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Purpose: We previously demonstrated that mitochondrial inhibitors' efficacy was restricted to a metabolic context in which mitochondrial respiration was the predominant energy source, a situation achievable by inducing vascular normalization/hypoxia correction with antiangiogenics. Vascular normalization can be tracked with 2[18F]fluoro-2-deoxy-d-glucose (FDG)-PET. We tested the efficacy of the mitochondrial inhibitor ME-344 or placebo added to bevacizumab in early breast cancer. Patients and Methods: Treatment-naïve HER2-negative patients with T > 1 cm (any N) underwent a breast-centered 18F-fluorodeoxyglucose (FDG)-PET (day 1) and received a single dose of bevacizumab (15 mg/kg), followed by a second FDG-PET (day 8). Patients were then randomized (1:1) to Arm A (ME-344 10 mg/kg intravenous on days 8, 15, and 21) or Arm B (placebo). Tumors were biopsied on days 0 and 29. Succinate dehydrogenase enzyme histochemistry (SDH-EHC), confocal microscopy of vessel architecture, and HIF1α staining were performed in pre- and posttreatment biopsies to assess the pharmacodynamics, vessel normalization, and tissue re-oxygenation by bevacizumab, respectively. Results: ME-344 displayed significant biological activity versus placebo: compared with a 186% increase in Arm B, Ki67 decreased by 23.4% from days 0 to 28 in Arm A (P < 0.001) (N = 42 patients). FDG-PET predicted vascular normalization in about one-third of the patients in each arm, which was confirmed using confocal microscopy and HIF1α staining. In the subgroup with vascular normalization, ME-344 induced a Ki67 decrease of 33.4% (placebo: 11.8 increase). SDH-EHC suggested on-target effects of ME-344. Conclusions: ME-344 has significant biological antitumor activity in HER2-negative breast cancer, particularly after induction of vascular normalization and tissue reoxygenation with bevacizumab.

This article belongs to the Special Issue Prosthetic Joint Infection: The Challenges of Prevention, Diagnosis and Treatment and Opportunities for Future Research. Fungal PJI is one of the most feared complications after arthroplasty. Although a rare finding, its high associated morbidity and mortality makes it an important object of study. The most frequent species causing fungal PJI is C. albicans. New technology to treat this type of PJI involves organic–inorganic sol-gels loaded with antifungals, as proposed in this study, in which anidulafungin is associated with organophosphates. This study aimed to evaluate the efficacy of an anidulafungin-loaded organic–inorganic sol-gel in preventing prosthetic joint infection (PJI), caused by Candida albicans using an in vivo murine model that evaluates many different variables. Fifty percent (3/6) of mice in the C. albicans-infected, non-coated, chemical-polished (CP)-implant group had positive culture and 100% of the animals in the C. albicans-infected, anidulafungin-loaded, sol-gel coated (CP + A)-implant group had a negative culture (0/6) (p = 0.023). Taking the microbiology and pathology results into account, 54.5% (6/11) of C. albicans-infected CP-implant mice were diagnosed with a PJI, whilst only 9.1% (1/11) of C. albicans-infected CP + A-implant mice were PJI-positive (p = 0.011). No differences were observed between the bone mineral content and bone mineral density of noninfected CP and noninfected CP + A (p = 0.835, and p = 0.181, respectively). No histological or histochemical differences were found in the tissue area occupied by the implant among CP and CP + A. Only 2 of the 6 behavioural variables evaluated exhibited changes during the study: limping and piloerection. In conclusion, the anidulafungin-loaded sol-gel coating showed an excellent antifungal response in vivo and can prevent PJI due to C. albicans in this experimental model. This research received financial support from the Mutua Madrileña Foundation (04078/001).

Nanotechnology changed the concept of treatment for a variety of diseases, producing a huge impact regarding drug and gene delivery. Among the different targeted diseases, osteoporosis has devastating clinical and economic consequences. Since current osteoporosis treatments present several side effects, new treatment approaches are needed. Recently, the application of small interfering RNA (siRNA) has become a promising alternative. Wnt/β‐catenin signaling pathway controls bone development and formation. This pathway is negatively regulated by sclerostin, which knock‐down through siRNA application would potentially promote bone formation. However, the major bottleneck for siRNA‐based treatments is the necessity of a delivery vector, bringing nanotechnology as a potential solution. Among the available nanocarriers, mesoporous silica nanoparticles (MSNs) have attracted great attention for intracellular delivery of siRNAs. The mesoporous structure of MSNs permits the delivery of siRNAs together with another biomolecule, achieving a combination therapy. Here, the effectiveness of a new potential osteoporosis treatment based on MSNs is evaluated. The proposed system is effective in delivering SOST siRNA and osteostatin through systemic injection to bone tissue. The nanoparticle administration produced an increase expression of osteogenic related genes improving the bone microarchitecture. The treated osteoporotic mice recovered values of a healthy situation approaching to osteoporosis remission., A mesoporous silica nanoparticles‐based system is designed for co‐delivering small interfering RNAs and an osteogenic peptide (osteostatin). The system is able to protect and deliver both biomolecules in the target tissue with promising results. It modifies gene expression and improves bone microarchitecture recovering healthy values. The application of nanoparticles can be considered a new potential alternative for osteoporosis remission.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagAGTP mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state., The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient and hormonal signals to control metabolism. Here the authors investigate the effects of constitutive nutrient signaling through genetic activation of RagA in adult mice and show that constitutive nutrient signaling regulates the response to feeding-fasting cycles and does not drive liver cancer.

