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Abstract Back pain is the leading cause of disability worldwide. Its emergence relates not only to the musculoskeletal degeneration biological substrate but also to psychosocial factors; emotional components play a pivotal role. In modern society, people are significantly informed by the Internet; in turn, they contribute social validation to a “successful” digital information subset in a dynamic interplay. The Affective component of medical pages has not been previously investigated, a significant gap in knowledge since they represent a critical biopsychosocial feature. We tested the hypothesis that successful pages related to spine pathology embed a consistent emotional pattern, allowing discrimination from a control group. The pool of web pages related to spine or hip/knee pathology was automatically selected by relevance and popularity and submitted to automated sentiment analysis to generate emotional patterns. Machine Learning (ML) algorithms were trained to predict page original topics from patterns with binary classification. ML showed high discrimination accuracy; disgust emerged as a discriminating emotion. The findings suggest that the digital affective “successful content” (collective consciousness) integrates patients’ biopsychosocial ecosystem, with potential implications for the emergence of chronic pain, and the endorsement of health-relevant specific behaviors. Awareness of such effects raises practical and ethical issues for health information providers.

Since the first studies, the mouse models have provided crucial support for the most important discoveries on NK cells, on their development, function, and circulation within normal and tumor tissues. Murine tumor models were initially set to study murine NK cells, then, ever more sophisticated human-in-mice models have been developed to investigate the behavior of human NK cells and minimize the interferences from the murine environment. This review presents an overview of the models that have been used along time to study NK cells, focusing on the most popular NOG and NSG models, which work as recipients for the preparation of human-in-mice tumor models, the study of transferred human NK cells, and the evaluation of various enhancers of human NK cell function, including cytokines and chimeric molecules. Finally, an overview of the next generation humanized mice is also provided along with a discussion on how traditional and innovative in-vivo and in-vitro approaches could be integrated to optimize effective pre-clinical studies.

Osteoarthritis is a leading cause of disability in the world. The scientific literature highlights the critical importance of epigenetic regulatory effects, intertwined with biomechanical and biochemical peculiar conditions within each musculoskeletal district. While the contribution of genetic and epigenetic factors to knee OA is well-recognized, their precise role in disease management remains an area of active research. Such a field is particularly heterogeneous, calling for regular analysis and summarizing of the data that constantly emerge in the scientific literature, often sparse and scant of integration. The aim of this study was to systematically identify and synthesize all new evidence that emerged in human and animal model studies published between 2020 and 2023. This was necessary because, to the best of our knowledge, articles published before 2019 (and partly 2020) had already been included in systematic reviews that allowed to identify the ones concerning the knee joint. The review was carried out in accordance with Preferential Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Only peer-reviewed articles were considered for inclusion. A total of 40 studies were identified, showing promising results in terms either of biomarker identification, new insight in mechanism of action or potential therapeutic targets for knee OA. DNA methylation, histone modification and ncRNA were all mechanisms involved in epigenetic regulation of the knee. Most recent evidence suggests that epigenetics is a most promising field with the long-term goal of improving understanding and management of knee OA, but a variety of research approaches need greater consolidation.

Periodontal ligament (PDL) has become an elective source of mesenchymal stem cells (PDLSCs) in dentistry. This research aimed to compare healthy PDLSCs (hPDLSCs) and periodontitis PDLSCs (pPDLSCs) to ascertain any possible functional differences owing to their milieux of origin. Cells were tested in terms of colony-forming unit efficiency; multi differentiating capacity; immunophenotype, stemness, and senescent state were studied by flow cytometry, immunofluorescence, and β-galactosidase staining; gene expression using RT-PCR. Both hPDLSCs and pPDLSCs were comparable in terms of their immunophenotype and multilineage differentiation capabilities, but pPDLSCs showed a senescent phenotype more frequently. Thus, a selective small molecule inhibitor of DNA methyltransferase (DNMT), RG108, known for its effect on senescence, was used to possibly reverse this phenotype. RG108 did not affect the proliferation and apoptosis of PDLSCs, and it showed little effect on hPDLSCs, while a significant reduction of both p16 and p21 was detected along with an increase of SOX2 and OCT4 in pPDLSCs after treatment at 100 μM RG108. Moreover, the subset of PDLSCs co-expressing OCT4 and p21 decreased, and adipogenic potential increased in pPDLSCs after treatment. pPDLSCs displayed a senescent phenotype that could be reversed, opening new perspectives for the treatment of periodontitis.

