Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades Narginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib by increasing: 1) killing of human MM cells by stimulating both bortezomib mediated apoptosis and necroptosis, a process regulated by p62; and 2) preservation of bone mass by stimulating osteoblasts differentiation and inhibiting osteoclastic bone destruction. Co-administration of bortezomib and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, coadministration of bortezomib and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti-MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Pharmacological Inhibition of NFκB Reduces Prostate Cancer Related Osteoclastogenesis In Vitro and Osteolysis Ex Vivo.

NFκB is implicated in cancer and bone remodelling, and we have recently reported that the verified NFκB inhibitor Parthenolide (PTN) reduced osteolysis and skeletal tumour growth in models of metastatic breast cancer. Here, we took advantage of in vitro and ex vivo bone cell and organ cultures to study the effects of PTN on the ability of prostate cancer cells and their derived factors to regulate bone cell activity and osteolysis. PTN inhibited the in vitro growth of a panel of human, mouse and rat prostate cancer cells in a concentration-dependent manner with a varying degree of potency. In prostate cancer cell-osteoclast co-cultures, the rat Mat-Ly-Lu, but not human PC3 or mouse RM1-BT, enhanced RANKL stimulated osteoclast formation and PTN reduced these effects without affecting prostate cancer cell viability. In the absence of cancer cells, PTN reduced the support of Mat-Ly-Lu conditioned medium for the adhesion and spreading of osteoclast precursors, and survival of mature osteoclasts. Pre-exposure of osteoblasts to PTN prior to the addition of conditioned medium from Mat-Ly-Lu cells suppressed their ability to support the formation of osteoclasts by inhibition of RANKL/OPG ratio. PTN enhanced the ability of Mat-Ly-Lu derived factors to increase calvarial osteoblast differentiation and growth. Ex vivo, PTN enhanced bone volume in calvaria organ-Mat-Ly-Lu cell co-culture, without affecting Mat-Ly-Lu viability or apoptosis. Mechanistic studies in osteoclasts and osteoblasts confirmed that PTN inhibit NFκB activation related to derived factors from Mat-Ly-Lu cells. Collectively, these findings suggest that pharmacological inhibition of the skeletal NFκB signalling pathway reduces prostate cancer related osteolysis, but further studies in the therapeutic implications of NFκB inhibition in cells of the osteoblastic lineage are needed.

Pharmacological Inhibition of the Skeletal IKKß Reduces Breast Cancer-Induced Osteolysis.

IKKß has previously been implicated in breast cancer bone metastasis and bone remodelling. However, the contribution of IKKß expressed by bone cells of the tumour microenvironment to breast cancer-induced osteolysis has yet to be investigated. Here, we studied the effects of the verified selective IKKß inhibitors IKKßIII or IKKßV on osteoclast formation and osteoblast differentiation in vitro and in vivo, human and mouse breast cancer cells' support for osteoclast formation and signalling in vitro and osteolysis ex vivo and in immunocompetent mice after supracalvarial injection of human MDA-MB-231 conditioned medium or intra-cardiac injection of syngeneic 4T1 breast cancer cells. Pre-treatment with IKKßIII or IKKßV prior to exposure to tumour-derived factors from human and mouse breast cancer cell lines protected against breast cancer-induced osteolysis in two independent immunocompetent mouse models of osteolysis and the ex vivo calvarial bone organ system. Detailed functional and mechanistic studies showed that direct inhibition of IKKß kinase activity in osteoblasts and osteoclasts was associated with significant reduction of osteoclast formation, enhanced osteoclast apoptosis and reduced the ability of osteoblasts to support osteoclastogenesis in vitro. When combined with previous findings that suggest NFκB inhibition reduces breast cancer tumorigenesis and metastasis our present findings have an important clinical implication on raising the possibility that IKKß inhibitors, as bone anabolics, osteoclast inhibitors as well as anti-metastatic agents, may have advantages over anti-osteoclasts agents in the treatment of both skeletal and non-skeletal complications associated with metastatic breast cancer.

The bone ecosystem facilitates multiple myeloma relapse and the evolution of heterogeneous drug resistant disease.

