Injectable three-dimensional tumor microenvironments to study mechanobiology in ovarian cancer.

Epithelial ovarian cancers are among the most aggressive forms of gynecological malignancies. Despite the advent of poly adenosine diphosphate-ribose polymerase (PARP) and checkpoint inhibitors, improvement to patient survival has been modest. Limited in part by clinical translation, beneficial therapeutic strategies remain elusive in ovarian cancers. Although elevated levels of extracellular proteins, including collagens, proteoglycans, and glycoproteins, have been linked to chemoresistance, they are often missing from the processes of drug- development and screening. Biophysical and biochemical signaling from the extracellular matrix (ECM) determine cellular phenotype and affect both tumor progression and therapeutic response. However, many state-of-the-art tumor models fail to mimic the complexities of the tumor microenvironment (TME) and omit key signaling components. In this article, two interpenetrating network (IPN) hydrogel scaffold platforms, comprising of alginate-collagen or agarose-collagen, have been characterized for use as 3D in vitro models of epithelial ovarian cancer ECM. These highly tunable, injection mold compatible, and inexpensive IPNs replicate the critical governing physical and chemical signaling present within the ovarian TME. Additionally, an effective and cell-friendly live-cell retrieval method has been established to recover cells post-encapsulation. Lastly, functional mechanotransduction in ovarian cancers was demonstrated by increasing scaffold stiffness within the 3D in vitro ECM models. With these features, the agarose-collagen and alginate-collagen hydrogels provide a robust TME for the study of mechanobiology in epithelial cancers. STATEMENT OF SIGNIFICANCE: Ovarian cancer is the most lethal gynecologic cancer afflicting women today. Here we present the development, characterization, and validation of 3D interpenetrating platforms to shift the paradigm in standard in vitro modeling. These models help elucidate the roles of biophysical and biochemical cues in ovarian cancer progression. The agarose-collagen and alginate-collagen interpenetrating network (IPN) hydrogels are simple to fabricate, inexpensive, and can be modified to create custom mechanical stiffnesses and concentrations of bio-adhesive motifs. Given that investigations into the roles of biophysical characteristics in ovarian cancers have provided incongruent results, we believe that the IPN platforms will be critically important to uncovering molecular drivers. We also expect these platforms to be broadly applicable to studies involving mechanobiology in solid tumors.

A Bioreactor for 3D In Vitro Modeling of the Mechanical Stimulation of Osteocytes.

The bone is a mechanosensitive organ that is also a common metastatic site for prostate cancer. However, the mechanism by which the tumor interacts with the bone microenvironment to further promote disease progression remains to be fully understood. This is largely due to a lack of physiological yet user-friendly models that limit our ability to perform in-depth mechanistic studies. Here, we report a tunable bioreactor which facilitates the 3D culture of the osteocyte cell line, MLO-Y4, in a hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold under constant fluidic shear stress and tunable hydrostatic pressure within physiological parameters. Increasing hydrostatic pressure was sufficient to induce a change in the expression of several bone remodeling genes such as Dmp1, Rankl, and Runx2. Furthermore, increased hydrostatic pressure induced the osteocytes to promote the differentiation of the murine macrophage cell line RAW264.7 toward osteoclast-like cells. These results demonstrate that the bioreactor recapitulates the mechanotransduction response of osteocytes to pressure including the measurement of their functional ability in a 3D environment. In conclusion, the bioreactor would be useful for exploring the mechanisms of osteocytes in bone health and disease.

Pharmacokinetics and Safety of Intravenous Ferric Pyrophosphate Citrate: Equivalence to Administration via Dialysate.

Ferric pyrophosphate citrate (FPC) is indicated to maintain hemoglobin in patients with stage 5 hemodialysis-dependent chronic kidney disease on chronic hemodialysis by addition to the dialysate. An intravenous (IV) FPC presentation containing 6.75 mg of iron in 4.5 mL was developed. The objective was to establish the equivalence of iron delivery via dialysate and IV infusion using a pharmacokinetic approach. An open-label, randomized, multiple-period, single-dose, crossover study was conducted in 27 patients with CKD-5HD. Each patient received (1) a basal iron profile over 12 hours, (2) FPC 6.75 mg Fe IV predialyzer, (3) FPC 6.75 mg Fe IV postdialyzer, and (4) FPC 2 µM (110 µg Fe/L of hemodialysate). Serum and plasma iron was analyzed for total Fe and transferrin bound iron (TBI). Equivalence was determined by comparing maximum observed concentration and area under the concentration-time curve from time 0 to the last observation of 110 µg Fe/L of hemodialysate (reference) and test treatments Fe predialyzer and postdialyzer iron profiles. The main outcome measure was the measurement of bioequivalence between the reference and test treatments. Bioequivalence parameters showed that infusion of FPC iron IV, predialyzer and postdialyzer delivered equivalent iron as via hemodialysate. The increment in serum total Fe from predialysis to postdialysis was the same as observed in the long-term clinical studies of FPC. FPC IV was well tolerated. IV infusion of 6.75 mg iron as FPC during 3 hours of HD delivers an equivalent amount of iron as when Triferic is delivered via hemodialysate. The IV presentation of FPC extends the ability to provide FPC iron to all patients receiving hemodialysis or hemodiafiltration.

