Browsing by Author "Munson, Jennifer M."

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    2D or 3D? How cell motility measurements are conserved across dimensions in vitro and translate in vivo
    Galarza, Sualyneth; Kim, Hyuna; Atay, Naciye; Peyton, Shelly R.; Munson, Jennifer M. (2019-11-19)
    Cell motility is a critical aspect of several processes, such as wound healing and immunity; however, it is dysregulated in cancer. Current limitations of imaging tools make it difficult to study cell migration in vivo. To overcome this, and to identify drivers from the microenvironment that regulate cell migration, bioengineers have developed 2D (two-dimensional) and 3D (three-dimensional) tissue model systems in which to study cell motility in vitro, with the aim of mimicking elements of the environments in which cells move in vivo. However, there has been no systematic study to explicitly relate and compare cell motility measurements between these geometries or systems. Here, we provide such analysis on our own data, as well as across data in existing literature to understand whether, and which, metrics are conserved across systems. To our surprise, only one metric of cell movement on 2D surfaces significantly and positively correlates with cell migration in 3D environments (percent migrating cells), and cell invasion in 3D has a weak, negative correlation with glioblastoma invasion in vivo. Finally, to compare across complex model systems, in vivo data, and data from different labs, we suggest that groups report an effect size, a statistical tool that is most translatable across experiments and labs, when conducting experiments that affect cellular motility.
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    Attributes of Astrocyte Response to Mechano-Stimulation by High-Rate Overpressure
    Hlavac, Nora (Virginia Tech, 2018-11-29)
    Blast neurotrauma represents a significant mode of traumatic injury to the brain. The incidence of blast neurotrauma is particularly high amongst military combat personnel and can be debilitating and endure clinically for years after injury is sustained. Mechanically, blast represents a unique and complex loading paradigm associated with compressive shock waves that propagate out from an explosive event and interact with the head and other organs through high-rate loading. When subjected to such insult, brain cells undergo characteristic injury responses which include neuroinflammation, oxidative stress, edema and persistent glial activation. These features of the injury have emerged as important mediators of the chronic brain damage that results from blast. Astrocytes have emerged as a potential therapeutic target because of their ubiquitous roles in brain homeostasis, tissue integrity and cognitive function. This glial subtype has a characteristic reactive response to mechanical trauma of various modes. In this work, custom in vitro injury devices were used to characterize functional models of astrocyte reactivity to high-rate insult to study mechano-stimulation mechanisms associated with the reactive phenotype. The working hypothesis was that high-rate overpressure exposure would cause metabolic aberrations, cell junction changes, and adhesion signal transduction activation, all of which would contribute to astrocyte response and reactivity. Astrocyte cultures were exposed to a 20 psi high-rate overpressure scheme using an underwater explosion-driven device. Astrocytes experienced dynamic energetic fluctuations in response to overpressure which were followed by the assumption of a classically defined reactive phenotype. Results indicated specific roles for cationic transduction, cell junction dynamics (gap junction and anchoring junctions) and downstream signal transduction mechanisms associated with adhesion alterations in onset of the astrocyte reactive phenotype. Investigation into adhesion signaling regulation by focal adhesion kinase in 2D and 3D cultures was also explored to better understand cellular reactivity as a function of extracellular environment. Additionally, another underwater in vitro device was built to study combination effects from overpressure and fluid shear associated with insult. Overall, the combined studies offer multiple mechanisms by which to explore molecular targets for harnessing astrocytes' potential for repair after traumatic injury to the brain.
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    Augmentation of brain tumor interstitial flow via focused ultrasound promotes brain-penetrating nanoparticle dispersion and transfection
    Curley, Colleen T.; Mead, Brian P.; Negron, Karina; Kim, Namho; Garrison, William J.; Miller, G. Wilson; Kingsmore, Kathryn M.; Thim, E. Andrew; Song, Ji; Munson, Jennifer M.; Klibanov, Alexander L.; Suk, Jung Soo; Hanes, Justin; Price, Richard J. (2020-04)
    The delivery of systemically administered gene therapies to brain tumors is exceptionally difficult because of the blood-brain barrier (BBB) and blood-tumor barrier (BTB). In addition, the adhesive and nanoporous tumor extra-cellular matrix hinders therapeutic dispersion. We first developed the use of magnetic resonance image (MRI)-guided focused ultrasound (FUS) and microbubbles as a platform approach for transfecting brain tumors by targeting the delivery of systemically administered "brain-penetrating" nanoparticle (BPN) gene vectors across the BTB/BBB. Next, using an MRI-based transport analysis, we determined that after FUS-mediated BTB/BBB opening, mean interstitial flow velocity magnitude doubled, with "per voxel" flow directions changing by an average of similar to 70 degrees to 80 degrees. Last, we observed that FUS-mediated BTB/BBB opening increased the dispersion of directly injected BPNs through tumor tissue by >100%. We conclude that FUS-mediated BTB/BBB opening yields markedly augmented interstitial tumor flow that, in turn, plays a critical role in enhancing BPN transport through tumor tissue.