Adenosinergic signaling is an important regulator of tissue homeostasis and extracellular accumulation of adenosine (Ado) and is associated with different pathologies, such as cancer. In non-small-cell lung cancer (NSCLC), a subset of CD133/CXCR4+ cancer stem cell (CSCs) has been demonstrated to initiate bone metastases. Here we investigated how NSCLC CSCs interact with osteoclasts (OCs) and osteoblasts (OBs) by modulating Ado production and OC activity. We proved that CSC-spheres, generated in vitro from NSCLC cell lines, express CD38, PC-1, and CD73, enzymes of the non-canonical adenosinergic pathway, produce high level of Ado, and down-regulate A1R and A3R inhibitory receptors, while expressing A2AR and A2BR. To address the Ado role and modulation of the in-bone pre-metastatic niche, we performed co-cultures of CSC-spheres with OCs and OBs cells. Firstly, we verified that active OCs do not activate non-canonical the adenosinergic pathway, conversely to OBs. OCs co-cultured with CSC-spheres increase Ado production that is related to the OC resorption activity and contributes to T-cell suppression. Finally, we proved the efficacy of anti-CD73 agents in blocking NSCLC cell migration. Overall, we assessed the importance of adenosinergic signaling in the interaction between CSCs and OCs at the pre-metastatic niche, with therapeutic implications related to Ado production.

Cancer stem cells (CSCs) are functionally defined as the cell subset with greater potential to initiate and propagate tumors. Within the heterogeneous population of lung CSCs, we previously identified highly disseminating CD133+CXCR4+ cells able to initiate distant metastasis (metastasis initiating cells-MICs) and to resist conventional chemotherapy. The establishment of an immunosuppressive microenvironment by tumor cells is crucial to sustain and foster metastasis formation, and CSCs deeply interfere with immune responses against tumors. How lung MICs can elude and educate immune cells surveillance to efficiently complete the metastasis cascade is, however, currently unknown. We show here in primary tumors from non-small cell lung cancer (NSCLC) patients that MICs express higher levels of immunoregulatory molecules compared to tumor bulk, namely PD-L1 and CD73, an ectoenzyme that catalyzes the production of immunosuppressive adenosine, suggesting an enhanced ability of MICs to escape immune responses. To investigate in vitro the immunosuppressive ability of MICs, we derived lung spheroids from cultures of adherent lung cancer cell lines, showing enrichment in CD133+CXCR4+MICs, and increased expression of CD73 and CD38, an enzyme that also concurs in adenosine production. MICs-enriched spheroids release high levels of adenosine and express the immunosuppressive cytokine IL-10, undetectable in an adherent cell counterpart. To prevent dissemination of MICs, we tested peptide R, a novel CXCR4 inhibitor that effectively controls in vitro lung tumor cell migration/invasion. Notably, we observed a decreased expression of CD73, CD38, and IL-10 following CXCR4 inhibition. We also functionally proved that conditioned medium from MICs-enriched spheroids compared to adherent cells has an enhanced ability to suppress CD8+ T cell activity, increase Treg population, and induce the polarization of tumor-associated macrophages (TAMs), which participate in suppression of T cells. Treatment of spheroids with anti-CXCR4 rescued T cell cytotoxic activity and prevented TAM polarization, likely by causing the decrease of adenosine and IL-10 production. Overall, we provide evidence that the subset of lung MICs shows high potential to escape immune control and that inhibition of CXCR4 can impair both MICs dissemination and their immunosuppressive activity, therefore potentially providing a novel therapeutic target in combination therapies to improve efficacy of NSCLC treatment.