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.

Personalized models of heterogeneous 3D epithelial tumor microenvironments: Ovarian cancer as a model.

Intractable human diseases such as cancers, are context dependent, unique to both the individual patient and to the specific tumor microenvironment. However, conventional cancer treatments are often nonspecific, targeting global similarities rather than unique drivers. This limits treatment efficacy across heterogeneous patient populations and even at different tumor locations within the same patient. Ultimately, this poor efficacy can lead to adverse clinical outcomes and the development of treatment-resistant relapse. To prevent this and improve outcomes, it is necessary to be selective when choosing a patient's optimal adjuvant treatment. In this review, we posit the use of personalized, tumor-specific models (TSM) as tools to achieve this remarkable feat. First, using ovarian cancer as a model disease, we outline the heterogeneity and complexity of both the cellular and extracellular components in the tumor microenvironment. Then we examine the advantages and disadvantages of contemporary cancer models and the rationale for personalized TSM. We discuss how to generate precision 3D models through careful and detailed analysis of patient biopsies. Finally, we provide clinically relevant applications of these versatile personalized cancer models to highlight their potential impact. These models are ideal for a myriad of fundamental cancer biology and translational studies. Importantly, these approaches can be extended to other carcinomas, facilitating the discovery of new therapeutics that more effectively target the unique aspects of each individual patient's TME. STATEMENT OF SIGNIFICANCE: In this article, we have presented the case for the application of biomaterials in developing personalized models of complex diseases such as cancers. TSM could bring about breakthroughs in the promise of precision medicine. The critical components of the diverse tumor microenvironments, that lead to treatment failures, include cellular- and extracellular matrix- heterogeneity, and biophysical signals to the cells. Therefore, we have described these dynamic components of the tumor microenvironments, and have highlighted how contemporary biomaterials can be utilized to create personalized in vitro models of cancers. We have also described the application of the TSM to predict the dynamic patterns of disease progression, and predict effective therapies that can produce durable responses, limit relapses, and treat any minimal residual disease.

Compressive Stimulation Enhances Ovarian Cancer Proliferation, Invasion, Chemoresistance, and Mechanotransduction via CDC42 in a 3D Bioreactor.

This report investigates the role of compressive stress on ovarian cancer in a 3D custom built bioreactor. Cells within the ovarian tumor microenvironment experience a range of compressive stimuli that contribute to mechanotransduction. As the ovarian tumor expands, cells are exposed to chronic load from hydrostatic pressure, displacement of surrounding cells, and growth induced stress. External dynamic stimuli have been correlated with an increase in metastasis, cancer stem cell marker expression, chemoresistance, and proliferation in a variety of cancers. However, how these compressive stimuli contribute to ovarian cancer progression is not fully understood. In this report, high grade serous ovarian cancer cell lines were encapsulated within an ECM mimicking hydrogel comprising of agarose and collagen type I, and stimulated with confined cyclic or static compressive stresses for 24 and 72 h. Compression stimulation resulted in a significant increase in proliferation, invasive morphology, and chemoresistance. Additionally, CDC42 was upregulated in compression stimulated conditions, and was necessary to drive increased proliferation and chemoresistance. Inhibition of CDC42 lead to significant decrease in proliferation, survival, and increased chemosensitivity. In summary, the dynamic in vitro 3D platform developed in this report, is ideal for understanding the influence of compressive stimuli, and can be widely applicable to any epithelial cancers. This work reinforces the critical need to consider compressive stimulation in basic cancer biology and therapeutic developments.

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

The Role of Cancer Stem Cells and Mechanical Forces in Ovarian Cancer Metastasis.

