Osteosarcoma (OS) is the most prevalent primary malignant bone cancer in children, adolescents, and canines, yet the standard treatment has remained unchanged for 5 decades and offers less than a 20% 5-year survival rate with metastatic disease. This highlights a critical need to improve our mechanistic understanding of the factors that drive OS progression and metastasis, which in turn will facilitate the discovery of novel prognostic indicators and therapeutic targets. Current OS research heavily relies on incomplete and inaccurate models, which limit treatment improvement and outcomes. In vitro models often fail to account for important components of the native tumor environment, including the cellular and microarchitectural complexity of the bone marrow niche, the three-dimensional (3D) nature of sarcomas, and the physiological oxygen tension of bone marrow (5% O2). Thus, there remains a critical lack of biomimetic models that reproduce the complexity of the native tumor, which has greatly hindered the study of OS. Additionally, macrophages are implicated in OS disease progression based on clinical histology, but there is only limited characterization of the interactions between OS and macrophages. Within oncology, tumor-promoting macrophages (M2) are thought to create a regenerative environment that aids tumorigenesis. However, studies have yet to discern the mechanisms by which macrophages influence OS and are often restricted by many of the same shortcomings in current models.

To overcome this lack of accurate models, this dissertation aims to study OS at the intersection of tissue engineering and immunology to both establish biomimetic models for OS and elucidate the role of macrophages in OS progression. I hypothesize that OS will exhibit increased tumorigenic behavior as a function of metastatic potential when cultured within physiologically relevant models. Tumorigenesis is characterized throughout this work by proliferation, immunomodulation of macrophages, and chemotherapeutic resistance.

We utilize models of increasing complexity, from spheroids, to hydrogels, to an engineered bone marrow, to study highly metastatic (K7M2) and less metastatic (K12) OS cell line behaviors. We examine the effects of oxygen tension, 3D culture, and the bone marrow niche on OS progression and interactions with macrophages. We find that all of these biologically relevant factors affect cell line behavior in such a way that increases mimicry of clinical primary tumors compared to traditional monolayer culture. The work presented is among the first to mechanistically investigate the bidirectional cross talk between OS and infiltrating macrophages by studying the effects of macrophage phenotypes and paracrine signaling on OS behavior. It is also the first work, to our knowledge, to interrogate the role of tissue resident macrophages in OS progression and pulmonary metastasis. Interestingly, we observed that across models, OS does not appear to follow the traditional pro-tumorigenic, M2 macrophage paradigm, and instead modulates anti-tumorigenic macrophage phenotypes more readily. In summary, the work presented in this dissertation has contributed foundational knowledge to the fields of immunology, tissue engineering, and oncology that emphasize the relevance of biologically accurate models and the importance of interdisciplinary research.