ASU Scholarship Showcase

Five immunocompetent C57BL/6-cBrd/cBrd/Cr (albino C57BL/6) mice were injected with GL261-luc2 cells, a cell line sharing characteristics of human glioblastoma multiforme (GBM). The mice were imaged using magnetic resonance (MR) at five separate time points to characterize growth and development of the tumor. After 25 days, the final tumor volumes of the mice varied from 12 mm³ to 62 mm³, even though mice were inoculated from the same tumor cell line under carefully controlled conditions. We generated hypotheses to explore large variances in final tumor size and tested them with our simple reaction-diffusion model in both a 3-dimensional (3D) finite difference method and a 2-dimensional (2D) level set method. The parameters obtained from a best-fit procedure, designed to yield simulated tumors as close as possible to the observed ones, vary by an order of magnitude between the three mice analyzed in detail. These differences may reflect morphological and biological variability in tumor growth, as well as errors in the mathematical model, perhaps from an oversimplification of the tumor dynamics or nonidentifiability of parameters. Our results generate parameters that match other experimental in vitro and in vivo measurements. Additionally, we calculate wave speed, which matches with other rat and human measurements.

Background:
Data assimilation refers to methods for updating the state vector (initial condition) of a complex spatiotemporal model (such as a numerical weather model) by combining new observations with one or more prior forecasts. We consider the potential feasibility of this approach for making short-term (60-day) forecasts of the growth and spread of a malignant brain cancer (glioblastoma multiforme) in individual patient cases, where the observations are synthetic magnetic resonance images of a hypothetical tumor.

Results:
We apply a modern state estimation algorithm (the Local Ensemble Transform Kalman Filter), previously developed for numerical weather prediction, to two different mathematical models of glioblastoma, taking into account likely errors in model parameters and measurement uncertainties in magnetic resonance imaging. The filter can accurately shadow the growth of a representative synthetic tumor for 360 days (six 60-day forecast/update cycles) in the presence of a moderate degree of systematic model error and measurement noise.

Conclusions:
The mathematical methodology described here may prove useful for other modeling efforts in biology and oncology. An accurate forecast system for glioblastoma may prove useful in clinical settings for treatment planning and patient counseling.

Introduction: Fluorescence-guided surgery is one of the rapidly emerging methods of surgical “theranostics.” In this review, we summarize current fluorescence techniques used in neurosurgical practice for brain tumor patients as well as future applications of recent laboratory and translational studies.

Methods: Review of the literature.

Results: A wide spectrum of fluorophores that have been tested for brain surgery is reviewed. Beginning with a fluorescein sodium application in 1948 by Moore, fluorescence-guided brain tumor surgery is either routinely applied in some centers or is under active study in clinical trials. Besides the trinity of commonly used drugs (fluorescein sodium, 5-aminolevulinic acid, and indocyanine green), less studied fluorescent stains, such as tetracyclines, cancer-selective alkylphosphocholine analogs, cresyl violet, acridine orange, and acriflavine, can be used for rapid tumor detection and pathological tissue examination. Other emerging agents, such as activity-based probes and targeted molecular probes that can provide biomolecular specificity for surgical visualization and treatment, are reviewed. Furthermore, we review available engineering and optical solutions for fluorescent surgical visualization. Instruments for fluorescent-guided surgery are divided into wide-field imaging systems and hand-held probes. Recent advancements in quantitative fluorescence-guided surgery are discussed.

Conclusion: We are standing on the threshold of the era of marker-assisted tumor management. Innovations in the fields of surgical optics, computer image analysis, and molecular bioengineering are advancing fluorescence-guided tumor resection paradigms, leading to cell-level approaches to visualization and resection of brain tumors.

Precise spatial positioning and isolation of mammalian cells is a critical component of many single cell experimental methods and biological engineering applications. Although a variety of cell patterning methods have been demonstrated, many of these methods subject cells to high stress environments, discriminate against certain phenotypes, or are a challenge to implement. Here, we demonstrate a rapid, simple, indiscriminate, and minimally perturbing cell patterning method using a laser fabricated polymer stencil. The stencil fabrication process requires no stencil-substrate alignment, and is readily adaptable to various substrate geometries and experiments.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers.

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett’s esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF). Functional assays revealed both cell types showed repressed proliferation and significant changes in motility (speed, displacement and directionality) as a result of interactions between the two cell types. Cellular interactions appear to be mediated through both direct cell-cell contact and secreted ligands. The findings of this study are important in that they reveal, for the first time, the effects of cellular communication on gene expression and cellular function between premalignant (dysplastic) epithelial cells and their normal counterparts.