Kevin Bennewith


Relevant Thesis-Based Degree Programs


Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Investigating the role of myeloid cells within the tumor microenvironment (2023)

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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Investigating intrinsic and extrinsic factors that regulate cancer cell motility (2022)

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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The development and role of hypoxia in tumor-reactive germinal centers (2021)

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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Oxygenation and radiosensitization consequences of solid tumour perfusion (2020)

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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Recruitment, phenotype and function of tumour-infiltrating regulatory T cells in the lung microenvironment (2018)

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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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Clinically relevant conventional dose of ionizing radiation enhances tumour cell migration in human breast cancer cell lines (2017)

It is estimated that >90% of cancer-related deaths are associated with the development and growth of tumour metastases. While tumour cell migration can be enhanced by high doses of ionizing radiation (IR) in vitro, the effect of lower, clinically relevant conventional IR doses on tumour cell migration and metastasis is unclear. I hypothesize that tumour cells that survive radiation therapy have a higher propensity to migrate in vitro and extravasate into the lungs in vivo, independent from radiation-induced changes in the solid tumour microenvironment. Breast cancer cell lines treated with 2.3Gy IR were imaged in real-time over 72h to quantify changes in single cell migration. EMT statuses of cell lines were determined using Western blot and flow cytometry. We used conditioned medium from irradiated cells to determine whether cellular migration was influenced by secreted factors. TGF-β ELISAs were used to elucidate its role in enhancing cell migration after IR. Pre-irradiated and sham treated breast tumour cells were IV-injected into mice to examine changes in lung extravasation. The mesenchymal MDA-MB-231 and LM2-4 cell lines treated with 2.3Gy of IR migrated a greater total distance and/or displaced further from the point of origin compared to untreated cells. No induction of EMT by 2.3Gy irradiation was observed, although MCF-7 cells migrated further from the point of origin after IR. Conditioned media from 2.3Gy treated tumour cells enhanced migration and displacement of untreated tumour cells. TGF-β ELISA analysis of supernatants from sham and 2.3Gy treated MDA-MB-231 cells revealed an almost two-fold increase in TGF-β1 72h post treatment. Chemokine antibody arrays revealed a number of up-regulated proteins after 2.3Gy treatment. 8 hours after IV injection, 2.3Gy pre-irradiated tumour cells was observed with enhanced lung colonization compared to sham controls. IR dose of 2.3Gy are sufficient to enhance migration of both non-metastatic and metastatic breast cancer cell lines independent of EMT. By quantifying changes in the metastatic ability of tumour cells treated with a clinically relevant dose of radiation, my findings will help to determine whether there is a need for additional administration of targeted secondary therapy to minimize tumour cell dissemination.

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Investigation into the role of eosinophils in pulmonary metastasis and primary tumours in mouse models of breast cancer (2017)

Eosinophils are multifunctional granulocytes with potent immune modulatory and cytotoxic capabilities. Despite the presence of eosinophils in various solid tumours and eosinophilia being a prognosistic indicator in some cancers, the role of this innate immune cell has been largely overlooked in the context of cancer. Specifically, the role of eosinophils in pulmonary metastasis is poorly described despite their prevalence in the lung and association with various pulmonary diseases. I sought to delineate the role of eosinophils in murine models of metastatic breast cancer using novel mouse models of genetic eosinophil deficiency (ddGATA) and by immunodepletion in orthotopic mouse models of cancer using an anti-IL-5 antibody. I hypothesize that eosinophils enhance pulmonary metastasis in mouse models of breast cancer, but do not affect the vascularization of the primary tumour. Eosinophils are increased 5.8-fold in the lungs of mice bearing 4T1 metastatic mammary tumours (p
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Effect of hypoxia-inducible secreted protein, tenascin C, on 4TI tumour cells in vitro and in vivo (2014)

Introduction: Metastatic cancer is responsible for 90% of cancer related deaths. Past research focused mainly on the primary tumour, leaving the process of metastasis poorly understood. Poorly oxygenated (hypoxic) tumour cells express hypoxia inducible proteins and have a more aggressive and invasive phenotype correlated with poorer prognosis. Hypoxic tumour cells are responsible for increased angiogenesis, invasion, matrix deposition and remodelling along with many other functions.We hypothesize hypoxic tumour secreted proteins are responsible for promoting metastasis. Our aim is to identify these hypoxia inducible secreted proteins and determine mechanisms promoting metastasis. Methods: Mammary carcinoma cells (4T1 – metastatic and 67NR – non-metastatic) were placed in 1% O₂ (hypoxic) or 21% O₂ (normoxic) for 24 hours. Stable Isotope Labelling of Amino acids in Cell culture (SILAC) and mass spectrometry were used to perform a quantitative proteomic screen of conditioned medium. Proteins in 4T1 conditioned media, up-regulated in hypoxia and absent from the 67NR results represented candidate secreted proteins in metastasis. Tenascin C (TNC), a candidate protein identified from the proteomic screen was stably knocked down and over-expressed. In vitro, the Boyden chamber and wound healing assay were used to study invasion and migration. In vivo, metastasis was assessed using flow cytometry-based quantification of metastasized tumour cells in the murine lungs.Results: (TNC) was identified as a secreted protein with a role promoting metastasis in vivo through enhanced migratory ability. In vitro, knockdown of TNC in 4T1 enhanced migratory ability whereas over-expression decreased migratory ability. These results were contradictory to the expected results based on the hypothesized in vivo role. However, in vivo knockdown of TNC in 4T1 tumour cells resulted in a significant decrease in lung metastases. These results are consistent with the expected role of TNC in vivo. Conclusions: Despite the contradictory results in vitro, TNC had a positive metastatic role potentially through a migratory mechanism. TNC represents a potential new therapeutic drug target. Given the 4T1 cell line results, these data support further examination of the migratory role of TNC and how it promotes metastasis. In addition, TNC expression in other tumour cell lines including human breast cancer should be examined.

