12b80 - Hydroxybisphosphonate Linked Doxorubicin: Bone Targeted Strategy for Treatment of Osteosarcoma.

To reply to as yet unmet medical needs to treat osteosarcoma, a form of primary bone cancer, we conceived the 12b80 compound by covalently conjugating antineoplastic compound doxorubicin to a bone targeting hydroxybisphosphonate vector and turned it into a prodrug through a custom linker designed to specifically trigger doxorubicin release in acidic bone tumor microenvironment. Synthesis of 12b80 was thoroughly optimized to be produced at gram scale. 12b80 was evaluated in vitro for high bone support affinity, specific release of doxorubicin in acidic condition, lower cytotoxicity, and cellular uptake of the prodrug. In vivo in rodents, 12b80 displayed rapid and sustained targeting of bone tissue and tumor-associated heterotopic bone and permitted a higher doxorubicin payload in tumor bone environment compared to nonvectorized doxorubicin. Consequently, 12b80 showed much lower toxicity compared to doxorubicin, promoted strong antitumor effects on rodent orthotopic osteosarcoma, displayed a dose-response therapeutic effect, and was more potent than doxorubicin/zoledronate combination.

New chondrosarcoma cell lines and mouse models to study the link between chondrogenesis and chemoresistance.

Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast to osteosarcoma they are resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance. Between 2007 and 2012, 10 chondrosarcoma biopsies were collected and used for cell culture and transplantation into nude mice. Only one transplanted biopsy and one injected cell line has engrafted successfully leading to conventional central high-grade chondrosarcoma similar to the original biopsies. In culture, two new stable cell lines were obtained, one from a dedifferentiated and one from a grade III conventional central chondrosarcoma biopsy. Their genetic characterization revealed triploid karyotypes, mutations in IDH1, IDH2, and TP53, deletion in CDKN2A and/or MDM2 amplification. These cell lines expressed mesenchymal membrane markers (CD44, 73, 90, 105) and were able to produce a hyaline cartilaginous matrix when cultured in chondrogenic three-dimensional (3D) pellets. Using a high-throughput quantitative RT-PCR approach, we observed that cell lines cultured in monolayer had lost expression of several genes implicated in cartilage development (COL2A1, COMP, ACAN) but restored their expression in 3D cultures. Chondrosarcoma cells in monolayer were sensitive to several conventional chemotherapeutic agents but became resistant to low doses of mafosfamide or doxorubicin when cultured in 3D pellets, in parallel with an altered nucleic accumulation of the drug. Our results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of several drugs and thus contribute to chemoresistance. Therefore, 3D chondrogenic cell pellets constitute a more relevant model to study chondrosarcoma chemoresistance and may be a valuable alternative to animal experimentations.

Oncostatin M is a growth factor for Ewing sarcoma.

Primary bone tumors, osteosarcomas and chondrosarcomas, derive from mesenchymal stem cells committed into osteoblasts and chondrocytes; in Ewing sarcomas (ESs), the oncogenic fusion protein EWS-FLI1 prevents mesenchymal differentiation and induces neuroectodermic features. Oncostatin M (OSM) is a cytokine from the IL-6 family that modulates proliferation and differentiation in numerous cells. The basis for inhibition versus induction of proliferation by this cytokine is obscure, although MYC was described as a potent molecular switch in OSM signaling. We show herein that, in contrast to osteosarcomas and chondrosarcomas, for which OSM was cytostatic, OSM induced proliferation of ES cell lines. Knockdown experiments demonstrated that growth induction by OSM depends on both types I [leukemia inhibitory factor receptor (LIFR)] and II [OSM receptor (OSMR)] receptors, high STAT3 activation, and induction of MYC to a high expression level. Indeed, ES cell lines, mice xenografts, and patient biopsy specimens poorly expressed LIF, precluding LIFR lysosomal degradation and OSMR transcriptional induction, thus leading to a high LIFR/OSMR ratio. Because other neuroectodermic tumors (ie, glioma, medulloblastoma, and neuroblastoma) had a similar expression profile, the main role of EWS-FLI1 could be through maintenance of stemness and neuroectodermic features, characterized by a low LIF, a high LIFR/OSMR ratio, and high MYC expression. Thus, this study on rare bone malignancies gives valuable insights on more common cancer regulatory mechanisms and could provide new therapeutic opportunities.

Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling.

Bone resorption by osteoclasts and bone formation by osteoblasts are tightly coupled processes implicating factors in TNF, bone morphogenetic protein, and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating bone formation by osteoblasts but the coupling factors were not identified. Using a high-throughput approach, we identified here Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages that induce osteoblast differentiation and matrix mineralization from human mesenchymal stem cells while inhibiting adipogenesis. Upon activation of toll-like receptors (TLRs) by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA to OSM, OSM receptor subunits gp130 and OSMR, or to the downstream transcription factor STAT3. The induced osteoblast differentiation program culminated with enhanced expression of CCAAT-enhancer-binding protein δ, Cbfa1, and alkaline phosphatase. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines have also a potent role in bone formation induced by monocytes/macrophages and activation of TLRs: IL-6 and leukemia inhibitory factor. We propose that during bone inflammation, infection, or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies.

Quaternary ammonium-melphalan conjugate for anticancer therapy of chondrosarcoma: in vitro and in vivo preclinical studies.

Cartilage tumours present ongoing therapeutic challenges due to their chondrogenic extracellular matrix that potentially hampers drug delivery, their low percentage of dividing cells, and their poor vascularity. In this context, and based on the affinity of the quaternary ammonium moiety for proteoglycans (PG), we developed a strategy that uses the quaternary ammonium function to selectively deliver DNA alkylating agents to the cartilage tumour tissue. We engineered the quaternary ammonium derivative of melphalan (Mel-AQ) and assessed its antitumoural activity in vitro and in vivo. In vitro, micromolar concentrations of Mel-AQ inhibited the proliferation of human HEMC-SS chondrosarcoma and Saos-2 osteosarcoma cell lines. Moreover, 24-h incubation with 20 µM Mel-AQ induced a 2.5-fold increase in S population and a 1.5-fold increase in subG0G1 population compared to controls. In vivo, Mel-AQ demonstrated antitumour activity in the orthotopic model of primary Swarm rat chondrosarcoma. When given to chondrosarcoma-bearing rats (three doses of 16 µmol/kg at days 8, 12 and 16 post-implant), Mel-AQ demonstrated an optimal antitumour effect at day 43, when tumour cell growth inhibition peaked at 69%. Interestingly, the treatment protocol was proved well tolerated, since the animals showed no weight loss over the course of the study. This antitumoural effect was assessed in vivo by scintigraphic imaging using (99m)Tc-NTP 15-5 developed in our lab as a PG-targeting radiotracer, and tumour tissue was analyzed at study-end by biochemical PG assay with Alcian blue staining. Mel-AQ treatment led to a significant decrease in the PG content of tumoural tissue. These experimental results highlighted the promising antitumour potential of Mel-AQ as a PG-targeting strategy for therapeutic management of chondrosarcoma.

Mortality associated with Behçet's disease in France assessed by multiple-cause-of-death analysis.

OBJECTIVES: To examine the mortality rates and causes of death among French decedents with Behçet's disease (BD). METHODS: Data collected between 1979 and 2016 by the French Epidemiological Center for the Medical Causes of Death was used to calculate BD-related mortality rates and examine differences according to age and sex, determine underlying and non-underlying causes of death (UCD/NUCD), and compare with the general population (observed/expected ratios, O/E). RESULTS: A total of 328 death certificates mentioned BD as UCD (n = 171) or NUCD (n = 157). The mean (± SD) age at death was 52.8 (± 11.3) years in men and 58.4 (± 16.5) in women (vs. 71.2 and 79.8 in the general population, respectively). The age-standardized mortality rate was 0.15/million person-years and significantly increased over the study period (p < 0.01). When BD was the UCD, the most frequent associated causes were infections (25.1%), underlying organ dysfunction (21.6%), and arterial events (15.2%). As compared to the general population, BD decedents < 45 years were more likely to die from a cardiovascular event (O/E = 6.25, p < 0.001); decedents > 45 years were more likely to die from infection (O/E = 9.69, p < 0.001). Between 1979 and 2016, BD as UCD decreases whereas NUCD increased. CONCLUSION: This study found an earlier mean age at death for BD compared to the general population; young patients were more prone to die from cardiovascular complication whereas older patients died more frequently from infections. Over the years, the mean age at death increased and BD was less frequently considered as an UCD, suggesting a better knowledge of the disease and complications. Key Points • Patients with Behçet's disease die earlier than the general population. • Young patients were more prone to die from cardiovascular complication. • Older patients died more frequently from infections.

