IntroductionTumor-associated angiogenesis is a critical driver of tumor progression and is frequently characterized by excessive branching and structural disorganization. These abnormalities arise from dynamic interactions between tumor cells and the microenvironment, where metabolic stressors such as hypoxia and extracellular acidosis promote the release of pro-angiogenic factors. Among these, sphingosine-1-phosphate (S1P) has emerged as a key bioactive lipid involved in vascular development. We previously demonstrated that acidosis promotes sphingomyelin turnover and S1P secretion in osteosarcoma cells, enhancing tumor cell survival and migration. In this study, we investigated the role of S1P in osteosarcoma-associated angiogenesis and the contribution of tumor acidosis to this process.MethodsMatrigel®-based angiogenesis assays, HUVEC cultures, 3D osteosarcoma spheroids, and microfluidic systems were employed to evaluate endothelial sprouting and tubulogenesis. S1P signaling was pharmacologically inhibited using the FDA-approved S1P modulator FTY720 (Fingolimod). Conditioned media from osteosarcoma spheroids cultured under neutral or acidic conditions were analyzed for their pro-angiogenic activity. Soluble and extracellular vesicle-associated angiogenic mediators were also assessed.ResultsS1P dose-dependently impaired endothelial tubulogenesis while strongly promoting endothelial sprouting. Conditioned media derived from acid-stimulated osteosarcoma spheroids significantly increased endothelial tubule length and branching compared with conditioned media from spheroids maintained at neutral pH. These effects were markedly reduced by FTY720 treatment. Furthermore, tumor-derived S1P activated autocrine signaling in osteosarcoma cells, enhancing the secretion of soluble and extracellular vesicle-associated pro-angiogenic mediators, including bFGF and the TGF-β co-receptor Endoglin (CD105).DiscussionThese findings identify a previously unrecognized acidosis–S1P axis that contributes to angiogenic remodeling in osteosarcoma. Our results highlight the multifaceted role of S1P in regulating endothelial behavior and suggest that targeting S1P signaling may represent a promising strategy to disrupt pathological neoangiogenesis in osteosarcoma.

Perilipin 2 (PLIN2) is a lipid droplet (LD)-associated protein broadly expressed across tissues, where it regulates fatty acid uptake, lipid storage, and LD dynamics. During adipogenesis, PLIN2 is localized to small LDs in preadipocytes but is absent in mature adipocytes, where it is replaced by other perilipins. Whether PLIN2 loss represents a driver or a consequence of adipogenic differentiation remains unresolved. To address this question, we performed in vitro and ex vivo analyses. Adipogenic differentiation was induced in human primary dermal fibroblasts (DFs), and PLIN2 function was investigated using small interfering RNA (siRNA)-mediated silencing and treatment with Brefeldin A and Monensin (B + M). In parallel, plasma samples from both physically active healthy subjects and patients with mobility impairment were analyzed to assess PLIN2 expression. PLIN2 silencing accelerated adipogenic differentiation, whereas its higher expression induced by B + M markedly delayed this process. Notably, although Plin2 mRNA became nearly undetectable during differentiation, PLIN2 protein continued to be synthesized and, at variance with DFs, released into the culture supernatant as a circulating form (cPLIN2), partially associated with extracellular vesicles. Consistent with these findings, plasma cPLIN2 levels in vivo positively correlated with increased adiposity and low physical activity. Moreover, skeletal muscle samples from healthy subjects exhibited higher expression of CD36, a recently identified PLIN2 interactor, compared with patients. Collectively, these data identify PLIN2 as a negative regulator of the early phases of adipogenic differentiation and suggest that its secretion may represent a mechanism of interorgan communication potentially mediated by extracellular vesicles.

