ) signaling as well as by epigenetic mechanisms. By binding to PD-1 on cytotoxic T cells, PD-L1 inhibits T cell-mediated antitumor responses, resulting in immune escape. This chapter describes analysis of the surface PD-L1 expression in ovarian cancer (OC) cells using flow cytometry (FC). Our data demonstrate that the surface PD-L1 expression in OC cells is induced by IFNγ as well as by the class I histone deacetylase (HDAC) inhibition by romidepsin, suggesting that class I HDAC inhibition might provide a useful strategy to modulate the PD-L1 levels on OC cells.

Immunoblotting Analysis of Intracellular PD-L1 Levels in Interferon-γ-Treated Ovarian Cancer Cells Stably Transfected with Bcl3 shRNA

Expression of the immune checkpoint programmed death ligand 1 (PD-L1, CD274) is increased in many types of cancer, including ovarian cancer (OC), but the mechanisms that regulate the PD-L1 expression are not fully understood. In addition to binding to PD-1 on T cells, thus inhibiting T cell-mediated antitumor responses, PD-L1 has also tumor-intrinsic effects that include increased cancer cell survival and proliferation, and that might be in part mediated by the intracellular PD-L1. In this chapter, we describe a protocol for the analysis of the intracellular PD-L1 protein levels in OC cells by immunoblotting. Our results show that interferon-γ (IFNγ) induces the intracellular levels of PD-L1 and the proto-oncogene Bcl3 in OC cells. However, the PD-L1 expression is significantly decreased in OC cells stably transfected with Bcl3 shRNA, demonstrating that the IFNγ-induced PD-L1 expression in OC cells is mediated by Bcl3. These data identify the IFNγ-Bcl3-PD-L1 axis as a novel therapeutic target in OC, and suggest that targeting Bcl3 may provide a novel strategy to regulate the PD-L1 expression, and especially the tumor-intrinsic PD-L1 effects mediated by the intracellular PD-L1 in OC cells.

Analysis of IFNγ-Induced Migration of Ovarian Cancer Cells

IFNγ is a pleiotropic cytokine that has both antitumor functions and pro-tumorigenic effects. Recent studies have shown that IFNγ induces expression of the immune checkpoint PD-L1 in ovarian cancer (OC) cells, resulting in their increased proliferation and tumor growth. Here, we tested the hypothesis that IFNγ induces migration of OC cells. Using the scratch wound healing assay, our results demonstrate that IFNγ promotes OC cell migration, thus adding to the complexities of IFNγ pro-tumorigenic mechanisms. This chapter describes analysis of the IFNγ-induced migration of OC cells by the wound healing assay followed by quantification of the obtained images using ImageJ software.

Probing Metabolic Changes in IFNγ-Treated Ovarian Cancer Cells

Interferon-γ (IFNγ) is a pleiotropic cytokine that signals to many different cell types. IFNγ has both antitumor functions and pro-tumorigenic effects and regulates different aspects of cell physiology, including metabolism. Cancer cells undergo a complex rearrangement of metabolic pathways that allows them to satisfy the needs of increased proliferation, and many cancer cells redirect glucose metabolism from oxidative phosphorylation to aerobic glycolysis. In this chapter, we describe a protocol that utilizes the Agilent Seahorse XFp Analyzer to assess mitochondrial respiration and glycolysis in ovarian cancer cells treated with IFNγ.

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.

Methods for Evaluation of a Snake Venom-Derived Disintegrin in Animal Models of Human Cancer

Integrin targeting has been shown to be an effective approach for anticancer therapy. We engineered a recombinant disintegrin, vicrostatin (VCN), that binds with high affinity and specificity to the Arg-Gly-Asp (RGD) class of integrins, including αvβ3, αvβ5, and α5β1, involved in tumor invasion and metastasis. We used three different delivery modalities to examine anticancer activity of VCN in mouse models of human ovarian cancer, glioma, and prostate cancer. A female mouse model was used to examine the treatment of established ovarian cancer (OC) using VCN delivered intraperitoneally (IP) weekly either in saline or impregnated in a viscoelastic gel. SKOV3

Techniques Associated with Exosome Isolation for Biomarker Development: Liquid Biopsies for Ovarian Cancer Detection

