Intratumor heterogeneity poses a significant challenge for the diagnosis and treatment of patients with breast cancer because distinct sub-populations of tumor cells contribute significantly more to therapy resistance and tumor recurrence than others. Consequently, understanding the mechanisms that contribute to this heterogeneity and identifying sub-populations responsible for aggressive behavior is a significant and timely problem. Considerable evidence indicates that a subpopulation of tumor cells with stem/progenitor-like characteristics, termed cancer stem cells (CSCs), is responsible for therapy resistance and recurrence, sparking interest in characterizing novel biomarkers and therapeutic targets for this aggressive population of cells. Unfortunately, CSCs share many protein markers with normal mammary stem/progenitor populations, minimizing potential targets for diagnostic and therapeutic purposes. Therefore, in my thesis research, I investigated novel ways to identify CSC populations based on their glycome. I observed that breast CSCs have a unique glycosylation pattern that can be used to distinguish them from other tumor populations. Specifically, I discovered a novel α2,3 sialoglycan on Core2 O-linked glycans expressed on CSCs that can identified using the lectin SLBR-N. I found that SLBR-N can be used to distinguish CSCs from bulk tumor cells in multiple in vitro and in vivo models. I also discovered that the CSC marker, CD44s, expresses O-linked α2,3 sialoglycan and that this glycan alters CD44s function by promoting the activation of the PDGFRβ/STAT3 pathway. In contrast, the fucosyltransferase FUT3 and its glycan sialyl Lewis X (sLeX) are expressed on non-CSCs and they function to impede stemness by inhibiting CD44s-mediated PDGFRβ/STAT3 signaling. In summary, this thesis provides insights into glycan heterogeneity in breast cancer and novel ways to identify CSCs using the glycome.

Structured abstract Hypothesis Cancer stem cells (CSCs) are subpopulations of cells which could contribute to tumor growth, metastasis formation and chemoresistance. CSCs can be detected by surface markers assessed by immunohistochemistry methods. A typical surface marker for CSCs is CD44 (standard form). We assumed, that CD44(s) could serve as a prognostic factor and marker of chemoresistance in patients with epithelial ovarian cancer. The aim of study 1. To recruit group of patients with histologically verified epithelial ovarian carcinoma. 2. To evaluate prognostic significance of known prognostic factors in our series of patients. 3. To assess the expression of CD44 in specimens of primary tumors and specimens of implantation metastasis using immnunohistochemistry and analyze their correlation. 4. To evaluate the expression of CD44 in relation to known prognostic factors. To analyze the significance of CD44 expression evaluation for overall survival, disease-free interval and chemoresistance. To find CD44 positivity cut-off by using statistical methods Materials and Methods A retrospective study was performed on 87 patients with histologically verified EOC. All patients were tested for primary tumor specimens, 48 of them were tested with regard to both specimens of primary tumor and implantation...