Over the past decade, much attention has been directed towards the gut microbiota (GM) as a contributing factor to a wide range of diseases, especially in the aging population. In this study, we analyzed the GM of elderly patients with fragility fractures (age range 71-90 years, n = 13), and patients with severe osteoarthritis (age range 65-87 years, n = 11) using 16S rRNA amplicon sequencing of fecal samples and compared these profiles with those of healthy individuals (age range 65-100 years, n = 20). The aim was to improve understanding of GM alterations in skeletal diseases and to inform the future development of personalized therapeutic strategies. Analysis of microbial composition revealed that patients with skeletal disorders showed a dysbiotic GM profile, characterized by enrichment of Peptostreptococcaceae and Butyricicoccaceae, and other potentially pathogenic taxa, alongside depletion of healthy-associated, short-chain fatty acids (SCFA)-producing bacteria. Moreover, analysis of gut metabolic modules identified multiple functional differences between patients' groups and healthy controls, particularly in pathways related to SCFA production, amino acid metabolism, and energy metabolism. Overall, these findings demonstrate both shared and disease-specific dysbiotic signatures in the GM of elderly patients with fragility fractures or severe osteoarthritis compared with healthy individuals. Once validated in larger cohorts and supported by mechanistic studies, these results may pave the way for the development of novel GM-based intervention strategies for the management of skeletal diseases.

Prostate cancer (PCa) is the second leading cause of cancer-related mortality in men, with bone representing the predominant metastatic site. Progress in treating bone metastatic disease is hindered by lack of preclinical models that faithfully recapitulate the bone microenvironment while reducing animal use. Biomaterial-based platforms offer a powerful alternative, enabling controlled reconstruction of bone composition, metabolic cues, and tumour-matrix interactions. A defining feature of PCa progression is citrate-centred metabolic reprogramming. While healthy prostate epithelial cells accumulate and secrete citrate, aggressive PCa cells import and oxidise it to sustain growth. Given the citrate-rich nature of prostate tissue and bone, we hypothesised that bone-derived citrate may be exploited by metastatic PCa cells to support bone colonisation. We developed a bone-mimetic platform by functionalising hydroxyapatite nanocrystals with citrate (HA-Nc-Cit) and incorporating them into collagen-based 3D matrices within a microfluidic chip. HA-Nc-Cit were characterised and citrate release quantified. Metastatic PCa cells were analysed for migration, viability, clonogenicity, metabolic reprogramming, and citrate transporter expression. HA-Nc-Cit released physiologically relevant citrate levels. Citrate exposure enhanced migration of androgen-independent PC3 cells and, within collagen type I-enriched matrices, increased clonogenicity, upregulated plasma membrane citrate transporter, suppressed glycolysis, and promoted lactate fermentation and mitochondrial biogenesis, without affecting respiratory chain or lipid metabolism. Citrate buffering supported PC3 clonal survival under acidic stress mimicking tumour acidification. In conclusion, citrate-functionalised HA-Nc promotes bone tropism of aggressive PCa by enhancing migratory potential, modulating tumour metabolism, and buffering extracellular acidification, underscoring the value of biomaterial-based models for studying bone-tumour interactions and guiding therapeutic development. STATEMENT OF SIGNIFICANCE: PCa often spreads to bone, but current models fail to capture the complexity of bone environment, limiting progress in treatment development. In this study, we created a 3D bone-mimicking system by binding citrate, a key bone metabolite, to hydroxyapatite nanocrystals mimicking bone mineral and embedding them in collagen-based matrices. This platform shows how citrate not only fuels PCa cells but also buffers the acidic conditions they create, making bone more prone to tumour growth. Unlike traditional models, such biomaterial-based approach combines mineral chemistry, metabolism, and pH regulation in a controlled setting. This work introduces a tool to study bone-tumour interactions and guide future therapies for metastatic PCa.

Human bone tissue is composed of a mineral component, mainly carbonate hydroxyapatite, an organic component, and water. Pathological conditions can alter the chemical and physical characteristics of hydroxyapatite, affecting the mechanical properties of bone. Fourier transform infrared spectroscopy is a valuable tool for investigating the chemical properties of bone tissue and detecting possible alterations through the assessment of specific bone quality indexes. In this work, a MATLAB tool was developed to enable rapid and efficient quantification of these indexes applied to mineral component of human bone samples. Comparison of the results obtained using the software with those derived by expert and beginner users demonstrated that the tool provides reliable outcomes and supports less experienced users, while also significantly reducing analysis time when processing large datasets. As methodological proof of concept, the tool was applied to samples derived from healthy, osteoporotic, and osteoarthritic human bone tissues, enabling the detection of significant differences in both the degree and type of carbonate group substitution between healthy and pathological samples. This highlights the tool's potential to assist researchers in investigating the chemical properties of bone tissue, contributing to the development of new therapeutic strategies and facilitating early-stage assessment of bone tissue samples.

