Kevin Bennewith

The solid tumor microenvironment contains structural and cellular components that can dictate cancer progression and therapy response. These cellular components can include cells of the innate immune system, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and eosinophils, as well as cells of the adaptive immune system, including CD4⁺ T regulatory cells (Tregs) and cytotoxic CD8⁺ T cells. The aim of this thesis work is to interrogate the pro- and anti- tumorigenic roles myeloid cells play in primary and metastatic disease, and how these myeloid populations interact with cells of the adaptive immune system to influence anti-tumor immunity. In Chapter 2, I examined how proteins secreted by cancer cells can promote a tumor-permissive microenvironment through the recruitment of suppressive innate and adaptive immune cells to the lungs. I demonstrated that tumor cells secreted proteins that drove the recruitment and/or expansion of pulmonary TAMs, Tregs, and granulocytic-MDSCs (G-MDSCs), resulting in increased primary lung tumor growth and pulmonary metastasis of mammary carcinoma cells, respectively. Targeting tumor-secreted proteins, or the suppressive immune cells that are expanded in response to said proteins, led to a decrease in primary and metastatic pulmonary disease. In Chapter 3, I utilized several transgenic mouse models of eosinophilia and eosinophil-deficiency to assess the role of eosinophils in breast cancer pulmonary metastasis. I found that eosinophils were directly cytotoxic towards mammary carcinoma cells, likely through degranulation, and that eosinophils decreased pulmonary metastatic growth of breast cancer cells. In Chapter 4, I profiled the dynamic myeloid compartment following antigen specific CD8⁺ T cell-mediated tumor regression and recurrence. I demonstrated that using glucose restriction of CD8⁺ T cells in vitro led to improved tumor regression in vivo. I identified several potentially suppressive myeloid cell subsets, including TAMs and arginase-1ʰⁱ monocytic-MDSCs (M-MDSCs) that expand during CD8⁺ T cell-mediated tumor regression and subsequent recurrence, which may play a role in tumor regrowth. This thesis work aims to examine the diverse phenotype and function of myeloid cells in primary and metastatic disease, ultimately contributing to an improved understanding of the interplay between the innate and adaptive immune system in the solid tumor microenvironment.

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Mechanisms of cellular motility are highly conserved and regulate many physiological processes including embryonic development, wound healing, and angiogenesis. Cancer cells can exploit normal mechanisms of cellular motility to facilitate invasion and metastasis, the process by which cancer cells disseminate to distant organs. Invasion can be triggered by a host of intrinsic and extrinsic factors, many of which remain unknown. Here, I investigate the role of one intrinsic factor (receptor-type protein tyrosine phosphatase alpha) and one extrinsic factor (ionizing radiation) in facilitating cancer cell invasion. Receptor-type protein tyrosine phosphatase alpha (PTPα is a widely expressed transmembrane-bound protein that has been implicated in integrin signaling, focal adhesion formation, and normal cell migration. During normal cell migration, cells use focal adhesions to facilitate cycles of cell adhesion to, and release from, the extracellular matrix (ECM). Focal adhesion structures bear resemblance to invadopodia, which are dynamic actin-based protrusions that form on the plasma membrane of cancer cells. In this study, I hypothesized that PTPα promotes invadopodia-mediated triple-negative breast cancer (TNBC) cell invasion. My work involving the depletion of PTPα in TNBC reveals PTPα as a regulator of ECM degradation and invasion in vitro and in vivo. My studies suggest that PTPα is an intrinsic regulator of TNBC cell invasion. Radiation therapy is among the most common treatments for cancer. While radiation is an effective treatment option, there have been reports showing the surviving fraction of cells may exhibit increased invasiveness. Since the precise mechanisms that modulate ionizing radiation (IR)-induced invasion remain largely unknown, the goal of this study was to investigate the role of IR in upregulating key signaling mechanisms associated with invadopodia activity. Our studies revealed IR upregulates a key invadopodium protein, TKS5, expression while decreasing Glioblastoma multiforme (GBM) cell invasion in vitro, suggesting GBM cells invade independently of invadopodia. I then developed an ex vivo brain slice invasion assay to investigate mechanisms of GBM cell invasion into brain tissue. Using this model, I found a subset of GBM spheroids exhibited increased invasion post-irradiation. These studies suggest IR is a potential extrinsic regulator of GBM invasion when cells are within the brain microenvironment.

