(P 75) METABOLIC EVALUATION OF THE RELATIONSHIP BETWEEN MACROPHAGES AND OSTEOSARCOMA TUMOR CELLS

Objective: Osteosarcoma is considered as the first cause of bone cancer affecting predominantly adolescents and young adults. This cancer is thought resulting from a modulated balance between bone-producing osteoblastic cells and bone-destroying osteoclastic cells. It remains up to date a challenge to treat insofar as some patients' disease progresses, develops resistance and metastasizes despite first line strategy. It is therefore necessary to understand the full range of cellular and molecular mechanisms to understand tumor cells interplay with their microenvironment. In fact, tumor cellular microenvironment seems to play a role in the growth of these malignant osteoblastic cells and favors the expression of genes involved in the proliferation, migration, and metabolic adaptation of this cancer. By the past, multiple works including ours showed beyond the identification of hypoxic areas within the diagnostic biopsy sample, giant osteoclastic cells associated to infiltration of macrophages expressing CD68+ and/or CD163+ markers. Those microenvironmental cells are modulated the tumoral balance between pro-inflammatory and pro-tumoral markers modulating the osteosarcoma growth and invasion. These macrophagic cells have also been correlated with poor prognosis and increased risk of relapse in osteosarcomas when associated to hypoxic modulation. Based on this preclinical work, our team hypothesized that the presence of these biphenotypic macrophages and the hypoxia modulation would lead to alter metabolic pathways within tumor cells. Then, the objective of this study was to differentiate macrophages expressing these different biomarkers and culture them in presence of patient-derived osteosarcoma cells and be able to study their relationship at the level of tumor escape as well as at the metabolic level.

Methods: To investigate this objective, we started by differentiating monocytes into macrophages CD68+, CD163+ and presenting both positivity (e.g., CD68+/CD163) using various stimulatory factors. Thereafter, these immune cells were co-cultured in 3D spheroids with several established osteosarcoma patient-derived cell lines (PDCL). This co-culture models enabled us to study cell phenotypic characteristics, their proliferation and migration under normoxic or hypoxic conditions. To differentiate both cells in those experiments, we were using nanoparticle cell labeling. Finally, we studied in those 3D co-cultures metabolites’ quantification using HRMAS NMR (High Resolution with Magic Angle Rotation), as well as at spatial level using non-targeted MALDI mass spectrometry.

Results: Based on different stimulation factors, two types of macrophages were derived from blood monocytes expressing CD68 or both CD68+/CD163+ membrane markers. By co-culturing metastatic osteosarcoma tumor cells with biphenotypic macrophages, we were able to evidence that macrophages were accelerating a rapid tumor cell invasion in normoxia, but also in hypoxia. The HRMAS NMR study highlighted major metabolic switches in tumor cells varying differently in both oxygen concentrations but also in presence of macrophages. Combining hypoxia and a high concentration of macrophages in close vicinity to the osteosarcoma cells was favoring significantly the anaerobic glycolytic pathway, glutaminolysis and a methionine salvage pathway. The non-targeted MALDI spectrometry approach identified two significant compounds. One was distinguished in normoxic conditions and was a witness of proinflammatory tumor cell profile in the center of the 3D co-culture models. Another one was evidenced in hypoxic conditions heterogeneously distributed from the periphery to the center of the spheroid and correlated to sphingolipid metabolism.

Conclusion: In addition to recreating in 3D models the macrophagic microenvironment present in osteosarcomas, we were able to link macrophages’ presence, oxygen modulation and tumor escape and invasion probably through metabolic pathway spatial modulation.