Luminescent nanoparticles are innovative tools for medicine, allowing the imaging of cells and tissues, and, at the same time, carrying and releasing different types of molecules. We explored and compared the loading/release ability of diclofenac (COX-2 antagonist), in both undoped- and luminescent Terbium3+ (Tb3+)-doped citrate-coated carbonated apatite nanoparticles at different temperatures (25, 37, 40 °C) and pHs (7.4, 5.2). The cytocompatibility was evaluated on two osteosarcoma cell lines and primary human osteoblasts. Biological effects of diclofenac-loaded-nanoparticles were monitored in an in vitro osteoblast’s cytokine–induced inflammation model by evaluating COX-2 mRNA expression and production of PGE2. Adsorption isotherms fitted the multilayer Langmuir-Freundlich model. The maximum adsorbed amounts at 37 °C were higher than at 25 °C, and particularly when using the Tb3+ -doped particles. Diclofenac-release efficiencies were higher at pH 5.2, a condition simulating a local inflammation. The luminescence properties of diclofenac-loaded Tb3+ -doped particles were affected by pH, being the relative luminescence intensity higher at pH 5.2 and the luminescence lifetime higher at pH 7.4, but not influenced either by the temperature or by the diclofenac-loaded amount. Both undoped and Tb3+-doped nanoparticles were cytocompatible. In addition, diclofenac release increased COX-2 mRNA expression and decreased PGE2 production in an in vitro inflammation model. These findings evidence the potential of these nanoparticles for osteo-localized delivery of anti-inflammatory drugs and the possibility to localize the inflammation, characterized by a decrease in pH, by changes in luminescence.

The synergistic crosstalk between osteodifferentiating stem cells and endothelial cells (ECs) gained the deserved consideration, shedding light on the role of angiogenesis for bone formation and healing. A deep understanding of the molecular basis underlying the mutual influence of mesenchymal stem cells (MSCs) and ECs in the osteogenic process may help improve greatly bone regeneration. Here, the authors demonstrated that osteodifferentiating MSCs co-cultured with ECs promote angiogenesis and ECs recruitment. Moreover, through the use of 3D co-culture systems, we showed that ECs are in turn able to further stimulate the osteodifferentiation of MSCs, thus enhancing bone production. These findings highlighted the existence of a virtuous loop between MSCs and ECs that is central to the osteogenic process. Unraveling the molecular mechanisms governing the functional interaction MSCs and ECs holds great potential in the field of regenerative medicine.

SmartBone® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.

(1) Background: Recently, surgical treatment of distal radius fractures has increased exponentially. Many locking plates’ fixation systems have been developed allowing a more stable reduction and early mobilization. Sometimes, open reduction and fixation of distal radius fractures may leave a residual bone loss requiring grafting. This retrospective study reports clinical and radiologic outcomes of distal radius fractures treated with xenohybrid bone grafting in order to assess (i) the safety of the investigated bone graft; (ii) its radiological integration and biomechanical performances, and (iii) clinical outcomes of the patients; (2) Methods: We performed a retrospective study on a cohort of 19 patients. Preoperative X-ray and CT scan were performed. The mean clinical and radiographical follow-up was two years. Safety of the xenohybrid bone graft was constantly evaluated. Clinical results were assessed through the DASH score and Mayo wrist score; (3) Results: No adverse reactions, infections, and local or general complication were related to the use of xenohybrid bone graft. The radiolucency of the xenografts suggested progressive osteointegration. No evidence of bone graft resorption was detected. All the patients reached consolidation with good to excellent clinical results; and (4) Conclusions: Clinical and radiological data demonstrated that xenohybrid bone grafting promotes new bone formation and healing in osteopenic areas caused by fracture reduction.

Adipose tissue-derived stem cells (ASCs) are a promising tool for the treatment of bone diseases or skeletal lesions, thanks to their ability to potentially repair damaged tissue. One of the major limitations of ASCs is represented by the necessity to be isolated and expanded through in vitro culture; thus, a strong interest was generated by the adipose stromal vascular fraction (SVF), the noncultured fraction of ASCs. SVF is a heterogeneous cell population, directly obtained after collagenase treatment of adipose tissue. In order to investigate and compare the bone-regenerative potential of SVF and ASCs, they were plated on SmartBone®, a xenohybrid bone scaffold, already used in clinical practice with successful results. We showed that SVF plated on SmartBone, in the presence of osteogenic factors, had better osteoinductive capabilities than ASCs, in terms of differentiation into bone cells, mineralization, and secretion of soluble factors stimulating osteoblasts. Indeed, we observed an increasing area of new tissue over time, with and without OM. These data strongly support an innovative idea for the use of adipose SVF and bone scaffolds to promote tissue regeneration and repair, also thanks to an easier cell management preparation that allows a potentially larger use in clinical applications.