The clinical management of COVID-19 is challenging. Medical imaging plays a critical role in the early detection, clinical monitoring and outcomes assessment of this disease. Chest x-ray radiography and computed tomography) are the standard imaging modalities used for the structural assessment of the disease status, while functional imaging (namely, positron emission tomography) has had limited application. Artificial intelligence can enhance the predictive power and utilization of these imaging approaches and new approaches focusing on detection, stratification and prognostication are showing encouraging results. We review the current landscape of these imaging modalities and artificial intelligence approaches as applied in COVID-19 management.

Interleukin 12 is a promising anti-cancer agent; however, IL12 systemic administration is hampered by side-effects. Although intratumoral administration of IL12 is giving promising results in clinical trials, only a small percentage of patients show a complete therapeutic response. This outcome could be improved by controlling the IL12 expression window. In this work we have tested the efficacy of a self-processing P2A and codon optimized murine IL12 (mIL12Pop) using inflammation-regulated lentivectors in a syngeneic tumor model. Our results show that implantation of cells expressing mIL12Pop employing either the strong constitutive SFFV promoter or a NFkB-based promoter reduced tumor growth, caused CD8+ T cell activation and increased IFNγ production. Importantly, the use of NFkBp-mIL12Pop increased the number of CD8+ TILs and improved the remission rate without increasing IL12-serum concentration. Further experiments suggest that there is a threshold intratumoral IL12 concentration that must be reached to trigger an efficient antitumor response and a limit that once surpassed causes detrimental systemic side effects. Altogether, these results demonstrate that using NFKBp-mIL12Pop significantly increases the overall survival of the mice. In summary, this new inflammation-regulated expression system might be useful for the development of new IL12 delivery systems with improved anti-tumor activity and limited toxicity., AR is supported by the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2012-32166) and the Comunidad Autonoma de Madrid, Spain (S2010/BMD-2312). FM is supported by the Comunidad Autonoma de Madrid, Spain (S2017/BMD-3867) and co-financed by European Structural and Investment Funds. AA and JMZ are supported by the Instituto de Salud Carlos III, Spain (PI15/01491 and PI16/00895, respectively). H.A. holds a research fellowship from Spanish Ministry of Education, Culture and Sports (MECD).

Myocardial uptake can hamper visualization of lung tumors, atherosclerotic plaques, and inflammatory diseases in 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) studies because it leads to spillover in adjacent structures. Several preparatory pre-imaging protocols (including dietary restrictions and drugs) have been proposed to decrease physiological [18F]FDG uptake by the heart, although their effect on tumor glucose metabolism remains largely unknown. The objective of this study was to assess the effects of a ketogenic diet (as an alternative protocol to fasting) on tumor glucose metabolism assessed by [18F]FDG positron emission tomography (PET) in a mouse model of lung cancer. PET scans were performed 60 min after injection of 18.5 MBq of [18F]FDG. PET data were collected for 45 min, and an x-ray computed tomograph (CT) image was acquired after the PET scan. A PET/CT study was obtained for each mouse after fasting and after the ketogenic diet. Quantitative data were obtained from regions of interest in the left ventricular myocardium and lung tumor. Three days on a ketogenic diet decreased mean standard uptake value (SUVmean) in the myocardium (SUVmean 0.95 ± 0.36) more than one night of fasting (SUVmean 1.64 ± 0.93). Tumor uptake did not change under either dietary condition. These results show that 3 days on high-fat diets prior to [18F]FDG-PET imaging does not change tumor glucose metabolism compared with one night of fasting, although high-fat diets suppress myocardial [18F]FDG uptake better than fasting.

Evidence suggests that chiropractic manipulation might exert positive effects in osteoporotic patients. The aim of this study was to evaluate the effects of chiropractic manipulation on bone structure and skeletal muscle in rats with bone loss caused by ovariectomy (OVX). The 6-month old Sprague-Dawley rats at 10 weeks following OVX or sham operation (Sh) did not suffer chiropractic manipulation (NM group) or were submitted to true chiropractic manipulation using the chiropractic adjusting instrument Activator V® three times/week for 6 weeks as follows: Force 1 setting was applied onto the tibial tubercle of the rat right hind limb (TM group), whereas the corresponding left hind limb received a false manipulation (FM group) consisting of ActivatorV® firing in the air and slightly touching the tibial tubercle. Bone mineral density (BMD) and bone mineral content (BMC) were determined in long bones and L3–L4 vertebrae in all rats. Femora and tibia were analyzed by μCT. Mechano growth factor (MGF) was detected in long bones and soleus, quadriceps and tibial muscles by immunohistochemistry and Western blot. The decrease of BMD and BMC as well as trabecular bone impairment in the long bones of OVX rats vs Sh controls was partially reversed in the TM group versus FM or NM rats. This bone improvement by chiropractic manipulation was associated with an increased MGF expression in the quadriceps and the anterior tibial muscle in OVX rats. These findings support the notion that chiropractic manipulation can ameliorate osteoporotic bone at least partly by targeting skeletal muscle.

