Developing a 3-dimensional multicellular bone model of osteosarcoma

Abstract

The main treatment for osteosarcoma patients remains a combination of surgery followed by neoadjuvant chemotherapy. The drug Mifamurtide, introduced in 2001, is routinely given concurrently with chemotherapy in the UK, and works by activating macrophages in an inflammatory manner. Unfortunately, there are mixed reviews on the therapeutic benefit of Mifamurtide, and few new drugs and therapies show efficacy in osteosarcoma clinical trials. Greater understanding of the osteosarcoma microenvironment, and the cellular interactions involved in tumour proliferation, could introduce novel targets for future therapies. To explore this a 3D multicellular model of osteosarcoma was developed, replicating some of the cellular interactions of stromal and immune cells in a human bone structure.
A range of stromal, myeloid and osteosarcoma cells were initially characterised in 2D assays for inclusion in the 3D bone model. These included; human bone marrow stromal cells (HBMSCs), macrophages derived from bone marrow and peripheral blood mononuclear cells (PBMCs), as well as two osteosarcoma cell lines, Saos-2 and MG63. The human bone structure of the 3D bone models were made from the femoral head of patients undergoing hip replacement surgery, and were cultured either in conditioned media for 20 days or incubated on the chorioallantoic membrane (CAM) of fertilised chicken eggs. Micro-computed tomography (μCT) and histological techniques were used to validate the 3D bone models; to determine whether bone remodelling occurred, as well as characterising the migration and biological activity of the inserted cells.
Characterisation of the two osteosarcoma cell lines showed distinct differences in tri-lineage differentiation, with Saos-2 cells promoting an osteogenic phenotype and consequently being used in the 3D bone models. Skeletal location also proved to be important in the tri-lineage characterisation of the HBMSCs, with cells isolated from the femoral diaphysis (FD) and femoral epiphysis (FE) demonstrating distinct phenotypes. HBMSCs from the FD were used in the development of the 3D bone models due to the higher osteogenic potential, which is also found in osteosarcoma patients. The third cell type included in the 3D model were monocyte-derived macrophages (MDMs) differentiated from PBMCs, which showed a similar phenotype to human bone marrow-derived macrophages, but could be produced in greater quantities. The Saos-2 cells,
FD HBMSCs and MDMs were introduced into the 3D bone models. Changes in the expression of CD68, CD105 and RANK were found between combinations of the three cell types, as well as those treated with and without Mifamurtide. No significant changes in bone formation or resorption were detected.
In summary, a 3D multicellular bone model of osteosarcoma has been developed that holds the potential to be used to test new drugs and therapies. While further analysis is needed to determine whether the stromal and immune cell interactions replicate those found in the osteosarcoma microenvironment, it has been shown that multiple cell types can be combined in a 3D human bone scaffold, cultured on the CAM and remain viable.

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ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

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