Elettra | XRD1, SYRMEP Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents and remains one of the most challenging diseases in pediatric oncology. Although relatively rare, OS is highly aggressive and often unresponsive to standard therapies. Despite decades of research, patient survival rates have seen little improvement, largely due to the tumor’s marked heterogeneity and an incomplete understanding of how it interacts with the surrounding tumor microenvironment (TME). In particular, OS effects on the structural and chemical properties of bone mineralized matrix remain poorly characterized in human tissue due to the limited availability of well-preserved clinical samples and the scarcity of multiscale analytical studies performed directly on patient-derived material. The aim of this study was to address this gap by examining how different OS subtypes influence bone mineralization through a multiscale approach using three synchrotron-based techniques. X-ray micro-computed tomography (XmCT, SYRMEP beamline, Elettra, Trieste) was used for 3D analysis of bone morphology. To investigate calcium chemistry within the mineral phase, we performed Micro-Xray Absorption Near-Edge Spectroscopy (µXANES) and X-ray Fluorescence Microscopy (XRFM) at the Ca K-edge on the ID21 beamline at ESRF, Grenoble. In addition, X-ray Diffraction (XRD) on the XRD1 beamline (Elettra) was used to Examine Hydroxyapatite (HA) crystallinity. Tissue samples were collected from tumor and control areas, stored at –20 °C, and imaged in their frozen state using XmCT. Subsequently, the samples were dehydrated, resin-embedded, sectioned (60–100 μm), and analyzed via µXANES and XRD. Specifically, we investigated the influence of three distinct OS subtypes, high-grade osteoblastic OS (HG-OS), parosteal OS (PA-OS), and periosteal OS (PE-OS), on bone tissue morphology, mineral structure, and composition. The analysis focused on how tumor development affects both the organization of bone matrix and the characteristics of HA, the principal mineral phase of bone. Importantly, alterations were assessed in comparison to adjacent non-tumoral tissue from the same patients, providing an internal reference (Fig. 1 a–c, tumor and control regions indicated in red and green, respectively). XCmT was employed to assess the 3D microarchitecture of tumor and control bone tissues across osteosarcoma subtypes. In HG-OS, reconstructions revealed moderate structural disruption, including trabecular thinning and irregular bone margins in tumor tissue with respect to control regions. In contrast, PE-O samples, characterized by a predominant cartilaginous component, exhibited hypomineralized bone and streamers of lytic bone matrix. The PA-OS sample displayed an interesting morphological profile. Originating from the outer periosteal layer, this subtype formed a lobulated, exophytic mass with central dense mineralization adjacent to the cortical bone. XCmT reconstructions revealed cortical thickening and mineralized protrusions containing disorganized, abnormal trabeculae (Fig. 1 d) —features absent in the control tissue, which maintained a compact and homogeneous structure (Fig. 1 e). µXANES revealed the occurrence of a dysregulated biomineralization in the PA-OS and PE-OS subtypes, marked by the presence of calcium compounds different from HA (such as amorphous calcium carbonate and crystalline calcium carbonate), in contrast to the mature mineral state found in the HG-OS variant. XRD was used to investigate the structural organization of HA in bone tissue. By analyzing the 002-diffraction peak, associated with the longitudinal axis of HA crystals, we mapped the local presence and crystalline structure of the mineral phase. This provided spatially resolved information on mineral content in tumors and control samples (Fig. 1 f,g). Particular attention was given to the interplanar spacing of HA along the c-axis. While control and HG-OS samples showed similar distributions (Fig. 1 h), both PA-OS and PE-OS exhibited significant shifts in the interplanar distance, indicating a shortening of the HA crystal along the crystal c-axis (Fig. 1 i, l). This study demonstrates the power of synchrotron-based techniques in uncovering subtle structural and compositional changes that are otherwise undetectable with conventional histology. The use of paired tumor and control samples from the same individuals is a notable strength, allowing us to isolate disease-specific effects while controlling for interpatient variability. Although limited by the rarity of OS and sample availability, our approach lays the foundation for more extensive studies. In conclusion, we provide novel evidence that OS subtypes influence bone mineralization in distinct ways, affecting not only morphology but also the crystalline and chemical state of the mineral matrix. These insights into the tumor microenvironment expand our understanding of OS biology and open avenues for the development of mineral-based biomarkers and targeted therapeutic strategies. Ultimately, this knowledge may contribute to improved diagnosis, stratification, and treatment outcomes for patients affected by this aggressive pediatric malignancy. Bone mineral disruption in osteosarcoma revealed by micro-computed tomography and x-ray diffraction Elettra Highlights 2024–2025 68
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