Purpose: We previously detected promising efficacy of neoadjuvant nintedanib (a multityrosine kinase inhibitor, TKI) in early HER2-negative breast cancer. In a preclinical study, we monitored stromal hypoxia with 18F-fluoromisonidazole-positron emission tomography (18F-FMISO-PET); we found that reoxygenation of tumors (or lack of it) during a window-of-opportunity (WoO) treatment with TKIs correlated with the benefit (or lack of it) from TKI-plus-chemotherapy combinations. We studied the predictive role of 18F-FMISO-PET for the TKI nintedanib in the neoadjuvant setting in a phase II WoO randomized trial. Experimental Design: Patients were randomized to a 14-day WoO of nintedanib preceded and followed by an 18F-FMISO-PET, followed by nintedanib plus weekly paclitaxel (Arm A) or an 18F-FMISO-PET followed by weekly paclitaxel (Arm B) before surgery. The endpoint was residual cancer burden (RCB). The objective was to detect the patients with no response (RCB-III) on the basis of the baseline or evolutive 18F-FMISO-PET values/changes. Results: One-hundred and thirty HER2-negative patients were randomized. Seventeen (27.9%), 34 (55.7%), and 8 (13.1%) patients had an RCB of III, II, and I/0, respectively, in Arm A. In this arm, baseline hypoxic tumors had a 4.4-fold higher chance of experiencing RCB = 3 (P = 0.036) compared with baseline normoxic tumors. Nintedanib WoO induced tumor reoxygenation in 24.5% of the patients; those not reoxygenating showed a trend toward higher chance of experiencing RCB-III (6.4-fold; P = 0.09). In Arm B, 18F-FMISO-PET lacked predictive/prognostic value. Conclusions: Baseline hypoxic tumors (measured with 18F-FMISO-PET) do not benefit from neoadjuvant nintedanib. Clin Cancer Res; 23(6); 1432–41. ©2016 AACR.

Osteoarthritis (OA) is a degenerative disease characterized by injury of all joint tissues. Our previous study showed that in experimental osteoporosis, chiropractic manipulation (CM) exerts protective effects on bone. We here assessed whether CM might ameliorate OA by improving subchondral bone sclerosis, cartilage integrity and synovitis. Male New-Zealand rabbits underwent knee surgery to induce OA by anterior cruciate ligament injury. CM was performed using the chiropractic instrument ActivatorV 3 times/week for 8 weeks as follows: force 2 setting was applied to the tibial tubercle of the rabbit right hind limb (TM-OA), whereas the corresponding left hind limb received a false manipulation (FM-OA) consisting of ActivatorV firing in the air and slightly touching the tibial tubercle. After sacrifice, subchondral bone integrity was assessed in the tibiae by microCT and histology. Cartilage damage and synovitis were estimated by Mankin's and Krenn's scores, respectively, and histological techniques. Bone mineral density and content in both cortical and trabecular compartments of subchondral bone decreased in OA rabbits compared to controls, but partially reversed in the TM-OA group. Trabecular bone parameters in the latter group also showed a significant improvement compared to FM-OA group. Moreover RANKL, OPG, ALP and TRAP protein expression in subchondral bone significantly decreased in TM-OA rabbits with respect to FM-OA group. CM was associated with lower Mankin's and Krenn's scores and macrophage infiltrate together with a decreased protein expression of pro-inflammatory, fibrotic and angiogenic factors, in TM-OA rabbits with respect to FM-OA. Our results suggest that CM may mitigate OA progression by improving subchondral bone as well as cartilage and synovial membrane status. AODM was supported by grants from the Spanish Chiropractic Association (AEQ). AM was supported by grants from Spanish Ministry of Economy and Competitiveness and Carlos III Institute of Health (CP15/00053 and PI16/00991). We thank Dr. Carlos Guillén-Viejo (School of Pharmacy, Universidad Complutense de Madrid) for his help in advising in molecular biology methods. The authors are also grateful to Mark S. Davis for his assistance with editing and proofreading the article. Sí

The aim of this study was to evaluate the effect of a moxifloxacin-loaded organicinorganic sol-gel with different antibiotic concentration in the in vitro biofilm development and treatment against Staphylococcus aureus, S. epidermidis, and Escherichia coli, cytotoxicity and cell proliferation of MC3T3-E1 osteoblasts; and its efficacy in preventing the prosthetic joint infection (PJI) caused by clinical strains of S. aureus and E. coli using an in vivo murine model. Three bacterial strains, S. epidermidis ATCC 35984, S. aureus 15981, and, E. coli ATCC 25922, were used for microbiological studies. Biofilm formation was induced using tryptic-soy supplemented with glucose for 24 h, and then, adhered and planktonic bacteria were estimated using drop plate method and absorbance, respectively. A 24-h-mature biofilm of each species growth in a 96- well plate was treated for 24 h using a MBECTM biofilm Incubator lid with pegs coated with the different types of sol-gel, after incubation, biofilm viability was estimated using alamrBlue. MC3T3-E1 cellular cytotoxicity and proliferation were evaluated using CytoTox 96 Non-Radioactive Cytotoxicity Assay and alamarBlue, respectively. The microbiological studies showed that sol-gel coatings inhibited the biofilm development and treated to a mature biofilm of three evaluated bacterial species. The cell studies showed that the sol-gel both with and without moxifloxacin were non-cytotoxic and that cell proliferation was inversely proportional to the antibiotic concentration containing by sol-gel. In the in vivo study, mice weight increased over time, except in the E. coliinfected group without coating. The most frequent symptoms associated with infection were limping and piloerection; these symptoms were more frequent in infected groups with non-coated implants than infected groups with coated implants. The response of moxifloxacin-loaded sol-gel to infection was either total or completely absent. No differences in bone mineral density were observed between groups with coated and non-coated implants and macrophage presence lightly increased in the bone grown directly in contact with the antibiotic-loaded sol-gel. In conclusion, moxifloxacinloaded sol-gel coating is capable of preventing PJI caused by both Gram-positive and Gram-negative species The authors acknowledge the financial support from the Regional Government of Madrid through the program MULTIMAT-CHALLENGE (S2013/MIT-2862), and from the Mutua Madrileña Foundation (04078/001). JA-C was funded by an FPI grant from the Spanish Ministry of Economics and Competitiveness (BES-2014-069007). AM was funded by grants from Instituto de Salud Carlos III through the “Miguel Servet” program (CP15/00053). DR was funded by a grant from the Fundación Conchita Rábago