Human Papilloma Virus Vaccination in Patients with Rheumatic Diseases in France: A Study of Vaccination Coverage and Drivers of Vaccination.

Objectives: To describe human papillomavirus (HPV) vaccination practices in adolescent girls with systemic lupus erythematosus (SLE) and juvenile idiopathic arthritis (JIA) and to identify barriers to and motivators for vaccination. Methods: Cross-sectional, multicenter study on girls aged 9 to 19 years and their accompanying adults. The measurement criteria were the proportion of girls who were vaccinated against HPV, compliance with the vaccination schedule, factors associated with vaccination, and reasons for vaccination and non-vaccination through a self-administered questionnaire. Results: Seventy-one patients (16 with SLE and 55 with JIA) were included with a mean age of 13 years old (rank 11−18). According to parental questioning, 39% of patients were vaccinated against HPV or in progress (44% and 38% of SLE and JIA, respectively). This rate was 82% for the 22 patients ≥ 15 years of age. The vaccine was administered as often by a general practitioner (39%) as by a hospital pediatrician (also 39%). Two factors were significantly associated with vaccination: Older age (OR 53.68, 95% CI 5.85−429.29, p < 0.001) and previous hepatitis B vaccination (OR 4.97, 95% CI 1.03−24.01, p = 0.040). Recommendation of the vaccine by a health professional and fear of HPV-related diseases were the main facilitators. Lack of knowledge about the vaccine, lack of recommendation by a health professional, and fear of vaccine side effects were the main barriers. Conclusions: HPV vaccination coverage remains insufficient among patients with autoimmune disease. Education and awareness of health professionals about HPV infections are crucial elements in vaccine acceptance.

Direct anti-cancer effect of oncostatin M on chondrosarcoma.

The cytokine Oncostatin M (OSM) is cytostatic, pro-apoptotic and induces differentiation of osteosarcoma cells into osteocytes, suggesting new adjuvant treatment for these bone-forming sarcomas. However, OSM systemic over-expression could lead to adverse side effects such as generalized inflammation, neoangiogenesis and osteolysis. We determine here the effect of OSM on chondrosarcoma, another primary bone sarcoma characterized by the production of cartilage matrix and altered bone remodelling. Chondrosarcomas are resistant to conventional chemotherapy and radiotherapy, and wide surgical excision remains the only available treatment. We found that OSM blocked the cell cycle in four of five chondrosarcoma cell lines, independently of p53 and presumably through the JAK3/STAT1 pathway. In two tested cell lines, OSM induced a hypertrophic chondrocyte differentiation, with an induced Cbfa1/SOX9 ratio and induced Coll10, matrix metalloproteinase 13 (MMP13) and RANKL expression. Adenoviral gene transfer of OSM (AdOSM) in the Swarm rat chondrosarcoma (SRC) model indicated that local intra-tumoral OSM over-expression reduces chondrosarcoma development not only with reduced tumor proliferation and enhanced apoptosis but also with enhanced RANKL expression, osteoclast formation and reduced bone volumes. Flu-like symptoms were induced by the AdOSM, but there was no effect on tumor angiogenesis. Therefore, OSM could be considered as a new adjuvant anti-cancer agent for chondrosarcomas. A local application of this cytokine is presumably needed to overcome the poor vascularization of these tumors and to limit the deleterious effect on other tissues. Its side effect on bone remodeling could be managed with anti-resorption agents, thus offering potential new lines of therapeutic interventions.

Phase I dose escalation study of 12b80 (hydroxybisphosphonate linked doxorubicin) in naturally occurring osteosarcoma.

