Browsing by Subject "Hypoxia"

Osteoporosis and low bone density affect an estimated 54 million adults of 50 years and over in the United States, resulting in $19 billion in costs for osteoporosis-related bone breaks. Current treatments include the use of antiresorptive and anabolic drugs to decrease the rate of bone resorption and increase the rate of bone formation, respectively. However, these current treatments are unable to completely normalize skeletal integrity. As bone diseases become increasingly prevalent, there is an urgent need to identify novel therapies to improve quality of life and reduce economic burden on the healthcare system.

To identify novel therapeutic targets, we must first begin to understand the cellular complexity of the bone marrow niche and how cellular function is regulated within the bone tissue. Bone-resident cells, such as skeletal progenitors and their descendants, are critically influenced by extrinsic signals derived from the local microenvironment. Previous studies have identified hypoxia as a key microenvironment factor in bone. Thus, the ability to target the hypoxic bone marrow niche presents an attractive and untapped potential for regenerative medicine.

Much of the work investigating the role of hypoxia and HIF signaling have focused on mature osteoblast and chondrocyte populations. In contrast, studies investigating the contribution of HIF signaling on skeletal progenitors and marrow adipocyte populations are scarce. In this dissertation, I investigate the role of hypoxia and HIF signaling in skeletal lineage cells, chiefly skeletal progenitor cells and marrow adipogenic lineage cells. Using cellular, genetic, and pharmacological-based approaches, I characterize the roles of HIF-1α and HIF-2α in both homeostatic and pathological contexts in the aforementioned cell populations.

First, I propose an optimized cell-based system to investigate the function of skeletal progenitors in vitro. Here, I highlight the limitations of current in vitro isolation techniques and introduce a relatively simple method of bone marrow stromal cell purification using hypoxia. Using this system, I assess how skeletal progenitors respond to hypoxic cues and interrogate skeletal progenitor cell differentiation and functional responses in my subsequent research. Next, using genetic and pharmacological approaches, I investigate the role of HIF-2α in bone formation following radiation-injury where I identify HIF-2α as a negative regulator of bone recovery. Additionally, with the assistance of my collaborators, I develop and characterize a bone-targeting nanocarrier to ameliorate radiation-induced bone loss. Lastly, I detail early work I conducted to investigate the role of HIF signaling in marrow adipogenic lineage cells. Here, I establish and characterize animal models to determine how hypoxia and HIF signaling influences adipogenic lineage commitment and expansion in an early and mature marrow adipogenic population.

In summary, this dissertation aims to expand our limited understanding on how the hypoxic bone microenvironment and HIF signaling regulate skeletal lineage cells in vivo, with a special focus on skeletal progenitor and marrow adipogenic populations. In terms of boarder impacts, understanding the signaling networks that regulate bone homeostasis and recovery processes will not only expand our basic understanding of the molecular mechanisms underlying skeletal development, but also provide novel insights for developing therapies to treat bone loss.

Regulation of cell growth is controlled by a variety of factors, including a number of oncogenes and tumor suppressors. PTEN is an inositol phosphatase that regulates cell growth by hydrolyzing the phospholipid products of PI3K. PTEN is mutated in a number of cancers, leading to its characterization as an important tumor suppressor. Recent data indicate that PTEN may also perform important functions that are independent of its phosphatase activity, most notably within the nucleus. Studies in this thesis addressed a novel role for PTEN in the regulation of the cellular response to hypoxia.