Total hip arthroplasty (THA) is a surgical procedure for the replacement of hip joints with artificial prostheses. Several approaches are currently employed in the treatment of this kind of defect. Overall, the most common method involves using a quite invasive metallic support (a Burch−Schneider ring). Moreover, valid alternatives and less invasive techniques still need to be supported by novel material development. In this work, we evaluated the performance of SmartBone®, a xenohybrid bone graft composed of a bovine bone matrix reinforced with biodegradable polymers and collagen, as an effective support in acetabular prosthesis reconstruction. Specifically, the material’s mechanical properties were experimentally determined (E = ~1.25 GPa, Ef = ~0.34 GPa, and Et = ~0.49 GPa) and used for simulation of the hip joint system with a SmartBone® insert. Moreover, a comparison with a similar case treated with a Burch−Schneider ring was also conducted. It was found that it is possible to perform THA revision surgeries without the insertion of an invasive metal support and it can be nicely combined with SmartBone®’s osteointegration characteristics. The material can withstand the loads independently (σmax = ~12 MPa) or be supported by a thinner titanium plate in contact with the bone in the worst cases. This way, improved bone regeneration can be achieved.

Introduction: Tibial plateau fractures represent a common challenge for orthopaedic surgeons, sometimes representing complex cases to manage, where augmentation using bone grafts is required for stabilisation. Autologous iliac bone graft (AIBG) is the current gold standard for bone grafting. In order to overcome limitations related to the procedure, alternative strategies, like allogenic and xenogeneic bone substitutes have been investigated. Here, within the framework of an observational clinical study, we report clinical and radiological outcomes of patients treated for tibial plateau fractures with a composite xenohybrid bone graft, aiming at assessing clinical and radiological outcomes. Materials and Methods: We performed a cohort retrospective study of patients treated for tibial plateau fractures from May 2017 to January 2018. Thirty-four patients, i.e. 100% of those having received the bone graft under investigation for tibial plateaux fracture treatment, met the inclusion criteria and were enrolled in the study. Patients were assessed at 2 weeks, and then at a 1-, 3-, and 6-months, and 1-year follow-up. At each evaluation patients filled a visual analogue scale (VAS) for the level of pain during the day life activities and underwent physical exam and anteroposterior and lateral projection radiographs of the knee. At 1 year the Tegner Lysholm Scoring Scale, International Knee Document Committee 2000 (IKDC 2000), and Short Form (36) Health Survey (SF-36) were administered. Results: At 1-year, mean VAS decreased from 6.33 ± 1.40 to 1 ± 0.79 (P < 0.0001); Tegner Lysholm Scoring Scale was 89 ± 4.10 and mean IKDC 2000 was 78.67 ± 3.31. No infections, neurovascular complications or adverse effects related to implants were reported during the clinical exams at follow-up. Mean ROM was 124 ± 6°. Radiographs did not show defects of consolidation or progressive post-surgical subsidence and demonstrated a good grade of integration of the implant. Conclusions: Clinical and radiological outcomes, and scores of questionnaires, were good. The xenograft has demonstrated to be a safe biomaterial, with satisfactory mechanical and biological performances in the mid-term period. It also showed a high grade of osteointegration and remodelling.

(1) Background: Bone tissue engineering is a promising tool to develop new smart solutions for regeneration of complex bone districts, from orthopedic to oral and maxillo-facial fields. In this respect, a crucial characteristic for biomaterials is the ability to fully integrate within the patient body. In this work, we developed a novel radiological approach, in substitution to invasive histology, for evaluating the level of osteointegration and osteogenesis, in both qualitative and quantitative manners. (2) SmartBone®, a composite xeno-hybrid bone graft, was selected as the base material because of its remarkable effectiveness in clinical practice. Using pre- and post-surgery computed tomography (CT), we built 3D models that faithfully represented the patient’s anatomy, with special attention to the bone defects. (3) Results: This way, it was possible to assess whether the new bone formation respected the natural geometry of the healthy bone. In all cases of the study (four dental, one maxillo-facial, and one orthopedic) we evaluated the presence of new bone formation and volumetric increase. (4) Conclusion: The newly established radiological protocol allowed the tracking of SmartBone® effective integration and bone regeneration. Moreover, the patient’s anatomy was completely restored in the defect area and functionality completely rehabilitated without foreign body reaction or inflammation.

Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at −80°C and −196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at −80°C and −196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.

Although bone has a high potential to regenerate itself after damage and injury, the efficacious repair of large bone defects resulting from resection, trauma or non-union fractures still requires the implantation of bone grafts. Materials science, in conjunction with biotechnology, can satisfy these needs by developing artificial bones, synthetic substitutes and organ implants. In particular, recent advances in materials science have provided several innovations, underlying the increasing importance of biomaterials in this field. To address the increasing need for improved bone substitutes, tissue engineering seeks to create synthetic, three-dimensional scaffolds made from organic or inorganic materials, incorporating drugs and growth factors, to induce new bone tissue formation. This review emphasizes recent progress in materials science that allows reliable scaffolds to be synthesized for targeted drug delivery in bone regeneration, also with respect to past directions no longer considered promising. A general overview concerning modeling approaches suitable for the discussed systems is also provided.

Vγ9Vδ2 T cells have a natural inclination to recognize malignant B cells in vitro via receptors for stress-induced self-ligands and TCR-dependent recognition of phosphoantigens (pAgs) generated in the mevalonate (Mev) pathway. This inclination is continuously challenged in vivo by the immune suppression operated by tumor cells. Multiple myeloma (MM) is a prototypic B-cell malignancy in which myeloma cells subvert the local microenvironment to reshape antitumor immune responses. In this study, we have investigated the immune competence of bone marrow (BM) Vγ9Vδ2 T cells in a large series of MM patients. We have found that the BM microenvironment significantly hampers the pAg-reactivity of BM Vγ9Vδ2 T cells, which become largely PD-1+ and are surrounded by PD-L1+ myeloma cells and increased numbers of PD-L1+ myeloid-derived suppressor cells (MDSC). Vγ9Vδ2 T-cell dysfunction is an early event that can be already detected in individuals with monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even when MM patients achieve clinical remission. Anti-PD-1 treatment increases the cytotoxic potential of Vγ9Vδ2 T cells by almost 5-fold after pAg stimulation, and appears to be a promising strategy for effective immune interventions in MM.

Conflicting data have been reported about the frequency and function of regulatory T cells in multiple myeloma. Most studies have investigated peripheral blood rather than bone marrow Tregs and side-by-side comparisons with bone marrow from healthy donors have still not been made. In this study, we show that regulatory T-cells total count, subset distribution, and expression of chemokine receptors are similar in the bone marrow of myeloma patients and healthy donors. Regulatory T cells are not recruited by myeloma cells in the bone marrow and their counts are unaffected by the tumor burden and the disease status. The diversity of T-cell receptor repertoire is highly preserved ensuring broad reactivity and effective suppressor function. Our results indicate that regulatory T cells may not be the main players of immunological tolerance to myeloma cells under base-line conditions, but their fully preserved immune competence may promote their inadvertent activation and blunt T-cell driven anti-myeloma immune interventions even after myeloma cells have successfully been cleared by chemotherapy.

BACKGROUND: Phenylketonuria (PKU) is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. METHODOLOGY: Peripheral blood mononuclear cell (PBMC) cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). Flow cytometry was utilized to analyze osteoclast precursors (OCPs) and T cell phenotype. Tumour necrosis factor α (TNF-α), RANKL and osteoprotegerin (OPG) were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated. PRINCIPAL FINDINGS: Several in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS). This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia. CONCLUSIONS: Our results indicate that PKU spontaneous osteoclastogenesis depends on the circulating OCP increase and the activation of T cells. Osteoclastogenesis correlates with clinical parameters, suggesting its value as a diagnostic tool for an early assessment of an increased bone resorption in PKU patients.

