Karla Williams

Breast cancer is the leading cause of cancer-related deaths in females. In the breast tumor microenvironment, tumor infiltrating leukocyte cells play a crucial role in eliciting tumor cell death. Immunotherapy has emerged as a new therapeutic option to treat breast cancer by stimulating the anticancer activity of immune cells. To improve on current immunotherapy strategies, we must identify new immune-inhibitory factors in breast cancer that can be targeted with blocking therapeutics. Recently, it was found that expression of the glycan polysialic acid on tumor-associated leukocytes is prognostically significant in breast cancer patients. Polysialic acid regulation or function in the human immune system has not been researched in depth, and further studies are required to understand how polysialic acid regulates the anticancer activity of immune cells. While glycans play a central role in regulating immune recognition, the role of polysialic acid specifically in driving breast cancer immune evasion is comparatively understudied. We propose that polysialic acid plays a role in the anticancer immune cell response. In this study, we performed extensive immunophenotyping of polysialic acid expression on immune cell subsets using flow cytometry. Monocytes, B-cells and naïve T-cells expressed low levels of polysialic acid. However, stimulated T-cells expressed polysialic acid at higher levels after one week of activation, and the expression of polysialic acid decreased by two weeks. In activated T-cells, NCAM was found to be polysialylated as well as other unknown protein carriers, as assessed by western blot and flow cytometry. Furthermore, functional killing assays to assess the role of polysialic acid in immune-mediated anticancer function found that T-cell cytotoxicity is not regulated by polysialic acid, however data suggests macrophage phagocytosis is impacted by polysialic acid.

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Breast cancer is the most commonly diagnosed cancer globally, where around 34% of patients will develop metastatic breast cancer with only 22% survival rate over five years. Individuals who develop metastatic breast cancer patients have limited effective treatment options. To improve treatment options, a better understanding of metastatic disease is needed. Studies focused on understanding the process of tumor cell metastasis will result in improvements to our understanding of tumor metastasis and may identify novel disease drivers that can be drug targets. Here, this thesis aims to investigate tumor cell dissemination as regulated by small sub-cellular structures called invadopodia. Invadopodia are specialized actin-rich membrane protrusions that aid in tumor cell dissemination and invasion. Invadopodia have been shown to degrade the extracellular matrix to invade surrounding tissues and facilitate the entry and exit through blood vessels. We investigated the role of invadopodia in driving tumor cell invasion through a model of the lymphatic endothelium. Invadopodia were found to mediate this process and further investigation into invadopodia-based drivers of this process identified CCR7 as a mediator. CCR7 is a chemokine receptor that is known to home the dendritic cells to the lymph nodes, and has two endogenous ligands CCL19 and CCL21. In our model, tumor cells were responsive to CCL19 and this promoted invadopodia formation and increased invasion through a lymphatic endothelium model. Overall, this study has advanced our understanding of invadopodia-associated tumor cell dissemination via lymphatics, and provides insight into a mechanism of CCR7-mediated invadopodia invasion.

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Cancer is a leading cause of death globally and most cancer-related deaths are attributable to cancer metastasis, a process describing the movement of cells from primary tumours to distant sites within the body. As metastasis is the primary cause of cancer patient death, understanding the process of metastasis is critical for the development of new therapeutics to treat or prevent metastasis.Gamma-aminobutyric acid (GABA), a prominent neurotransmitter, has been linked to processes modulating tumour cell proliferation and metastasis. GABA is expressed at elevated levels in many tumour types including triple negative breast cancers (TNBC), where it is associated with tumour progression and poor prognosis. However, the precise molecular mechanism underlying GABA-induced pro-metastatic phenotypes in tumour cells is largely undescribed. Recently, our lab described a role for GABA in promoting tumour cell extravasation into GABA-rich environments, with early evidence suggesting a role for GABA in promoting invadopodia formation. Invadopodia, are actin-rich membrane protrusions that have been observed in vitro and in vivo in many metastatic cell lines and are critical to the progression of cancer metastasis.Here, we demonstrate GABA’s role in invadopodia formation and function in three TNBC cell lines. We evaluated endogenous GABA levels in a panel of TNBC cell lines through dot-blot analysis; incubated cells with exogenous GABA and the GABA receptor (GABAR) inhibitors, gabazine and 2-OH-saclofen, then assessed their effects on invadopodia formation. We observed that stimulation of cells with exogenous GABA significantly increased invadopodia formation and inhibition of GABAARs using gabazine significantly reduced invadopodia formation. Conversely, our control had no significant differences relative to cells treated with the GABABR antagonist, 2-OH-Saclofen. Collectively, our findings indicate GABA is important for invadopodia formation and function; the reduction in invadopodia formation in response to gabazine suggests GABA signals through GABAARs to mediate invadopodia formation. Lastly, our dot-blots confirmed endogenous GABA expression in our TNBC cells, providing further evidence to suggest TNBC cells utilize GABA to promote invadopodia formation, local-tumour invasion and increase extravasation into GABA-rich microenvironments. Understanding how GABA mediates tumor metastasis in TNBC has the potential to identify novel therapeutic targets with clinical utility for TNBC treatment.

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