Ewing sarcoma (EwS) shows a limited clinical response to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi), despite promising preclinical data. In this study, we compared five PARPi with different PARP-trapping capacities in PDX-derived cell lines and mouse models. Talazoparib, the strongest PARP-trapping agent, showed markedly greater efficacy than olaparib or veliparib. It triggered extensive DNA damage, micronuclei formation, and activation of the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, leading to robust type I interferon and pro-inflammatory cytokine release, an effect not seen in osteosarcoma. In vivo, talazoparib also reshaped the tumor microenvironment, increasing macrophage infiltration and reducing tumor growth. In vitro, conditioned media from treated EwS cells promoted M0-like macrophage polarization towards an inflammatory M1-like status. These immunostimulatory effects were initiated by tumor-derived interferons and were absent in talazoparib-resistant and olaparib-treated EwS cells, underscoring the importance of the PARP trapping activity of PARPi rather than catalytic inhibition. Combination of talazoparib with exogenous 2'-3'-cyclic GMP-AMP (cGAMP) does not further increase phagocytosis of EwS cells when co-cultured with macrophages, and no additive effects were observed under the tested conditions. Thus, talazoparib is a potent cytotoxic agent with innate immune activation/macrophage-mediated effects, prompting further clinical evaluation in this tumor type.

AIM:The tumor microenvironment in solid tumors is characterized by extracellular acidosis, which promotes cancer aggressiveness. In osteosarcoma, the most common primary bone cancer, a highly acidic tumor microenvironment is associated with metastasis and poor prognosis, partly due to metabolic rewiring, including changes in lipid pathways such as those involving sphingosine-1-phosphate, a bioactive sphingolipid. Sphingosine-1-phosphate has been previously implicated in histone deacetylase inhibition and gene activation. Here, we investigated whether acidosis induces nuclear sphingosine-1-phosphate accumulation via sphingosine kinase 2, leading to epigenetic activation of oncogenes like FOS in osteosarcoma. METHODS:Osteosarcoma spheroids were cultured under neutral or acidic conditions. Histone H3 acetylation was assessed by capillary Western blotting. FOS expression and FOS nuclear localization were analyzed. Sphingosine-1-phosphate's role was addressed through sphingosine kinase 2 silencing and inhibition (ABC294640). Functional effects were measured using colony formation assays. Patient-derived OS tissues (n = 7) were analyzed for correlations between acidity markers (LAMP2, V-ATPase), sphingosine kinase 2, and FOS expression. RESULTS:Acidosis increased both sphingosine kinase 2 mRNA expression after 24 h and histone H3 acetylation, which followed progressive FOS upregulation and nuclear FOS accumulation. Sphingosine kinase 2 inhibition or silencing reduced these effects and impaired clonogenicity. In patient tissues, sphingosine kinase 2 levels correlated with acidosis markers and FOS expression. CONCLUSIONS:We identified a novel mechanism where acidosis stimulates both nuclear sphingosine kinase 2 to synthesize sphingosine-1-phosphate and histone H3 acetylation, ultimately leading to FOS transcription. Targeting this axis decreased clonogenesis, underscoring its therapeutic potential in osteosarcoma and potentially other acid-adapted cancers.

The gut microbiome (GM) interacts with bone metabolism through several mechanisms, including food digestion, nutrient adsorption, immune regulation, and maintenance of the intestinal epithelium, building a close relationship with the host. Therefore, when dysbiosis occurs, it can negatively affect bones and joints. In this pilot study, the GM of patients with bone fragility fractures of the hip (OP group, n=11) was analyzed and compared to that of patients with osteoarthrosis (OA group, n=11) undergoing total hip replacement as well as that of healthy subjects (H group, n=21) to explore differences in GM composition in different settings. To this end, stool samples were collected and characterized by 16S rRNA amplicon sequencing. Analyses at phylum, family and genus level showed few differences between OP and OA, while a remarkable difference was observed between OP/OA patients and H, suggesting a potential relationship between GM dysbiosis and age-related bone disorders. The main differences in the taxonomic composition among OP, OA and H groups are displayed in where taxa are hierarchically clustered and sorted based on their Euclidean distances. The OA and OP groups displayed lower Shannon diversity compared to the H group, reflecting a less diverse and potentially unbalanced GM. Taxonomically, OP and OA had a lower relative abundance of Firmicutes and higher levels of Actinobacteria compared to the H group. Furthermore, Peptostreptococcaceae, Collinsella, and Ruminococcus were higher in the OP/OA group compared to H, while short-chain fatty acid-producing bacteria such as Eubacterium, Lachnospira, and Butyricicoccaceae, were less abundant in OP/OA compared to H. The data collected highlights a potential unique GM signature in OP and OA patients, which may affect bone and joint tissue quality and health. Further evaluations are required to validate and extend these findings, with the ultimate aim of determining the impact of GM dysbiosis on bone and joint tissue homeostasis.