BACKGROUND: Osteosarcoma (OSA) is lethal when metastatic after chemotherapy and/or surgical treatment. Thus animal models are necessary to study the OSA metastatic spread and to validate novel therapies able to control the systemic disease. We report the development of a syngeneic (Balb/c) murine OSA model, using a cell line derived from a spontaneous murine tumor. METHODOLOGY: The tumorigenic and metastatic ability of OSA cell lines were assayed after orthotopic injection in mice distal femur. Expression profiling was carried out to characterize the parental and metastatic cell lines. Cells from metastases were propagated and engineered to express Luciferase, in order to follow metastases in vivo. PRINCIPAL FINDINGS: Luciferase bioluminescence allowed to monitor the primary tumor growth and revealed the appearance of spontaneous pulmonary metastases. In vivo assays showed that metastasis is a stable property of metastatic OSA cell lines after both propagation in culture and luciferase trasduction. When compared to parental cell line, both unmodified and genetically marked metastatic cells, showed comparable and stable differential expression of the enpp4, pfn2 and prkcd genes, already associated to the metastatic phenotype in human cancer. CONCLUSIONS: This OSA animal model faithfully recapitulates some of the most important features of the human malignancy, such as lung metastatization. Moreover, the non-invasive imaging allows monitoring the tumor progression in living mice. A great asset of this model is the metastatic phenotype, which is a stable property, not modifiable after genetic manipulation.

BACKGROUND: Bone forming metastases are a common and disabling consequence of prostate cancer (CaP). The potential role of osteoclast activity in CaP bone metastases is not completely explained. In this study, we investigated ex vivo whether the osteolytic activity is present and how it is ruled in CaP patients with bone forming metastases. METHODOLOGY: Forty-six patients affected by newly diagnosed CaP and healthy controls were enrolled. At diagnosis, 37 patients had a primary tumour only, while 9 had primary tumour and concomitant bone forming metastases. In all patients there was no evidence of metastasis to other non-bone sites. For all patients and controls we collected blood and urinary samples. We evaluated patients' bone homeostasis; we made peripheral blood mononuclear cell (PBMC) cultures to detect in vitro osteoclastogenesis; we dosed serum expression of molecules involved in cancer induced osteoclatogenesis, such as RANKL, OPG, TNF-alpha, DKK-1 and IL-7. By Real-Time PCR, we quantified DKK-1 and IL-7 gene expression on micro-dissected tumour and healthy tissue sections. PRINCIPAL FINDINGS: CaP bone metastatic patients showed bone metabolism disruption with increased bone resorption and formation compared to non-bone metastatic patients and healthy controls. The CaP PBMC cultures showed an enhanced osteoclastogenesis in bone metastatic patients, due to an increase of RANKL/OPG ratio. We detected increased DKK-1 serum levels and tissue gene expression in patients compared to controls. IL-7 resulted high in patients' sera, but its tissue gene expression was comparable in patients and controls. CONCLUSIONS: We demonstrated ex vivo that osteoclastogenesis is an active mechanism in tumour nesting of bone forming metastatic cancer and that serum DKK-1 levels are increased in CaP patients, suggesting to deeply investigate its role as tumour marker.

BACKGROUND: Interleukin-7 (IL-7) is a potent regulator of lymphocyte development, which has also significant effects on bone; in fact it is a potent osteoclastogenic factor. Some human solid tumors produce high IL-7 levels, suggesting a potential IL-7 role on tumor development and progression. METHODOLOGY: We studied 50 male patients affected by solid tumors, and their blood samples were collected at tumor diagnosis. PBMCs were isolated and cultured with/without IL-7 to study its influence on osteoclastogenesis. Serum and cell culture supernatant IL-7 levels were measured by ELISA. The quantitative analysis of IL-7 expression on T and B cells was performed by Real-Time PCR. PRINCIPAL FINDINGS: Serum IL-7 levels were highest in osteolytic cancer patients, followed by cancer patients without bone lesions, and then healthy controls. We showed the IL-7 production in PBMC cultures and particularly in monocyte and B cell co-cultures. A quantitative analysis of IL-7 expression in T and B cells confirmed that B cells had a high IL-7 expression. In all cell culture conditions, IL-7 significantly increased osteoclastogenesis and an anti-IL-7 antibody inhibited it. We demonstrated that IL-7 supports OC formation by inducing the TNF-alpha production and low RANKL levels, which synergize in promoting osteoclastogenesis. CONCLUSIONS: We demonstrated the presence of high serum IL-7 levels in patients with bone metastasis, suggesting the use of serum IL-7 level as a clinical marker of disease progression and of bone involvement. Moreover, we showed the capability of IL-7 to stimulate spontaneous osteoclastogenesis of bone metastatic patients and to induce osteoclastogenesis in cancer patients without bone involvement. These findings add further details to the disclosure of the mechanisms controlling bone metastasis in solid tumors.