(P 283) A TRIDIMENSIONAL PRECLINICAL MODEL TO STUDY HUMAN AND CANINE OSTEOSARCOMA DEVELOPMENT AND INTERACTION WITH TUMOR MICROENVIRONMENT

Objective: Osteosarcoma (OS) is a rare primary bone tumor affecting humans and pets, particularly large bread dogs, that develops mainly in the medullary cavity of the axial skeleton. OS tumor microenvironment is characterized by the formation of woven bone with defective biomineralization, and its rapid growth may cause osteolytic lesions, which can free growth factors stored in the bone matrix and undermine bone strength. The gold standard treatment in both species resides in chemotherapy and surgery, which are not curative in all cases, entailing the risk of developing a local recurrence or distant metastases. Animal models still represent the optimum for pathology and pharmacological studies, but the Italian legislation has placed special attention on developing alternative models of laboratory animals. Our aim is to develop a 3D in vitro model of human and canine OS by culturing cancer cells encased within alginate-based capsules in the presence or absence of demineralized bone matrix (DBM), which simulates the matrix in osteolytic lesions and woven bone. Our goals are to assess the ability of the model to "recreate" a similar tissue to that of origin and to evaluate the influence of part of the tumor microenvironment, such as the DBM. Finally, we aim to highlight differences or similarities between human and dog cancer cells.

Methods: Alginate capsules were produced by dripping a mixture of cells in culture medium, xanthan gum, and CaCl2 with/without DBM in an alginate solution in agitation. The cell lines were 143B-GL (aggressive metastatic, +GFP/Luciferase) and MG63-GL (fibroblastic non-metastatic, +GFP/Luciferase) for the human model, and D17 cells for canine OS. Capsules were cultivated for 21 days in static conditions. On days 1, 7, 14, and 21, we performed cellular viability, gene expression, immunohistochemical (SATB2, alcian blue, alizarin red staining), and fluorescence/confocal microscopy analyses.

Results: All cell lines display a sustained growth inside the capsules for at least 14 days, forming 3D aggregates of different sizes. Between day 14 and day 21, while human OS cells enter growth arrest, canine D17 cells continue proliferating. The 143B-GL cells show a slight surge in proliferation in the presence of DBM, while MG63-GL and D-17 cells have no effects. All cell lines tend to grow around the DBM. Interestingly, in human OS models, the analysis of cell viability at day 21 displays a significant higher number of MG63-GL cells evading the capsules in the presence of DBM, reaching the potential of the invasive 143B-GL cell line. Both fluorescence and confocal microscopy analyses show a structural change of DBM in all samples, suggesting that cancer cells could remodel the matrix. Histochemical staining reveal that from day 7, all cells deposit calcium and collagen, indicating moderate osteoinductive properties and initial mineralization. This is more evident for MG63-GL cells, which are defined as osteoblast-like cells. In all samples, the addition of DBM induces a major calcium deposition, indicating a good reproduction of the tumor microenvironment. Finally, the positivity to SATB2 confirms the consistency of our model after 21 days of culture.

Conclusion: Our 3D models demonstrated good potential to recreate the human and canine OS microenvironment in vitro. The presence of DBM not only influenced the cells' way of growing, but the results on the MG63-GL cell line suggest that cells that are known in the literature to represent an indolent form of OS acquire the ability of motility in the presence of a component that mimics tumor microenvironment. Further analyses are needed to better define the accuracy and reliability of our model with respect to the in vivo tumors. Once optimized, our model will allow to test various drugs, such as antiproliferative chemotherapeutics from 7 to 14 days of culture, when cells are still in their exponential growth phase, and pro-differentiating drugs to study and induce OS cell osteo-differentiation.