(P 86) DECIPHERING CELL-MATRIX INTERACTIONS IN OSTEOSARCOMA RESPONSE TO CHEMOTHERAPY IN A 3D TISSUE-ENGINEERED IN VITRO MODEL

Objective: Osteosarcoma (OS), the most prevalent bone tumor diagnosed in the pediatric population, presents a grim 5-year survival rate of 60% in primary disease and a mere 20% in refractory and metastatic disease. Gold-standard chemotherapy was first introduced in the 1970’s and involves multiple cycles of doxorubicin, cisplatin, and methotrexate. However, despite efforts in creating molecular-targeted drugs and immune-based cellular therapies, treatment regimens and survival rates remain unchanged. One major hurdle hindering the advancement of novel therapeutics is the lack of representative models that recapitulate the extracellular matrix (ECM) of the tumor. The intricate structural and signaling components of the ECM play a pivotal role in driving OS growth, development, and influencing response to chemotherapy.

Methods: We engineered a 3D tissue-engineered model for OS to investigate the effect of ECM on OS function, as well as the influence of cell-matrix interactions in determining the response to chemotherapy. The 143B OS cell line was encapsulated within hydrogels composed of collagen type 1, the primary ECM of bone tissue, through a pH and thermo-sensitive reaction. Traditional 2D culture on tissue culture plastic served as a control. Cell viability and cell morphology after 7 days of culture were assessed through Live/Dead staining. Cell phenotype was characterized through the expression of alkaline phosphatase activity and cell proliferation markers. Response to chemotherapy was quantified by metabolic activity and the organization of cytoskeletal proteins.

Results: OS cells exhibited high viability (>90%) in both 2D and 3D culture conditions but altered their morphology to become more elongated and clustered into multicellular units in 3D, while cells remained rounded and dispersed in 2D (Fig. 1). Alkaline phosphatase, a biomarker of OS that is elevated at diagnosis and correlated with poor prognosis, was significantly increased in 3D compared to 2D at days 1, 3, and 7 in culture (Fig. 2). 2D cultures and 3D tumoroids were then grown for 7 days and exposed to chemotherapy for 24 hours followed by assessment of cellular metabolic activity to evaluate chemosensitivity. After exposure to doxorubicin, cisplatin, or a combination of doxorubicin and cisplatin, cellular metabolic activity was significantly higher in 3D tumoroids compared to 2D cultures (Fig. 3), suggesting that the cell phenotype in 2D is more chemosensitive, while the cell phenotype in 3D is more chemoresistant. As chemotherapy attacks the rapidly dividing cells in the body, we assessed for expression of Ki67, a nuclear protein, that is present in the active phases of the cell cycle (G1, S, G2) and is used as a marker of cell proliferation. Ki67 expression was significantly higher in 3D compared to 2D indicating that cells were actively proliferating at the time of chemotherapy exposure. We hypothesized that targeting the cytoskeletal microtubules of these elongated cells in 3D would rescue chemosensitivity, as these structures play a critical role in chromosome segregation during mitosis. To test this hypothesis, we added vinorelbine, a microtubule destabilizing agent used as chemotherapy for non-small cell lung cancer, to our 3D OS cultures. Immunocytochemistry confirmed the degradation of microtubules and significant alteration of the cell cytoskeleton along with a significant decrease in metabolic activity for the combinatorial therapy compared to doxorubicin and cisplatin alone (Fig. 4).

Conclusion: These data validate the need to study OS cells within a 3D ECM of collagen to capture cell-matrix interactions that are critical to the native tumor microenvironment. Our data suggest that combinatorial treatment with traditional front-line chemotherapy and a microtubule destabilizing agent may be beneficial. Future work will delve into the underlying molecular mechanisms governing OS cell-ECM interactions. While further exploration with patient-derived cell lines and patient-derived xenografts is necessary to validate the clinical relevance of this model, these results strongly support the use of 3D tissue engineered models as a valuable culture system for studying OS.