Background: Angiogenesis, the process of new blood vessel formation, is critically regulated by a balance of pro- and anti-angiogenic factors. This process plays a central role in tumor progression and is modulated by tumor cells. Sphingosine-1-phosphate (S1P), a bioactive lipid signaling molecule acting via G-protein-coupled receptors (S1PR1–5), has emerged as a key mediator of vascular development and pathological angiogenesis in cancer. Consequently, targeting the S1P-S1PRs axis represents a promising strategy for antiangiogenic therapies. This study explores S1PR3 as a potential therapeutic target in osteosarcoma, the most common primary bone malignancy, which we have previously demonstrated to secrete S1P within the acidic tumor microenvironment. Methods: The effects of KRX-725-II and its derivatives, Tic-4-KRX-725-II and [D-Tic]4-KRX-725-II—pepducins acting as S1PR3 antagonists as allosteric modulators of GPCR activity—were tested on metastatic osteosarcoma cells (143B) for proliferation and migration inhibition. Anti-angiogenic activity was assessed using endothelial cells (HUVEC) through proliferation and tubulogenesis assays in 2D, alongside sprouting and migration analyses in a 3D passively perfused microfluidic chip. Results: S1PR3 inhibition did not alter osteosarcoma cell growth or migration. However, it impaired endothelial cell tubulogenesis up to 75% and sprouting up to 30% in respect to controls. Conventional 2D assays revealed reduced tubule nodes and length, while 3D microfluidic models demonstrated diminished sprouting area and maximum migration distance, indicating S1PR3’s role in driving endothelial cell differentiation. Conclusions: These findings highlight S1PR3 as a critical regulator of angiogenesis and posit its targeting as a novel anti-angiogenic strategy, particularly for aggressive, S1P-secreting tumors with pronounced metastatic potential and an acidic microenvironment.

Culture and other conventional diagnostic tests are hindered in their practicality as they are time- and labour intensive to perform. Point-of-care (POC) are designed for rapid, on-site pathogen detection and are increasingly recognized as a crucial tool for timely clinical decisions, especially in clinical settings in which the collection of the correct specimen requires invasive procedures, helping in defining effective therapies and optimizing patient management, particularly in perioperative settings. Accurate and timely detection of bacterial infection is critical for defining effective therapies and optimizing patient management, particularly in perioperative settings. Staphylococcus aureus, a leading cause of periprosthetic joint infection, requires rapid and reliable detection in order to define proper surgical treatment. We present an innovative portable point-of-care (PoC) device based on a fluorescent sensor array for the direct detection of S. aureus, via its secretome, in biological fluids, without the need for sample pre-treatment or signal amplification, minimizing false positives. Experimentally trained on bacterial supernatants, the sensor array delivers results within 40 min, achieving a low detection limit (102 CFU/mL) and a high selectivity, effectively discriminating S. aureus from other Gram-positive and -negative species. With a cost of ∼ € 5 per sample, this tool is suitable for clinical as well as decentralized settings. Preliminary evaluations in simulated synovial fluid further highlighted its potential in clinical settings.