Ovarian cancer is the leading gynecological malignancy worldwide. This is attributed to the fact that the disease is often diagnosed at an advanced stage, where the survival rates drop from approximately 90% (detection at an early stage) to 20%. Furthermore, ovarian cancer is not associated with overt physical symptoms. Thus, there is an urgent need for a highly sensitive and minimally invasive biomarker for the early detection of ovarian cancer. However, this continues to remain an unmet clinical need, as several proposed techniques have shown low sensitivity and specificity, with poor positive and negative predictive values. The quest for an ideal biomarker has bought exosomes to the forefront. Exosomes are small extracellular vesicles of an endocytic origin, which can encapsulate genetic information, in the form of proteins and miRNAs. They are released by multiple cell types and are involved in intercellular communication, through the transfer of their cargo. The process of exosome biogenesis allows for the packaging of molecules from both membranous and cytosolic origins. Therefore, exosomes are representations of the releasing cell, and thus provide an insight into the cellular environment. Furthermore, exosomal encapsulation of molecules such as proteins and miRNAs can prevent degradation, making exosomes an ideal biomarker source. Thus, this chapter provides an overview of ovarian cancer, the potential of exosomes as an early detection biomarker, and the different methods associated with the isolation of different vesicle subpopulations, and exosome enrichment.

Thermal Ablation Treatment for Cervical Precancer (Cervical Intraepithelial Neoplasia Grade 2 or Higher [CIN2+])

Cervical cancer is a leading cause of mortality for women in low- and middle-income countries (LMICs). Invasive disease can be prevented through the treatment of high-grade cervical precancer lesions. Types of treatment for cervical precancer include excisional procedures that surgically remove the affected tissue and ablation treatments which utilize extreme temperatures to destroy precancerous cells. Excision is the first-line treatment in higher income countries, but requires specialized training and equipment that make it unsuitable for low-income settings. The most common treatment globally is cryotherapy, which utilizes cryogenic gas to freeze the area. However, the need for gas presents significant procurement and logistical challenges. The World Health Organization (WHO) has recently endorsed the use of thermal ablation, a method that utilizes heat to destroy precancerous tissue. This review describes three existing thermal ablation devices and protocols for their use, including step-by-step instruction guides to perform a successful treatment with each device and observations specific to each machine.

Evaluation of Metabolic Characteristics of Cancer Cells

Evaluating Nuclear Levels of PD-L1 in Ovarian Cancer Cells by Western Blotting

Flow Cytometry Analysis of IFNγ-Induced PD-L1 Cell Surface Expression in Ovarian Cancer Cells

Analysis of IFNγ-Induced Invasion of Ovarian Cancer Cells Using 3D Spheroids

The Use of APC/C Antagonists to Promote Mitotic Catastrophe in Cancer Cells

Assessing Gelatinase Activity in Normal and Disease Uterine Tissue and Cells Via Gelatin Zymography

Matrix metalloproteinases (MMPs) are critical for the maintenance and remodeling of the extracellular matrix (ECM) under normal physiological conditions such as pregnancy and wound healing. However, an increase of MMPs in uterine diseases, such as adenomyosis, endometrial cancer, endometriosis, and uterine fibroids, has been observed and suspected to contribute to important pathophysiology phenotypes like invasion and migration. Of note, MMP-2 (also referred to as gelatinase A) and MMP-9 (also referred to as gelatinase B) are common gelatinases that have demonstrated increased activity in uterine diseases and in several cancer types, such as breast cancer, to promote cancerous phenotypes like increased invasion and migration. In-gel zymography is a useful technique for the detection of MMP activity via degradation of gelatin in gelatin-based gels. Using zymography, it is possible to assess the activity levels of MMP-2 and MMP-9 via gelatin degradation during the zymography process. Here, we will describe the process of zymography and assessment of MMP activity levels (MMP-2 and MMP-9) for both uterine tissues and cancerous cell lines.

Generation of Polyploid Giant Cancer Cells (PGCCs) from Hey Ovarian Cancer Cells and Analysis of Embryonic Properties

Polyploid giant cancer cells (PGCCs) play a fundamental role in tumor initiation, dormancy, drug resistance, and metastasis, although the detailed biology of PGCCs remains poorly understood. The lack of literature on establishing a reproducible in vitro system for generating PGCCs is the leading technological obstacle to studying the biology of PGCCs. Here we provide a detailed protocol for generating stable PGCCs from Hey cancer cells and studying the PGCCs' embryonic stemness. This protocol includes (1) generating PGCCs of high purity in 2D culture by exposing Hey cells to paclitaxel, monitoring the cell cycle and amitotic budding of daughter cells from PGCCs, and collecting and studying the daughter cells; (2) inducing PGCCs to form spheroids expressing embryonic stemness markers and observing the spheroids' cleavage and blastocyst-like structure; and (3) inducing redifferentiation of PGCCs into different lineages of differentiated cells.