PURPOSE: 12b80 combines doxorubicin bound to a bone targeting hydroxybisphosphonate vector using a pH-sensitive linker, designed to specifically trigger doxorubicin release in an acidic bone tumor microenvironment. This phase I study aimed to determine the safety and toxicity profiles of 12b80 in dogs with naturally occurring osteosarcoma, with the objective to translate findings from dogs to humans. EXPERIMENTAL DESIGN: Ten client-owned dogs with osteosarcoma were enrolled in an accelerated dose-titration design followed by 3 + 3 design. Dogs received three cycles of 12b80 intravenous injection at 4 mg/kg (n = 1), 6 mg/kg (n = 2), 8 mg/kg (n = 3), and 10 mg/kg (n = 4). Endpoints included safety, tolerability, maximum tolerated dose (MTD), and dose-limiting toxicity (DLT). RESULTS: The MTD of 12b80 was 8 mg/kg (i.e., equivalent dose of doxorubicin of 110 mg/m2, range: 93-126). Most adverse events included grade ≤ 2 gastrointestinal disorders and hypersensitivity reactions. No hematological or cardiac DLT were observed at any dose tested. CONCLUSIONS: In dogs, 12b80 is overall well tolerated and expends the MTD of doxorubicin up to four times the standard dose of 30 mg/m2. These results demonstrate the potential therapeutic benefit of 12b80 in canine and human osteosarcoma.

99mTc-NTP 15-5 assessment of the early therapeutic response of chondrosarcoma to zoledronic acid in the Swarm rat orthotopic model.

BACKGROUND: Since proteoglycans (PGs) appear as key partners in chondrosarcoma biology, PG-targeted imaging using the radiotracer 99mTc-N-(triethylammonium)-3-propyl-[15]ane-N5 (99mTc-NTP 15-5) developed by our group was previously demonstrated to be a good single-photon emission computed tomography tracer for cartilage neoplasms. We therefore initiated this new preclinical study to evaluate the relevance of 99mTc-NTP 15-5 imaging for the in vivo monitoring and quantitative assessment of chondrosarcoma response to zoledronic acid (ZOL) in the Swarm rat orthotopic model. FINDINGS: Rats bearing chondrosarcoma in the orthotopic paratibial location were treated by ZOL (100 µg/kg, subcutaneously) or phosphate-buffered saline, twice a week, from day 4 to day 48 post-tumor implantation. 99mTc-NTP 15-5 imaging was performed at regular intervals with the target-to-background ratio (TBR) determined. Tumor volume was monitored using a calliper, and histology was performed at the end of the study. From day 11 to day 48, mean TBR values ranged from 1.7 ± 0.6 to 2.3 ± 0.6 in ZOL-treated rats and from 2.1 ± 1.0 to 4.9 ± 0.9 in controls. Tumor growth inhibition was evidenced using a calliper from day 24 and associated to a decrease in PG content in treated tumor tissues (confirmed by histology). CONCLUSIONS: This work demonstrated two proofs of concept: (1) biphosphonate therapy could be a promising therapeutic approach for chondrosarcoma; (2) 99mTc-NTP 15-5 is expected to offer a novel imaging modality for the in vivo evaluation of the extracellular matrix features of chondrosarcoma, which could be useful for the follow-up and quantitative assessment of proteoglycan 'downregulation' associated to the response to therapeutic attempts.

Long term oncostatin M treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells.

Previous in vitro studies on primary osteoblastic and osteosarcoma cells (normal and transformed osteoblasts) have shown that oncostatin M (OSM), a member of the interleukin-6 family, possesses cytostatic and pro-apoptotic effects in association with complex and poorly understood activities on osteoblast differentiation. In this study, we use rat osteosarcoma cells transduced with lentiviral particles encoding OSM (lvOSM) to stably produce this cytokine. We show that after several weeks of culture, transduced OSRGA and ROS 17/2.8 cells are growth inhibited and sensitized to apoptosis induced by the kinase inhibitor Staurosporine (Sts). Moreover, this long term OSM treatment induces (i) a decrease in osteoblastic markers, (ii) morphological changes leading to an elongated and/or stellate shape and (iii) an increase in osteocytic markers (sclerostin and/or E11), suggesting an osteocyte-like differentiation. We also show that non transformed rat calvaria cells transduced with lvOSM differentiate into stellate shaped cells expressing sclerostin, E11, Phex and functional hemichannels. Together, these results indicate that osteosarcoma cells stably producing OSM do not develop resistance to this cytokine and thus could be a valuable new tool to study the anti-cancer effect of OSM in vivo. Moreover, OSM-over-expressing osteoblastic cells differentiate into osteocyte-like cells, the major cellular contingent in bone, providing new culture conditions for this cell type which is difficult to obtain in vitro.

Síndrome de tako-tsubo probablemente asociado con la administración de sulprostona tras una cesárea / Tako-tsubo cardiomyopathy probably associated with administration of sulprostone following cesarean delivery

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