Extracellular acidosis stemming from altered tumor metabolism promotes cancer progression by enabling tumor cell adaptation to the hostile microenvironment. In osteosarcoma, we have previously shown that acidosis increases tumor cell survival alongside substantial lipid droplet accumulation. In this study, we explored the role of lipid droplet formation in mitigating cellular stress induced by extracellular acidosis in osteosarcoma cells, thereby enhancing tumor survival during progression. Specifically, we examined how lipid droplets shield against reactive oxygen species induced by extracellular acidosis. We demonstrated that lipid droplet biogenesis is critical for acid-exposed tumor cell survival, as it starts shortly after acid exposure (24 h) and inversely correlates with ROS levels (DCFH-DA assay), lipid peroxidation (Bodipy assay), and the antioxidant response, as also revealed by NRF2 transcript. Additionally, extracellular metabolites, such as lactate, and interaction with mesenchymal stromal cells within the tumor microenvironment intensify lipid droplet build-up in osteosarcoma cells. Critically, upon targeting two key proteins implicated in LD formation - PLIN2 and DGAT1 - cell viability significantly declined while ROS production escalated. In summary, our findings underscore the vital reliance of acid-exposed tumor cells on lipid droplet formation to scavenge oxidative stress. We conclude that the rewiring of lipid metabolism driven by microenvironmental cues is of paramount importance for the survival of metabolically altered osteosarcoma cells in acidic condition. Overall, we suggest that targeting key members of lipid droplet biogenesis may eradicate more aggressive and resistant tumor cells, uncovering potential new treatment strategies for osteosarcoma.

Sarcomas are a rare and heterogeneous group of malignant tumors that pose significant clinical challenges, including delayed diagnosis, therapeutic resistance, and lack of reliable biomarkers. Despite advances in surgery and chemotherapy, effective treatment options for advanced disease remain limited, underscoring the urgent need to identify novel therapeutic vulnerabilities. The unfolded protein response (UPR), a conserved cellular stress pathway that maintains proteostasis under conditions of endoplasmic reticulum stress, has emerged as a critical modulator of cancer cell fate. By regulating protein folding, redox balance, and survival pathways, the UPR exerts a dual role in tumor biology, supporting tumor growth under stress while triggering apoptosis when stress becomes sustained or severe. In sarcomas, accumulating evidence indicates that UPR activation contributes to metabolic adaptation, angiogenesis, immune evasion, and chemoresistance. Drawing on the current literature encompassing preclinical models, recent translational research (PubMed from 2000 to 2025), and registered clinical trials, this narrative review synthesizes current knowledge on the multifaceted role of the UPR in sarcoma pathogenesis, with a particular focus on osteosarcoma. Furthermore, it explores the feasibility of UPR-targeted strategies as adjuvant or combinatorial approaches. In conclusion, this review provides an integrated and in-depth analysis of UPR-mediated mechanisms in sarcomas, offering perspectives on how targeting this pathway could accelerate the development of more effective and personalized treatments.

Osteosarcoma (OS), the most common primary bone malignancy, is a leading cause of cancer-related mortality in children and adolescents. Besides genomic abnormalities, several features of tumour microenvironment (TME), including cancer-associated mesenchymal stromal cells (MSC), have been recognized to play a key role in OS progression. The pathogenetic function of de novo innervation in TME has been extensively studied in carcinomas but is still an unexplored area of investigation in sarcomas, including OS. We evaluated nerve infiltration in tissue samples from a small cohort of human OS (n = 5) and from canine OS (n = 11), a translational model for the human disease, by βIII-tubulin immunostaining. We then analysed nerve-stroma-tumour crosstalk using direct and indirect co-cultures of dorsal root ganglion (DRG) neurons with OS/tumour-associated mesenchymal stromal cells (MSC and cancer-associated fibroblasts, CAF), both under standard and microfluidic conditions. In particular, we investigated the effects of tumour and stromal cells on axonal tropism and outgrowth by measuring neurite recruitment, length, and branches and, vice versa, the impact of neuron-derived secretome on OS cell proliferation and migration. Finally, we assessed the secretion of pro-neurotrophic mediators, including brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), and nerve growth factor (NGF), by MSC, CAF, and OS cells. The functional roles of IL-6 and BDNF were also verified by the blocking antibody Tocilizumab (TCZ) and the neutralizing Anti-BDNF antibody. We provided evidence of OS innervation within and surrounding the tumour in association with mesenchymal stroma that also corresponded to the most proliferative area of the tumour (Ki-67+). In vitro, both MSC and, to a lesser extent, OS cells promoted axonal growth through cytokine (IL-6) and neuromodulator (BDNF) secretion. Extracellular acidosis – a hallmark of OS aggressiveness – amplified IL-6 release by stromal cells, and its pro-neurogenic effect was prevented by IL-6 blockade. In turn, tumour-associated innervation stimulated OS cell proliferation and migration, eventually driving tumour aggressiveness. We showed, for the first time, that bone-associated nerves, fostered by the OS microenvironment, promote tumour aggressiveness. Interfering with the nerve-tumour axis, particularly with the signalling associated with mesenchymal stroma, offers novel opportunities for OS treatment.