Patient-Derived Xenograft Models for Ovarian Cancer

Patient-derived xenograft (PDX) models play a crucial role for in vivo research. They maintain the original molecular characteristics of the human tumor and provide a more accurate tumor microenvironment, which cannot be replicated by in vitro models. This chapter describes four different transplantation methods, namely, intra-bursal, intrarenal capsule, intraperitoneal, and subcutaneous, to develop PDX models for ovarian cancer research.

Pathologic Classification of Ovarian Cancer

Optimal use of human tissue for research requires an understanding of basic pathologic principles. Given that the physical assessment of tissue must occur as part of standard clinical examination, it cannot be handled directly by investigators unless they are also a part of the care team. The purpose of this chapter is to provide an overview of the clinical analytic process, from initial gross handling to histologic examination by light microscopy and the use of ancillary studies, in order to provide context for samples that are used in research and to highlight specific considerations that are relevant for obtaining appropriate tissue for experimental purposes. Given that they comprise >95% of ovarian malignancies, there is an emphasis on epithelial tumors.

Hypoxic 3D Tumor Model for Evaluating of CAR-T Cell Therapy In Vitro

A 3D Co-Culture Model of Endometrial Carcinoma Cells with Peripheral Blood Immune Cells (PBMCs) to Study the Activity of Immune Checkpoint Inhibitors

Abstract The 3D co-culture model we describe was developed to study the potency of immune checkpoint inhibitors. In the assay, the first step is to provide 3D cell growth of endometrial carcinoma RL95-2 cells on a hydrogel, LifeGel ® , and then to co-culture cancer cells with PBMCs isolated from healthy blood donors. After 5 days of the co-culture, in the presence of anti-PD-1/PD-L1 or anti-VISTA immune checkpoint inhibitors, the cancer cells’ viability is measured using a colorimetric assay and fluorescence readout. The key features of the presented model are (1) a direct 3D co-culture model that involves cancer cells and immune cells represented by PBMCs, (2) the use of LifeGel ® hydrogel, where cancer cells form spheroids on the surface, allowing immune cells and larger molecules, such as monoclonal antibodies, to penetrate the cancerous structures, and (3) the possible reactivation of immune cells by immune checkpoint inhibitors may be observed via killing/destroying effects on cancer structures.

Flow Cytometric Analyses of p53-Mediated Cell Cycle Arrest and Apoptosis in Cancer Cells

Phenotypic analysis of the effects of a gene of interest may be limited because stable expression of some genes leads to adverse consequences in cell survival, such as disturbance of cell cycle progression, senescence, autophagy, and programmed cell death. One of the best examples is tumor suppressor p53. p53 functions as a tumor suppressor by inducing cell cycle arrest and apoptosis in response to genotoxic and environmental insults. The choice and timing of either pathways induced by p53 depend on cellular context, cell types, and the degree of cellular/genomic damage (For review, see (Chen J, Cold Spring Harb Perspect Med 6:a026104, 2016)). The uncertainty makes the studies on the long-term effects of p53 in cells challenging. This chapter describes a method of flow cytometric analysis of ectopic expression of p53 to better quantify cell cycle distribution and apoptosis in cells treated with DNA damaging agents. The method can be easily adapted to other genes of interest to study their contributions to the fate of variety of cell types in response to endogenous or exogenous stresses.

Subcellular Fractionation to Demonstrate Activation of Intrinsic Apoptotic Pathway

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.

Determination of Caspase Activation by Western Blot

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.

Detection of Unfolded Protein Response by Polymerase Chain Reaction

The unfolded protein response is a cellular adaptive mechanism localized in the endoplasmic reticulum. It involves three phases: the detection of increased presence of unfolded proteins as a result of cellular stressors; the execution of an adaptive cascade of events aimed at the enhancement of proper protein folding and degradation of improperly folded proteins; and finally, when stress is not alleviated, the execution of programmed cell death. The main effectors of the UPR are transcription factors involved in the upregulation of either chaperone proteins or proapoptotic proteins. Two of these transcription factors are CHOP and the spliced variant of XBP-1 (XBP1s). In this chapter, we describe a quantitative PCR method to detect the upregulation of CHOP and XBP1s mRNA during Tunicamycin-induced UPR.

