Insights into the osteosarcoma microenvironment: Multiscale analysis of structural and mineral heterogeneity

Osteosarcoma (OS) is a malignant and heterogeneous disease that typically originates in the long bones of children and adolescents. It is characterized by the presence of immature cells having an aggressive phenotype and rapid uncontrolled proliferation. OS progression induces significant molecular and cellular changes locally within the bone, resulting in the development of an abnormal tumor microenvironment (TME). The OS TME plays a crucial role in tumor progression and development, however, the precise effects of OS on bone structure and mineralization still remain poorly understood. In this study, we examined the OS TME by analyzing samples from osteoblastic, parosteal, and periosteal osteosarcomas. Employing advanced synchrotron-based X-ray techniques, we performed a multiscale analysis to evaluate the structural and mineral complexity of tumor-affected bone. Our results revealed marked morphological differences among the osteosarcoma subtypes, while confirming that biomineralization remains active through the production of hydroxyapatite (HA). X-ray diffraction identified two distinct hydroxyapatite crystalline phases across all samples, suggesting a critical behavior of minerals in bone. Additionally, we observed that the bone mineral structure in periosteal and parosteal osteosarcomas exhibited crystal deformations along the c-axis, whereas the osteoblastic osteosarcoma displayed a mineral profile comparable to control bone. Micro-X-ray absorption near-edge spectroscopy revealed the occurrence of a dysregulated biomineralization in the parosteal and periosteal subtypes, marked by the presence of calcium compounds different from HA, in contrast to the mature mineral state found in the osteoblastic variant. These findings highlight the complexity of osteosarcoma repercussion on bone tissue, offering new insights into the interactions within the OS TME. STATEMENT OF SIGNIFICANCE: This study investigates the tumor microenvironment (TME) of osteosarcoma (OS), a rare and aggressive bone cancer mainly affecting children and adolescents. Using advanced synchrotron-based X-ray techniques, we analyzed structural and mineral alterations in bone from three OS subtypes: osteoblastic, parosteal, and periosteal. The results reveal distinct subtype-specific differences in bone mineralization and crystallinity, highlighting the heterogeneity of OS and the pivotal role of its microenvironment in driving disease progression. This research contributes to our understanding of OS pathophysiology and provides foundation for future studies aimed at developing targeted therapies and improving diagnostic approaches.