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During humoral immune responses to foreign antigens, activated B cells are clonally selected and expanded within the germinal center (GC) reactions of secondary lymphoid organs. Upon exposure to commonly used model antigens such as chicken ovalbumin, GCs can develop regions of low oxygen (hypoxia) that affect the phenotype of exposed B cells. It is currently unknown whether GC hypoxia also develops during humoral immune responses to cancer, and how this poorly oxygenated microenvironment impacts anti-tumor immunity. Using mouse models of breast cancer, we assessed whether GC hypoxia was present in tumor-draining lymph nodes (TDLNs) and sought to determine the role of GC hypoxia during tumor-targeted B cell responses. LNs draining mammary tumors contained hypoxia amongst GC B cells, which was not detected in contralateral LNs, or in LNs from tumor-free mice. Exposure to lethally irradiated cells was sufficient to induce GC hypoxia, and we found that a hypoxic microenvironment promoted antibody-secreting cell differentiation in exposed B cells. To better delineate the functional role of GC hypoxia during tumor-targeted immunity, we developed and validated a Cre-Lox mouse model with deletion of pVHL, a negative regulator of the hypoxia-inducible transcription factor (HIF), in class-switched B cells. pVHL deletion resulted in decreased GC B cells in TDLNs, along with reduced antibody class-switching and lower levels of tumor-binding antibodies in the serum. Accompanying the reduction in GC responses, pVHL deletion in class-switched B cells reduced the growth of 4T1 murine mammary tumors, and had no effect on the tumor growth of the related 4T07 mammary carcinoma line. Samples of sentinel and non-sentinel LNs from breast cancer patients contained hypoxia inducible CA9 within their GC reactions, suggesting that GC hypoxia also develops in humans. An automated image analysis of CA9-demarcated GCs from patients showed that co-occurrence of fewer, but larger GCs in patient’s LNs associated with improved overall survival relative to patients with more abundant, but smaller GCs. We conclude that hypoxia can develop in tumor-reactive GCs, and that the hypoxia response system negatively regulates the GC reaction and humoral immunity against tumors.

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Solid tumours often develop regions that are poorly oxygenated (hypoxic). Hypoxic tumour cells are resistant to radiation and are associated with poor patient outcome. “Chronic hypoxia” develops where vascular density is insufficient, causing tumour cells beyond ~70-100 μm from blood vessels to receive little oxygen. “Transient hypoxia” occurs in solid tumours where fluctuating microregional perfusion exposes tumour cells to cycles of hypoxia and reoxygenation. The fundamental understanding of transient hypoxia in vivo is lacking, and there are no available strategies to eliminate transient hypoxia. This thesis hypothesizes that transiently hypoxic tumour cells are long-lived radiation resistant cells and that eliminating the development of transient hypoxia will improve tumour radiation response. This thesis first investigated the rate of hypoxic cell turnover in vivo. Exogenous hypoxia reporters were used to label hypoxic tumour cells, while loss of labelled cells over time indicated loss of hypoxic cells. The perfusion-modifying drug pentoxifylline was used to control which hypoxic populations were labelled and therefore which populations were lost over time. We find that transiently hypoxic tumour cells survive longer than neighbouring chronically hypoxic cells, further justifying transient hypoxia as an important therapeutic target.To better study tumour perfusion-modifying interventions, this thesis next validated 2-18F-fluoroethanol as a novel reporter of solid tumour perfusion compatible with positron emission tomography. We found 2-18F-fluoroethanol to effectively respond to established tumour perfusion-modifying interventions and applied 2-18F-fluoroethanol to characterize novel interventions aiming to modify tumour perfusion and transient hypoxia. This thesis then focused on the angiotensin II type 1 receptor blocker telmisartan. Telmisartan treatment reduced tumour collagen 1 content, increased and stabilized tumour perfusion, reduced the development of transient hypoxia, and improved tumour radiation response. This presents the target of telmisartan, cancer associated fibroblast activity and tumour collagen 1 content, as a potential microenvironmental cause of transient hypoxia. Overall, this thesis provides insight into the basic biology of transient hypoxia in vivo, validates a novel non-invasive reporter of solid tumour perfusion, and identifies telmisartan as a clinically relevant treatment to stably reduce the development of transient hypoxia in solid tumours and improve radiation response.

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Despite the crucial role of regulatory T cells (Tregs) in the regulation of immunity toward innocuous antigens, these cells may contribute to the growth and metastasis of a variety of malignancies, either directly by production of cell growth and angiogenic factors, or indirectly by suppression of anti-tumour immunity. Tregs may support the development of both primary and metastatic tumours; however, the therapeutic inhibition of these cells is complicated by the presence of tissue-specific phenotypes and functions, which require further characterization based on the type of cancer and local microenvironment. We have observed that Treg accumulation in primary and metastatic tumours in the lung microenvironment is significantly increased in a clinical and pre-clinical setting, respectively. Therefore, in this thesis we hypothesize that Tregs promote tumour growth in the lungs. To explore this hypothesis, we examine the phenotype and function of tumour-infiltrating Tregs and inhibit their homing to observe the impact on tumour growth. Herein, we report that Tregs isolated from mice bearing metastatic tumours are functionally immune suppressive and enriched for expression of the homing receptor C-C chemokine receptor type 5 (CCR5). Inhibition of Tregs with the CCR5 antagonist, Maraviroc, reduced CCR5⁺ Treg accumulation and tumour burden in the lungs. We identify that Tregs accumulate during early stage disease in lung cancer patients, and show that oncogene-induced chemokine production promotes Treg migration. Further, we identify that oncogene-induced Treg migration is significantly reduced by antibody-mediated neutralization of the CCR5 ligand, CCL5 ex vivo. Finally, we report that a subset of Tregs expressing the interleukin 1 family receptor, ST2, may directly promote tumour progression by production of the growth factor amphiregulin (AREG) in response to the cognate ligand, IL-33 in the lung microenvironment. Taken together, these results provide evidence that Tregs are important contributors to tumour development in the lung microenvironment and identify signaling axes required for their recruitment and function. Our findings highlight the viability of homing inhibitors as an alternative mechanism to systemically ablating Tregs for the treatment of lung metastases.

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