PTEN overexpression significantly increased hypoxic gene expression independent of its catalytic activity, while shRNA-mediated silencing of PTEN significantly inhibited hypoxia-mediated HRE-luciferase activity. Nuclear-localized PTEN was more effective in promoting HRE activity than nuclear-excluded PTEN. These results suggested a scaffolding function of PTEN in the hypoxic nucleus. To identify specific gene targets regulated by PTEN in hypoxia, a custom oligo-array consisting of 46 hypoxia-responsive genes was utilized following both gain- and loss-of- PTEN function. Based on real-time quantitative results, PTEN positively regulated genes involved in metabolism (PFKFB3, PFKFB4, ALDOA, PGK-1), oxygen supply (VEGFA, EPO), cell growth (Tgf-a, TERT, cyclin D1, BNIP3), motility (E-cadherin) and transcription (DEC2). A single missense mutation at isoleucine 224 (I224M) of PTEN, however, abrogated the ability of PTEN to regulate the hypoxia response without affecting its lipid phosphatase activity. PTEN has previously been shown to bind to the co-activator p300 and to affect p53 acetylation and stabilization. As p300 is also a co-activator for the HIF proteins, we hypothesized that PTEN's association with p300 would promote the HIF/p300 complex to positively regulate hypoxic gene transcription. Overexpression of PTEN-WT extended the half-life of p300 and histone acetyltransferase activity of p300 in hypoxia, while overexpression of PTEN-I224M or PTEN silencing decreased both. In vivo, these effects resulted in a significant increase in hypoxic area in PTEN-null tumors compared to tumors expressing endogenous levels of PTEN, suggesting an inability to mount a hypoxia response necessary for revascularization of the tissue. PTEN's effect on p300 extended to other functions of p300 outside of the hypoxia response, most notably p300's role in p53 stability and p53-mediated gene transcription. Overexpression of PTEN resulted in an increase in p53 reporter activity following DNA damage (mitomycin C treatment). PTEN silencing or overexpression of PTEN-I224M resulted in abrogation of these effects. Taken together, these findings demonstrate that PTEN is required for the hypoxia response and they suggest that PTEN acts as a scaffold for p300 and the HIF machinery in the hypoxic nucleus independent of its canonical lipid phosphatase activity. These results may have important implications for the treatment of tumors in which PTEN is lost or mutated. The potential use of PTEN-I224M as a therapeutic is also discussed

Hypoxia is a profound stressor of the central nervous system implicated in numerous neurodegenerative diseases. While it is increasingly evident that the early effects of hypoxia cause impairment at the level of the axon, the precise mechanisms through which hypoxia compromises axonal structure and function remain unclear. However, links between hypoxia-induced axonopathic disease and the amyloid cascade, as well as the upregulation of amyloid precursor protein (APP) and amyloid beta (Aβ) by hypoxic stress, give rise to the hypothesis that proteolytic cleavage of APP into Aβ may be specifically responsible for axonopathy under conditions of hypoxia.

The goal of this dissertation was thus to understand dependence of hypoxia-induced axonal morphological and functional impairment on APP cleavage and the production of Aβ. I have developed a model of hypoxia-induced axonopathy in retinal explants. Using this model, I have experimentally addressed the core hypothesis that APP cleavage, and in particular the formation of Aβ, is necessary and sufficient to mediate morphological and functional axonopathy caused by hypoxia. I have found that there is a dissociation between the mechanisms responsible for hypoxia-induced morphological and functional impairment of the axon in the explanted retina, with the former being dependent on APP-to-Aβ processing and the latter likely being dependent on cleavage of a non-APP substrate by the enzyme BACE1. These findings shed light on mechanisms of hypoxia-induced axonopathy.

Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.

Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions.

The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.

The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.

In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. Additionally, cancer cells exposed to these stresses are more resistant to therapies, more likely to metastasize and often are worse for patient prognosis. While the presence of these stresses is generally negative for cancer patients, since these stresses are mostly unique to the TME, they also offer an opportunity to develop more selective therapeutics. If we achieve a better understanding of the adaptive mechanisms cancer cells employ to survive the TME stresses, then hopefully we, as a scientific community, can devise more effective cancer therapeutics specifically targeting cancer cells under stress. To systematically identify genes that modulate cancer cell survival under stresses, we performed shRNA screens under hypoxia or lactic acidosis. From these screens, we discovered that genetic depletion of acetyl-CoA carboxylase alpha (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Furthermore, the loss of ACLY or ACC1 reduced the levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate was paradoxically increased under hypoxia when ACC1 or ACLY were depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulated the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