Radiomics can be defined as the extraction and analysis of large amounts of advanced quantitative imaging features with high throughput from medical images obtained with various modalities, including nuclear medicine modalities of Single photon emission computed tomography (SPECT) and Positron emission tomography (PET). We describe it as the process of transferring the medical imaging interpretation knowledge and skill set from humans to machines in a way that they can see more, process more information, and have deeper insights into what the disease is and how it behaves and might respond to therapeutic intervention. Radiomics methods can be applied across various cancers to identify tumor phenotype characteristics in the images that correlate with their likelihood of survival, as well as their association with the underlying driving biology. Identifying this characteristic set of features called tumor signature holds tremendous value in predicting cancer behavior and progression, which in turn has the potential to predict cancer’s response to various therapeutic options (Fig. 3.1). Moreover, we are beginning to see the application of radiomics principles in non-oncologic indications as well, such as cardiovascular disease. In allowing us to have this capacity, radiomics holds the promise of driving the engine of precision medicine. However, there are numerous challenges in the validation methods needed to establish radiomics as a clinically viable solution.

Background Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, although both subchondral bone deterioration and synovial inflammation are also hallmarks of the disease. Chiropractic manipulation (CM) is a therapeutic approach focused on the diagnosis, treatment and prevention of musculoskeletal disorders. It is essentially manual, allowing the chiropractor to restore the normal range of motion and function of the joints, muscles, and ligaments. Clinical evidences suggest that CM might exert positive effects in OA patients. Objectives The aim of this study was to evaluate the effects of CM on cartilage, subchondral bone and synovitis state in an OA rabbit model. Methods Ten (4 months old) male New Zealand rabbits underwent knee surgery to induce OA by transection of anterior cruciate ligament. One week after the surgery, CM was performed using the chiropractic adjusting instrument ActivatorV as follows: Force 2 setting was applied onto the tibial tubercle of the right hind limb (true manipulation, TM-OA group), at an angle of approximately 90°, from medial to lateral, whereas the corresponding left hind limb received a false manipulation (FM-OA group) consisting of ActivatorV firing in the air and slightly touching the tibial tubercle. These procedures were repeated 3 times a week for 8 weeks. Three healthy animals were used as control. Following sacrifice, μCT and histological damage evaluation (Mankin score) were done in femur and tibiae. RANKL/OPG protein expressions were studied by immunohistochemistry in tibia samples. Sinovitis was assessed by Krenn score and immunohistochemistry for macrophages (RAM11) and angiogenesis (CD31) were done. Protein expression of VEGF, COX2, TNFa, IL-1b and MMP3 were determinated by Western Blot. Results In the OA rabbits, subchondral BMD decreased in relation to control, been partially reversed in the tibiae of TM-OA group. When subchondral trabecular bone structural parameters were analyzed by microCT, a significant decrease of bone volume/trabecular volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) was observed in the OA rabbits, while trabecular separation (Tb.S) increased compared to control animals. TM-OA group showed a significant improvement of these parameters compare to FM-OA group. FM-OA joints had higher Mankin score (cartilage damage) than control joints, and TM decreased Mankin score compare to FM-OA (p Conclusion These results suggest that mechanical stimulus induced by CM may retard the pathologic progression of OA. The beneficial effects of CM might be associated with an improvement in bone and cartilage damage and also inflammatory state. Disclosure of Interests None declared

13 p.-5 fig., Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the deadliest cancers for which optimal diagnostic tools are still greatly needed. Identification of PDAC-specific molecular markers would be extremely useful to improve disease diagnosis and follow-up. MT1-MMP has long been involved in pancreatic cancer, especially in tumour invasion and metastasis. In this study, we aim to ascertain the suitability of MT1-MMP as a biomarker for positron emission tomography (PET) imaging. Two probes were assessed and compared for this purpose, a MT1-MMP-specific binding peptide, MT1-AF7p, and a specific antibody, LEM2/15, labelled respectively with 68Ga with 89Zr. PET imaging with both probes was conducted in patient-derived xenograft (PDX), subcutaneous and orthotopic, PDAC mouse models and in a cancer cell line (CAPAN-2)-derived xenograft (CDX) model. Both radiolabelled tracers were successful in identifying, by means of PET imaging techniques, tumour tissues expressing MT1-MMP although they did so at different uptake levels. The 89Zr-DFOLEM2/ 15 probe showed greater specific activity compared to the 68Ga-labelled peptide. The mean value of tumour uptake for the 89Zr-DFO-LEM 2/15 probe (5.67±1.11 %ID/g, n=28) was 25-30 times higher than that of the 68Ga-DOTA-AF7p ones. Tumour/blood ratios (1.13±0.51 and 1.44±0.43 at 5 and 7 days of 89Zr-DFO-LEM 2/15 post-injection were higher than those estimated for 68Ga-DOTAAF7p probes (of approximately tumour/blood ratio=0.5 at 90 min post-injection). Our findings strongly point out that i) the in vivo detection of MT1-MMP by PET imaging is a promising strategy for PDAC diagnosis and ii) labelled LEM2/15 antibody is a better candidate than MT1-AF7p for PDAC detection., This work was partially supported by a grant from the Regional Government of Madrid (CAM), Spain (S2017/BMD-3867 RENIM-CM), and cofinanced by European Structural and Investment Fund.

