Iron profiling in Glioblastoma

Author:

Shesh, Bhavyata B

Graduate Program:

Biomedical Sciences

Degree:

Doctor of Philosophy

Document Type:

Dissertation

Date of Defense:

September 14, 2023

Committee Members:

Jeffrey Sundstrom, Major Field Member
James Connor, Chair & Dissertation Advisor
Dhimant Desai, Outside Field Member
Mohammad Ali, Outside Unit Member
Alicia McDonald, Special Member
Rhoda Joseph, Special Member
Lisa M Shantz, Special Signatory

Keywords:

Glioblastoma
Iron
invasion
sex differences
T cells
CD8+ T cells

Abstract:

Ferritin is a 450 kDa protein composed of 24 subunit polypeptides of two kinds: heavy (H) and light (L) chains. The ratio of these chains varies depending on the tissue in which ferritin is found. Ferritin protein is expressed ubiquitously and was historically believed to be solely involved in cytosolic intracellular iron sequestration, preventing the availability of excess redox-active iron for Fenton chemistry and the production of reactive oxygen species that can damage cells. However, recent studies have revealed additional functions beyond iron sequestration. In addition to its role in iron sequestration, ferritin has been found to act as an iron-delivery molecule. It has been shown that extracellular ferritin can deliver iron to organs throughout the body, including the brain. Due to its ability to carry more iron atoms than the traditional iron delivery protein transferrin, ferritin uptake is believed to be occurring in tissues or conditions that require higher levels of iron, such as during developmental stages and cancer. However, the uptake of ferritin by cancer tissues from the extracellular environment and its role in cancer growth and progression are still poorly understood. In this thesis, I explore whether glioblastoma (GBM), a highly aggressive and malignant form of brain cancer, can uptake extracellular ferritin as an iron delivery agent and investigate the subsequent role of ferritin in tumor development. The first experimental section of this dissertation focuses on the uptake of extracellular H-ferritin by patient-derived glioblastoma cancer stem cell lines (GSCs). These cells, characterized by their ability to self-renew and differentiate within the tumor, contribute to tumor growth, recurrence, therapy resistance, and poor prognosis. Moreover, these cell types have been shown to acquire iron aggressively and preferentially from the tumor microenvironment. But the mechanism and mode of iron uptake are unclear. Thus, understanding the mechanism and functional implications of ferritin uptake in these cells is crucial. I demonstrate that GSCs can uptake extracellular H-ferritin as an iron delivery agent and that blocking transferrin receptor reduces ferritin uptake, suggesting the involvement of the receptor in this process. Moreover, I investigate the impact of extracellular ferritin uptake on the invasive potential of GSCs and find that H-ferritin reduces invasiveness without affecting proliferation capacity. This study sheds light on the H-ferritin mediated iron uptake mechanism in GSCs, providing valuable insights into GBM biology. The next experimental section of my thesis focuses on understanding the factors that affect the uptake of extracellular H-ferritin. Using in vivo mouse models of GBM, I investigate whether the Homeostatic Iron Regulator (HFE) and the sex of the mice have any effect on H-ferritin uptake. Differences in GBM occurrence, characteristics, and outcomes between sexes suggest that sex may regulate different mechanisms in tumors. Additionally, findings from the previous section indicated a potential sex difference in the binding of H-ferritin to human GBM tissues. However, my mouse model data reveals no significant difference in H-ferritin uptake between male and female GBM tissues. HFE protein is an iron sensor in cells, it monitors the levels of iron within the body and through its interaction with TfR helps regulate the uptake of iron at this receptor. Considering TfR’s association with H-ferritin uptake in the previous section, and the sex-specific role of HFE in GBM survival, I investigate whether the Homeostatic Iron Regulator (HFE) affects the uptake of extracellular H-ferritin. Downregulation of HFE does not significantly affect the uptake of H-ferritin or transferrin proteins in GBM tissues either. Interestingly, mice with HFE downregulation exhibit a significant survival benefit compared to control levels of HFE in tumors. Although there are differences between the mouse models and human GBM tissues, the survival benefit observed in these mice models provides a useful tool to study mechanisms affecting survival in male GBMs and explore their translatability in human patients. The final section of my thesis explores the role of H-ferritin beyond tumor expression levels. I investigate the impact of systemically downregulating H-ferritin in a novel mouse model of GBM. The results show that the extrinsic status of H-ferritin significantly impacts the survival of female GBM mice but not males. Sequencing of the tumors from these mice reveals differentially regulated pathways between male and female tumors. Female GBM tumors exhibit increased expression of genes related to immune system activation, cytokines, and chemokines, while male tumors show a reduction in immune system gene expression. Further analysis reveals decreased infiltration of T-cells and CD8+ T-cells but increased infiltration of mast cells in female mice, highlighting a novel role played by tumor extrinsic H-ferritin in regulating tumor-infiltrating T-cell populations and emphasizing the sex-biased nature of its functions. This section uncovers a sex-specific role of tumor extrinsic H-ferritin in modulating GBM tumors and elucidates sex-biased molecular mechanisms in tumor growth and development. In conclusion, this dissertation provides evidence of the complex role of H-ferritin in GBM. It demonstrates the dynamic nature of H-ferritin, from its role in delivering iron to regulating tumor-infiltrating cells. The findings suggest a sex-specific role of H-ferritin in GBM tumor modulation. Overall, this research offers deeper insights into GBM biology and provides a new frame of reference in furthering our understanding of these tumors.