Detection of Anoikis Using Cell Viability Dye and Quantitation of Caspase Activity

Anoikis is a type of programmed cell death triggered by the loss of cellular interaction with the extracellular matrix (ECM) and culminates in the activation of caspases. Specific interaction between cellular receptors such as integrins and the ECM is important to maintain cellular homeostasis in normal tissues through multiple cascades. This interaction provides not only physical attachment, but more importantly, vital interaction with the actin cytoskeleton and growth factors. Normal epithelial and endothelial cells require this interaction with ECM to survive. In cancer, the acquisition of anoikis resistance is a hallmark of malignant transformation and is required in the process of metastasis formation. As such, strategies to inhibit and/or counteract anoikis resistance are important in controlling cancer progression. In this chapter, we describe the method for detecting anoikis using cell viability and caspase activity assays.

Visualization of Necroptotic Cell Death through Transmission Electron Microscopy

Transmission electron microscopy (TEM) is an all-in-one tool to visualize the complex systems of any specimen that is 1 nm in size or smaller. The current chapter provides detailed guidelines for imaging morphological changes during programmed cell necrosis using TEM as a single-step methodology. In this protocol, a novel aldehyde dehydrogenase inhibitor is used to induce cell programmed necrosis in ovarian cancer cell lines (A2780 and SKOV3). This process is followed by gradient dehydration with ethanol, chemical fixation, sampled grid preparation, and staining with 0.75% uranyl formate. Following fixation and grid preparation, cells are imaged using TEM. The resulting images reveal morphological changes consistent with necrotic morphology, including swelling of cells and organelles, appearance of vacuoles, and plasma membrane rupture followed by leakage of cellular contents. The current approach allows a single-step methodology for characterization of cell-programmed necrosis in cells based on morphology.

A Triple-Parameter-Based Laboratory-Friendly Fluorescence Imaging to Identify Apoptosis in Live Cells

Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.

Interleukin-8 Induces Proliferation of Ovarian Cancer Cells in 3D Spheroids

Ovarian cancer (OC) is the most common cause of cancer deaths among gynecological malignancies. OC ascites contain multicellular spheroid aggregates, which exhibit increased pro-survival signaling, invasive behavior, and chemotherapeutic resistance. OC cells are characterized by an increased expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which increases their survival and migration, thus contributing to OC metastasis and angiogenesis. While previous studies have shown that IL-8 increases proliferation of OC cells grown in monolayer cultures, the effect of IL-8 on proliferation of OC cells grown in 3D spheroids has not been investigated. The spheroid 3D culture assays have been particularly useful in translational research since they allow cell-to-cell interactions that resemble tumor growth in vivo, while allowing easy cell manipulations and visualization. Here, we used the 3D spheroid culture assay to investigate the effect of IL-8 on OC cell proliferation. Using this assay, our results show that IL-8 significantly increases proliferation of OC cells grown in 3D spheroids.

PET Imaging of Estrogen Receptors Using 18F-Based Radioligands

In vivo molecular imaging of estrogen receptor alpha (ER) can be performed via positron emission tomography (PET) using ER-specific radioligands, such as 16α-[

Clinical Staging of Ovarian Cancer

Ovarian cancer may arise from any of the histologic portions of the ovary including the epithelium, stroma, or germ cells. Of these, high-grade serous carcinoma arising from the epithelium of the ovary is the most common type. The clinical management and prognosis of ovarian cancer depend upon the stage of the cancer. Cancer stage is the extent to which the cancer has spread from its site of origin. For ovarian cancer, staging guidelines are determined by FIGO, the Fédération Internationale de Gynécologie et d'Obstétrique. The stage of ovarian cancer is determined by performing surgery to remove the ovaries and other gynecologic organs as well as lymph nodes and other tissues where the cancer may have spread. The histologic specimens from this surgery provide information from which the stage can be determined. In more advanced cases, this surgery may also include procedures to remove other areas of cancer. The stage of ovarian cancer guides treatment and is also the most important factor in ovarian cancer prognosis. Most epithelial ovarian cancers are diagnosed in advanced stages and are treated with surgery and chemotherapy. Despite aggressive treatment, the survival of patients with advanced stage epithelial ovarian cancer remains low, and more effective diagnostic and therapeutic approaches are needed.

