(L-478) Osteosarcoma spheroids undergo dynamic rearrangement mediated by integrin and cadherin signaling

Introduction:: Osteosarcoma (OS) is the primary malignant bone cancer affecting children and adolescents, with high pulmonary metastasis rates and correspondingly poor clinical outcomes. Furthermore, treatment options have remained unchanged since the advent of modern chemotherapeutics. This is, in part, due to the difficulties in modeling OS. Most studies are performed in monolayer culture, which fails to mimic the three-dimensional models of in vivo tumors. Here, we culture 3D OS spheroids and interrogate the role of oxygen tension on OS cell behavior, health, and interaction with one another. Our findings suggest that our OS spheroid model will provide improved understanding of OS tumor behavior.

Materials and Methods:: Spheroids were made via forced aggregation using two murine osteosarcoma cell lines (K7M2 and K12 cells) cultured in D-MEM and a non-cancerous, pre-osteoblast control of MC3T3 cells cultured in -MEM. For each cell type, two densities of spheroids (10,000 cell and 5,000 cell) were formed over 72 h under two oxygen tensions (5% and 21%), cultured for an additional 24 h after formation, and then collected. Proliferation and metabolic activity were measured via PicoGreen and alamarBlue, respectively. To interrogate the roles of cellcell and cell-extracellular matrix (ECM) signaling, we measured spheroid stiffness via compression, as well as integrin and cadherin expression via qPCR.

Results, Conclusions, and Discussions:: It is well established that spheroids are held together by ECM, yet the cellular response within spheroids is poorly understood. Our data suggest that spheroids, regardless of size, undergo a selfregulated dynamic equilibrium, which is demonstrated by the similar DNA content of 5k and 10k spheroids (Figure 1). This data shows that even though the 10k spheroids have twice the number of cells as the 5k, PicoGreen results indicate that their DNA content is similar between the different sizes for all cell types across oxygen tensions. Therefore, we investigated cell-cell and cell-ECM interactions and their regulation of spheroid size by quantifying compressive modulus. We also evaluated integrin and cadherin gene expression via qPCR. Our data reveal a complex internal signaling dynamic within the spheroid. Future studies will also interrogate ECM deposition through collagen staining and further interrogate the role of oxygen tension throughout the tumor.

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