Spheroids are three-dimensional cell clusters that serve as reliable in vitro models for cancer drug screening, mimicking tumor microarchitecture and chemoresistance. Despite their potential, current spheroid culture systems lack essential but highly challenging features, such as automatic real-time imaging, treatment response assessment, and detailed live image capturing without disturbing the spheroid structure. To address these challenges, we developed a custom culture dish with a micro-patterned agarose structure, fabricated from a 3D-printed mold. This innovative tool facilitates spheroid growth and immobilization, enabling automated high-throughput imaging and data collection. It allows the microscope objective to approach the spheroid closely (within micrometers) while it floats in the culture medium, in a mapped position. Furthermore, the platform is compatible with several imaging systems, including standard, confocal and dual-photon microscopy. We successfully demonstrated the effectiveness of our platform by culturing and treating osteosarcoma spheroids with different concentrations of a standard chemotherapeutic agent and by capturing confocal images of extracellular matrix antigens in live spheroids. Additionally, we showed that the platform is compatible with viability and metabolic assays (e.g. Alamar blue and acid phosphatase), with minimal reagent consumption and cost-effective, simultaneous imaging-based assays. In conclusion, we propose an innovative platform for the study of tumor spheroids and other three-dimensional cellular structures, which allows tracking of size, shape, antigen expression, viability, and indirect monitoring of cell count over time. This advancement enhances our capacity to conduct in-depth investigations of cell behavior and therapeutic responses, contributing significantly to cancer research and drug development.

The high failure rate of anticancer drugs in clinical trials highlights the need for preclinical models that accurately reproduce the structural, biochemical, and mechanical complexity of human tumors. Conventional two-dimensional cultures and animal models often lack the physiological complexity required to predict clinical outcomes, driving the development of three-dimensional systems that better emulate the tumor microenvironment. Among these, microfluidic-based spheroid models have emerged as powerful tools for cancer research and drug screening. By integrating 3D spheroids with microfluidics, these platforms allow precise control of nutrient flow, oxygen gradients, shear stress, and interstitial pressure, while supporting co-culture with stromal, immune, and endothelial cells. Such systems enable the investigation of drug response, angiogenesis, metastasis, and immune interactions under dynamic and physiologically relevant conditions. This review summarizes recent advances in microfluidic spheroid models for cancer, covering both carcinomas and sarcomas, with an emphasis on device design, biomaterial integration, and translational validation. Key challenges remain, including technical complexity, scalability constraints, and the absence of standardized protocols. Overall, the merger of microfluidic technology with 3D spheroid culture provides a promising pathway toward predictive, ethical, and personalized preclinical testing, bridging the gap between in vitro modeling and clinical oncology.

Chondrosarcomas (CHS) constitute approximately 20% of all primary malignant bone tumors, characterized by a slow growth rate with initial manifestation of few signs and symptoms. These malignant cartilaginous neoplasms, particularly those with dedifferentiated histological subtypes, pose significant therapeutic challenges, as they exhibit high resistance to both radiation and chemotherapy. Ranging from relatively benign, low-grade tumors (grade I) to aggressive high-grade tumors with the potential for lung metastases and a grim prognosis, there is a critical need for innovative diagnostic and therapeutic approaches, particularly for patients with more aggressive forms. Herein, small extracellular vesicles (sEVs) derived from mesenchymal stem cells are presented as an efficient nanodelivery tool to enhance drug penetration in an in vitro 3D model of CHS. Employing high-pressure homogenization (HPH), we achieved unprecedented encapsulation efficiency of doxorubicin (DXR) in sEVs derived from mesenchymal stem cells (MSC-EVs). Subsequently, a comparative analysis between free DXR and MSC-EVs encapsulated with DXR (DXR-MSC-EVs) was conducted to assess their penetration and uptake efficacy in the 3D model. The results unveiled a higher incidence of necrotic cells and a more pronounced toxic effect with DXR-MSC-EVs compared to DXR alone. This underscores the remarkable ability of MSC-EVs to deliver drugs in complex environments, highlighting their potential application in the treatment of aggressive CHS.