Background Osteoarthritis (OA) is a degenerative joint disease characterised by the degradation and inflammation of cartilage and synovium with bone damage. Different approaches that decrease subchondral bone remodelling during OA have demonstrated to improve cartilage damage and synovial inflammation. Chiropractic is a therapeutic approach focused on the diagnosis, treatment and prevention of musculoskeletal disorders. Chiropractic manipulation (CM) is essentially manual, allowing the chiropractor to restore the normal range of motion and function of the joints, muscles, and ligaments. We have previously observed that CM is able to increase subchondral bone mineral density (BMD) in an experimental model of osteoporosis1. Objectives To evaluate if CM could prevent the subchondral BMD alterations induced by OA, in association to an improvement in synovial membrane inflammation and cartilage damage in an OA model in rabbits. Methods Ten male New Zealand rabbits were submited knee surgery to induce OA by transection of anterior cruciate ligament. CM was performed using the chiropractic adjusting instrument ActivatorV 3 times a week during 8 weeks as follows: Force 2 setting was applied onto the tibial tubercle of the rabbit right hind limb (CM-OA group), at an angle of 90°, whereas the corresponding left hind limb received a false manipulation (FM-OA group) consisting of ActivatorV firing in the air and touching the tibial tubercle. Three healthy animals were used as controls. Following sacrifice, tibiae and femora were removed for mCT and histological evaluation. Synovial inflammation was evaluated by Krenn’s score and the protein presence of VEGF, MMP3 and CollagenVI in the synovial membrane was evaluated by western blot. Results In the OA rabbits, subchondral BMD decreased in relation to control animals (OA 4729±193 vs Control 5181±209 mg/cc), been partially reversed in the tibiae of OA rabbits with CM (TM-OA 5055±216 vs FM-OA 4404±170 mg/cc). Subchondral trabecular bone structural parameters were analysed by microCT and a significant decrease of bone volume/trabecular volume (BV/TV), trabecular number (TbN) and trabecular thickness (TbTh) was observed in the OA rabbits, while trabecular separation (TbS) increased compared to control animals. TM-OA group showed a significant improvement of these parameters compare to FM-OA group. TM-OA had lower cartilage damage compare to FM-OA (TM-OA 4±0,67 AU vs FM-OA 8±1,25 AU). TM-OA synovial membranes presented a total Krenn score lower than FM-OA joints (TM-OA 3±0,35 vs FM-OA 4.5±0,38 AU). OA synovial membranes showed higher levels of CollagenVI respect to control ones; TM-OA synovial membranes presented less expression of CollagenVI than FM-OA group (TM-OA 1.4±0,13 vs FM-OA 2.2±0,3 AU), been this associated with a decrease of both MMP3 (TM-OA 1.2±0,1 vs FM-OA 1.7±0.2 AU) and VEGF (TM-OA 1.2±0.14 vs FM-OA 1.9±0.26 AU). Conclusions These results support the hypothesis that CM may ameliorate subchondral BMD alterations induced by OA, in association to an improvement on synoviopathy and cartilage degradation. Reference [1] Lopez-Herradon A, et al. Impact of Chiropractic Manipulation on Bone and Skeletal Muscle of Ovariectomized Rats.CalcifTissueInt2017;101(5):519–529. Disclosure of Interest F. Conesa: None declared, R. Fujikawa: None declared, A. Mediero Grant/research support from: CP15/00053 PI16/00991, P. Gratal: None declared, F. Mulero: None declared, G. Herrero-Beaumont Grant/research support from: PI16/00065, R. Largo Grant/research support from: PI15/00340, A. Ortega-de Mues: None declared

In the present study, the possibility that a diabetic (DM) status might worsen age-related bone deterioration was explored in mice. Male CD-1 mice aged 2 (young control group) or 16 months, nondiabetic or made diabetic by streptozotocin injections, were used. DM induced a decrease in bone volume, trabecular number, and eroded surface, and in mineral apposition and bone formation rates, but an increased trabecular separation, in L1-L3 vertebrae of aged mice. Three-point bending and reference point indentation tests showed slight changes pointing to increased frailty and brittleness in the mouse tibia of diabetic old mice. DM was related to a decreased expression of both vascular endothelial growth factor and its receptor 2, which paralleled that of femoral vasculature, and increased expression of the pro-adipogenic gene peroxisome proliferator-activated receptor γ and adipocyte number, without affecting β-catenin pathway in old mouse bone. Concomitant DM in old mice failed to affect total glutathione levels or activity of main anti-oxidative stress enzymes, although xanthine oxidase was slightly increased, in the bone marrow, but increased the senescence marker caveolin-1 gene. In conclusion, DM worsens bone alterations of aged mice, related to decreased bone turnover and bone vasculature and increased senescence, independently of the anti-oxidative stress machinery.

Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.

Osteosarcoma (OS) is a highly aggressive bone tumor that usually arises intramedullary at the extremities of long bones. Due to the fact that the peak of incidence is in the growth spurt of adolescence, the specific anatomical location, and the heterogeneity of cells, it is believed that osteosarcomagenesis is a process associated with bone development. Different studies in murine models showed that the tumor-initiating cell in OS could be an uncommitted mesenchymal stem cell (MSC) developing in a specific bone microenvironment. However, only a few studies have reported transgene-induced human MSCs transformation and mostly obtained undifferentiated sarcomas. In our study, we demonstrate that activator protein 1 family members induce osteosarcomagenesis in immortalized hMSC. c-JUN or c-JUN/c-FOS overexpression act as tumorigenic factors generating OS with fibroblastic or pleomorphic osteoblastic phenotypes, respectively. Stem Cells 2018;36:1487-1500. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 [to J.G.-C.]; and RTICC: RD12/0036/0027 [to J.G.-C.], RD12/0036/0020 [to S.N.]; Consorcio CIBERONC CB16/12/00390 [to R.R.] and CB16/12/00484 [to S.N.]; Miguel Servet II Program CPII16/00049 [to R.R.]; and Sara Borrell CD16/13/00103 [to S.T.M.] and CD11/00132 [to A.A.]); The Juan de la Cierva program JCI2010-06123 [to A.A]); the Agencia Estatal de Investigación (AEI) (MINECO/Fondo Europeo de Desarrollo Regional [FEDER]: SAF2016-75286-R [to R.R.]); and the Madrid Regional Government (CellCAM; P2010/BMD-2420 [to J.G.-C.]) in Spain. Sí

