Browsing by Author "Kelly, John James Patrick"

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    Characterization and Therapeutic Manipulation of Glioblastoma-Associated Microglia, Macrophages, and Cytokines to Improve Prognosis
    (2020-01-30) Poon, Candice Cassia; Kelly, John James Patrick; Yong, Voon Wee; Cairncross, Gregory J.; Chan, Jennifer A. W.
    Glioblastoma comprises the majority of malignant primary adult brain tumors and has one of the worst survival rates of all cancers. The poor prognosis is a product of the transformed cells acting in collusion with the tumor microenvironment, a large component of which are inflammatory infiltrates. Glioblastoma-associated microglia and macrophages (GAMMs) predominate this immune infiltrate, making them important considerations for tumor biology and therapy. These innate immune cells are meant to participate in tumor surveillance and eradication, but they become compromised by glioblastoma and exploited in the process. However, the phenotypic variability of GAMMs has not been well characterized in the two major groups of glioblastoma, isocitrate dehydrogenase (IDH)-mutated and wildtype tumors. Furthermore, whether pro-inflammatory, anti-tumor cytokines secreted by healthy microglia and macrophages can alter the progression of glioblastoma by targeting cancer stem cells known as brain tumor-initiating cells (BTICs) is not well-developed as a therapy. Lastly, there have been a paucity of glioblastoma-associated microglia and macrophage (GAMM) studies conducted with human tissue in favor of more accessible murine models. In this thesis, we sought to address these understudied topics. We developed an immunofluorescent imaging protocol that quantifies the expression of GAMM inflammatory markers and identifiers at the individual-cell level, although this method could be adapted to quantitate any immunofluorescent marker in slide-mounted tissues. Using this technique, flow cytometry, and analysis of publicly available single-cell RNA sequencing databases we discerned similarities and differences between human GAMMs in IDH-mutant and wildtype-glioblastomas. We found that macrophages may be the drivers of the anti-inflammatory, pro-tumor phenotype displayed by immune cells in the glioblastoma microenvironment. IDH-mutant tumors which have more favorable prognoses in contrast to wildtype tumors possessed more pro-inflammatory microglia. Not only does this suggest immunostimulants may be particularly promising therapies, but peripheral monocyte-derived macrophages may be targeted systemically instead of needing to create a blood brain barrier penetrant drug. We also investigated tumor necrosis factor alpha (TNF), a pro-inflammatory cytokine secreted by microglia and macrophages, as an anti-brain tumor-initiating cell (BTIC) therapy. Not only does TNF directly inhibit BTICs as a monotherapy, but in combination with the frontline chemotherapy for glioblastoma, temozolomide, acts in a combinatorial fashion to channel BTICs towards cellular demise. Significant challenges remain but treatments that affect and/or are derived from glioblastoma-associated microglia and macrophages hold considerable promise to improve the prognosis for patients with this disease.
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    Culture and characterization of human brain tumor stem cells
    (2011) Kelly, John James Patrick; Weiss, Samuel
    Diffusely infiltrating gliomas, of which glioblastoma (GBM) is most common, are highly aggressive brain tumors with dismal outcomes. Application of the Cancer Stem Cell (CSC) hypothesis to glioma led to the discovery of Brain Tumor Stem Cells (BTSCs) and a direct link to Neural Stem Cells (NSCs). In this thesis I begin by trying to identify characteristics that distinguish BTSCs from NSCs. I cultured cells isolated from GBMs, using the neurosphere culture system, in order to understand their growth requirements. GBM BTSCs proliferated in the absence of exogenous mitogenic stimulation and gave rise to multipotent GBM spheres that were capable of self-renewal. Epidermal growth factor and fibroblast growth factor-2 enhanced GBM BTSC survival and proliferation. Implantation of exogenous mitogen independent GBM BTSCs led to the formation of highly invasive intracranial tumors in immunocompromised mice. Thus, exogenous mitogen independence is one characteristic that distinguishes GBM BTSCs from NSCs. I then ask whether BTSCs can be isolated from other gliomas, specifically oligodendroglioma. Investigating the biology of oligodendroglioma, and its characteristic combined deletion of chromosomal arms 1 p and 19q, has been hampered by the lack of cell lines that harbor these traits. Cells from anaplastic oligodendrogliomas cultured in serum-free conditions, followed by serial propagation and expansion, led to the establishment of permanent cell lines that maintained the genetic signature of the parent tumors. Furthermore, these oligodendroglioma cells displayed features of BTSCs in vitro. These lines may be important tools for understanding the biology of oligodendroglioma and the function of their defining genetic traits. Finally, I employed the neurosphere culture system in order to generate 70 cell lines that collectively comprise a cellular model system of GBM. This robust model system harbors characteristic genetic abnormalities of GBM that permit sub-grouping of the cell lines. Importantly, this system recapitulates the heterogeneity of GBM and facilitates both in vitro and in vivo studies of phenotypic and molecular sub-groups. By coupling cell line generation with molecular characteristics and patient outcome, the model system has direct clinical relevance. In sum, the results demonstrate that glioma heterogeneity is evident in BTSCs isolated from individual tumors. This unique experimental system will permit elucidation of mechanisms that underlie glioma BTSC biology together with preclinical studies that evaluate novel therapeutic strategies.
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    Magnetic Resonance Imaging of Tumor Growth and Leukocyte Infiltration in Brain Tumors after Stimulation of the Innate Immune System
    (2018-06-26) Yang, Run Ze; Yong, Voon Wee; Dunn, Jeffrey F.; Pike, G. Bruce; Kelly, John James Patrick
    Glioblastoma (GBM) is one of the deadliest brain cancers, and immunotherapy may be a promising treatment option. One of the challenges with immunotherapy in GBM is the lack of biomarkers that can be used to detect treatment response early on during treatment. The goal of this thesis is to use MRI to determine whether vitamin B3 (niacin) can suppress GBM growth, and develop a MRI method capable of detecting whether niacin stimulates monocyte trafficking to the brain tumor. We used human and animal derived brain tumor initiating cells for the studies. We used conventional anatomical MRI to show that niacin is capable of reducing tumor growth in the mouse. We showed that innate immune cells can be labeled by ferumoxytol, an iron based MRI contrast agent. This can be used to detect the effects of the innate immune stimulating drugs (Amphotericin B and niacin) in a mouse model of GBM. This is done by intravenously injecting ferumoxytol, and then quantifying iron changes in the tumor with susceptibility MRI. We expanded upon our results to investigate the mechanism of the iron changes after ferumoxytol injection. We used clodronate liposomes to deplete monocytes in the blood and Kupffer cells in the liver and showed that there is minimal passive leakage of ferumoxytol into the tumor, suggesting that ferumoxytol must be carried into the tumor by a phagocytic cell such as monocytes. We implemented another method of measuring ferumoxytol, quantitative susceptibility mapping (QSM), and used it in conjunction with T2* mapping to investigate whether the magnitude of ferumoxytol enhancement is related to future tumor control in niacin treated animals. We found that the degree of ferumoxytol enhancement is directly related to niacin’s ability to control tumor growth, with high ferumoxytol enhancement corresponding to smaller tumors. This thesis showed that niacin treatment can reduce tumor growth by stimulating monocytes. In addition, using ferumoxytol to track the innate immune system is a promising imaging tool to aid the assessment of immunotherapy treatment response in GBM.