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Myeloid-derived suppressor cell accumulation in secondary target organs promotes metastatic growth in breast cancer (2014)

A role for bone marrow-derived cells (BMDCs) in promoting metastatic tumour growth is emerging. Previous work has shown accumulation of CD11b⁺ BMDCs in pre-metastatic niches in the lungs of mice bearing metastatic breast tumours, although questions remain about the precise identity of these cells and their potential long-term influence on metastatic growth. We studied the induction, identity, longevity, and function of CD11b⁺ BMDCs in tissues of mice bearing murine mammary tumours. Metastatic, but not non-metastatic, mammary tumours induced the accumulation of CD11b⁺Gr1⁺ cells, which we functionally identified as immunosuppressive myeloid-derived suppressor cells (MDSCs) using ex vivo assays. Unlike BMDCs associated with pre-metastatic niches in other breast tumour models, MDSCs were induced systemically, with levels increasing in metastatic target organs (lung, liver, bone-marrow) and in tissues that do not harbor metastases from these tumours (kidney, spleen). We also found that circulating MDSC levels can be used as a surrogate marker for monitoring MDSC accumulation in tissues. Primary tumour resection caused decreased serum levels of granulocyte-colony stimulating factor and MDSCs, but functional MDSCs remained elevated in the lungs for several weeks after tumour resection. These MDSCs were associated with enhanced subsequent pulmonary metastatic growth, providing evidence that MDSC induction by metastatic primary tumours helps create a long-lasting metastasis-promoting environment in lung tissue. In addition to surgery, we utilized gemcitabine (GEM), 5-fluorouracil (5-FU) and tirapazamine (TPZ) to target MDSCs in the lungs and spleen of tumour bearing mice. We administered GEM to mice with resected tumours in order to target residual MDSCs. Our data demonstrates that GEM significantly eliminates residual MDSC levels in the lungs which resulted in a decreased ability of 4T1 tumour cells to colonize the metastatic target organ. Taken together, our findings suggest that metastatic murine mammary carcinomas induce systemic elevation of MDSCs and highlight the potential importance of identifying patients with elevated MDSC levels. Additionally, our research provides support for therapeutic targeting of MDSCs in patients at risk of developing or re-developing metastatic disease.

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The Effect of Heme Oxygenase-1 Breast Cancer Metastasis In Vivo and In Vitro (2014)

The metastatic spread of cancer is linked to over 90% of cancer-related deaths. Therapies designed to prevent the dissemination of metastatic cells from the primary tumour is a therapeutic strategy to improve outcome for patients at risk of developing metastatic disease. Heme oxygenase-1 (HMOX1) is the rate-limiting enzyme in heme catabolism and is induced by various stress stimuli. The role of HMOX1 in breast cancer metastasis is conflicting with some groups indicating that HMOX1 promotes metastasis while others indicating that HMOX1 reduces metastatic spread. Previously in our laboratory, HMOX1 expression was chemically induced with hemin, a potent and effective HMOX1 inducer, in murine mammary carcinoma cells to determine the effect of HMOX1 on tumour cell migration and invasion. Hemin reduced migration and invasion of all three cell lines when assessed by Boyden chamber transwell assays. Therefore, we hypothesize that HMOX1 reduces breast cancer metastasis by decreasing tumour cell migration and invasion.We used three different murine mammary carcinoma cell lines as models for breast cancer: 67NR – noninvasive/nonmetastatic, 4TO7 – invasive/metastatic, 4T1 - highly aggressive metastatic behavior. We increased HMOX1 expression genetically or by chemical induction with another HMOX1 inducer, cobalt protoporphyrin (CoPP), to determine the effect of HMOX1 on migration and invasion. CoPP and HMOX1 overexpression reduced migration of all tumour cells in vitro. Interestingly, HMOX1 overexpression decreased invasion of 4T1 cells, but increased the invasion of 67NR and 4TO7 cells, modeling the dichotomy of HMOX1’s influence on breast cancer cell invasion. We also assessed the effect of HMOX1 overexpression and knockdown on lung metastases in vivo. In 4T1 tumours, HMOX1 overexpression had no effect on primary tumour growth or lung metastases. On the other hand, in 4T1 tumours with HMOX1 knockdown, there was a reduction in tumour growth and in lung metastases.

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