Browsing by Subject "Mouse model"
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Item Open Access Dissecting Tumor Response to Radiation Therapy Using Genetically Engineered Mouse Models(2015) Moding, Everett JamesApproximately 50% of all patients with cancer receive radiation therapy at some point during the course of their illness. Despite advances in radiation delivery and treatment planning, normal tissue toxicity often limits the ability of radiation to eradicate tumors. The tumor microenvironment consists of tumor cells and stromal cells such as endothelial cells that contribute to tumor initiation, progression and response to therapy. Although endothelial cells can contribute to normal tissue injury following radiation, the contribution of stromal cells to tumor response to radiation therapy remains controversial. To investigate the contribution of endothelial cells to the radiation response of primary tumors, we have developed the technology to contemporaneously mutate different genes in the tumor cells and stromal cells of a genetically engineered mouse model of soft tissue sarcoma. Using this dual recombinase technology, we deleted the DNA damage response gene Atm in sarcoma and heart endothelial cells. Although deletion of Atm increased cell death of proliferating tumor endothelial cells, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. In addition, the ATM inhibitor NVP-BEZ235 selectively radiosensitized primary sarcomas, demonstrating a therapeutic window for inhibiting ATM during radiation therapy. Sensitizing tumor endothelial cells to radiation by deleting Atm prolonged tumor growth delay following a non-curative dose of radiation, but failed to increase local control. In contrast, deletion of Atm in tumor parenchymal cells increased the probability of tumor eradication. These results demonstrate that tumor parenchymal cells rather than endothelial cells are the critical targets that regulate tumor eradicaiton by radiation therapy.
Item Open Access Improved Pre-clinical Radiation Treatment Techniques for a Novel Mouse Model of Head-and-neck Cancer(2019) Chen, DeqiMice are the predominant animal model used in radiation therapy research for investigating radiobiological kinetics and evaluating new therapeutics to achieve a higher therapeutic ratio in the clinic. A novel carcinogen-induced and genetically engineered head and neck squamous cell carcinoma mouse model was developed at Duke to study head and neck cancer, one of the most widely spread cancers in the world. However, platforms that are able to perform precise and reproducible radiation therapy on these mice to mimic human radiation therapy are lacking. To address this issue, a platform based on the X-RAD 225Cx orthovoltage irradiator was developed. 3D printing technique was used to generate imaging phantoms, immobilization devices, and blocks. A simulation was conducted to optimize imaging protocol. Results were verified on the measurement on both the 3D-printed phantom and the actual mouse. Prior to irradiation, mice were placed on the immobilization device in a supine position, and the isocenter was determined by the position of the device since the irradiator does not have a laser localizer system. The performance of the immobilization was obtained by scanning several mice separately at various time points, ranging from several hours post-imaging to two months post-imaging. In order to make up the deficiency that irradiator only have rectangular and circular collimators which cannot provide moderate protection for organs at risk. Blocks with 3% transmission were designed based on the contours of central nervous system by a state-of-art program, BlockGen.
A protocol was developed for immobilization and image acquisition. 60 kVp was found to give the highest contrast of iodine, so it was set as the tube voltage for image acquisition. The deviations of positioning, i.e. the same mouse in separate scanning, are measured as 0.22±0.44 mm in LR axis, 0.15±0.30 mm in PA axis, and -0.24±0.25 mm in IS axis. Blocks with a 1.5 mm margin which can shield brain and spinal cord even in the worst case, were printed for opposed lateral beams; they were verified on fluoroscopy.
The block system was modified to eliminate potential human errors. Comparison on brain and spinal cord among different mice showed the largest deviation in 2.6 mm, however, with manually selection of the middle one, 1.5 mm margin is enough to shield central nervous system. Indicating that a generic block could be used in the experiment that does not require a very accurate treatment. The generic block can significant save time and effort for preclinical radiation treatment experiment. In this study, a platform that is capable of enhancing contrast imaging and allowing precise radiation therapy to be performed on genetically-engineered mice with head and neck cancer has been developed. This paves the way for more accurate head and neck mice model radiation therapy studies. In addition, the platform could be used in other types of preclinical studies.