Assessment of Tumor Heterogeneity in High-Grade Serous Ovarian Cancer: Mass Cytometry to Understand the Complex Tumor Biology

Ovarian cancer (OC) is the most deadly gynecological malignancy worldwide. OC patients undergo debulking surgery followed by platinum/taxane-based chemotherapy; however, despite recent development of new therapeutic approaches based on combination of chemotherapy and innovative targeted-therapies, most of them relapse due to chemoresistance. Many studies have been carried out to decipher the high heterogeneity of ovarian cancer cells that drives tumor treatment failure. Here, we describe our experience in the characterization of ovarian cancer cell subsets through a high-resolution technology in multiparametric analysis, such as mass cytometry (MC).

A Three-Dimensional Culture-Based Assay to Detect Early Stages of Vasculogenic Mimicry in Ovarian Cancer Cells

Vasculogenic mimicry is a cellular mechanism in which tumor cells grow and align forming complex three-dimensional (3D) channel-like structures in a hypoxic microenvironment. This phenomenon represents a novel oxygen, nutrient, and blood supply, in a similar way as occurs in classic angiogenesis. Vasculogenic mimicry has been described in numerous clinical tumors including breast, prostate, lung, and ovarian cancers where it is associated with poor prognosis; thus, it is considered as a hallmark of highly aggressive and metastatic tumors. Here, we describe a simple method to model the in vitro formation of three-dimensional cellular networks over Matrigel in SKOV3 ovarian cancer cells representing the early stages of vasculogenic mimicry.

Photodynamic Treatments for Disseminated Cancer Metastases Using Fiber-Optic Technologies

Tumor-targeted and -activatable photosensitizer delivery platforms are creating new opportunities to develop photodynamic therapy (PDT) of metastatic disease. This is possible by confining the activity of the photosensitizing chemical (i.e., the PDT agent) to the tumor in combination with diffuse near-infrared light irradiation for wide-field treatment. This chapter outlines protocols and research tools for preclinical development of light-activated therapies of cancer metastases using advanced-stage ovarian cancer as a model system. We also describe an in vivo molecular imaging approach that uniquely enables tracking intraperitoneal micrometastatic burden and responses to treatment using fluorescence microendoscopy.

A Perfusion Model to Evaluate Response to Photodynamic Therapy in 3D Tumors

Numerous cancer models have been developed to investigate the effects of mechanical stress on the biology of cells. Here we describe a protocol to fabricate a perfusion model to culture 3-dimensional (3D) ovarian cancer nodules under constant flow. The modular design of this model allows for a wide range of treatment regimens and combinations, including PDT and chemotherapy. Finally, methods for a number of readouts are detailed, allowing researchers to investigate a variety of biological and cytotoxic parameters related to mechanical stress and therapeutic modalities.

An Orthotopic Murine Model of Peritoneal Carcinomatosis of Ovarian Origin for Intraoperative PDT

Advanced ovarian cancer is the most serious among gynecological malignancies and is associated with 35% five-year overall survival. Surgery is the first therapeutic indication, and the absence of remaining macroscopic lesions is the most important prognostic factor. However, tumor dissemination over the whole abdominal cavity largely contributes to the difficulty of complete surgical resection. Consequently, any therapeutic approach that may complete surgical resection should improve patient survival. Considering that some sites are not suitable for surgery because of their close location to vital organs, intraoperative photodynamic therapy (ioPDT) appears to be a complementary therapeutic approach to surgery to obtain the lowest residual disease.Relevant in vivo cancer models that closely resemble human ovarian cancer are essential for preclinical research of alternative antitumor therapeutic strategies. Thus, we propose a comprehensive protocol to set up an orthotopic ovarian xenograft in mice leading to peritoneal carcinomatosis that could be harnessed for antitumor therapeutic application and evaluation.

Confocal Imaging of Single-Cell Signaling in Orthotopic Models of Ovarian Cancer

Ovarian cancer (OVCA) is frequently detected at late stages of disease, often with dissemination throughout the peritoneal cavity surface, abdomen, and ascites fluid. Tumor signaling via mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways can promote OVCA progression and depend on local microenvironmental cues. To better study OVCA in situ within native tissue contexts, here we describe confocal microscopy techniques to image mouse models of intraperitoneal disease at a single-cell resolution. As a proof of principle demonstration, examples are highlighted for simultaneously imaging tumor vascularization, infiltrating and often immunosuppressive immune cells (tumor-associated macrophages), and OVCA kinase activity.