Multiple myeloma (MM) is an osteolytic malignancy that is incurable due to the emergence of treatment resistant disease. Defining how, when and where myeloma cell intrinsic and extrinsic bone microenvironmental mechanisms cause relapse is challenging with current biological approaches. Here, we report a biology-driven spatiotemporal hybrid agent-based model of the MM-bone microenvironment. Results indicate MM intrinsic mechanisms drive the evolution of treatment resistant disease but that the protective effects of bone microenvironment mediated drug resistance (EMDR) significantly enhances the probability and heterogeneity of resistant clones arising under treatment. Further, the model predicts that targeting of EMDR deepens therapy response by eliminating sensitive clones proximal to stroma and bone, a finding supported by in vivo studies. Altogether, our model allows for the study of MM clonal evolution over time in the bone microenvironment and will be beneficial for optimizing treatment efficacy so as to significantly delay disease relapse.

Iron Pill-Induced Chemical Laryngitis.

OBJECTIVE: To discuss the presentation and management of pill-induced chemical laryngitis by illustrating a rare case. METHODS: We report a unique case of a patient with iron pill-induced laryngitis. RESULTS: A 71-year-old male presented for evaluation of dysphonia. Five weeks prior, the patient had reportedly aspirated an iron pill. The pill was lodged in his throat for several hours before being coughed up, soft but still intact. Since that event, the patient noted complete voice loss and in clinic was found to have a very breathy and asthenic voice. Stroboscopy revealed aperiodicity with severe false fold compression and significant ulceration of the infraglottic region associated with thick exudate. Vocal folds were mobile but atrophic, with overlying crusted secretions. A sensory deficit was suspected based on scope tolerance. The patient was treated with nebulized ciprodex and humidified air with some improvement in mucosal crusting but had persistent glottic insufficiency and dysphonia, prompting bilateral hyaluronic acid injection. CONCLUSIONS: Pill-induced laryngitis is an extremely rare phenomenon. While typically associated with bisphosphonates, this condition should be considered in any patient presenting with dysphonia and history of aspiration of a pill, including iron supplements. Regardless of the inciting medication, pill-induced laryngitis may be treated with humidified air, nebulized steroids, and antibiotics. Injection augmentation of the vocal folds may be made considered when glottic insufficiency and weak cough contribute to the presentation.

Medical Student Mentorship in the COVID-19 Era.

Background: The Covid-19 pandemic led to a reduction of in-person, guided mentorship due to social distancing and an emphasis on virtual meetings. The effect of these changes on medical students' experiences and specialty choice has yet to be studied in a large-scale manner. Objective: To determine the perspective of third- and fourth-year medical students regarding the impact of the COVID-19 pandemic on mentorship. Design: The authors distributed a modified Likert scale questionnaire (score: 1-10) to assess responses. Participants: Third- and fourth-year medical students at two large US allopathic medical schools. Main Measures: Responses to each survey item were analyzed to characterize the impact of the COVID-19 pandemic on mentorship relationships in medical school. A score of 1-5 was considered "disagree" and a score of 6-10 was considered "agree." Key Results: A total of 144 responses were collected with a response rate of 16.2%. Overall, 80.6% (n = 116) of respondents agree that the COVID-19 pandemic has had a negative impact on their medical school experience. Nearly half (41.0%, n = 59) expressed concern over the lack of mentorship opportunities, and 66.0% (n = 95) reported that the pandemic has made it more difficult to form or maintain connections with their mentors. Importantly, 43.6% (n = 61) of respondents reported that having close mentoring relationships reduced the impact of the pandemic on their medical training. While many respondents (79.9%, n = 114) did not change career plans due to the pandemic, most students are concerned about evaluating prospective residency programs (88.9%, n = 128). Notably, M3s have much lower confidence than M4s in their ability to choose a specialty (5.9 vs. 8.2, p = 6.43e - 08). Conclusions: This investigation illustrates the concerns that medical students have regarding access to mentorship opportunities due to the COVID-19 pandemic. We hope that these findings encourage medical schools to evaluate and expand their current mentorship programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-023-01838-4.

γδ-Enriched CAR-T cell therapy for bone metastatic castrate-resistant prostate cancer.

