Water transport regulates nucleus volume, cell density, Young's modulus, and E-cadherin expression in tumor spheroids.

Cell volume is maintained by the balance of water and solutes across the cell membrane and plays an important role in mechanics and biochemical signaling in cells. Here, we assess the relationship between cell volume, mechanical properties, and E-cadherin expression in three-dimensional cultures for ovarian cancer. To determine the effect of water transport in multi-cellular tumors, ovarian cancer spheroids were subjected to hypotonic and hypertonic shock using water and sucrose mixtures, respectively. Increased osmolality resulted in decreased nucleus volume, increased Young's modulus, and increased tumor cell density in ovarian cancer spheroids. Next, we looked at the reversibility of mechanics and morphology after 5 min of osmotic shock and found that spheroids had a robust ability to return to their original state. Finally, we quantified the size of E-cadherin clusters at cell-cell junctions and observed a significant increase in aggregate size following 30 min of hypertonic and hypotonic osmotic shocks. Yet, these effects were not apparent after 5 min of osmotic shock, illustrating a temporal difference between E-cadherin regulation and the immediate mechanical and morphology changes. Still, the osmotically induced E-cadherin aggregates which formed at the 30-minute timepoint was reversible when spheroids were replenished with isotonic medium. Altogether, this work demonstrated an important role of osmolality in transforming mechanical, morphology, and molecular states.

Mechanical Modulation of Ovarian Cancer Tumor Nodules Under Flow.

OBJECTIVE: Perfusion models are valuable tools to mimic complex features of the tumor microenvironment and to study cell behavior. In ovarian cancer, mimicking disease pathology of ascites has been achieved by seeding tumor nodules on a basement membrane and subjecting them to long-term continuous flow. In this scenario it is particularly important to study the role of mechanical stress on cancer progression. Mechanical cues are already known to be important in key cancer processes such as survival, proliferation, and migration. However, probing cell mechanical properties within microfluidic platforms has not been achievable with current technologies since samples are not easily accessible within most microfluidic channels. METHODS: Here, to analyze the mechanical properties of cells within a perfusion chamber, we use Brillouin confocal microscopy, an all-optical technique that requires no contact or perturbation to the sample. RESULTS: Our results indicate that ovarian cancer nodules under long-term continuous flow have a significantly lower longitudinal modulus compared to nodules maintained in a static condition. CONCLUSION: We further dissect the role of distinct mechanical perturbations (e.g., shear flow, osmolality) on tumor nodule properties. SIGNIFICANCE: In summary, the unique combination of a long-term microfluidic culture and noninvasive mechanical analysis technique provides insights on the effects of physical forces in ovarian cancer pathology.

Malignant Ascites in Ovarian Cancer: Cellular, Acellular, and Biophysical Determinants of Molecular Characteristics and Therapy Response.

Ascites refers to the abnormal accumulation of fluid in the peritoneum resulting from an underlying pathology, such as metastatic cancer. Among all cancers, advanced-stage epithelial ovarian cancer is most frequently associated with the production of malignant ascites and is the leading cause of death from gynecologic malignancies. Despite decades of evidence showing that the accumulation of peritoneal fluid portends the poorest outcomes for cancer patients, the role of malignant ascites in promoting metastasis and therapy resistance remains poorly understood. This review summarizes the current understanding of malignant ascites, with a focus on ovarian cancer. The first section provides an overview of heterogeneity in ovarian cancer and the pathophysiology of malignant ascites. Next, analytical methods used to characterize the cellular and acellular components of malignant ascites, as well the role of these components in modulating cell biology, are discussed. The review then provides a perspective on the pressures and forces that tumors are subjected to in the presence of malignant ascites and the impact of physical stress on therapy resistance. Treatment options for malignant ascites, including surgical, pharmacological and photochemical interventions are then discussed to highlight challenges and opportunities at the interface of drug discovery, device development and physical sciences in oncology.

A model for in situ plan of care for a critically unstable pediatric patient following I-131 MIBG infusion.

Recent clinical trials have moved iodine-131 (I-131) metaiodobenzylguanidine (MIBG) therapy into frontline management of high-risk neuroblastoma. With this expansion, it is reasonable to anticipate the need for intensive care level resuscitations. Radiation exposure remains the greatest risk to health care professionals managing these patients. We combined shock simulation scenario data with actual radiation dosimetry data to create a care model allowing for aggressive, prolonged in situ resuscitation of a critically ill pediatric patient after I-131 MIBG administration. This model will maintain a critical care provider's radiation level below 10% of the annual occupational dose limit (5 mSv, 500 mrem) per patient managed.