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    Autologous Gradient Formation under Differential Interstitial Fluid Flow Environments
    Stine, Caleb A.; Munson, Jennifer M. (MDPI, 2022-01-04)
    Fluid flow and chemokine gradients play a large part in not only regulating homeostatic processes in the brain, but also in pathologic conditions by directing cell migration. Tumor cells in particular are superior at invading into the brain resulting in tumor recurrence. One mechanism that governs cellular invasion is autologous chemotaxis, whereby pericellular chemokine gradients form due to interstitial fluid flow (IFF) leading cells to migrate up the gradient. Glioma cells have been shown to specifically use CXCL12 to increase their invasion under heightened interstitial flow. Computational modeling of this gradient offers better insight into the extent of its development around single cells, yet very few conditions have been modelled. In this paper, a computational model is developed to investigate how a CXCL12 gradient may form around a tumor cell and what conditions are necessary to affect its formation. Through finite element analysis using COMSOL and coupled convection-diffusion/mass transport equations, we show that velocity (IFF magnitude) has the largest parametric effect on gradient formation, multidirectional fluid flow causes gradient formation in the direction of the resultant which is governed by IFF magnitude, common treatments and flow patterns have a spatiotemporal effect on pericellular gradients, exogenous background concentrations can abrogate the autologous effect depending on how close the cell is to the source, that there is a minimum distance away from the tumor border required for a single cell to establish an autologous gradient, and finally that the development of a gradient formation is highly dependent on specific cell morphology.
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    Convection-Enhanced Delivery: Connection to and Impact of Interstitial Fluid Flow
    Stine, Caleb A.; Munson, Jennifer M. (Frontiers, 2019-10-02)
    Convection-enhanced delivery (CED) is a method used to increase transport of therapeutics in and around brain tumors. CED works through locally applying a pressure differential to drive fluid flow throughout the tumor, such that convective forces dominate over diffusive transport. This allows therapies to bypass the blood brain barrier that would otherwise be too large or solely rely on passive diffusion. However, this also drives fluid flow out through the tumor bulk into surrounding brain parenchyma, which results in increased interstitial fluid (IF) flow, or fluid flow within extracellular spaces in the tissue. IF flow has been associated with altered transport of molecules, extracellular matrix rearrangement, and triggering of cellularmotility through a number ofmechanisms. Thus, the results of a simple method to increase drug delivery may have unintended consequences on tissue morphology. Clinically, prediction of dispersal of agents via CED is important to catheter design, placement, and implementation to optimize contact of tumor cells with therapeutic agent. Prediction software can aid in this problem, yet we wonder if there is a better way to predict therapeutic distribution based simply on IF flow pathways as determined from pre-intervention imaging. Overall, CED based therapy has seen limited success and we posit that integration and appreciation of altered IF flow may enhance outcomes. Thus, in this manuscript we both review the current state of the art in CED and IF flow mechanistic understanding and relate these two elements to each other in a clinical context.