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the “Miguel Servet” program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees. Sí

Polo-like kinase 1 (PLK1), an essential regulator of cell division, is currently undergoing clinical evaluation as a target for cancer therapy. We report an unexpected function of Plk1 in sustaining cardiovascular homeostasis. Plk1 haploinsufficiency in mice did not induce obvious cell proliferation defects but did result in arterial structural alterations, which frequently led to aortic rupture and death. Specific ablation of Plk1 in vascular smooth muscle cells (VSMCs) led to reduced arterial elasticity, hypotension, and an impaired arterial response to angiotensin II in vivo. Mechanistically, we found that Plk1 regulated angiotensin II-dependent activation of RhoA and actomyosin dynamics in VSMCs in a mitosis-independent manner. This regulation depended on Plk1 kinase activity, and the administration of small-molecule Plk1 inhibitors to angiotensin II-treated mice led to reduced arterial fitness and an elevated risk of aneurysm and aortic rupture. We thus conclude that a partial reduction of Plk1 activity that does not block cell division can nevertheless impair aortic homeostasis. Our findings have potentially important implications for current approaches aimed at PLK1 inhibition for cancer therapy. This work-was supported by the Marie Curie activities of the European Commission (Oncotrain program; fellowship to P.W), the Spanish Ministry of Economy and Competitiveness (MINECO; fellowship to A.G.-L.), the CENIT AMIT Project "Advanced Molecular Imaging Technologies" (TEC2008-06715-C02-1, RD07/0014/2009 to F.M.), the Red de investigacion Cardiovascular (RIC), cofunded by FEDER (grant RD12/004240022 to J.M.R.; grant RD12/0042/0056 to L.J.J.-B), Fundacio La Marato TV3 (grant 20151331 to J.M.R.), the Castilla-Leon Autonomous Government (BIO/SA01/15, CS049U16 to X.R.B.), the Solorzano and Ramon Areces Foundations (to X.R.B.), MINECO (grants RD12/0036/0002 and SAF2015-64556-R to X.R.B.; SAF2015-63633-R to J.M.R.; and SAF2015-69920-R to M.M.), Consolider-Ingenio 2010 Programme (grant SAF2014-57791-REDC to M.M.), Red Tematica CellSYS (grant BFU2014-52125-REDT to M.M.), Comunidad de Madrid (OncoCycle Programme; grant S2010/BMD-2470 to M.M.), Worldwide Cancer Research (grants 14-1248 to X.R.B., and 15-0278 to M.M.) and the MitoSys project (European Union Seventh Framework Programme; grant HEALTH-F5-2010-241548 to M.M.). CNIC is supported by MINECO and the Pro-CNIC Foundation. CNIO and CNIC are Severo Ochoa Centers of Excellence (MINECO awards SEV-2015-0510 and SEV-2015-0505, respectively). Sí

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunomodulatory properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Previous studies have demonstrated that MSCs, administered systemically, migrate to lymphoid tissues associated with the inflammatory site where functional MSC-induced immune cells with a regulatory phenotype were increased mediating the immunomodulatory effects of MSCs. These results suggest that homing of MSCs to the lymphatic system plays an important role in the mechanism of action of MSCs in vivo. Thus, we hypothesized that direct intralymphatic (IL) (also referred as intranodal) administration of MSCs could be an alternative and effective route of administration for MSC-based therapy. Here, we report the feasibility and efficacy of the IL administration of human expanded adipose mesenchymal stem cells (eASCs) in a mouse model of collagen-induced arthritis (CIA). IL administration of eASCs attenuated the severity and progression of arthritis, reduced bone destruction and increased the levels of regulatory T cells (CD25+Foxp3+CD4+ cells) and Tr1 cells (IL10+CD4+), in spleen and draining lymph nodes. Taken together, these results indicate that IL administration of eASCs is very effective in modulating established CIA and may represent an alternative treatment modality for cell therapy with eASCs. This project has received funding from the Spanish Ministerio de Economía y Competitividad and Comunidad Autónoma de Madrid to TiGenix and the European Union’s Seventh Programme for research, technological development and demonstration under grant agreement No 279174 to TiGenix, CIEMAT, and Farma-Cros. Sí

Cancer evolution is a process that is still poorly understood because of the lack of versatile in vivo longitudinal studies. By generating murine non–small cell lung cancer (NSCLC) orthoallobanks and paired primary cell lines, we provide a detailed description of an in vivo, time-dependent cancer malignization process. We identify the acquisition of metastatic dissemination potential, the selection of co-driver mutations, and the appearance of naturally occurring intratumor heterogeneity, thus recapitulating the stochastic nature of human cancer development. This approach combines the robustness of genetically engineered cancer models with the flexibility of allograft methodology. We have applied this tool for the preclinical evaluation of therapeutic approaches. This system can be implemented to improve the design of future treatments for patients with NSCLC. Cancer Res; 74(21); 5978–88. ©2014 AACR.

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-Ras(V14I), a recurrent KRAS mutation in NS patients. K-Ras(V14I)-mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-Ras(V14I)-mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome.