Humanized Patient-Derived Xenograft Models of Ovarian Cancer

In vivo modeling of cancer is a critical step in testing novel therapeutic strategies to advance patient care. Here we describe how to develop a humanized patient-derived xenograft (PDX) model of ovarian cancer that uses orthotopically transplanted patient ovarian tumors with autologous transfer of expanded tumor infiltrating T cells (TILs) as a model that can be utilized to test immunomodulating therapeutics in vivo.

Xenograft Models of Ovarian Cancer for Therapy Evaluation

The evaluation of novel treatment regimes in ovarian cancer, ranging from cytotoxic agents and targeted therapy to surgery, demands clinically relevant mouse models to mimic human disease. These more advanced preclinical models provide a tool to obtain robust data on the mechanism of action, cytotoxicity and therapeutic efficacy of newly emerging antitumor therapies.In this chapter, we describe how to generate ovarian cancer xenograft models through injection of human tumor cell lines in immunocompromised mice. Detailed methodological descriptions are provided for both the commonly applied subcutaneous model and the more technically challenging orthotopic tumor model that involves inoculation of cancer cells in the ovarian bursa. We demonstrate how to monitor tumor growth and metastases in orthotopic ovarian models through noninvasive optical imaging and the procedures for treatment strategy, including administration of test compounds and debulking surgery. We comment on the strengths, limitations, and procedural challenges associated with each of the models.

In Vivo and Ex Vivo Analysis of Omental Adhesion in Ovarian Cancer

In vivo and ex vivo analyses of omental adhesion in ovarian cancer (OvCa) are necessary to understand the dynamics of OvCa metastasis. Here we describe methods to analyze OvCa omental adhesion, including in vivo and ex vivo fluorescent imaging, advanced microscopy, and histological techniques. The use of fluorescently tagged OvCa cells allows for omental tumor visualization and quantification in adhesion and tumor studies. Additionally, advanced microscopy modalities allow for visualization and multiplexed analysis of OvCa omental adhesion. Second harmonic generation microscopy permits the visualization and analysis of omental collagen, specifically the tumor-associated collagen signature that forms as the tumor progresses. Scanning electron microscopy is used for the observation of microscopic details between OvCa cells and the omentum, such as tunneling nanotubes or microvilli. Histological methods are used to investigate several intratumoral properties including visualizing tumor structure using hematoxylin and eosin stain; quantifying collagen with Masson's trichrome stain; analyzing collagen structure with a collagen hybridizing peptide; and identifying a number of markers including, but not limited to proliferation, immune cell types, adhesion molecules, and fibroblasts with immunohistochemistry. Both the in vivo and ex vivo imaging modalities and subsequent analysis can provide insight into the interaction of metastasizing OvCa cells and the omentum.

Isolation of Primary Normal and Cancer-Associated Adipocytes from the Omentum

The omentum is the metastatic site for intra-abdominal cancers such as colon, stomach, and ovarian (where it is the primary site for metastasis). Adipocytes are the primary cell type of the omentum, and they aid in cancer cell proliferation, migration, and invasion. Therefore, systematic characterization of adipocyte-cancer cell interactions will help in understanding the metastatic spread of intra-abdominal cancer. Here, a detailed mechanical-enzymatic digestion method describes the isolation of both normal and cancer-associated adipocyte from omental tissues.

Establishment of In Vivo Ovarian Cancer Mouse Models Using Intraperitoneal Tumor Cell Injection

Mouse models-xenograft models, syngeneic models (directly implanted or chemically or virally induced), and genetically engineered mice (including transgenic and knockout methods) are invaluable for preclinical studies of ovarian cancer as they recapitulate the structures and microenvironments of tumors, which in vitro studies are unable to accomplish.This chapter describes the methodology and approaches for generating various murine models currently employed in ovarian cancer research. It covers the implantation of cells from ovarian cancer cell lines into mice by intraperitoneal injection.