Ovarian cancer is an extremely lethal gynecologic disease; with the high-grade serous subtype predominantly associated with poor survival rates. Lack of early diagnostic biomarkers and prevalence of post-treatment recurrence, present substantial challenges in treating ovarian cancers. These cancers are also characterized by a high degree of heterogeneity and protracted metastasis, further complicating treatment. Within the ovarian tumor microenvironment, cancer stem-like cells and mechanical stimuli are two underappreciated key elements that play a crucial role in facilitating these outcomes. In this review article, we highlight their roles in modulating ovarian cancer metastasis. Specifically, we outline the clinical relevance of cancer stem-like cells, and challenges associated with their identification and characterization and summarize the ways in which they modulate ovarian cancer metastasis. Further, we review the mechanical cues in the ovarian tumor microenvironment, including, tension, shear, compression and matrix stiffness, that influence (cancer stem-like cells and) metastasis in ovarian cancers. Lastly, we outline the challenges associated with probing these important modulators of ovarian cancer metastasis and provide suggestions for incorporating these cues in basic biology and translational research focused on metastasis. We conclude that future studies on ovarian cancer metastasis will benefit from the careful consideration of mechanical stimuli and cancer stem cells, ultimately allowing for the development of more effective therapies.

Fluid shear stress stimulates breast cancer cells to display invasive and chemoresistant phenotypes while upregulating PLAU in a 3D bioreactor.

Breast cancer cells experience a range of shear stresses in the tumor microenvironment (TME). However most current in vitro three-dimensional (3D) models fail to systematically probe the effects of this biophysical stimuli on cancer cell metastasis, proliferation, and chemoresistance. To investigate the roles of shear stress within the mammary and lung pleural effusion TME, a bioreactor capable of applying shear stress to cells within a 3D extracellular matrix was designed and characterized. Breast cancer cells were encapsulated within an interpenetrating network hydrogel and subjected to shear stress of 5.4 dynes cm-2 for 72 hr. Finite element modeling assessed shear stress profiles within the bioreactor. Cells exposed to shear stress had significantly higher cellular area and significantly lower circularity, indicating a motile phenotype. Stimulated cells were more proliferative than static controls and showed higher rates of chemoresistance to the anti-neoplastic drug paclitaxel. Fluid shear stress-induced significant upregulation of the PLAU gene and elevated urokinase activity was confirmed through zymography and activity assay. Overall, these results indicate that pulsatile shear stress promotes breast cancer cell proliferation, invasive potential, chemoresistance, and PLAU signaling.

Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance.

Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. METHOD: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. RESULTS: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. CONCLUSION: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

Integrated cancer tissue engineering models for precision medicine.

Tumors are not merely cancerous cells that undergo mindless proliferation. Rather, they are highly organized and interconnected organ systems. Tumor cells reside in complex microenvironments in which they are subjected to a variety of physical and chemical stimuli that influence cell behavior and ultimately the progression and maintenance of the tumor. As cancer bioengineers, it is our responsibility to create physiologic models that enable accurate understanding of the multi-dimensional structure, organization, and complex relationships in diverse tumor microenvironments. Such models can greatly expedite clinical discovery and translation by closely replicating the physiological conditions while maintaining high tunability and control of extrinsic factors. In this review, we discuss the current models that target key aspects of the tumor microenvironment and their role in cancer progression. In order to address sources of experimental variation and model limitations, we also make recommendations for methods to improve overall physiologic reproducibility, experimental repeatability, and rigor within the field. Improvements can be made through an enhanced emphasis on mathematical modeling, standardized in vitro model characterization, transparent reporting of methodologies, and designing experiments with physiological metrics. Taken together these considerations will enhance the relevance of in vitro tumor models, biological understanding, and accelerate treatment exploration ultimately leading to improved clinical outcomes. Moreover, the development of robust, user-friendly models that integrate important stimuli will allow for the in-depth study of tumors as they undergo progression from non-transformed primary cells to metastatic disease and facilitate translation to a wide variety of biological and clinical studies.

Morphometric and computational assessments to evaluate neuron survival and maturation within compartmentalized microfluidic devices: The influence of design variation on diffusion-driven nutrient transport.

Burgeoning use of segregated microfluidic platforms that parse somas and neurites into discrete compartments is fueling unique examinations of neuronal structure and physiology in a manner impossible to achieve with non-compartmentalized systems. However, even though this line of axon-soma polarizing microfluidic devices stems from the same general design of a Campenot chamber set-up, slight deviations in device geometry appear to induce vastly different nutrient transport profiles that influence neuron survival and maturation. Here we examine the uptake of nerve growth factor (NGF) by a pheochromocytoma PC12 cell line cultured using two Campenot-like device designs, a "Standard" layout, representative of a commercial device, and a custom "Notch" layout, predicted to encourage more efficient nutrient transfer that gives rise to sustained neuron viability and extensive neurite elaboration. Exploiting in vitro culture schemes coupled with computational analyses, we identify the influence of device design geometry on the interplay between neuronal survival and maturation, gauged from morphometric assessments and the spatiotemporal distribution of NGF. Computer simulations of NGF transport within the devices revealed that the microfluidic neuron culture system is highly sensitive to change, where nutrient transport is intricately linked to device geometry and cell plating density, and premature depletion of nutrients is observed if specific design criteria are not met. This study underscores the importance of validating specific device geometries for a particular neuro-based assessment, while showcasing computational modeling as a powerful tool to achieve this goal.

