Rebecca M. Williams

Altered metabolism enables adaptive advantages for cancer cells, driving the need for improved methods for noninvasive long-term monitoring of cellular metabolism. Here, we present a fast live-cell NADH imaging method that provides a real-time measurement of the fractional level of unbound NADH and show that it is a robust indicator of a cell’s metabolic status. The method, two-photon fluorescence polarization ratiometric microscopy (FPRM), is easy and inexpensive to implement and more than an order of magnitude faster than fluorescence lifetime imaging microscopy (FLIM), a common means of assessing bound and unbound NADH levels. We show that FPRM returns instrument-independent ratiometric parameters that correlate with the expected metabolic changes arising from pharmaceutical and environmental perturbations. By correlating FPRM-returned parameters with cell morphology and migration in two- and three-dimensional collagen matrices, we demonstrate the technique’s versatility in typical bioengineered platforms used in cancer metabolism research.

To effectively assess ovarian vascular function and understand its role in ovarian physiology and pathology, non-invasive in vivo approaches are essential for capturing real-time changes without disrupting normal blood flow. Doppler ultrasonography is a well-established, non-invasive tool for assessing ovarian blood flow in larger species, but its application has been limited in mice, which are one of the most widely used animal models for research in ovarian biology, such as ovulation. Ovulation is a tightly regulated process that depends on coordinated follicular maturation stimulated by follicle-stimulating hormone, followed by a preovulatory luteinizing hormone (LH) surge that leads to rupture of the follicle wall and release of oocytes for fertilization. The LH surge also triggers a series of structural and functional changes in the ovarian vasculature (vascular remodeling), such as angiogenesis and constriction of capillaries at the follicular rupture site shortly before ovulation. In addition, a rapid increase in ovarian blood flow following the LH surge has been reported in multiple species but not in mice. This protocol utilizes Doppler ultrasonography to visualize the murine ovarian vasculature and quantify hemodynamic parameters. The protocol presented here reports detailed methods for hormone priming, anesthesia, positioning of the mouse, identification of the ovary and its vasculature using power and color Doppler, and measurement of blood flow velocity and resistance parameters. This method enables real-time, longitudinal assessment of ovarian vascular function in live mice, providing a powerful tool for studying ovarian vascular function in both physiological and pathological contexts.

To effectively assess ovarian vascular function and understand its role in ovarian physiology and pathology, non-invasive in vivo approaches are essential for capturing real-time changes without disrupting normal blood flow. Doppler ultrasonography is a well-established, non-invasive tool for assessing ovarian blood flow in larger species, but its application has been limited in mice, which are one of the most widely used animal models for research in ovarian biology, such as ovulation. Ovulation is a tightly regulated process that depends on coordinated follicular maturation stimulated by follicle-stimulating hormone, followed by a preovulatory luteinizing hormone (LH) surge that leads to rupture of the follicle wall and release of oocytes for fertilization. The LH surge also triggers a series of structural and functional changes in the ovarian vasculature (vascular remodeling), such as angiogenesis and constriction of capillaries at the follicular rupture site shortly before ovulation. In addition, a rapid increase in ovarian blood flow following the LH surge has been reported in multiple species but not in mice. This protocol utilizes Doppler ultrasonography to visualize the murine ovarian vasculature and quantify hemodynamic parameters. The protocol presented here reports detailed methods for hormone priming, anesthesia, positioning of the mouse, identification of the ovary and its vasculature using power and color Doppler, and measurement of blood flow velocity and resistance parameters. This method enables real-time, longitudinal assessment of ovarian vascular function in live mice, providing a powerful tool for studying ovarian vascular function in both physiological and pathological contexts.

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O -glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism.

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting complicated and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardised 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes cause minimal spherical aberration with objective numerical apertures less than 0.65. Furthermore, we describe methods for aligning and assessing the light sheets, discriminating fluorescence from autofluorescence, identifying chromatic artefacts due to differential scattering and removing streak artefacts such that they do not confound downstream 3D object segmentation analyses, with mouse embryo, liver and heart imaging as demonstrated examples.

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting glitchy and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardized 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes should be non-aberrating with objective numerical apertures less than 0.65, and present an inexpensive tool for alignment and calibration of standard lightsheet parameters. Mouse embryo, liver and heart imaging are demonstrated as examples with practical recommendations for acquisition and post-processing.

During breast cancer bone metastasis, tumor cells interact with bone microenvironment components including inorganic minerals. Bone mineralization is a dynamic process and varies spatiotemporally as a function of cancer‐promoting conditions such as age and diet. The functional relationship between skeletal dissemination of tumor cells and bone mineralization, however, is unclear. Standard histological analysis of bone metastasis frequently relies on prior demineralization of bone, while methods that maintain mineral are often harsh and damage fluorophores commonly used to label tumor cells. Here, fluorescent silica nanoparticles (SNPs) are introduced as a robust and versatile labeling strategy to analyze tumor cells within mineralized bone. SNP uptake and labeling efficiency of MDA‐MB‐231 breast cancer cells is characterized with cryo‐scanning electron microscopy and different tissue processing methods. Using a 3D in vitro model of marrow‐containing, mineralized bone as well as an in vivo model of bone metastasis, SNPs are demonstrated to allow visualization of labeled tumor cells in mineralized bone using various imaging modalities including widefield, confocal, and light sheet microscopy. This work suggests that SNPs are valuable tools to analyze tumor cells within mineralized bone using a broad range of bone processing and imaging techniques with the potential to increase the understanding of bone metastasis.

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

Despite its relevance in numerous natural and industrial processes, the solubility of molecular oxygen has never been directly measured in capillary-condensed liquid water. In this article, we measure oxygen solubility in liquid water trapped within nanoporous samples, in metastable equilibrium with a subsaturated vapor. We show that solubility increases two fold at moderate subsaturations (relative humidity ∼0.55). This evolution with relative humidity is in good agreement with a simple thermodynamic prediction using properties of bulk water, previously verified experimentally at positive pressure. Our measurement thus verifies the validity of this macroscopic thermodynamic theory to strong confinement and large negative pressures, where significant nonidealities are expected. This effect has strong implications for important oxygen-dependent chemistries in natural and technological contexts.