Aquatic systems are impacted by a wide variety of natural and anthropogenic stressors. Laboratory studies often focus on impacts of a single stressor, ignoring how these stressors may interact. This dissertation focuses on the interactions between polycyclic aromatic hydrocarbons (PAHs) and two physical stressors (hypoxia and solar radiation). PAHs are ubiquitous environmental pollutants that occur in the environment as complex mixtures, the components of which may interact. Some PAHs are agonists of the aryl hydrocarbon receptor (AhR), which regulates expression of a number of genes (such as CYP1A) involved in metabolism, often of the same compounds that induced the AhR. PAHs that are AhR agonists have been shown to interact synergistically with PAHs that inhibit activity of CYP1A, inducing developmental deformities in fish. AhR shares a dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT), with the protein responsible for regulating hypoxia-induced gene expression, hypoxia-inducible factor 1á (HIF1α), possibly resulting in competition between the two pathways. Competition would result in decreased induction of CYP1A under hypoxic conditions, possibly resulting in synergistic embryonic toxicity between AhR agonist PAHs and hypoxia. In this dissertation, the results are presented of experiments regarding the occurrence of AhR/HIF1α crosstalk in fish and the developmental consequences of co-exposure to hypoxia and PAHs. In vitro testing revealed competition for ARNT in which HIF1α appeared to out-compete AhR. Induction of an AhR-responsive luciferase reporter by several AhR agonists (benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF), and polychlorinated biphenyl (PCB126)) was reduced under hypoxia and this effect was eliminated by overexpression of ARNT. Induction of a HIF1α-responsive reporter was unaffected by BkF and PCB126. BaP caused an ARNT-independent decrease in both basal and induced HIF1α reporter activity. Attempts to verify this crosstalk pattern in vivo revealed that BaP-induced CYP1A expression was further increased under hypoxic conditions. Induction of hypoxia-inducible genes VEGF and LDHa were unaffected by BaP. The effect of hypoxia on CYP1A expression was not repeated with BkF or pyrene (PY) and the exact conditions that result in hypoxic changes in CYP1A expression remain to be determined. Embryonic toxicity in zebrafish (Danio rerio), including pericardial edema, heart malformations, and death were synergistically induced by hypoxia and BaP, BkF or complex, environmentally relevant PAH mixtures. There was no effect of hypoxia on PCB126 toxicity and hypoxia protected from the pericardial edema induced by PY. CYP1A knockdown mimicked the effects of hypoxia on BkF and PY toxicity and even further exacerbated BkF toxicity under hypoxic conditions, suggesting a role for metabolism in the toxicity. Additionally, since two CYP1A inhibitors (fluoranthene (FL) and á-naphthoflavone (ANF)) had been previously reported to interact synergistically with hypoxia, three other CYP1A inhibitors (dibenzothiophene, carbazole and 2-aminoanthracene) were tested. None of them induced toxicity in combination with hypoxia, indicating that CYP1A inhibition was not the reason for the interaction of FL and ANF with hypoxia.

A second stressor known to interact with PAHs to induce toxicity is solar radiation. While most studies of the effects of solar radiation on PAH toxicity have focused on the effects of UV light on PAHs already absorbed by an organism (photoactivation), less attention has been paid to the toxic effects of photomodified PAHs. We exposed carbazole to ambient sunlight and subsequently exposed developing zebrafish to the resulting mixture. Photo-exposed carbazole caused developmental toxicity including edema, heart malformations, craniofacial malformations and death that were not caused by parent carbazole; these effects were severely exacerbated by hypoxia and significant mortality was also observed. Additionally, photo-exposed carbazole induced expression of CYP1A and GSTp, likely resulting from agonism of the AhR and toxicity of this mixture was alleviated by morpholino knockdown of AhR. Some photoproducts were identified, but none of them appeared to be involved in the toxicity or supposed AhR induction observed with photoexposed carbazole. The results of these experiments underscore the importance of consideration of the interactive effects of physical and chemical stressors when assessing risks to wildlife populations inhabiting polluted areas.

At the present time, here is no approved medical countermeasure (MCM) for mitigating or treating pneumonitis/fibrosis following acute radiation exposure. Since it is neither ethical nor feasible to evaluate potential MCMs against radiation injury in the clinical setting, the FDA permits MCM licensure under an alternative drug development pathway ("Animal Efficacy Rule") that relies on the predictive validity of animal models. The purpose of the current project was to design a research platform that addresses many of the critical knowledge gaps associated with successful adherence to the FDA Animal Rule.

In these studies, we evaluated the dose-response relationship for survival and function injury among CBA/J, C57L/J, and C57BL/6J mouse strains. These strains were previously identified to represent the full spectrum of pulmonary pathology associated with acute radiation exposure to the thorax. We next evaluated ultrastructural pathology to identify differences in tissue response among strains as early as twenty-four hours after radiation. Global differential gene expression analysis was utilized to identify the major signaling pathways and genes associated with development of radiation pneumonitis and/or fibrosis by exploiting the phenotypic differences in radiation-injury among strains. Genes with significant differences were validated by quantitative real-time PCR and their protein products validated by western blot. Finally, we performed longitudinal analysis of hypoxia-associated gene expression to elucidate the natural history of disease progression in "fibrosis prone" C57BL/6J mice.