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    Convective forces increase CXCR4-dependent glioblastoma cell invasion in GL261 murine model
    Cornelison, R. Chase; Brennan, Caroline E.; Kingsmore, Kathryn M.; Munson, Jennifer M. (Nature Publishing Group, 2019-11-18)
    Glioblastoma is the most common and malignant form of brain cancer. Its invasive nature limits treatment efficacy and promotes inevitable recurrence. Previous in vitro studies showed that interstitial fluid flow, a factor characteristically increased in cancer, increases glioma cell invasion through CXCR4- CXCL12 signaling. It is currently unknown if these effects translate in vivo. We used the therapeutic technique of convection enhanced delivery (CED) to test if convective flow alters glioma invasion in a syngeneic GL261 mouse model of glioblastoma. The GL261 cell line was flow responsive in vitro, dependent upon CXCR4 and CXCL12. Additionally, transplanting GL261 intracranially increased the populations of CXCR4+ and double positive cells versus 3D culture. We showed that inducing convective flow within implanted tumors indeed increased invasion over untreated controls, and administering the CXCR4 antagonist AMD3100 (5 mg/kg) effectively eliminated this response. These data confirm that glioma invasion is stimulated by convective flow in vivo and depends on CXCR4 signaling. We also showed that expression of CXCR4 and CXCL12 is increased in patients having received standard therapy, when CED might be elected. Hence, targeting flow-stimulated invasion may prove beneficial as a second line of therapy, particularly in patients chosen to receive treatment by convection enhanced delivery.
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    Delivery strategies for cell-based therapies in the brain: overcoming multiple barriers
    Turk, Olivia M.; Woodall, Ryan C.; Gutova, Margarita; Brown, Christine E.; Rockne, Russell C.; Munson, Jennifer M. (Springer, 2021-10-30)
    Cell-based therapies to the brain are promising for the treatment of multiple brain disorders including neurodegeneration and cancers. In order to access the brain parenchyma, there are multiple physiological barriers that must be overcome depending on the route of delivery. Specifically, the blood–brain barrier has been a major difficulty in drug delivery for decades, and it still presents a challenge for the delivery of therapeutic cells. Other barriers, including the blood-cerebrospinal fluid barrier and lymphatic-brain barrier, are less explored, but may offer specific challenges or opportunities for therapeutic delivery. Here we discuss the barriers to the brain and the strategies currently in place to deliver cell-based therapies, including engineered T cells, dendritic cells, and stem cells, to treat diseases. With a particular focus on cancers, we also highlight the current ongoing clinical trials that use cell-based therapies to treat disease, many of which show promise at treating some of the deadliest illnesses. Graphical abstract: [Figure not available: see fulltext.].
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    Designing Patient-Driven, Tissue-Engineered Models of Primary and Metastatic Breast Cancer
    Beeghly, Garrett F.; Thomas, Candace; Yuan, Jessica X.; Harris, Alexandra R.; Munson, Jennifer M. (MDPI, 2022-01-18)
    The rising survival rate for early-stage breast cancer in the United States has created an expanding population of women in remission at risk for distant recurrence, with metastatic spread to the brain demonstrating an especially poor prognosis. The current standard of care for breast cancer brain metastases is not well defined or differentiated from the treatment of brain metastases from other primary sites. Here, we present tissue-engineered models of the primary and brain metastatic breast cancer microenvironments informed by analysis of patient tumor resections. We find that metastatic resections demonstrate distinct cellular and matrix components compared with primary resections or non-cancerous controls. Using our model systems, we find that the observed deposition of collagen I after metastasis to the brain may enhance breast cancer invasion. Future optimization of these models will present a novel platform to examine tumor-stroma interactions and screen therapeutics for the management of metastatic breast cancer.
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    Developing Teaching Proficiencies for New Instructors Through a Learning Community
    Hall, Tracy Michelle; Barb, Christopher; Gilmore, Tracy; Hall, Monena; Henshaw, Neal; Lawrence, Anne S.; Meier, Carolyn; Miller, Rebecca K.; Moyo, Lesley; Munson, Jennifer M.; Ogier, Andrea; Thum, Sara (2013-02-06)
    Preparation and training for teaching roles within the Virginia Tech library environment.