Immune checkpoint blockade has been largely unsuccessful for the treatment of bone metastatic castrate-resistant prostate cancer (mCRPC). Here, we report a combinatorial strategy to treat mCRPC using γδ-enriched chimeric antigen receptor (CAR) T cells and zoledronate (ZOL). In a preclinical murine model of bone mCRPC, γδ CAR-T cells targeting prostate stem cell antigen (PSCA) induced a rapid and significant regression of established tumors, combined with increased survival and reduced cancer-associated bone disease. Pretreatment with ZOL, a U.S. Food and Drug Administration-approved bisphosphonate prescribed to mitigate pathological fracture in mCRPC patients, resulted in CAR-independent activation of γδ CAR-T cells, increased cytokine secretion, and enhanced antitumor efficacy. These data show that the activity of the endogenous Vγ9Vδ2 T cell receptor is preserved in CAR-T cells, allowing for dual-receptor recognition of tumor cells. Collectively, our findings support the use of γδ CAR-T cell therapy for mCRPC treatment.

Recognition and Management of Nonaccidental Esophageal Perforation.

Nonaccidental trauma is a common pediatric concern that often goes unrecognized. Although most patients present with bruising, burns, fractures, and head trauma, it is critical that physicians be able to diagnose and treat atypical presentations such as pharyngeal and esophageal trauma. In this report, we describe the presentation and management of a 5-week-old girl with an inflicted esophageal perforation.

Effect of injection laryngoplasty material on outcomes in pediatric vocal fold paralysis.

Background: While injection laryngoplasty is becoming increasingly common in children, there has not been clearly established guidelines for the choice of injection material. This study evaluates for variability in post-surgical outcomes between different materials used for injection laryngoplasty in the treatment of pediatric unilateral vocal cord paralysis. Methods: In this cohort study, a retrospective chart review was performed for all patients undergoing injection laryngoplasty for unilateral vocal cord paralysis at our tertiary-care children's hospital between January 2010 and December 2019. Patients with vocal cord paresis or bilateral vocal cord paralysis were excluded from this study. Demographics, pre- and post-injection clinic visits, and operative reports were reviewed to compare outcomes between injection materials, including the number of injections required, inter-surgical interval, and rate of vocal improvement. Results: Forty-four patients were included in the analysis. Half of the patients were female, and half were male. A total of 97 injections were observed, with 32 patients receiving multiple injections. The mean age at first injection was 7 years. The most common causes of vocal fold paralysis were iatrogenic (n=21, 48%) and idiopathic (n=9, 20%). Thirty-nine percent (n=17) had a history of cardiac surgery. Forty-five percent of injections used Radiesse® voice/Prolaryn® plus, 35% used Radiesse®/Prolaryn® voice Gel, and 20% used Cymetra™. The material used was not associated with a difference in post-operative outcomes, including number of injections, (P=0.10; 0.29), inter-surgical interval (P=0.27; 0.29), or rate of voice improvement (P=0.86; 0.36). Conclusions: Neither material choice nor demographic factors were associated with a difference in outcomes following injection laryngoplasty or a change in the inter-surgical interval. Further research is needed to develop standardized protocols for injection laryngoplasty in this population.

GFI1-Dependent Repression of SGPP1 Increases Multiple Myeloma Cell Survival.

Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5' end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.

Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis.

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1ß-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.

Petrous apex lesion following meningitis: management and discussion.

A 9-year-old male with history of mixed hearing loss presented with petrous apex lesion following episode of meningitis. Serial imaging revealed persistence of the lesion necessitating biopsy to rule out malignancy. Biopsy revealed inflammatory changes. The management of petrous apex lesions following meningitis can be conservative but repeat imaging is necessary to rule out progression and to rule out neoplastic process.

Management of infected nasal dermoid cysts and sinuses.

This study investigates outcomes of surgical management of pediatric patients with nasal dermoids with prior infection. A retrospective review at Nationwide Children's Hospital, a large free-standing pediatric hospital in the Midwestern USA, was performed. Patients were identified by the Current Procedural Terminology codes 30124 (simple excision of dermoid cyst) and 30125 (complex excision of nasal dermoid cyst) from 2011 to 2016. Demographic, imaging data, surgical findings, microbiological data and recurrence rates were collected for these patients. Descriptive statistical investigation was performed. In total, 14 patients were identified, 4 of the 14 patients (28.5%) had recurrent infection and required additional surgery. Three of seven patients required incision and drainage prior to definitive excision. One of seven patients in the infected group had recurrence. Prior infection does not increase the recurrence rate and almost half of the patients required I&D prior to definitive management.