Item Open Access Leveraging copper chelators as targeted therapy for BRAFV600E mutant thyroid cancer(2018) Xu, MengMengThe incidence of thyroid cancer, and in particular papillary thyroid cancer (PTC), is rising faster than that of any other malignancy in the United States. Thyroid cancer is now the most common endocrine cancer and the fifth most common cancer in women. While most thyroid cancers are treated effectively with surgical resection and radioiodine therapy, survival drops precipitously in metastatic or radioiodine-resistant disease. 60% of papillary thyroid cancers (PTC) have an oncogenic V600E mutation in the kinase BRAF, which leads to a constitutively active and oncogenic kinase. This mutation has been associated with as well as a 2.14-fold increase in recurrent/persistent disease. Excitingly, inhibitors against this mutant kinase or its substrates, the MEK1/2 kinases, can prolong progression free survival or stabilize disease in radioiodine-refractory thyroid cancer patients. However, the indolent nature of PTC may be a challenge to the clinical adaption of these inhibitors, as the financial and physical toxicities of these treatments may be amplified over the prolonged time-course typical of PTC. MEK1/2 require copper (Cu) for kinase activity and can be inhibited with the well-tolerated and economical Cu chelator tetrathiomolybdate (TM). Cu chelators have been in use for decades in patients with Wilson’s Disease, a disease of Cu accumulation. Unlike current BRAF and MEK inhibitors, Cu chelators can be well tolerated for decades with few side effects, and thus may find use in long-term inhibition of BRAFV600E signaling in PTC.
Here I test the ability of Cu chelator TM to inhibit tumor growth in BRAFV600E-mutant PTC. TM inhibited MEK1/2 kinase activity and transformed growth of human BRAFV600E-mutant PTC cells as well as or more potently than standard-of-care drugs. Consistent with TM deriving its antineoplastic activity by inhibiting MEK1/2, expression of activated ERK2, a substrate of MEK1/2, overcame the ability of TM to suppress growth of BRAFV600E-mutant PTC cells. TM was also effective in a genetically engineered mouse model of BrafV600E-mutant PTC; oral TM reduced tumor burden as well as a clinical BRAF inhibitor. This in vivo effect was attributed to a reduction of phospho-Erk1/2 signaling in the tumors. Additionally, long-term maintenance therapy using TM after cessation of a clinical BRAF inhibitor reduced tumor volume in the same mouse model. Genetic reduction of the Cu transporter CTR1 in developing tumors also trended towards a survival advantage in mice with BrafV600E-mutant PTC. Finally, TM also enhanced the antineoplastic activity of standard of care clinical BBRAF inhibitor drugs. These results support the clinical evaluation of Cu chelation as targeted therapy for BrafV600E-mutant PTC and suggests three possible avenues for clinical exploration: i) as a less toxic monotherapy for BrafV600E-mutant PTC, ii) as long-term maintenance therapy after initial treatment, and iii) as a combination-therapy amplifying the antineoplastic effects of other treatment modalities.
Item Open Access Mycobacterium tuberculosis Surface-binding Antibodies Influence Early Infection Events(2015) Perley, CaseyMycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), is among the leading causes of death from infectious disease world-wide. An intracellular pathogen, M. tuberculosis infects phagocytic cells, and subverts the host immune response, preventing eradication once infection has been established. Even after successful chemotherapy, exogenous re-infection occurs, indicating that sterilizing immune responses are not generated during natural infection. While a TB vaccine exists, it does not alter M. tuberculosis infection rate, rather it prevents the progression from latent TB infection to active TB disease. Vaccines against Haemophilus influenzae and Streptococcus pneumonia protect from bacterial colonization and infection through the induction of antibodies to capsular surface components. This dissertation explores if antibodies to the surface of M. tuberculosis can alter the initial interaction between a bacterium and host cell, leading to a reduction in infection rate.
When pre-mixed with M. tuberculosis prior to in vitro infection of macrophages, or retropharyngeal instillation of mice, monoclonal surface-binding, but not non-surface-binding antibodies, decrease bacterial burden and the number of infected cells within the first twenty-four hour of infection. If administered retropharyngeally prior to aerosol exposure, surface-binding antibodies decreased pulmonary bacterial burden at twenty-four hours post infection in an FcγR independent manner. Despite decreasing early bacterial burden, pre-administration of surface-binding antibodies prior to ultra-low dose aerosol infection did not alter infection rate compared to mice instilled with PBS (Chapters 4 and 5).
Infected humans do not produce high-titer, high-avidity surface-binding antibodies. Plasma from uninfected controls, individuals with latent TB infection, and active TB disease was assayed by ELISA to determine the titer, avidity and IgG/IgM ratio for antibodies to the surface and additional bacterial fraction. In contrast to antibodies to bacterial fractions, individuals with active TB disease had decreased avidity, and no augmentation of the IgG/IgM ratio for antibodies to the live M. tuberculosis surface, as compared to uninfected controls (Chapter 3).