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    The Development of a Printable Device with Gravity-Driven Flow for Live Imaging Glioma Stem Cell Motility
    Macias-Orihuela, Yamilet (Virginia Tech, 2023-01-25)
    The post-prognosis lifespan for those suffering with Glioblastoma (GBM) is approximately 13 months with current standard of care. Intratumoral heterogeneity is a common characteristic that hinders GBM treatment in the form of therapy resistant cell subsets and influence on cellular phenotypes. One cell subset in particular, glioma stem cells (GSCs), is frequently left behind in the brain parenchyma once the bulk of the tumor has been resected. Previous research has found that patient-derived GSCs displayed varying invasion responses with and without the presence of interstitial flow. Interestingly, GSCs from a single patient are heterogeneous, displaying differences among sub-colonies derived from the same parental line. To study the motility of cells under flow, PDMS microfluidics are commonly used. Unfortunately, this setup often involves active flow generation using pumps, limiting the number of cell lines that can be imaged at a time. To increase the throughput of GSC sub-colonies imaged simultaneously, we developed a bio-compatible, printable device fabricated to allow for passive, gravity-driven flow through a hydrogel that recapitulates the brain microenvironment, eliminating the need for pumps. Stereo lithography 3D printing was chosen as the manufacturing method for the device, and this facilitated design feature modification when prototyping, increased the potential complexity of future iterations, and avoided some of the hurdles associated with fabricating PDMS microfluidics. This printable imaging device allows for higher throughput live-imaging of cell lines to aid in the understanding of the relationships between intratumoral heterogeneity, invasion dynamics, and interstitial flow.
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    Development of a Synthetic, Injectable Hydrogel to Capture Residual Glioblastoma and Glioblastoma Stem-Like Cells with CXCL12-Mediated Chemotaxis
    Khan, Zerin Mahzabin; Munson, Jennifer M.; Long, Timothy E.; Vlaisavljevich, Eli; Verbridge, Scott S. (Wiley, 2023-06)
    Glioblastoma (GBM), characterized by high infiltrative capacity, is the most common and deadly type of primary brain tumor in adults. GBM cells, including therapy-resistant glioblastoma stem-like cells (GSCs), invade the healthy brain parenchyma to form secondary tumors even after patients undergo surgical resection and chemoradiotherapy. New techniques are therefore urgently needed to eradicate these residual tumor cells. A thiol-Michael addition injectable hydrogel for compatibility with GBM therapy is previously characterized and optimized. This study aims to develop the hydrogel further to capture GBM/GSCs through CXCL12-mediated chemotaxis. The release kinetics of hydrogel payloads are investigated, migration and invasion assays in response to chemoattractants are performed, and the GBM-hydrogel interactions in vitro are studied. With a novel dual-layer hydrogel platform, it is demonstrated that CXCL12 released from the synthetic hydrogel can induce the migration of U251 GBM cells and GSCs from the extracellular matrix microenvironment and promote invasion into the synthetic hydrogel via amoeboid migration. The survival of GBM cells entrapped deep into the synthetic hydrogel is limited, while live cells near the surface reinforce the hydrogel through fibronectin deposition. This synthetic hydrogel, therefore, demonstrates a promising method to attract and capture migratory GBM cells and GSCs responsive to CXCL12 chemotaxis.
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    Development of an Injectable Hydrogel Platform to Capture and Eradicate Glioblastoma Cells with Chemical and Physical Stimuli
    Khan, Zerin Mahzabin (Virginia Tech, 2023-05-15)
    Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumor. Even after patients undergo maximum and safe surgical resection followed by adjuvant chemotherapy and radiation therapy, residual GBM cells form secondary tumors which lead to poor survival times and prognoses for patients. This tumor recurrence can be attributed to the inherent GBM heterogeneity that makes it difficult to eradicate the therapy-resistant and tumorigenic subpopulation of GBM cells with stem cell-like properties, referred to as glioma stem cells (GSCs). Additionally, the migratory nature of GBM/GSCs enable them to invade into the healthy brain parenchyma beyond the resection cavity to generate new tumors. In an effort to address these challenges of GBM recurrence, this research aimed to develop a biomaterials-based approach to attract, capture, and eradicate GBM cells and GSCs with chemical and physical stimuli. Specifically, it is proposed that after surgical removal of the primary GBM tumor mass, an injectable hydrogel can be dispensed into the resection cavity for crosslinking in situ. A combination of chemical and physical cues can then induce