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.

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.

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.

The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an ‘epigenetic’ drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat’s differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.

Improved tools for providing specific intraoperative diagnoses could improve patient care. In neurosurgery, intraoperatively differentiating non-operative lesions such as CNS B-cell lymphoma from operative lesions can be challenging, often necessitating immunohistochemical (IHC) procedures which require up to 24-48 hours. Here, we evaluate the feasibility of generating rapid ex vivo specific labeling using a novel lymphoma-specific fluorescent switchable aptamer. Our B-cell lymphoma-specific switchable aptamer produced only low-level fluorescence in its unbound conformation and generated an 8-fold increase in fluorescence once bound to its target on CD20-positive lymphoma cells. The aptamer demonstrated strong binding to B-cell lymphoma cells within 15 minutes of incubation as observed by flow cytometry. We applied the switchable aptamer to ex vivo xenograft tissue harboring B-cell lymphoma and astrocytoma, and within one hour specific visual identification of lymphoma was routinely possible. In this proof-of-concept study in human cell culture and orthotopic xenografts, we conclude that a fluorescent switchable aptamer can provide rapid and specific labeling of B-cell lymphoma, and that developing aptamer-based labeling approaches could simplify tissue staining and drastically reduce time to histopathological diagnoses compared with IHC-based methods. We propose that switchable aptamers could enhance expeditious, accurate intraoperative decision-making.

Background: The successful treatment of malignant gliomas remains a challenge despite the current standard of care, which consists of surgery, radiation and temozolomide. Advances in the survival of brain cancer patients require the design of new therapeutic approaches that take advantage of common phenotypes such as the altered metabolism found in cancer cells. It has therefore been postulated that the high-fat, low-carbohydrate, adequate protein ketogenic diet (KD) may be useful in the treatment of brain tumors. We have demonstrated that the KD enhances survival and potentiates standard therapy in a mouse model of malignant glioma, yet the mechanisms are not fully understood.

Methods: To explore the effects of the KD on various aspects of tumor growth and progression, we used the immunocompetent, syngeneic GL261-Luc2 mouse model of malignant glioma.

Results: Tumors from animals maintained on KD showed reduced expression of the hypoxia marker carbonic anhydrase 9, hypoxia inducible factor 1-alpha, and decreased activation of nuclear factor kappa B. Additionally, tumors from animals maintained on KD had reduced tumor microvasculature and decreased expression of vascular endothelial growth factor receptor 2, matrix metalloproteinase-2 and vimentin. Peritumoral edema was significantly reduced in animals fed the KD and protein analyses showed altered expression of zona occludens-1 and aquaporin-4.

Conclusions: The KD directly or indirectly alters the expression of several proteins involved in malignant progression and may be a useful tool for the treatment of gliomas.

Introduction: The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that alters metabolism by increasing the level of ketone bodies in the blood. KetoCal® (KC) is a nutritionally complete, commercially available 4∶1 (fat∶ carbohydrate+protein) ketogenic formula that is an effective non-pharmacologic treatment for the management of refractory pediatric epilepsy. Diet-induced ketosis causes changes to brain homeostasis that have potential for the treatment of other neurological diseases such as malignant gliomas.

Methods: We used an intracranial bioluminescent mouse model of malignant glioma. Following implantation animals were maintained on standard diet (SD) or KC. The mice received 2×4 Gy of whole brain radiation and tumor growth was followed by in vivo imaging.

Results: Animals fed KC had elevated levels of β-hydroxybutyrate (p = 0.0173) and an increased median survival of approximately 5 days relative to animals maintained on SD. KC plus radiation treatment were more than additive, and in 9 of 11 irradiated animals maintained on KC the bioluminescent signal from the tumor cells diminished below the level of detection (p<0.0001). Animals were switched to SD 101 days after implantation and no signs of tumor recurrence were seen for over 200 days.

Conclusions: KC significantly enhances the anti-tumor effect of radiation. This suggests that cellular metabolic alterations induced through KC may be useful as an adjuvant to the current standard of care for the treatment of human malignant gliomas.