Overall these findings demonstrate that surface-binding monoclonal antibodies alter early infection events, both in vivo and in vitro, though the magnitude of protection was not sufficient to decrease M. tuberculosis infection rate. Additionally, the failure of humans to generate high-titer, high-avidity surface-binding antibodies after infection indicates and that induction of surface-binding antibodies may be an appropriate target for future vaccines.
Item Open Access The Development of an In Vivo Mobile Dynamic Microscopy System that Images the Hypoxic Microenvironments of Cancerous Tumors via Fluorescent and Phosphorescent Nanoparticles(2017) Rickard, AshlynHypoxic tumor microenvironments have a clear correlation with a lack of radiosensitivity and diminished therapy response. This relationship can be described through the use of fluorescent and phosphorescent nanoparticles optically imaged in a mouse model. Through the use of this ratiometric oxygen sensing, the hypoxic state of the cancerous tumor can be compared. Normally, the microscope imaging system requires the mouse to be imaged under anesthesia and data recorded for a short amount of time. This has led to challenges in clearly defining the oxygen saturation levels in hemoglobin because the anesthesia can affect the tumor vascular dynamics. Moreover, it is desirable to track blood flow and oxygenation changes over a longer period of time in order to characterize the dynamics of cycling hypoxia and therapeutic response. Therefore, a mobile imaging apparatus has been designed and built to directly attach to the dorsal skinfold window chamber installed on nude murine models. Current progress includes quantifiable ratiometric oxygenation in boron nanoparticle solutions imaged under UV light with the mobile unit. The concept has also been successful in in vivo studies for anesthetized mice. The mobile unit is capable of resolving vasculature and is sensitive enough to record nanoparticle emissions originating from tissue in a mouse window chamber model. This system will use dynamic microscopy to image the tumor’s hypoxic environment on un-anesthetized mice and yield insight into tumor biology and therapeutic response.
Item Open Access The Genetic and Epigenetic Landscape of Oxytocin Signaling in the Social Brain of Humans and Mice(2021) Siecinski, Stephen KennethHuman beings are inherently social. As we grow, the interactions we have with those around us become the foundation of the relationships with our families, friends, educators, and caretakers. Aberrant social behavioral traits can put a strain on these relationships and negatively impact an individual's quality of life. When severe, these disruptions represent core etiological features of many neurodevelopmental and behavioral disorders, including addiction, schizophrenia, and autism spectrum disorder (ASD).
Advances in the fields of psychology, psychiatry, neuroscience, and the behavioral sciences have developed a wide range of techniques to disentangle the biological and behavioral components of complex social traits, often with the goal of developing targeted interventions to improve outcomes for affected individuals. Still, there remains a massive unmet need for pharmaceutical interventions that ameliorate these aberrant social phenotypes, and current interventions are costly and labor intensive. A particularly promising candidate to address this unmet need is \Oxy, an endogenously expressed neuropeptide, with demonstrated pro-social effects in both humans and animal models, and low incidence of adverse effects. However, clinical trials of \oxy in ASD have had mixed results, likely due to a range of issues from inadequately powered study designs, difficulty in quantifying changes in social behavior over time, heterogeneous study populations, and an inadequate understanding of the underlying mechanisms of \oxy signaling in the brain.
The work described in this dissertation aims to address some of these gaps in knowledge. First, the results of a collaboration with a phase-2 clinical trial of \inoxy are discussed, in which I contributed to identifying a number of biological markers that were associated with \enoxy production in human participants. I discuss how this may be an important component of predicting an individuals response to \exoxy.
I will then discuss our findings in the \cd{} mouse model of ASD-like behaviors. These mice exhibit a unique socially divergent phenotype in which some mice will display very little social motivation within litters while the others behave normally. Importantly, these antisocial mice are responsive to treatment with \exoxy, but the mechanisms behind their unique social phenotype and responsiveness to \oxy remain unknown. I quantified broad patterns of differential gene expression and DNA methylation in the brains of \cd{} mice and their closely related but highly social \cs{} counterparts. This work identified a pattern of differential gene expression in the hippocampus of \cs{} mice that may offer insights into the nature of their naturally occurring social divergence.
Collectively, the findings of these two projects provide valuable information to the field of \oxy and ASD research. The results of the human trial can be used to guide new studies into the endogenous regulation of \oxy, providing potential targets for future interventions to responsive candidates. The results of the mouse experiments identified a myriad of underlying biological pathways that distinguish \cd{} from \cs{} mice, which can serve as the foundation for new targeted hypotheses and to test expanded pharmaceutical interventions to enhance the effects of \exoxy.