the migration of the residual GBM/GSCs into the injectable hydrogel to localize and concentrate the malignant cells prior to non-invasively abating them. In order to develop this proposed treatment, this dissertation focused on 1) characterizing and optimizing the thiol-Michael addition injectable hydrogel, 2) attracting and entrapping GBM/GSCs into the hydrogel with CXCL12-mediated chemotaxis, and 3) assessing the feasibility of utilizing histotripsy to mechanically and non-invasively ablate cells entrapped in the hydrogel. The results revealed that hydrogel formulations comprising 0.175 M NaHCO3(aq) and 50 wt% water content were the most optimal for physical, chemical, and biological compatibility with the GBM microenvironment on the basis of their swelling characteristics, sufficiently crosslinked polymer networks, degradation rates, viscoelastic properties, and interactions with normal human astrocytes. Loading the hydrogel with 5 µg/mL of CXCL12 was optimal for the slow, sustained release of the chemokine payload. A dual layer hydrogel platform demonstrated in vitro that the resulting chemotactic gradient induced the invasion of GBM cells and GSCs from the extracellular matrix and into the synthetic hydrogel with ameboid migration and myosin IIA activation. This injectable hydrogel also demonstrated direct therapeutic benefits by passively eradicating entrapped GBM cells through matrix diffusion limitations as well as decreasing the GBM malignancy and GSC stemness upon cancer cell-hydrogel interactions. Research findings revealed the hydrogels can be synthesized under clinically relevant conditions mimicking GBM resection in vitro, and hydrogels were distinguishable with ultrasound imaging. Furthermore, the synthetic hydrogel was acoustically active to generate a stable cavitation bubble cloud with histotripsy treatment for ablation of entrapped red blood cells with well-defined, uniform lesion areas. Overall, the results from this research demonstrate this injectable hydrogel is a promising platform to attract and entrap malignant GBM/GSCs for subsequent eradication with chemical and physical stimuli. Further development of this platform, such as by integrating electric cues for electrotaxis-directed cell migration, may help to improve the cancer cell trapping capabilities and thereby mitigate GBM tumor recurrences in patients.
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    Docetaxel facilitates lymphatic-tumor crosstalk to promote lymphangiogenesis and cancer progression
    Harris, Alexandra R.; Perez, Matthew J.; Munson, Jennifer M. (2018-07-06)
    Background Infiltration into lymphatic vessels is a critical step in breast cancer metastasis. Lymphatics undergo changes that facilitate metastasis as a result of activation of the cells lining lymphatic vessels, lymphatic endothelial cells (LECs). Inhibition of activation by targeting VEGFR3 can reduce invasion toward lymphatics. To best benefit patients, this approach should be coupled with standard of care that slows tumor growth, such as chemotherapy. Little is known about how chemotherapies, like docetaxel, may influence lymphatics and conversely, how lymphatics can alter responses to therapy. Methods A novel 3D in vitro co-culture model of the human breast tumor microenvironment was employed to examine the contribution of LECs to tumor invasion and viability with docetaxel and anti-VEGFR3, using three cell lines, MDA-MB-231, HCC38, and HCC1806. In vivo, the 4T1 mouse model of breast carcinoma was used to examine the efficacy of combinatorial therapy with docetaxel and anti-VEGFR3 on lymph node metastasis and tumor growth. Lymphangiogenesis in these mice was analyzed by immunohistochemistry and flow cytometry. Luminex analysis was used to measure expression of lymphangiogenic cytokines. Results In vitro, tumor cell invasion significantly increased with docetaxel when LECs were present; this effect was attenuated by inhibition of VEGFR3. LECs reduced docetaxel-induced cell death independent of VEGFR3. In vivo, docetaxel significantly increased breast cancer metastasis to the lymph node. Docetaxel and anti-VEGFR3 combination therapy reduced lymph node and lung metastasis in 4T1 and synergized to reduce tumor growth. Docetaxel induced VEGFR3-dependent vessel enlargement, lymphangiogenesis, and expansion of the LEC population in the peritumoral microenvironment, but not tumor-free stroma. Docetaxel caused an upregulation in pro-lymphangiogenic factors including VEGFC and TNF-α in the tumor microenvironment in vivo. Conclusions Here we present a counter-therapeutic effect of docetaxel chemotherapy that triggers cancer cells to elicit lymphangiogenesis. In turn, lymphatics reduce cancer response to docetaxel by altering the cytokine milieu in breast cancer. These changes lead to an increase in tumor cell invasion and survival under docetaxel treatment, ultimately reducing docetaxel efficacy. These docetaxel-induced effects can be mitigated by anti-VEGFR3 therapy, resulting in a synergism between these treatments that reduces tumor growth and metastasis.