Pediatric nasal septoplasty outcomes.

BACKGROUND: Corrective nasal surgery has historically been avoided in the pediatric population out of concerns surrounding the potential disruption of nasal growth centers. There is a paucity of data on the rate of complications or revision surgery following septoplasty in this population. As such, the purpose of this study is to review the long-term outcomes of a large cohort of children who underwent nasal septoplasty and to compare outcomes of septoplasty patients under the age of 14 to those 14 years and older. METHODS: A retrospective review was performed on all patients who received nasal septoplasty at our tertiary care pediatric referral center between October 2009 and September 2016. All patients who underwent septoplasty for a deviated nasal septum and were 0-18 years of age at the time of surgery were included in this analysis. Outcomes were compared between patients under the age of 14 to those 14 years and older. Demographic, surgical, and follow-up data were collected including complications and the need for revision surgery. RESULTS: A total of 194 pediatric patients were identified as meeting inclusion criteria for the study. Mean age for the total cohort was 14.6 years (0-18 years), with a mean of 15.9 years in the older group and 10.6 years in the younger group. Revision septoplasty was performed more frequently in the younger group. However, no significant difference in the rate of complications was seen between the two groups. CONCLUSIONS: To the best of our knowledge, this is the largest retrospective study examining outcomes following septoplasty in pediatric patients. We also specifically examine outcomes of very young septoplasty patients, a population for which limited evidence exists. Further retrospective studies are needed to validate the use of nasal septoplasty in the pediatric population.

Systematic Literature Review and Critical Appraisal of Health Economic Models Used in Cost-Effectiveness Analyses in Non-Alcoholic Steatohepatitis: Potential for Improvements.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a severe, typically progressive form of non-alcoholic fatty liver disease (NAFLD). The global prevalence of NASH is increasing, driven partly by the global increase in obesity and type 2 diabetes mellitus (T2DM), such that NASH is now a leading cause of cirrhosis. There is currently an unmet clinical need for efficacious and cost-effective treatments for NASH; no pharmacologic agents have an approved indication for NASH. OBJECTIVE: Our objective was to summarise and critically appraise published health economic models of NASH, to evaluate their quality and suitability for use in the assessment of novel treatments for NASH, and to identify knowledge gaps, challenges and opportunities for future modelling. METHODS: A systematic literature review was performed using the MEDLINE, Embase, Cochrane Library and EconLit databases to identify published health economic analyses in patients with NAFLD or NASH. Supplementary hand searches of grey literature were also performed. Articles published up to November 2019 were included in the review. Quality assessment of identified studies was also performed. RESULTS: A total of 19 articles comprising 16 unique models including either NAFLD as a whole or NASH alone were included in the review. Structurally, most models had a state-transition component; in terms of health states, two different approaches to early disease states were used, modelling either progression through fibrosis stages or NAFLD/NASH-specific health states. Conditions that frequently co-exist with NASH, such as obesity, T2DM and cardiovascular disease were not captured in models identified here. Late-stage complications such as cirrhosis, decompensated cirrhosis and hepatocellular carcinoma were consistently included, but input data (e.g. costs, utilities and transition probabilities) for late-stage complications were frequently sourced from other liver disease areas. The quality of included studies was heterogenous, and only a small proportion of studies reported internal and external validation processes. CONCLUSION: The health economic models identified in this review are associated with limitations primarily driven by a lack of NASH-specific data. Identified models also largely overlooked the intricate association between NASH and other conditions, including obesity and T2DM, and did not capture the increased risk of cardiovascular events associated with NASH. High-quality, transparent, validated health economic models of NASH will be required to evaluate the cost effectiveness of treatments currently in development, particularly compounds that may target other non-hepatic outcomes.

Combined administration of a small-molecule inhibitor of TRAF6 and Docetaxel reduces breast cancer skeletal metastasis and osteolysis.