Review: Mechanotransduction in ovarian cancer: Shearing into the unknown.

Ovarian cancer remains a deadly diagnosis with an 85% recurrence rate and a 5-year survival rate of only 46%. The poor outlook of this disease has improved little over the past 50 years owing to the lack of early detection, chemoresistance and the complex tumor microenvironment. Within the peritoneal cavity, the presence of ascites stimulates ovarian tumors with shear stresses. The stiff environment found within the tumor extracellular matrix and the peritoneal membrane are also implicated in the metastatic potential and epithelial to mesenchymal transition (EMT) of ovarian cancer. Though these mechanical cues remain highly relevant to the understanding and treatment of ovarian cancers, our current knowledge of their biological processes and their clinical relevance is deeply lacking. Seminal studies on ovarian cancer mechanotransduction have demonstrated close ties between mechanotransduction and ovarian cancer chemoresistance, EMT, enhanced cancer stem cell populations, and metastasis. This review summarizes our current understanding of ovarian cancer mechanotransduction and the gaps in knowledge that exist. Future investigations on ovarian cancer mechanotransduction will greatly improve clinical outcomes via systematic studies that determine shear stress magnitude and its influence on ovarian cancer progression, metastasis, and treatment.

Prevesical Calcification and Hydronephrosis in a Girl Treated for Vesicoureteral Reflux.

The endoscopic STING procedure using Deflux is a common and minimal invasive treatment for vesicoureteral reflux. Herein we present the case of an 11-year-old girl with loin pain and de novo hydronephrosis and megaureter on the left. Ultrasound and plain abdominal X-ray demonstrated a calcification at the ureterovesical junction. She had been treated with Deflux injections 5 years before. The clinical quiz addresses the differential diagnosis, workup, and pathogenesis of calcifications at the ureterovesical junction following endoscopic reflux therapy.

Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity.

Multicellular tumor spheroids are powerful in vitro models to perform preclinical chemosensitivity assays. We compare different methodologies to generate tumor spheroids in terms of resultant spheroid morphology, cellular arrangement and chemosensitivity. We used two cancer cell lines (MCF7 and OVCAR8) to generate spheroids using i) hanging drop array plates; ii) liquid overlay on ultra-low attachment plates; iii) liquid overlay on ultra-low attachment plates with rotating mixing (nutator plates). Analysis of spheroid morphometry indicated that cellular compaction was increased in spheroids generated on nutator and hanging drop array plates. Collagen staining also indicated higher compaction and remodeling in tumor spheroids on nutator and hanging drop arrays compared to conventional liquid overlay. Consequently, spheroids generated on nutator or hanging drop plates had increased chemoresistance to cisplatin treatment (20-60% viability) compared to spheroids on ultra low attachment plates (10-20% viability). Lastly, we used a mathematical model to demonstrate minimal changes in oxygen and cisplatin diffusion within experimentally generated spheroids. Our results demonstrate that in vitro methods of tumor spheroid generation result in varied cellular arrangement and chemosensitivity.

Introducing the HOPE (Hypospadias Objective Penile Evaluation)-score: a validation study of an objective scoring system for evaluating cosmetic appearance in hypospadias patients.

OBJECTIVE: To determine the reliability and internal validity of the Hypospadias Objective Penile Evaluation (HOPE)-score, a newly developed scoring system assessing the cosmetic outcome in hypospadias. PATIENTS AND METHODS: The HOPE scoring system incorporates all surgically-correctable items: position of meatus, shape of meatus, shape of glans, shape of penile skin and penile axis. Objectivity was established with standardized photographs, anonymously coded patients, independent assessment by a panel, standards for a "normal" penile appearance, reference pictures and assessment of the degree of abnormality. A panel of 13 pediatric urologists completed 2 questionnaires, each consisting of 45 series of photographs, at an interval of at least 1 week. The inter-observer reliability, intra-observer reliability and internal validity were analyzed. RESULTS: The correlation coefficients for the HOPE-score were as follows: intra-observer reliability 0.817, inter-observer reliability 0.790, "non-parametric" internal validity 0.849 and "parametric" internal validity 0.842. These values reflect good reproducibility, sufficient agreement among observers and a valid measurement of differences and similarities in cosmetic appearance. CONCLUSIONS: The HOPE-score is the first scoring system that fulfills the criteria of a valid measurement tool: objectivity, reliability and validity. These favorable properties support its use as an objective outcome measure of the cosmetic result after hypospadias surgery.