Changes in glycosylation have been well-documented with regard to the onset and progression of cancer. Pervasive amongst these is the overexpression of cell surface sialic acid, a shared characteristic of myriad cancer types. Hypersialylation has been noted as a defining feature of the malignant phenotype, playing numerous functional roles in promoting cancer cell survival and invasivity, and a great body of research has sought to unravel its multifaceted contributions to metastatic advancement.
A complete understanding of the impact elevated sialic acid has on cancer progression has been hindered by a lack of tools for its direct study. Glycan biosynthesis is not genomically encoded, disqualifying commonplace molecular biology techniques such as mutagenesis and fluorescent protein fusions. This has motivated the development of novel chemical approaches to fill this void. In particular, bioorthogonal chemical methods have accomplished the direct tagging and imaging of sialylated glycans, enabling precise tracking within complex cellular milleu. As an additional challenge, the cancer disease state comprises intimate interactions between tumor and host cells; a comprehensive grasp of sialic acid’s involvement in cancer must be obtained through the use of animal models to recapture the physiological environment. Current in vivo models, primarily mice, are severely limited in their ability to observe cancer behavior at the cellular scale. An alternative model, the embryonic zebrafish, surmounts this barrier and permits the real-time detailed observation of cancer cell dynamics in vivo. Utilization of the zebrafish model’s imaging capabilities may deepen our mechanistic understanding of sialic acid’s role in cancer. The work presented in this dissertation catalogs efforts to study cancer sialylation in zebrafish using bioorthogonal chemistry and other approaches.
The first chapter reviews advancements made in the field of in vivo bioorthogonal cancer labeling. Recent years have seen the emergence of varied approaches to target cancer in mouse models for imaging and therapy, using both metabolic incorporation and antibody-pretargeting strategies. This survey of the literature highlights the potential for continually evolving bioorthogonal technology to play a role in human health, and also illuminates the challenges associated with bioorthogonal chemistry-based approaches within mice, creating a framework for discussion of bioorthogonal labeling as applied to a zebrafish model of cancer.
Chapter 2 describes the development of methods for the bioorthogonal labeling of cancer-associated sialic acid in a zebrafish xenograft model. Through the use of metabolic incorporation of modified sialic acid precursors and two widely popular bioorthogonal reactions, copper-free click chemistry and the tetrazine ligation, selective labeling of the cancer cell surface was achieved with greater sensitivity and spatial resolution than has been demonstrated in mouse tumor models. These efforts lay groundwork for the application of bioorthogonal chemistry to monitor sialic acid distribution on the subcellular scale in zebrafish throughout different stages of cancer progression.
Chapter 3 investigates the interplay between cancer cells and the innate immune system in zebrafish. Sialic acid-mediated interactions between cancer and immune cells have been observed previously and their biochemical basis is beginning to be understood. How these interactions impact the behavior of cancer and immune cells in vivo is an ongoing area of research. Embryonic zebrafish are a powerful tool in this regard, as they enable the facile monitoring of cancer and immune cell dynamics through real-time imaging. Through sialidase-treatment of cancer xenografts, we examined the role sialic acid plays in cancer cell survival and recruitment of macrophages and neutrophils. Sialic acid was found to have a significant influence on these processes, the mechanisms of which are currently being deciphered.
Finally, Chapter 4 represents an excursion outside the realm of zebrafish cancer biology. Genetically encodable self-labeling protein tags are an invaluable tool for protein imaging, and offer greater temporal control over traditional fluorescent protein tags. However, their large size, often in excess of 20 kDa, can interfere with native interactions. Described herein is a 10 kDa riboflavin-binding protein rationally engineered from riboflavin synthase. This protein undergoes a self-labeling reaction with novel acrylamide-modified flavin fluorophores, creating a covalently bound fluorescent protein tag. Preliminary results indicate that this system is suitable for labeling within cellular environments with a potential application as a small, genetically encodable tag for fluorescence anisotropy measurements of protein hydrodynamics.