Tumour necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in breast cancer and osteoclastic bone destruction. Here, we report that 6877002, a verified small-molecule inhibitor of TRAF6, reduced metastasis, osteolysis and osteoclastogenesis in models of osteotropic human and mouse breast cancer. First, we observed that TRAF6 is highly expressed in osteotropic breast cancer cells and its level of expression was higher in patients with bone metastasis. Pre-exposure of osteoclasts and osteoblasts to non-cytotoxic concentrations of 6877002 inhibited cytokine-induced NFκB activation and osteoclastogenesis, and reduced the ability of osteotropic human MDA-MB-231 and mouse 4T1 breast cancer cells to support bone cell activity. 6877002 inhibited human MDA-MB-231-induced osteolysis in the mouse calvaria organ system, and reduced soft tissue and bone metastases in immuno-competent mice following intra-cardiac injection of mouse 4T1-Luc2 cells. Of clinical relevance, combined administration of 6877002 with Docetaxel reduced metastasis and inhibited osteolytic bone damage in mice bearing 4T1-Luc2 cells. Thus, TRAF6 inhibitors such as 6877002 - alone or in combination with conventional chemotherapy - show promise for the treatment of metastatic breast cancer.

2D and 3D In Vitro Co-Culture for Cancer and Bone Cell Interaction Studies.

Co-culture assays are used to study the mutual interaction between cells in vitro. This chapter describes 2D and 3D co-culture systems used to study cell-cell signaling crosstalk between cancer cells and bone marrow adipocytes, osteoblasts, osteoclasts, and osteocytes. The chapter provides a step-by-step guide to the most commonly used cell culture techniques, functional assays, and gene expression.

Pharmacological inhibition of the IKKε/TBK-1 axis potentiates the anti-tumour and anti-metastatic effects of Docetaxel in mouse models of breast cancer.

IκB kinase subunit epsilon (IKKε), a key component of NFκB and interferon signalling, has been identified as a breast cancer oncogene. Here we report that the IKKε/TBK1 axis plays a role in the initiation and progression of breast cancer osteolytic metastasis. Cancer-specific knockdown of IKKε in the human MDA-MB-231-BT cells and treatment with the verified IKKε/TBK1 inhibitor Amlexanox reduced skeletal tumour growth and osteolysis in mice. In addition, combined administration of Amlexanox with Docetaxel reduced mammary tumour growth of syngeneic 4T1 cells, inhibited metastases and improved survival in mice after removal of the primary tumour. Functional and mechanistic studies in breast cancer cells, osteoclasts and osteoblasts revealed that IKKε inhibition reduces the ability of breast cancer cells to grow, move and enhance osteoclastogenesis by engaging both IRF and NFκB signalling pathways. Thus, therapeutic targeting of the IKKε/TBK1 axis may be of value in the treatment of advanced triple negative breast cancer.

Paradoxical effects of JZL184, an inhibitor of monoacylglycerol lipase, on bone remodelling in healthy and cancer-bearing mice.

BACKGROUND: Cancer-associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone, 2-arachidonoyl glycerol (2AG). METHODS: The effects of the verified MAGL inhibitor on bone remodelling were assessed in healthy mice and in mouse models of bone disease caused by prostate and breast cancers and osteosarcoma. FINDINGS: JZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors. INTERPRETATION: MAGL inhibitors such as JZL184, or its novel analogues, may be of value in the treatment of bone disease caused by primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit their usefulness as osteoprotective agents.

Bidirectional regulation of bone formation by exogenous and osteosarcoma-derived Sema3A.

Semaphorin 3A (Sema3A), a secreted member of the Semaphorin family, increases osteoblast differentiation, stimulates bone formation and enhances fracture healing. Here, we report a previously unknown role of Sema3A in the regulation of ectopic bone formation and osteolysis related to osteosarcoma. Human recombinant (exogenous) Sema3A promoted the expression of osteoblastic phenotype in a panel of human osteosarcoma cell lines and inhibited the ability of these cells to migrate and enhance osteoclastogenesis in vitro. In vivo, administration of exogenous Sema3A in mice after paratibial inoculation of KHOS cells increased bone volume in non-inoculated and tumour-bearing legs. In contrast, Sema3A overexpression reduced the ability of KHOS cells to cause ectopic bone formation in mice and to increase bone nodule formation by engaging DKK1/ß-catenin signalling. Thus, Sema3A is of potential therapeutic efficacy in osteosarcoma. However, inhibition of bone formation associated with continuous exposure to Sema3A may limit its long-term usefulness as therapeutic agent.