Flow-induced Shear Stress Confers Resistance to Carboplatin in an Adherent Three-Dimensional Model for Ovarian Cancer: A Role for EGFR-Targeted Photoimmunotherapy Informed by Physical Stress.

A key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.

Mechanical Characterization of 3D Ovarian Cancer Nodules Using Brillouin Confocal Microscopy.

INTRODUCTION: The mechanical interaction between cells and their microenvironment is emerging as an important determinant of cancer progression and sensitivity to treatment, including in ovarian cancer (OvCa). However, current technologies limit mechanical analysis in 3D culture systems. Brillouin Confocal Microscopy is an optical non-contact method to assess the mechanical properties of biological materials. Here, we validate the ability of this technology to assess the mechanical properties of 3D tumor nodules. METHODS: OvCa cells were cultured in 3D using two established methods: (1) overlay cultures on Matrigel; (2) spheroids in ultra-low attachment plates. To alter the mechanical state of these tumors, nodules were immersed in PBS with varying levels of sucrose to induce osmotic stress. Next, nodule mechanical properties were measured by Brillouin microscopy and validated with standard stress-strain tests: Atomic Force Microscopy (AFM) and a parallel plate compression device (Microsquisher). Finally, the nodules were treated with a chemotherapeutic commonly used to manage OvCa, carboplatin, to determine treatment-induced effects on tumor mechanical properties. RESULTS: Brillouin microscopy allows mechanical analysis with limited penetration depth (~ 92 µm for Matrigel method; ~ 54 µm for low attachment method). Brillouin microscopy metrics displayed the same trends as the corresponding "gold-standard" Young's moduli measured with stress-strain methods when the osmolality of the medium was increased. Nodules treated with carboplatin showed a decrease in Brillouin frequency shift. CONCLUSION: This validation study paves the way to evaluate the mechanics of 3D nodules, with micron-scale three-dimensional resolution and without contact, thus extending the experimental possibilities.

Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism.

Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.

Prehospital Analgesic Administration by Parents for Pain Relief in Children.

PURPOSE: Undertreatment of pain by caregivers before presentation to the pediatric emergency department (ED) has been well documented. What has yet to be elucidated are the reasons why caregivers fail to adequately treat pain before arrival in the ED and whether there are differences based on ethnic background or age of the child. The objectives of this study were to determine the barriers to giving pain medication for injuries before ED arrival and to determine if there are any ethnic- or age-related variations to giving pain relief at home. METHODS: This prospective descriptive study was conducted in the ED at a tertiary care, freestanding children's hospital with a current annual census of approximately 80,000. An anonymous prospective questionnaire was given to caregivers of children between 2 and 17 years of age presenting to the ED between August 2013 and September, 2014. The study population was obtained as a convenience sample. All were self-referred with chief complaints of head, ear, or extremity pain. The questionnaire asked about pain medications and doses given at home as well as the reasons parents gave medication or refused to give pain medication before arrival. Charts were then abstracted to obtain demographic information and care received in the ED. RESULTS: A total of 154 (45.6%) of the 338 patients enrolled did not receive pain relief before coming to the ED. There were no differences in pain medication received at home based on ethnicity (P = 0.423) or age (P = 0.580). Parents could choose from a list of multiple reasons as to why pain medications were given and/or free text their own answer. The main reasons given by parents were that the accident did not happen at home (28.6%) and that they did not have time to give pain relief before coming to the ED (13%). Other common answers were "had no pain relievers at home" (12.4%) or "afraid it would be wrong/harmful/did not want to mask symptoms" (9.2%). Seventeen parents responded that their child did not complain of pain. Overall, only 28.1% of participants stated lack of pain medications at home. CONCLUSIONS: In this study, approximately half of all children receive an analgesic for their painful condition before coming to the ED. Continued education regarding pain relief before coming to the ED is needed. Future studies will focus on educating parents to provide analgesia at home.

Epidemiology of Hyperbilirubinemia in a Quaternary Pediatric Emergency Department over a Three-Year Period.