In these studies, we identified significant differences in the dose-response, temporal onset, disease progression, and pathologic manifestations of radiation lung injury among murine strains. The severity of ultrastructural damage at twenty-four hours also differed among strains indicating the early tissue response to the radiation insult was dissimilar. A significant difference was found in gene expression among strains. The most interesting differences were associated with the acute-phase response, iron homeostasis, cell cycle/proliferation, and cell death. Lastly, hypoxia-associated gene expression, including HIF-1alpha; and HIF-2alpha; mRNA and protein stabilization, was dynamically altered during the temporal course of radiation pathogenesis in the "fibrosis-prone" C57BL/6J mice. As the C57BL/6J strain is more "resistant" to radiation-induced lung injury, a better understanding of the pathways involved in tissue response to radiation in this strain might elucidate the mechanisms that make the lungs of this strain significantly more radiotolerant than their counterparts.

The research platform developed in this project provides essential information to interpret and define the complex interrelationships in clinically relevant models of the human response to potentially lethal irradiation and treatment. The overall goal is to provide a rigorous scientific platform for MCM development under the Animal Efficacy Rule with reasonable expectation that MCMs acquired for the Strategic National Stockpile will effectively prevent, treat, or mitigate radiation-induced lung injury and improve survival among the exposed population.

Functional imaging gives clinicians the ability to monitor breast cancer progression and response to therapy. Modern techniques such as Positron Emission Tomography (PET) has allowed for clinicians to visualize the metabolic need of breast cancer and track it longitudinally. However, these techniques are expensive, technologically complex and not easily implemented in rural areas. To add to the difficulty, breast cancer is a highly heterogeneous disease. The heterogeneity means that a single therapy is not always applicable to all patients and every patient requires an individual treatment plan. Being able to first diagnose breast cancer, and then monitor its response to therapy in a cost-effective manner is imperative to improve the survival of patients with this disease.

Optical techniques such as fluorescence are ideal for these applications since they can be fast and implemented with portable technology. These techniques use differences in light interaction with tissue to allow for abnormality detection. This dissertation tests the hypothesis that the fluorescent molecularly specific agents, protoporphyrin IX (PpIX) and 2-NBDG, which utilize metabolic alterations caused by cancer, can be used for ubiquitous breast cancer differentiation and therapy monitoring. Confocal microscopy is used to demonstrate the applicability of both agents in vitro to breast cancer cells regardless of phenotype.

First, 5-aminolevulinic acid (ALA) was incubated with cells causing an increased cellular production of the heme prequel, protoporphyrin IX (PpIX). In cancer cells, the production of PpIX is higher and allows for detection from normal after a 2 hour incubation period. The PpIX was then detected via confocal microscopy and the change in fluorescence intensity between ALA-induced PpIX and controls was measured. A spectroscopy measurement is also completed on a second experimental set of cells to demonstrate that collection of single spectra, post-ALA administration, can discriminate breast cancer cells from normal mammary epithelium.

2-NBDG is a fluorescent glucose analogue that is follows the metabolic pathway of glycolysis, similarly to D-glucose and fluorodeoxyglucose (FDG). Greater accumulation of 2-NBDG can occur in as little as 20 minutes in cells with higher glycolytic demand, which is commonly associated with cancer and hypoxic cells. The shorter incubation period required for 2-NBDG makes it ideal for clinical use, and 2-NBDG was therefore tested further.

2-NBDG uptake was used to detect changes in cellular glycolysis after anti-cancer and endocrine therapy. The anti-cancer therapies, lonidamine and a-cyano-hydroxycinnamate (a-Cinn), which increased and decreased glycolysis, respectively were tested on a subset of breast cancer cells. Lonidamine directly inhibits the metabolism of 2-NBDG and inhibited its uptake. a-Cinn stimulates glycolysis by inhibiting the monocarboxylate transporter 1 preventing lactate from entering as a source for oxidative phosphorylation. 2-NBDG was concurrently increased after a-Cinn treatment. Observation of changes in downstream glycolysis has been determined after the estrogen receptor therapy, tamoxifen, in breast cancer cells. Sixty percent of all breast cancers are estrogen receptor positive (ER+) and have the potential to respond. Known ER+ cells, MCF7, and ER- cells, MDA-MB-435, were treated with tam. 2-NBDG was used to determine therapeutic responders from non-responders by measureable differences in fluorescence uptake.