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    Enhancing Brain Flow Visualization with Automated 3D Data Processing: A Study on DCE-MRI Data from Mice with Tumors
    Mohammed, Ayat; Polys, Nicholas F.; Cunningham, Jessica; Munson, Jennifer M.; Chutkowski, James; Liang, Hun; Park, Daniel; Rockne, Russell; Woodall, Ryan; Esparza, Cora (ACM, 2023-10-09)
    Enhancing the process of generating entirely automated visualization schemes of complex fluid flow patterns within brain tumors is critical for gaining insights into their movements and behaviors. This study focused on optimizing and automating the processing of 3D volumetric and vector field data sets obtained from DCE-MRI (Dynamic Contrast-Enhanced Magnetic Resonance Imaging) scans. It is crucial to maintain performance, preserve data quality and resolution, and provide an accessible platform for biomedical scientists. In this paper, we represent an innovative approach to enhance fluid flow visualization of brain tumors through scalable visualization techniques. New techniques have been designed, benchmarked, and authenticated to produce X3D visualizations in Web3D environments using Python, and ParaView. The proposed approach does not only enhance fluid flow visualization in the context of brain tumor research but also provides a reproducible and transparent framework for future studies with both human and mouse scans.
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    Evaluating Microglia Dynamics in Blast and Impact-Induced Neurotrauma and Assessing the Role of Hemostatic Nanoparticles in Microglia Activation
    White, Michelle Renee (Virginia Tech, 2022-10-03)
    Traumatic brain injury (TBI) is a major medical concern that has demonstrated to be particularly challenging to treat because of the disparity amongst injury modes and severities. Increased use of explosive devices during combat has caused blast TBI (bTBI) to become a widespread consequence in military and Veteran populations, and impact-related trauma from contact-related sports or motor vehicle accidents has made mild impact-induced TBIs (concussion) a major health problem. There is a high risk for those who have sustained a TBI to develop behavioral and cognitive disorders following injury, and these symptoms can present as delayed onset, causing diagnosis to be a major feat when planning for treatment and long-term healthcare. Both preclinical and clinical studies report the neuropathological changes following TBI, yet investigating the distinct mechanistic changes in blast and impact trauma that contribute to pathological disparities has yet to be elucidated. Microglia dynamics play a key role in initiating the inflammatory response after injury, as microglia become activated by undergoing morphological changes that influence their function in the injured brain, and unique signaling pathways influence their functional inflammatory states. While previous literature report on the unique responses of microglia, their mediated-inflammatory responses are still not well defined. This work aimed to investigate the acute and subacute responses of microglia to injury through their diverse activation states following blast and impact trauma. The work herein employed rodent models to investigate these changes, finding that microglia activation was spatially and temporally heterogeneous within and across injury paradigms. Three days following bTBI, activated microglia in the cortex displayed morphologies similar to microglia that are known to increase their interactions with dysfunctional synapses, while dystrophic microglia were prevalent in the hippocampus seven days following injury. Moreover, transhemispheric changes in microglia activation were noted following impact TBI, with stressed/primed microglia responding to immune challenges of the cortex at three days, whereas a unique morphological state that was markedly different from those traditionally reported in CNS injury and disease was present within the hippocampus three- and seven-days following injury. State-of-the-art cell sorting techniques were used for in vivo analysis of microglia, which also exhibited that functional changes of microglia vary between injury paradigms, providing insight into how differences in primary insult may elicit distinct signaling pathways involved in microglia-mediated inflammatory responses. These in vivo studies were then crucial in understanding the malleable responses of microglia to complex injuries such as "blast plus impact" TBI, indicating that phenotypic changes in microglia following this injury are also unique and spatially heterogeneous. To date, therapeutic efforts for TBI are limited due to the lack of understanding the underlying mechanisms that influence TBI pathology. This work also investigated novel therapeutic targets, noting that administration of polyester nanoparticles restored microglia to baseline levels following impact. The fundamental research presented in this study is innovative and advantageous as it can provide essential data into targeted and personalized treatments that can improve long-term healthcare and ultimately, the quality of life for those suffering from a TBI.
