Osteosarcoma remains a highly aggressive malignancy with limited therapeutic progress and poor outcomes, particularly in metastatic or recurrent cases. Conventional treatment approaches, primarily based on surgery and high-dose chemotherapy, are hindered by significant drawbacks, including severe toxicity, high relapse rates, and drug resistance, underscoring the inadequacy of current standard approaches. This review examines emerging advances in precision medicine and drug discovery, including targeted inhibitors, immunomodulatory agents, combination treatments, and advanced biomaterials, that promise to transform osteosarcoma care. Recent advances, such as combinations of immune checkpoint inhibitors with novel agents or nanoparticle-based drug delivery systems, as well as CRISPR-Cas9 gene-editing applications, offer new strategies to overcome the inherent challenges of conventional therapies. In addition, cutting-edge research leveraging multi-omics analyses and digital pathology is refining our understanding of the tumour microenvironment, paving the way for more individualised treatment strategies.

Cole–Carpenter syndrome (CCS) is a rare autosomal dominant skeletal disorder characterized by severe bone fragility, recurrent fractures, craniosynostosis, and distinctive craniofacial abnormalities. Pathogenic variants in P4HB, encoding protein disulfide isomerase A1 (PDIA1), represent the most frequent cause of CCS. The recurrent missense mutation p.Tyr393Cys (Y393C) has been identified in unrelated patients, yet the in vivo consequences of this mutation on skeletal biology and its therapeutic implications remain poorly understood. Here, we generated a mouse model carrying the mouse homolog P4hbY395C mutation. Comprehensive skeletal phenotyping revealed pronounced osteopenia and increased bone fragility in mutant mice across multiple ages. Consistently, CCS mice exhibited reduced circulating levels of procollagen type I N-terminal propeptide (PINP I), indicating decreased type I collagen biosynthesis. At the cellular level, primary osteoblasts isolated from CCS mice showed lower expression and secretion of type I collagen, along with decreased expression of osteocalcin, whereas osteoclast differentiation was increased. Given the lack of disease-modifying therapies for CCS, we explored different therapeutic strategies. We performed a functional screen of an FDA-approved drug library identifying four compounds that significantly enhanced type I collagen secretion. Finally, we developed an allele-specific RNA interference (siRNAs) approach and identified siRNA sequences capable of selectively silencing the mutant P4hb allele in vitro and ex vivo without cytotoxic effects. Collectively, our study establishes a robust preclinical model for P4hb-related CCS, defines defective type I collagen biosynthesis as a central pathogenic mechanism, and identifies promising therapeutic strategies with translational potential for this currently untreatable skeletal disorder.

Background/Objectives: Osteosarcoma is the most common primary malignant bone tumour, affecting children and young adults. Recent evidence suggests that extracellular vesicles (EVs), small membrane-bound nanoparticles released by all cell types, play a key role in intercellular communication within the tumour microenvironment. Therefore, we aimed to investigate the effects of osteoblast-derived EVs (OB-EVs) on osteosarcoma cell behaviour and to characterise the transcriptional and miRNA-mediated mechanisms underlying these effects. Methods: Phenotypic assays were performed to assess metabolic activity, proliferation, apoptosis, and invasion ability of human osteosarcoma cell lines after treatment with OB-EVs. Illumina-based RNAseq was conducted on RNA isolated from OB-EVs-treated cells, and qRT-PCR was assessed using commercially available TaqMan miRNA cards on RNA isolated from OB-EVs. Results: In U2OS cells, OB-EVs reduced metabolic activity (1.30-fold decrease, p = 0.0137) and proliferation (1.70-fold decrease, p = 0.017) while increasing apoptosis (1.15-fold increase, p = 0.014). In MG63, OB-EVs increased proliferation (4.9-fold increase, p = 0.020) without affecting tumour cell aggressiveness, while normal osteoblast behaviour was not affected by OB-EVs. MNNG/HOS cells treated with OB-EVs for 48 h showed substantial transcriptomic changes, with 296 differentially expressed genes (97 up- and 199 down-regulated in OB-EVs treated cells versus untreated cells), indicating a direct impact of OB-EVs on gene expression. Intriguingly, Gene Set Enrichment Analysis (GSEA) showed trends consistent with modulation of signalling pathways, including Wnt/β-catenin and NOTCH. Conversely, miRNA profiling of OB-EVs identified 13 highly expressed miRNA. Integration of transcriptomic and miRNA target prediction data highlighted convergent pathway-level signals, suggesting that OB-EVs may modulate tumour-associated regulatory networks. Conclusions: Taken together, these findings indicate that OB-EVs modulate osteosarcoma cell phenotype, with miRNA shuttling representing a potentially relevant contributing mechanism. The integrative analysis suggests that pathways associated with proliferation and cellular homeostasis, including Wnt/β-catenin signalling, may be involved, although further functional validation is required to confirm these mechanisms.

Background/Objectives: Bone metastases are a common complication of breast cancer. In our previous study, we reported that extracellular vesicles released by osteotropic human (MDA-MB-231) and murine (4T1) breast cancer cells disrupt bone homeostasis by enhancing osteoclast differentiation and impairing osteoblast function. Based on these findings, we investigated whether microRNAs contained within tumour-derived EVs could mediate these bone-altering effects. Methods: MDA-MB-231- and 4T1-EVs were tagged with the RNA-specific fluorophore SYTORNA and employed to treat mouse primary bone marrow macrophages (BMMs) and osteoblasts (OBs). We also performed RNAseq on MDA-MB-231- and 4T1-EVs to assess their miRNAs content. Finally, we evaluated the effect of selected miRNA-mimics on OBs, BMMs and HUVEC cells. Results: Fluorescence microscopy demonstrated EV-RNAs shuttling to recipient cells, while RNA sequencing on MDA-MB-231- and 4T1-EVs revealed that, of the top 20 expressed miRNAs, 10 were common. Among them, we first focused on the following four: miR-26a-5p, miR-24-3p, miR-29a-3p, and miR-29b-3p, which were linked to bone biology. We confirmed their presence in MDA-MB-231-/4T1-EVs by qPCR. Then, we evaluated their EV-mediated shuttling to BMMs and OBs using affinity tags. Among all the conditions tested, miR-29a and miR-29b were the best-shuttled miRNAs, with efficiency between 50–100% in both OBs and BMMs, both for MDA-MB-231- and 4T1-EVs. Finally, to test whether miR-29a and miR-29b could have a functional role in bone cells, OBs were transfected with miR-29a and 29b-mimics, discovering that this treatment reduced collagen1α1 and 1α2 mRNA as well as the OBs’ mineralisation ability, while the same miRNA mimics were found to have no effect on osteoclastogenesis or on in vitro angiogenesis. Conclusions: MDA-MB-231- and 4T1-EVs shuttle miRNAs to bone cells, which likely contributes to OBs’ activity impairment.