(L-475) Osteosarcoma proliferationis impacted by senescent cell load and oxygen tension

Introduction:: Osteosarcoma (OS) is the most common primary, malignant bone cancer in adolescents and young adults. The main chemotherapeutics used to treat OS, such as doxorubicin, have not changed in 40 years and primarily disrupt DNA synthesis and repair mechanisms. Recent studies have shown that doxorubicin can induce cellular senescence rather than apoptosis in many tumors and associated stromal cells, yet the role of senescence is poorly understood in OS. Quiescent cancer cells can repress tumors by inhibiting proliferation but can also lead to possible recurrence. In order to develop new therapeutics for OS, senescence and aging of cancer cells must be further understood. Here, we investigate the role of senescence on OS tumorigenesis using murine OS cell lines. We specifically characterize the impacts of oxygen tension and 3D culture on senescent cell phenotype.

Materials and Methods:: Two murine osteosarcoma cell lines, K7M2 and K12, and one non-cancerous pre-osteoblast control, MC3T3 cells, were cultured in monolayer or formed into 5,000 and 10,000 cell spheroids for three days at two separate oxygen tensions (21% and 5%). To accentuate the senescent population, we induced replicative senescence for these studies, using cells between p45-55, which is approximately 15 passages past normal use. These cells proliferate quite slowly compared to lower passage cells. Senescence was evaluated with β-galactosidase and Gamma H2AX staining, as well as PCR for p53 and p16. Live dead staining and cell proliferation were evaluated as markers of tumorigenesis.


Results, Conclusions, and Discussions:: Our data reveal that oxygen tension impacts senescent phenotype, with cells in 5% O2 exhibiting more intense staining for β-galactosidase compared to cells in ambient air. Increased β-galactosidase staining (Figure 1) and Gamma H2AX staining was observed in all three cell types at 5% O2 compared to ambient air (21% O2). These results suggest the possibility of OS tumor suppression at the physiological oxygen tension of bone marrow (5% O2). OS spheroids formed and cultured under 5% O2 also exhibited decreased DNA content compared to those cultured at 21%, suggesting impaired proliferation under these conditions. This may suggest that increased senescence burden decreases OS tumor proliferation. Future steps include β-gal staining on OS spheroids to further characterize the apoptotic vs senescent load. We will also treat the cells in both monolayer and spheroid form with different senolytic agents to directly evaluate the role of senescent cells in OS tumorigenesis. This will give us more information for feasible therapeutics in order to advance the treatment of OS. These data tell us more about the beneficial role senescence can play in possible tumor suppression.


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