3100 Background: We have shown that when Aas induce vascular normalization (VN), tumors escape upregulating mitochondrial metabolism. Mitochondrial inhibition with ME344 induced synergy with various Aas. We also found that VN could be traced by showing a 10% decrease in tumor FDG-PET SUV from day (d) 0 to d8 of Aa. We studied the activity of adding ME344 or placebo to Bev (Ki67 decrease) in E-HERNEBC in a phase 0 randomized trial. As a secondary objective we measured the activity of the combination in patients (Pts) showing VN according to FDG-PET. Methods: Untreated E-HERNEBC Pts with T > 1cm, any N, M0 underwent a baseline FDG-PET (d1) and received a single dose of Bev (15mg/kg) prior to randomization (1:1) to arm A (FDG-PET on d8 followed by ME344 10 mg/kg IV on d8, d15 and d21) or Arm B (FDG-PET on d8 followed by placebo on d8, d15 and d21). Tumors were biopsied on d0 and 28. A 40 Pts sample size was powered to detect a 30% relative difference between arms in digitally acquired Ki67 decrease from d0 to d28 (alpha 0.05, beta 0.2). Results: Arm A: 20 Pts; Arm B: 21 Pts. Baseline characteristics were in arm A vs B: age 58.4(41.5-75.3) vs 53.6(39-82.8); T1(30%)/T2(60%)/T3(10%) vs T1(52%)/T2(48%)/T3(0%); N0(80%)/N1(20%) vs N0(81%)/N1(19%); ER+(75%)/TNBC(25%) vs ER+(71.4%)/TNBC(28.6%); Ki67 31.6% (3.6%-70%) vs 25.2% (1.2% - 81.5%). PET-SUV decreased > 10% from d0 to d8 in 6/20 (arm A) and 6/21 (arm B) Pts. Two G3 adverse events (blood pressure) were reported (1/arm) and deemed related to Bev. Results of the primary endpoint: table. Conclusions: ME344 showed significant biologic activity, enhancing the effect in Ki67 decrease vs placebo when added to Bev in E-HERNEBC. The activity was greater in TNBC. A trend for greater activity in patients experiencing VN according to FDG-PET was observed. Clinical trial information: NCT02806817. [Table: see text]

Clinical interest on human mesenchymal progenitor cells (hMPC) relies on their potential applicability in cell-based therapies. An in vitro characterization is usually performed in order to define MPC potency. However, in vitro predictions not always correlate with in vivo results and thus there is no consensus in how to really assess cell potency. Our goal was to provide an in vivo testing method to define cell behavior before therapeutic usage, especially for bone tissue engineering applications. In this context, we wondered whether bone marrow stromal cells (hBMSC) would proceed in an osteogenic microenvironment. Based on previous approaches, we developed a fibrin/ceramic/BMP-2/hBMSCs compound. We implanted the compound during only 2 weeks in NOD-SCID mice, either orthotopically to assess its osteoinductive property or subcutaneously to analyze its adequacy as a cell potency testing method. Using fluorescent cell labeling and immunohistochemistry techniques, we could ascertain cell differentiation to bone, bone marrow, cartilage, adipocyte and fibrous tissue. We observed differences in cell potential among different batches of hBMSCs, which did not strictly correlate with in vitro analyses. Our data indicate that the method we have developed is reliable, rapid and reproducible to define cell potency, and may be useful for testing cells destined to bone tissue engineering purposes. Additionally, results obtained with hMPCs from other sources indicate that our method is suitable for testing any potentially implantable mesenchymal cell. Finally, we propose that this model could successfully be employed for bone marrow niche and bone tumor studies. This work was supported by Seventh Framework Programme of the European Commission (EuroNanomed ERA-NET initiative, REBONE project; PI10/02985FIS to J.G-C), Madrid regional government (CellCAM; P2010/BMD-2420 to J.G-C), Ministerio de Ciencia e Innovación (PI11/00377FIS to J.G-C, CSD2009-00088 to J.L.L-L.) and Mapfre foundation (SA/12/AYU/197 to Ar.A). We are grateful to Elena Andrés, David Castejón and María Encarnación Fernandez-Valle for their support in image techniques, to Daniel Baizan for his technical support in animal care, to Isabel Mirones for her technical support in immunohistochemistry and to Isabel Colmenero, pathologist, for her support in sample characterizations. Sí

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis1,2,3. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma4,5. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress6,7. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma8,9,10,11,12,13,14. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear2,14. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches15. Injectable lymphatic tracers have been developed7, but their limited diffusion precludes whole-body imaging at visceral sites16. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive ‘lymphoreporter’17 because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer17,18. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a ‘MetAlert’ discovery platform for drivers and inhibitors of metastasis., M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (project SAF2014-56868-R), the Asociación Española Contra el Cáncer (AECC), the Worldwide Cancer Research, an Established Investigator Award from the Melanoma Research Alliance (MRA), and a L’Oréal Paris USA-MRA Team Science Award for Woman in Scientific Research. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. N.I. and J.M. are funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. J.L.R.-P and P.O.-R are funded by grants FIS 2014/1737, 11/02568 and FIS 2014/01784, 11/1759, respectively, from the Spanish Ministry of Health. F.M. is funded by the AMIT Project/CDTI/CENIT Programme (MICINN), S.O. by SAF2013-44866-R (MINECO), and J.J.B.-C. by an NCI K22CA196750 grant and the TCI Young Scientist Cancer Research Award JJR Fund (P30 CA196521). J.D.M. is the recipient of a postdoctoral fellowship from the ARC Foundation and E.R.-F. from Fundación Científica de la Asociación Española Contra el Cáncer. D.C.-W. is the recipient of a predoctoral fellowship from Fundación La Caixa, and M.C.-A. and X.C. are recipients of the Immutrain Marie Skłodowska-Curie ITN Grant.

