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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.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1 -/- ) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1 -/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner., (© 2024. The Author(s).)

Disruption of the complex network that regulates redox homeostasis often underlies resistant phenotypes, which hinder effective and long-lasting cancer eradication. In addition, the RNA methylome-dependent control of gene expression also critically affects traits of cellular resistance to anti-cancer agents. However, few investigations aimed at establishing whether the epitranscriptome-directed adaptations underlying acquired and/or innate resistance traits in cancer could be implemented through the involvement of redox-dependent or -responsive signaling pathways. This is unexpected mainly because: i) the effectiveness of many anti-cancer approaches relies on their capacity to promote oxidative stress (OS); ii) altered redox milieu and reprogramming of mitochondrial function have been acknowledged as critical mediators of the RNA methylome-mediated response to OS. Here we summarize the current state of understanding on this topic, as well as we offer new perspectives that might lead to original approaches and strategies to delay or prevent the problem of refractory cancer and tumor recurrence.

Extracellular vesicles (EVs) are potent means of cell-to-cell communication. They are released in biological fluids, including blood, urine, and saliva, and can be exploited to identify new biomarkers of diseases. We hypothesized that EVs contain molecular cargos involved in bone metabolism, possibly mirroring biological differences between postmenopausal and disuse osteoporosis. We tested this hypothesis in primary murine osteoblasts subjected to steroid depletion or to unloading, and in the serum of animal models of osteoporosis induced by ovariectomy or hindlimb tail suspension. EVs were isolated by ultracentrifugation and analysed by transmission electron microscopy, cytofluorimetry, immunoblotting and RT-PCR. Large-scale analyses were performed by Real-Time arrays and Proteome Profiler™ Antibody arrays. Finally, precise titration of analytes was carried out by ELISA assay. In vitro, we confirmed an increased release of EVs enriched in surface RANKL by primary mouse osteoblasts subjected to steroid depletion or simulated microgravity compared to controls. In vivo, circulating EVs isolated from the sera of control female mice expressed RANKL along with other genes associated with bone metabolism. Serum EVs from ovariectomized or hindlimb tail-suspended mice showed distinct molecular profiles. They expressed RANKL with different kinetics, while transcriptomic and proteomic profiles uncovered unique molecular signatures that discriminated the two conditions, unveiling exclusive molecules expressed in time- and osteoporosis type-dependent manner. These results suggest that circulating EVs could represent a new tool for monitoring the onset and the progression of diverse types of the disease in mice, paving the way for their exploitation to diagnose human osteoporosis in liquid biopsies., (© 2022. The Author(s).)

Osteosarcoma is the most common primary bone malignancy. The crosstalk between osteosarcoma and the surrounding tumour microenvironment (TME) drives key events that lead to metastasization, one of the main obstacles for definitive cure of most malignancies. Extracellular vesicles (EVs), lipid bilayer nanoparticles used by cells for intercellular communication, are emerging as critical biological mediators that permit the interplay between neoplasms and the tumour microenvironment, modulating re-wiring of energy metabolism and redox homeostatic processes. We previously showed that EVs derived from the human osteosarcoma cells influence bone cells, including osteoblasts. We here investigated whether the opposite could also be true, studying how osteoblast-derived EVs (OB-EVs) could alter tumour phenotype, mitochondrial energy metabolism, redox status and oxidative damage in MNNG/HOS osteosarcoma cells.These were treated with EVs obtained from mouse primary osteoblasts, and the following endpoints were investigated: i) cell viability and proliferation; ii) apoptosis; iii) migration and invasive capacity; iv) stemness features; v) mitochondrial function and energy metabolism; vi) redox status, antioxidant capacity and oxidative molecular damage. OB-EVs decreased MNNG/HOS metabolic activity and viability, which however was not accompanied by impaired proliferation nor by increased apoptosis, with respect to control. In addition, OB-EV-treated cells exhibited a significant reduction of motility and in vitro invasion as compared to untreated cells. Although the antioxidant N-acetyl-L-cysteine reverted the cytotoxic effect of OB-EVs, no evidence of oxidative stress was observed in treated cells. However, the redox balance of glutathione was significantly shifted towards a pro-oxidant state, even though the major antioxidant enzymatic protection did not respond to the pro-oxidant challenge. We did not find strong evidence of mitochondrial involvement or major energy metabolic switches induced by OB-EVs, but a trend of reduction in seahorse assay basal respiration was observed, suggesting that OB-EVs could represent a mild metabolic challenge for osteosarcoma cells. In summary, our findings suggest that OB-EVs could serve as important means through which TME and osteosarcoma core cross-communicate. For the first time, we proved that OB-EVs reduced osteosarcoma cells' aggressiveness and viability through redox-dependent signalling pathways, even though mitochondrial dynamics and energy metabolism did not appear as processes critically needed to respond to OB-EVs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ponzetti, Ucci, Puri, Giacchi, Flati, Capece, Zazzeroni, Cappariello, Rucci and Falone.)

Automated microscope slide stainers are usually very expensive and unless the laboratory performs heavy histological work it is difficult to justify buying a 2000-10000€ machine. As a result, histology and pathology labs around the world lose thousands of working hours for following procedures that could be easily automated. Herein, we propose a simple modification of an open-source 3D printer, the Creality Ender-3, into an automated microscope slide autostainer, the HistoEnder. The HistoEnder is cheap (less than 200€), modular, and easy to set up, with only two 3D-printed parts needed. Additionally, the 3D printer retains its full functionality, and it can be reverted back into 3D printing in less than 1 min. The g-code associated with the procedure is extremely simple, and can be written by anyone. The HistoEnder can also be used in chemistry and material science laboratories for automating surface modifications and dip coating., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Liquid biopsies are a powerful tool to non-invasively analyze tumor phenotype and progression as well as drug resistance. In the bone oncology field, liquid biopsies would be particularly important to develop, since standard biopsies can be very painful, dangerous (e.g., when found in proximity to the spinal cord), and hard to collect. In this review, we explore the recent advances in liquid biopsies in both primary (osteosarcoma and Ewing sarcoma) and secondary bone cancers (breast, prostate, and lung cancer-induced bone metastases), presenting their current role and highlighting their unexpressed potential, as well as the barriers limiting their possible adoption, including costs, scalability, reproducibility, and isolation methods. We discuss the use of circulating tumor cells, cell-free circulating tumor DNA, and extracellular vesicles for the purpose of improving diagnosis, prognosis, evaluation of therapy resistance, and driving therapy decisions in both primary and secondary bone malignancies., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2022.)