The Role of the Tumor Microenvironment in CSC Enrichment and Chemoresistance: 3D Co-culture Methods

Cancer stem-like cells (CSC) are responsible for tumor progression, chemoresistance, recurrence, and poor outcomes in many cancers, making them critical research and therapeutic targets. One of the critical components potentiating CSC chemoresistance is the interactions between CSC and the surrounding cells in the tumor microenvironment. Our lab has developed several 3D co-culture models to study ovarian CSC interactions with stromal or immune cells found in ovarian tumor microenvironments. In this chapter, we use ovarian cancer as a model to describe the methodologies developed in our lab; however, these techniques are applicable to a wide range of cancers. First, we discuss our method for isolating CSC from heterogeneous tumors and for creating 3D self-assembled tumoroids in hanging drop plates, in either monoculture or co-culture with mesenchymal stem cells or monocytes/macrophages. We then discuss methods for analyzing these models with a focus on isolating cell-type-specific changes and mechanism investigation. Specifically, we describe lentiviral transduction and flow cytometry as established and robust methods to identify and separate each cell type for downstream analysis. We then describe methods to examine CSC functionality with transwell migration assays and colorimetric MTS-based proliferation assays. Finally, we demonstrate enzyme-linked immunosorbent assays (ELISA ) and quantitative polymerase chain reaction (qPCR) methods as mechanistic investigation tools to decouple paracrine and juxtacrine interactions. These methods have wide-reaching applications in cancer research from basic biological investigations, to drug discovery, and personalized drug screening for precision medicine.

Processing and Analysis of Ascites

The accumulation of peritoneal fluid, referred to as ascites, is common in ovarian cancer. This fluid is a complex mixture that may include cells as well as a diverse array of cytokines and growth factors. Here we describe a comprehensive method to process ascites to maximize data collection. The cellular fraction and fluid are first separated by centrifugation. The fluid can be frozen for later analysis of soluble factors or for use in in vitro experiments. The cellular fraction can be processed to analyze its composition or stored for future use.

Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors

Mass cytometry aka Cytometry by Time-Of-Flight (CyTOF) is one of several recently developed multiparametric single-cell technologies designed to address cellular heterogeneity within healthy and diseased tissue. Mass cytometry is an adaptation of flow cytometry in which antibodies are labeled with stable heavy metal isotopes and the readout is by time-of-flight mass spectrometry. With minimal spillover between channels, mass cytometry enables readouts of up to 60 parameters per single cell. Critically, mass cytometry can identify minority cell populations that are lost in bulk tissue analysis. Mass cytometry has been used to great effect for the study of immune cells. We have extended its use to examine single cells within disaggregated solid tissues, specifically freshly resected tubo-ovarian high-grade serous tumors. Here we detail our protocols designed to ensure the production of high-quality single-cell datasets. The methodology can be modified to accommodate the study of other solid tissues.

Preparation of Lipid-Conjugated siRNA Oligonucleotides for Enhanced Gene Inhibition in Mammalian Cells

Nucleic acid conjugates are promising drugs for treating gene-related diseases. Conjugating specific units like lipids, cell-penetrating peptides, polymers, antibodies, and aptamers either at the 3'- or 5'-termini of a siRNA duplex molecule has resulted in a plethora of siRNA bioconjugates with improved stabilities in bloodstream and better pharmacokinetic values than unmodified siRNAs. In this sense, lipid-siRNA conjugates have attracted a remarkable interest for their potential value in facilitating cellular uptake. In this chapter, we describe a series of protocols involving the synthesis of siRNA oligonucleotides carrying either neutral or cationic lipids at the 3'- and 5'-termini. The resulting lipid-siRNA conjugates are aimed to be used as exogenous effectors for inhibiting gene expression by RNA interference. A protocol for the formulation of lipid siRNA using sonication in the presence of serum is described yielding interesting transfection properties for cell culture without the use of transfecting agents.

OT-I TCR Transgenic Mice to Study the Role of PTPN22 in Anti-cancer Immunity

Development and Expansion of Patient-Derived Xenografts for Endometrial Cancer

Patient-Derived Xenografts (PDXs) are established by implanting a fragment of a patient tumor into rodents either subcutaneously or orthotopically. PDX models faithfully recapitulate the histologic and molecular profile of the donor patient's cancer and are regarded as authentic preclinical models for drug testing, understanding of tumor biology and biomarker discovery. This Chapter describes the detailed method for establishing robust PDXs for endometrial cancer and provide important notes for users of the protocol to consider during PDXs development.