Bioprinting shows promise for bioengineered scaffolds and three-dimensional (3D) disease models, but assessing the viability of embedded cells is challenging. Conventional assays are limited by the technical problems that derive from using multi-layered bioink matrices dispersing cells in three dimensions. In this study, we tested bioprinted osteogenic bioinks as a model system. Alginate- or gelatin-based bioinks were loaded with/without ceramic microparticles and osteogenic cells (bone tumor cells, with or without normal bone cells). Despite demonstrating 80%-90% viability through manual counting and live/dead staining, this was time-consuming and operator-dependent. Moreover, for the alginate-bioprinted scaffold, cell spheroids could not be distinguished from single cells. The indirect assay (alamarBlue), was faster but less accurate than live/dead staining due to dependence on hydrogel permeability. Automated confocal microscope acquisition and cell counting of live/dead staining was more reproducible, reliable, faster, efficient, and avoided overestimates compared to manual cell counting by optical microscopy. Finally, for 1.2 mm thick 3D bioprints, dual-photon confocal scanning with vital staining greatly improved the precision of the evaluation of cell distribution and viability and cell-cell interactions through the z-axis. In summary, automated confocal microscopy and cell counting provided superior accuracy for the assessment of cell viability and interactions in 3D bioprinted models compared to most commonly and currently used techniques.

Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research. Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms. To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.

Convolutional Neural Networks are being increasingly applied to the detection of anomalies in Computed Tomographies (CTs). The goal of this paper is to implement an automated Computer-Aided Detection (CADe) system for spinal lesions using CTs and Convolutional Neural Networks pre-trained on commercial datasets. The proposed pipeline works as follows. The CADe takes in input CT scans and is equipped with an intuitive GUI to allow physicians to use it as a support tool for diagnoses. From the CT scans, the CADe selects volumes containing the vertebrae and extracts 2D slices of these volumes. These slices are pre-processed and then analyzed using a VGG19 Convolutional Neural Network and a tailored, binary classifier. The neural network identifies healthy vertebrae and vertebrae containing lesions (e.g. metastases, primary tumors, lytic and sclerotic lesions). For training and testing purposes, we generated a dataset from CTs of vertebrae from patients treated at IRCCS Rizzoli Orthopaedics Institute of Bologna, Italy, between 2009 and 2019. Both healthy and lesioned vertebrae retrieved with different tomography machines and setups are considered. The dataset has been enlarged using data augmentation techniques and subsequently used to train a wide range of deep learning models. We perform an in-depth benchmark study to assess the performance of the considered classifier against different Convolutional Neural Networks pre-trained on the ImageNet dataset and exploiting Transfer Learning techniques. Leveraging Transfer Learning techniques, we reached 93.43% accuracy and a recall of 92.99%.

The advancement of personalized treatments in oncology has garnered increasing attention, particularly for rare and aggressive cancer with low survival rates like the bone tumors osteosarcoma and chondrosarcoma. This study introduces a novel PDMS–agarose microfluidic device tailored for generating patient-derived tumor spheroids and serving as a reliable tool for personalized drug screening. Using this platform in tandem with a custom imaging index, we evaluated the impact of the anticancer agent doxorubicin on spheroids from both tumor types. The device produces 20 spheroids, each around 300 µm in diameter, within a 24 h timeframe, facilitating assessments of characteristics and reproducibility. Following spheroid generation, we measured patient-derived spheroid diameters in bright-field images, calcein AM-positive areas/volume, and the binary fraction area, a metric analyzing fluorescence intensity. By employing a specially developed equation that combines viability signal extension and intensity, we observed a substantial decrease in spheroid viability of around 75% for both sarcomas at the highest dosage (10 µM). Osteosarcoma spheroids exhibited greater sensitivity to doxorubicin than chondrosarcoma spheroids within 48 h. This approach provides a reliable in vitro model for aggressive sarcomas, representing a personalized approach for drug screening that could lead to more effective cancer treatments tailored to individual patients, despite some implementation challenges.