PURPOSE: There is a lack of scholarly reports on pediatric emergency department (PED) exposure to hyperbilirubinemia. We aimed to describe the epidemiology of hyperbilirubinemia in patients presenting to a PED over a three-year period. METHODS: This was a retrospective cohort study, completed at an urban quaternary academic PED. Patients were included if they presented to the PED from 2010 to 2012, were 0 to 18 years in age, and had an elevated serum bilirubin for age. A chart review was completed to determine the incidence of hyperbilirubinemia, etiology, diagnostic work up and prognosis. The data set was stratified into four age ranges. RESULTS: We identified 1,534 visits where a patient was found to have hyperbilirubinemia (0.8% of all visits). In 47.7% of patients hyperbilirubinemia was determined to have arisen from an identifiable pathologic etiology (0.38% of all visits). First-time diagnosis of pathologic hyperbilirubinemia occurred in 14% of hyperbilirubinemia visits (0.11% of all visits). There were varying etiologies of hyperbilirubinemia across age groups but a male predominance in all (55.0%). 15 patients went on to have a liver transplant and 20 patients died. First-time pathologic hyperbilirubinemia patients had a mortality rate of 0.95% for their initial hospitalization. CONCLUSION: Hyperbilirubinemia was not a common presentation to the PED and a minority of cases were pathologic in etiology. The etiologies of hyperbilirubinemia varied across each of our study age groups. A new discovery of pathologic hyperbilirubinemia and progression to liver transplant or death during the initial presentation was extremely rare.

Noninvasive Imaging: Brillouin Confocal Microscopy.

In the past decades, there has been increased awareness that mechanical properties of tissues and cells are closely associated with disease physiology and pathology. Recognizing this importance, Brillouin spectroscopy instrumentation, already utilized in physics and material science, has been adopted for cell and tissue biomechanics. For biomedical applications, progress of Brillouin spectrometer technology has been crucial, mainly improvement in the acquisition speed and combination with confocal microscopy, to enable measurement of material longitudinal modulus in three dimensions with high spatial resolution. Micron spatial resolution and high sensitivity allow mapping intracellular modulus and distinguishing between nuclear and cytoplasmic mechanical properties as well as detecting changes due to perturbations of individual cellular components. In cancer, environmental mechanical factors and intracellular mechanics are expected to play an integral role in cancer progression and treatment success. Brillouin confocal microscopy is appealing for many studies in cancer mechanobiology involving both primary tumors and metastatic dissemination. Specifically, Brillouin technology is suitable for experimental scenarios where noncontact mechanical measurements are required such as 3D tumor models, interactions with the extracellular matrix (ECM), investigation of nuclear mechanical properties, or analysis of cells within microfluidic chips.

Complementary and Alternative Medicine in Palliative Care: A Comparison of Data From Surveys Among Patients and Professionals.

PURPOSE: Many cancer patients use complementary and alternative medicine (CAM) during or after their therapy. Because little is known about CAM in palliative care, we conducted 2 surveys among patients and professionals in the palliative setting. PARTICIPANTS AND METHODS: Patients of a German Comprehensive Cancer Center were interviewed, and an independent online survey was conducted among members of the German Society for Palliative Care (DGP). RESULTS: In all, 25 patients and 365 professional members of the DGP completed the survey (9.8% of all members); 40% of the patients, 85% of the physicians, and 99% of the nurses claimed to be interested in CAM. The most important source of information for professionals is education, whereas for patients it is radio, TV, and family and friends. Most patients are interested in biological-based methods, yet professionals prefer mind-body-based methods. Patients more often confirm scientific evidence to be important for CAM than professionals. CONCLUSIONS: To improve communication, physicians should be trained in evidence for those CAM methods in which patients are interested.

In vitro analysis and modification of aquaporin pore selectivity.

Aquaporins enable the passage of a diverse set of solutes besides water. Many novel aquaporin permeants, such as antimonite and arsenite, silicon, ammonia, and hydrogen peroxide, have been described very recently. By the same token, the number of available aquaporin sequences has rapidly increased. Yet, sequence analyses and structure models cannot reliably predict permeability properties. Even the contribution to pore selectivity of individual residues in the channel layout is not fully understood. Here, we describe and discuss established in vitro assays for water and solute permeability. Measurements of volume change due to flux along osmotic or chemical gradients yield quantitative biophysical data, whereas phenotypic growth assays can hint at the relevance of aquaporins in the physiological setting of a certain cell. We also summarize data on the modification of pore selectivity of the prototypical water-specific mammalian aquaporin-1. We show that replacing residues in the pore constriction region allows ammonia, urea, glycerol, and even protons to pass the aquaporin pore.