Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2^−/− mice (MDXxLcn2^−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2^−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

Bone is the preferential site of metastasis for the most common tumors, including breast cancer. On the other hand, osteosarcoma is the primary bone cancer that most commonly occurs and causes bone cancer-related deaths in children. Several treatment strategies have been developed so far, with little or no efficacy for patient survival and with the development of side effects. Therefore, there is an urgent need to develop more effective therapies for bone primary tumors and bone metastatic disease. This almost necessarily requires the use of in vivo animal models that better mimic human pathology and at the same time follow the ethical principles for the humane use of animal testing. In this review we aim to illustrate the main and more suitable in vivo strategies employed to model bone metastases and osteosarcoma. We will also take a look at the recent technologies implemented for a partial replacement of animal testing.

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.

The aim of this article is to use zeolitic imidazolate framework‐8 (ZIF‐8) metal–organic frameworks (MOFs) as drug delivery system for lidocaine, a local anesthetic with recently discovered antitumoral effects, in particular for the early‐stage treatment of breast cancer. A previously optimized ZIF‐8 synthetic method is employed, and the prepared material is deeply characterized with X‐ray powder diffraction (XRPD), surface area analysis, scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). After confirming the successful synthetic outcomes, a tailored loading procedure is developed to incorporate lidocaine within the ZIF‐8 pores. The effectiveness of this loading process is verified using UV–Vis spectroscopy and TGA, while SEM and attenuated total reflectance‐mid infrared spectroscopy further confirms the presence of lidocaine within the MOF pores. Afterwards, the pH‐responsive drug release by dialyzing the lidocaine@ZIF‐8 sample is tested either at physiological or at weakly acid pH values by UV spectroscopy on the dialysis solutions. Finally, the MDA‐MB‐231 breast cancer cells viability of lidocaine@ZIF‐8 system is tested and compared with the free drug and the MOF alone, confirming its significant capabilities in the reduction of cells viability.

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.