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    Ex Vivo Deformations of the Uterosacral Ligaments
    Donaldson, Kandace E. (Virginia Tech, 2023-02-24)
    The uterosacral ligaments (USLs) are important anatomical structures that support the uterus and apical vagina within the pelvis. As these structures are over-stretched, become weak, and exhibit laxity, pelvic floor disorders such as pelvic organ prolapse occur. Although several surgical procedures to treat pelvic floor disorders are directed toward the USLs, there is still a lot that is unknown about their function. These surgeries often result in poor outcomes, demonstrating the need for new surgical approaches and biomaterials. The first chapter of this dissertation presents a review of the current knowledge on the mechanical properties of the USLs. The anatomy, microstructure, and clinical significance of the USLs are first reviewed. Then, the results of published experimental studies on the {emph{in vivo}} and {emph{ex vivo}}, uniaxial and biaxial tensile tests are compiled. Based on the existing findings, research gaps are identified and future research directions are discussed. The second chapter proposes the use of planar biaxial testing, digital image correlation (DIC), and optical coherence tomography (OCT) to quantify the deformations of the USLs, both in-plane and out-of-plane. Using virgin swine as an animal model, the USLs were found to deform significantly less in their main direction (MD) of {emph{in vivo}} loading than in the direction perpendicular to it (PD) at increasing equibiaxial stresses. Under constant equibiaxial loading, the USLs deformed over time equally, at comparable rates in both the MD and PD. The thickness of the USLs decreased as the equibiaxial loading increased but, under constant equibiaxial loading, the thickness increased in some specimens and decreased in others. The third chapter presents new experimental methods for testing the {emph{ex vivo}} tensile properties of the uterosacral ligaments (USLs) in rats. USL specimens were carefully dissected to preserve their anatomical attachments, and they were loaded along their main {emph{in vivo}} loading direction (MD) using a custom-built uniaxial tensile testing device. This chapter reports the first mechanical data on the rat USLs in isolation from surrounding organs. It is also the first experimental study to provide measurements of the inhomogeneous deformations of the USLs during loading along their main textit{in vivo} loading direction, revealing that the USLs may behave as auxetic structures. The fourth and final chapter presents preliminary findings on novel imaging applications to characterize the evolving structure of the USLs before, during, and after tensile pulling along the ligaments' main textit{in vivo} axis of loading. Rat USLs were excised using the proposed novel dissection method and pulled uniaxially as was performed in the previous chapter. Before and after mechanical testing, second harmonic generation (SHG) was used to image collagen and muscle within the three anatomical regions of the USLs. During mechanical testing, OCT was used to collect out-of-plane images of the cervical/intermediate regions of the USL specimens, resulting in 3D volume scans of the regions. SHG images showed the USLs to have complex microstructures with significant wavy collagen bundles interwoven with muscle bundles. Preliminary observation of the microstructure during testing revealed interwoven sections of tissue with collagenous fibers that reoriented in all directions illustrating how the USLs may expand laterally during uniaxial loading, causing the auxetic properties documented in the previous chapter. Though more quantitative work remains to be done, the findings presented in this dissertation improve our understanding of how the USLs deform with increasing load, such as what occurs during pregnancy. Together, these studies serve as a springboard for future investigations on the supportive function of the USLs in animal models by offering guidelines on testing methods that capture their complex mechanical behavior.
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    Extracellular Matrix Contributions to Early Vascular Development and Pericyte Precursor Dynamics
    Hoque, Maruf M. (Virginia Tech, 2023-07-24)
    The vasculature is a highly intricate system of "highways" that shuttles blood from the heart to every tissue and organ in the human body. These vessels are responsible for carrying oxygen, trafficking hormones, delivering nutrients, and removing waste products from the body. The formation of a functioning vascular system depends on the close coordination of many cell types and, on the capillary level, specifically endothelial cells and pericytes as well as the surrounding protein microenvironment, known as the extracellular matrix (ECM). Impaired coordination amongst the cellular and protein constituents results in the improper functioning of the vascular network and can eventually contribute to the failure of organ systems. This dissertation research focuses on how improper ECM deposition affects vascular assembly. We utilized several approaches to affect ECM composition, specifically: 1) hypoxia exposure and 2) reducing ECM pharmacologically and utilizing lentiviral-mediated silencing of Type IV Collagen (Col-IV, gene Col4a1) expression. In these experimental settings, we observed downstream changes in the coordination between endothelial cells and pericytes while forming vascular networks. In short, this dissertation work suggests that excess ECM deposition, and particularly that of Col-IV, has unique deleterious effects on the developing vasculature as compared to reduced ECM deposition. The findings from this work suggest mechanisms underlying how the vasculature may be destabilized in hypoxia-associated pathologies, such as preeclampsia.
