Metastasis continues to be the leading cause of breast cancer-related deaths. Metastasis is a multistep process during which cancer cells leave the primary tumor, invade and seed inside secondary tissues, leading to lethal organ failure. Currently, no efficient method exists for the elimination of invasive cancer cells, which poses a major clinical challenge. Indeed, while localized cancers are clinically manageable, invasive breast cancer is largely an uncurable disease. Understanding how cancer cells sustain an invasive behavior may lead to the development of novel potent therapies.A vast genetic and phenotypic heterogeneity exists among invasive cancer cells. This heterogeneity is important to investigate as, in breast cancer patients, most metastatic nodules are polyclonal and consist of cancer cells that invaded and disseminated collectively. While recent studies demonstrated that collective invasion can be cooperative, where leader cells enable the invasion of otherwise non-invasive follower cells, the mechanisms regulating their cooperativity have been poorly investigated. Additionally, it is not known whether cooperative invasion can lead to metastasis, where metastatic cells enable the dissemination of non-metastatic cells. Our laboratory previously showed that breast cancer cells can locally degrade the extracellular matrix using invadopodia, which are membrane protrusions enriched in matrix metalloproteinases. Importantly, invadopodia are required for metastasis of single cancer cells. However, whether invadopodia enable the collective invasion and metastasis of cancer cell clusters remains unknown. Further, the role of invadopodia-mediated matrix degradation in leader vs. follower cells is unclear. In this dissertation, we aim to understand how cancer clones with and without invadopodia may cooperate during invasion and metastasis. Our results suggest that a few cells with invadopodia can drive the metastasis of cell clusters from multiclonal tumors. From this work, we believe that a more detailed understanding of how cancer cells cooperate may help in identifying new targets for preventing metastasis. / Bioengineering

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INTRODUCTION: Immunotherapy continues to garner strong support for use in the treatment of cancer. Adoptive transfer therapies offer a promising approach to combating metastatic disease. In addition, viruses can also be exploited to drive antitumor immunity and tumor destruction. While the use of chimeric antigen receptor (CAR)-engineered T cells has shown dramatic clinical benefit for use in blood-based cancers, solid tumors remain a significant hurdle. METHODS: We have investigated the use of multi-faceted immunotherapies combining CAR-T cells with oncolytic virotherapy. We have also evaluated how these therapies interact with pre-conditioning lymphodepletion regimes. RESULTS: In chapter 3, we investigated the differences between three similar chimeric receptors targeting NKG2DL. Upon adoptive transfer, we observed dramatic T cell-induced toxicity. In addition, there were stark differences in the severity of toxicity induced between different receptors or across different mouse strains, or if combined with pre-conditioning chemotherapy. In chapter 4, we tested the ability of oncolytic vaccines to boost engineered T cells through their natural antigen receptor. While CAR-T cells could be boosted via oncolytic vaccines, prolonged T cell engraftment and successful oncolytic vaccine boost required pre-conditioning chemotherapy. Further analysis revealed a lack of antitumor function of the CAR-T cells in vivo. iii In chapter 5, we evaluated loading CAR-T cells with oncolytic viruses (OVs). Loading of CAR-T cells with OV did not impair CAR expression or functionality of the T cells. In addition, CAR target recognition did not impact the ability of OV-loaded cells to deposit OVs onto tumor targets. CAR-T cells loaded with OV also displayed enhanced antitumor functions as compared to either treatment alone. CONCLUSIONS: The research described in this thesis reveals important information into the interactions between CAR-T cells and OVs, and how pre- conditioning regimes may influence responses from either or both therapies. Overall, our research offers novel insight into future CAR-T cell therapeutic developments. / Thesis / Doctor of Philosophy (PhD)

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