Finally, the effect of hypoxia, low oxygenation, on 2-NBDG uptake is discussed. The cellular response to hypoxia, known as the Pasteur Effect, causes an increase in glycolysis. Hypoxia is shown in vitro to increase 2-NBDG uptake. Simulated, chronic and cycling hypoxia were completed in vitro with subsequent increases in 2-NBDG as well. Cycling hypoxia has been previously shown to have a greater impact on tumor environment and was implemented in an in vivo murine dorsal window chamber mammary carcinoma model. The uptake of 2-NBDG in tumor and normal tumor-free tissue was tested and 2-NBDG discriminated normal from tumor in a normal oxygen environment. An increase in 2-NBDG was demonstrated after cycling hypoxia in tumor and normal tissue. However, by including hemoglobin saturation data, cycling hypoxic tumor tissue can be discriminated from cycling hypoxic normal tissue and normoxic tumor tissue. From these experiments, the applicability of 2-NBDG as a method to monitor changes in glycolysis and its increased potential by including hemoglobin

saturation measurements is demonstrated.

Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.

Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.

The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.

In vivo imaging is an important scientific tool for studying bio-molecular interactions, but lack of preserved functionality during imaging restricts scientists’ abilities to gain critical knowledge. Structure can be preserved while using high-resolution optical imaging by utilizing window chambers in murine models1, yet the use of anesthesia for immobilization is problematic. Anesthesia affects tissue oxygenation2, blood cell velocities3, immunosuppression4, and allowable duration of imaging5–thus its usage restricts in vivo bio-molecular imaging accuracy and duration.

Developing a portable imaging system that attaches to murine dorsal window chambers enables imaging without anesthesia, avoiding previous drawbacks of window chamber models. A raspberry pi camera (RPI-CAM-V2, Raspberry Pi) was modified for microscopy and used alongside 3D printed panels for attaching the camera, optical filters, and LED light source to murine window chambers. Multiple applications for the portable system were developed, each requiring their own setup of filters and stimulating LEDs. The system is powered by a Raspberry Pi 3 Model B single-board computer (RASPBERRYPI3-MODB-1GB, Raspberry Pi), allowing for stream-lined data acquisition.

Imaging tissue oxygenation was the first application developed for the portable system. Oxygen sensing boron nanoparticles were injected into window chambers, while a UV LED was used to stimulate fluorescent and phosphorescent signals. When stimulated by UV light, the boron nanoparticles emit fluorescence and phosphorescence. Fluorescence is stable regardless of oxygenation, while phosphorescence signal from the nanoparticles is quenched in the presence of higher oxygenations. The ratio of fluorescence to phosphorescence was used to calculate oxygen concentration maps of window chamber tissue. Tissue oxygenations in awake and anesthetized mice inhaling varied oxygen concentrations were analyzed. In 5 awake nude mice inhaling 20% O2, the median partial pressure of oxygen was measured as 49 mmHg within their window chambers. From a one-tailed t-test with a false positive correction, 3 of the mice had significantly higher (p ≤ 0.05) tissue oxygenation while anesthetized compared to the awake measurements.

Developing the portable systems ability to image blood cells was another focus of this project. Blood cells were visible with white LED exposure. A frame rate of 30 frames/second was adequate for tracking cell motion while allowing for the highest resolution possible with the system. Blood cell velocities in a mouse awake and anesthetized were analyzed, while also observing change in blood cell velocities during sepsis that was induced by cecal ligation puncture (CLP). Three days after CLP, the mean awake blood cell velocity was measured as 0.21 ± 0.03 mm/s, while the mean anesthetized blood cell velocity was measured as 0.080 ± 0.002 mm/s. Six days after CLP, the awake measurement had reduced to 0.019 ± 0.005 mm/s, while the anesthetized measurement was reduced to 0.031 ± 0.002 mm/s (91% decrease in awake measurement, 61% decrease in anesthetized measurement). A two-way ANOVA on the factors of anesthesia and time post-CLP performed on multiple vessel regions calculated significance (p ≤ 0.05) for both of these factors on blood cell velocities within the pilot mouse’s window chamber.

Noting the differences between data collected on awake and anesthetized mice, our system has been validated as a tool for real-time imaging of tissue without the observed effects of anesthesia. By avoiding anesthesia, the developed device allows for continual data acquisition to increase from hours to days. The system is generalizable, and while only two applications are presented in this study, the system could be modified for imaging fluorescently labeled cells/proteins for other bio-molecular interactions.

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