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To address the unmet clinical need to eradicate treatment-resistant CD133+ cancer stems within tumors, a CSC-targeted radionuclide therapy (TRT) and companion diagnostic imaging probes were developed utilizing CD133-targeting antibodies and antibody fragments. In Chapter 1, background research providing context for the work in this Thesis is presented. In Chapter 2, a CD133-targeting antibody, RW03IgG, underwent radiolabeling with lutetium-177 to synthesize [177Lu]Lu-DOTA-RW03IgG for CD133-TRT. The CD133-TRT was evaluated for pharmacokinetics and treatment of a CD133 expressing human colorectal tumor bearing mouse model. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG demonstrated notable uptake in the colorectal tumors and off-target organ uptake consistent with previously reported antibody-based TRTs. Confirmation that tumor uptake was mediated by antibody-antigen binding was verified through co-injection with an excess dose of unlabeled RW03IgG. A dose-escalation therapy trial using [177Lu]Lu-DOTA-RW03IgG for treatment of the colorectal cancer mouse model revealed a dose-dependent reduction in tumor growth rate at well-tolerated doses. The decrease in tumor growth rate observed due to [177Lu]Lu-DOTA-RW03IgG treatment, along with an improvement in overall mouse survival, demonstrate the therapeutic efficacy of CD133-TRT. Additionally, histopathological and immunohistochemical (IHC) analyses indicated low off-target organ toxicity and significant anti-tumor effects. These findings suggested the potential for enhanced overall survival benefits through multiple doses. However, results on multiple-dosed CD133-TRT on the tumor growth rate and overall mouse survival were inconclusive. In Chapter 3, an orthotopic patient-derived glioblastoma (GBM) mouse model was developed that replicates anatomical pharmacokinetic challenges and CSC populations observed in patient tumors. Stereotactic engraftment of the patient GBM cells was optimized to reproducibly deliver tumor cells to the thalamus and growth was monitored using bioluminescence imaging. Ex vivo analysis confirmed various key characteristics of patient GBM, including CD133 expression, hypercellularity, and invasiveness. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG using the PDX GBM mouse model indicate antibody-antigen driven tumor uptake, determined through co-injection an excess dose of unlabeled RW03IgG. Ex vivo autoradiography supported the biodistribution results and showed elevated uptake of [177Lu]Lu-DOTA-RW03IgG in tumor relative to non-tumor bearing brain tissue. Chapters 4 and 5 centered on the development and evaluation of companion diagnostic CD133-targeted immunoPET probes. Chapter 4 specifically explored probes derived from the full antibody, RW03IgG. The probes were synthesized by conjugating RW03IgG with DFO-NCS to produce DFO-RW03IgG at different chelator-to-antibody ratios. The various DFO-RW03IgG conjugates were then radiolabeled with zirconium-89 to obtain [89Zr]-DFO-RW03IgG. Biodistribution studies and PET imaging revealed promising tumor uptake of [89Zr]-DFO-RW03IgG, and it was observed that higher chelator-to-antibody ratios led to increased accumulation in off-target organs. Chapter 5 investigated a probe derived from an scFv-Fc fragment of RW03, [89Zr]-DFO-RW03scFv-Fc. Biodistribution studies and PET images of colorectal tumor-bearing mice administered [89Zr]-DFO-RW03scFv-Fc showed favorable tumor uptake and low off-target organ accumulation. In Chapter 6, a probe for CD133-Photoacoustic Imaging (PAI) was synthesized through conjugation of RW03IgG with IR-783, an organic dye recognized for its favorable photoacoustic properties. Challenges were encountered in isolating the product, (IR-783)-RW03IgG, at high degrees of labeling (DOL) due to product aggregation. In vitro binding assays indicated that (IR-783)-RW03IgG (DOL = 1) maintained a comparable binding affinity to native RW03IgG. In vivo, colorectal tumors in mice administered (IR-783)-RW03IgG (DOL = 1) did not exhibit significant contrast from the background tissue, and the tumor PA signal did not differ significantly compared to tumors in mice administered an IR-783 labeled isotype IgG. The results suggest that a higher concentration of dye is needed within colorectal tumors for effective tumor visualization than what was provided by IR-783-RW03IgG. Chapter 7 investigated the use of Imaging Mass Cytometry (IMC) to simultaneously visualize [177Lu]Lu-DOTA-RW03IgG and multiple tumor biomarkers in tissue specimens collected from colorectal tumor xenograft mice treated with CD133-TRT. IMC showed undetectable concentrations of hafnium-177 (the decay product of lutetium-177) in tumors treated with CD133-TRT. However, lutetium-176 and lutetium-175, sourced from the carrier-added [177Lu]LuCl3 used in the synthesis of [177Lu]Lu-DOTA-RW03IgG, were present at levels sufficient for IMC visualization. The distribution of lutetium-176, representing [177Lu]Lu-DOTA-RW03IgG, within tumors, was imaged concomitantly with CD133, DNA damage markers, and several additional biomarkers that describe elements of the tumor microenvironment. These collective results endorse IMC as a useful tool to assess the distribution of TRT within tumors and uncover changes to the microenvironment in response to treatment. / Thesis / Doctor of Philosophy (PhD) / Targeted radionuclide therapy (TRT) and molecular imaging strategies were developed to aid in the elimination of the rare and particularly resilient Cancer Stem Cell (CSC) population in tumors. A fully human monoclonal antibody and antibody fragments targeting CD133, a molecular biomarker for CSCs, provided the means to deliver radioactive isotopes for therapy and imaging to CD133+ cells in tumors. The therapeutic efficacy of CD133-TRT for treatment of a colorectal cell line-derived xenograft mouse model was promising, and the treatment showed uptake in orthotopic patient derived glioblastoma tumors engrafted in mice. ImmunoPET probes targeting CD133 were optimized and successfully delineated CD133 expressing tumors from background tissue, warranting further evaluation using patient-representative cancer models. A non-invasive CD133-targeting Photoacoustic Imaging (PAI) probe was synthesized through conjugation of the CD133-targeting antibody to an organic dye, IR-783, although further probe optimization is required to provide tumor contrast. Tumor specimens from mice treated with CD133-TRT were assessed by Imaging Mass Cytometry (IMC), which revealed detectable concentrations of carrier isotopes from the therapy in the tumors, implicating the discovery of a powerful new tool for multiplexed single-cell level resolution imaging for cellular-scale analysis of targeted radionuclide therapy. The CSC-therapy and select molecular imaging probes generated in this Thesis warrant further evaluation using patient-representative mouse models of cancer.

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<p>Breast cancer is the most common cancer among Canadian women with one in nine women expected to develop breast cancer in their lifetime. Until recently these breast tumors were thought to be a homogeneous cell population. Recent studies have shown that breast tumors contain a rare cell type termed breast tumor initiating cells (TICs) or cancer stem cells (CSCs) with the ability to elicit new tumor growth and metastases. These TICs exist apex of a tumor cell hierarchy and give rise to more TICs and non-tumorigenic cells. Traditionally, drugs were developed to target the highly proliferative cells population resulting in a decrease in tumor volume. However, these therapies spare the TICs, which results in tumor relapse demonstrating the need for new drugs that target the TICs. Because in cancer, mutated protein kinases are the controllers of cell proliferation, invasion and metastasis, they have become a target for drug development. Inhibition of these kinases could lead to the identification of compounds that selectively target breast TICs. Using mammary tumors from cancer prone mice propagated as non-adherent tumorspheres (TMS), which contain a high fraction of breast TICs and the same conditions to propagate the non-transformed mouse mammary epithelial stem and progenitor cells (MESC), as non-adherent mammospheres (MMS) a 240-kinase inhibitor library was screened using an AlamarBlue proliferation assay. Twenty percent of the compounds resulted in 75% decrease in proliferation of TMS derived cells and some of which were TMS-selective. Sunitinib, a multi-targeted kinase inhibitor, was one of the selective compounds identified and when administered to mice with subcutaneous mammary tumors resulted in tumor shrinkage. This was accompanied by an increase in apoptotic cells, decrease in proliferating cells and tumor vasculature, and a change in tumor morphology and composition. These findings show the efficacy of Sunitinib in shrinking mouse mammary tumors and suggest a potential use of Sunitinib for treatment of breast cancer.</p> / Master of Science (MSc)