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    In-vitro Glioblastoma Treatment Focusing on Convection Enhanced Delivery
    Brocke, Conner Ethan (Virginia Tech, 2022-05-25)
    Glioblastoma is a deadly brain cancer with discouraging standard of care. New methods like convection enhanced delivery and chimeric antigen receptor T cells (CAR-T) are promising treatments that can be translated to glioblastoma. In this study, CAR-T cell flow through a hydrogel was explored in the context of in-vitro convection enhanced delivery. A culture method to create large spheroids mimicking tumors from preexisting glioblastoma stem cell lines was fabricated, a convection enhanced delivery system for in-vitro testing was designed, and characterization of the CAR-T cells using the in-vitro system took place. The spheroid culture method was successfully optimized to produce spheroids large enough to act as a sufficient tumor in little time, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities through a medium as their injection velocity increased. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro.
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    Interstitial fluid flow in cancer: implications for disease progression and treatment
    Munson, Jennifer M.; Shieh, Adrian C. (Dove Medical Press, 2014-08-19)
    As cancer progresses, a dynamic microenvironment develops that creates and responds to cellular and biophysical cues. Increased intratumoral pressure and corresponding increases in interstitial flow from the tumor bulk to the healthy stroma is an observational hallmark of progressing cancers. Until recently, the role of interstitial flow was thought to be mostly passive in the transport and dissemination of cancer cells to metastatic sites. With research spanning the past decade, we have seen that interstitial flow has a promigratory effect on cancer cell invasion in multiple cancer types. This invasion is one mechanism by which cancers can resist therapeutics and recur, but the role of interstitial flow in cancer therapy is limited to the understanding of transport of therapeutics. Here we outline the current understanding of the role of interstitial flow in cancer and the tumor microenvironment through cancer progression and therapy. We also discuss the current role of fluid flow in the treatment of cancer, including drug transport and therapeutic strategies. By stating the current understanding of interstitial flow in cancer progression, we can begin exploring its role in therapeutic failure and treatment resistance.
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    Interstitial Fluid Flow Magnitude and Its Effects on Glioblastoma Invasion
    Stine, Caleb A. (Virginia Tech, 2022-06-13)
    Fluid flow is a complex and dynamic process in the brain, taking place at the macro- and microscopic level. Interstitial fluid in particular flows throughout the interstitial spaces within the tissue, interacting with cells and the extracellular matrix. We are coming to find that this interstitial fluid flow plays an important role in both homeostatic and pathologic conditions. It helps to transport chemokines and other molecules such as extracellular vesicles within the environment, clear waste from the brain, and provide biophysical cues to cells. When this flow is disrupted however, such as in glioblastoma or Alzheimer's disease, profound events can occur, for example the build-up of plaques or an increase in tumor cell invasion. While there has recently been an up-tick in interstitial fluid flow research, there is surprisingly little known about its exact nature within the interstitial space and its effects on brain pathology such as glioblastoma. In particular, ways to manipulate and measure brain IFF magnitude at the cellular level are lacking. In this dissertation, a set of tools is created and used to explore the role that interstitial fluid flow magnitude plays in the brain through the lens of glioma invasion. We developed and implemented a flow device that is used in conjunction with an established in vitro tissue culture insert assay to manipulate fluid flow rates through a 3D matrix of tumor cells. We showed that this flow device is biocompatible and accurately recreates flow rates that have been measured previously through the use of MRI. We quantified tumor cell invasion from several glioma cell lines using this device to show a nonlinear trend of invasion in response to increasing fluid flow magnitudes. In addition, we developed a computational model to explore one potential mechanism that fluid flow magnitude might be modulating: autologous chemotaxis. Through this model we showed that increased flow magnitudes such as those seen in gliomas cause an increase in the distribution of the chemokine gradient around a cell of interest, that the morphology of the cell is important to this gradient formation, that temporal effects should not be overlooked, and that within the tumor environment, a minimum distance is required for the invading cell to develop this gradient. Finally, we developed a novel in vivo surgical technique that allows for the manipulation and measurement of interstitial fluid flow within the brain through simultaneous multiphoton imaging. We showed that this technique can be used to modulate interstitial fluid flow, as a mechanism by which to label cells of interest, and as a means to implant and monitor glioma progression. Through these means we further characterize interstitial fluid flow in the brain, allowing for its manipulation and measurement, and examine the ability of increased interstitial fluid flow magnitudes to impact glioma invasion.