Background A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models. Methods An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for Zr-89 labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments. Results Zr-89 labeling of DFO-LEM2/ 15 was achieved with high yield (>90\%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that Zr-89-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent Zr-89-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP-MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. Zr-89-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models. Conclusion A new anti MT1-MMP-mAb tracer, Zr-89-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM. MAM JLMT AJS FMA: Crowdfunding platform ``Precipita´´ FECYT (Spanish Foundation for Science and Technology) [http://www.precipita.es/precipitado/deteccion-y-seguimiento-del-cance r-cerebral-mas-comun-y-danino.html]. AGA JLMT: Regional Government of Madrid Angiobodies Programme S2010/BMD-2312 [http://www.madrid.org/cs/Satellite?buscador=true\&c=CM\_ConvocaPresta c\_FA\&cid=1142619643182\&language=es\&pagename=ComunidadMadrid\%2FEstru ctura]. AJS: BBVA Foundation grant for Research, Innovation and Cultural Creation and M+Vision Advanced fellowship. MS: Marie Curie CIG grant \& Seve Ballesteros Foundation grant. Sí

Background: Rationalization of antiangiogenics requires biomarkers. Vascular re‐normalization is one widely accepted mechanism of action for this drug class. The interstitium of tumors with abnormal vasculature is hypoxic. We sought to track vascular normalization with 18F‐misonidazole ([18F]‐FMISO, a probe that detects hypoxia) PET, in response to window‐of‐opportunity (WoO) treatment with the antiangiogenic dovitinib. Methods: Two patient‐derived pancreas xenografts (PDXs; Panc215 and Panc286) and the spontaneous breast cancer model MMTV‐PyMT were used. Animals were treated during 1 week of WoO treatment with vehicle or dovitinib, preceded and followed by [18F]‐FMISO‐PET, [18F]‐FDG‐PET, and histologic assessment (dextran extravasation, hypoxia and microvessel staining, and necrosis, cleaved caspase‐3 and Ki67 measurements). After WoO treatment, gemcitabine (pancreas)/adriamycin (breast) or vehicle was added and animals were treated until the humane endpoint. Tumor growth inhibition (TGI) and survival were the parameters studied. Results: [18F]‐FMISO SUV did not change after dovitinib‐WoO treatment compared to vehicle‐WoO (0.54 vs. 0.6) treatment in Panc215, but it decreased significantly in Panc286 (0.58 vs. 1.18; P, Highlights We describe a new technique to monitor the efficacy of antiangiogenics.This drug class currently lacks biomarkers.Antiangiogenics exert their positive effect by inducing stromal normalization.18F‐fluoromisonidazole PET is able to monitor stromal normalization.Tumors showing stromal normalization by PET experience benefit from antiangiogenics.

13 páginas, 7 figuras, 7 figuras suplementarias, 7 tablas suplementarias.-- et al., Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Ptentg mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Ptentg mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Ptentg mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Ptentg fibroblasts programmed with Prdm16 and Cebpβ form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage, Work in the laboratory of A.M.V. was funded by grant SAF2009-08114 and by the CIBERDEM (ISCIII), and work in the laboratory of E. Rial was funded by grants SAF2010-20256 and Consolider-Ingenio CSD2007-00020. A.O.-M. was recipient of a predoctoral contract of the Regional Government of Madrid. E.L.-G. was recipient of a predoctoral contract from the Spanish Ministry of Education. M.M.G.-B. was supported by the “Ramon y Cajal” program of the Spanish Ministry of Science and Innovation.

An age-related bone loss occurs, apparently associated with the concomitant increase in an oxidative stress situation. However, the underlying mechanisms of age-related osteopenia are ill defined since these studies are time consuming and require the use of many animals (mainly rodents). Here, we aimed to characterize for the first time the bone status of prematurely aging mice (PAM), which exhibit an increased oxidative stress. Tibiae from adult (6 months) PAM show an increase in bone mineral density (BMD) and bone mineral content (assessed by bone densitometry) versus those in their normal counterparts (non-prematurely aging mice, NPAM) and similarly decreased in both kinds of mouse with age. However, at this bone site, trabecular BMD (determined by μ-computerized tomography) was similar in both adult PAM and old (18 months) NPAM. Femurs from these groups of mice present an increase in oxidative stress, inflammation, osteoclastogenic, and adipogenic markers, but a decrease in the gene expression of osteoblastic differentiation markers and of the Wnt/β-catenin pathway. Our findings show that adult PAM recapitulate various age-related bone features, and thus are a suitable model for premature bone senescence studies.

The possibility of imaging tumors in live mice has opened new opportunities for cancer research, particularly regarding the ability to perform longitudinal studies in combination with a therapeutic intervention. Here, we detail methods to optimize visualization of murine tumors by positron emission tomography (PET), computed tomography (CT), and combined PET-CT. Curr. Protoc. Mouse Biol. 1:85-103. © 2011 by John Wiley & Sons, Inc.

Directive 2010/63/EU on the protection of animals used for scientific purposes requires that the killing of mammal foetuses during the last third of their gestational period should be accomplished through effective and humane methods. The fact that murine foetuses are resistant to hypoxia-mediated euthanasia renders the current euthanasia methods ineffective or humane for the foetuses when these methods are applied to pregnant female mice. We have assessed the time to death of foetuses after performing either indirect (dam euthanasia) or direct (via intraplacental injection – a new approach to euthanasia) euthanasia methods in order to determine a euthanasia method that is appropriate, ethical and efficient for the killing of mouse foetuses. The respective times to death of foetuses after performing the three most commonly used euthanasia methods (namely cervical dislocation, CO2 inhalation and intraperitoneal sodium pentobarbital administration) were recorded. Absence of foetal heartbeat was monitored via ultrasound. We consider that the most effective and humane method of foetal euthanasia was the one able to achieve foetal death within the shortest possible period of time. Among the indirect euthanasia methods assessed, the administration of a sodium pentobarbital overdose to pregnant female mice was found to be the fastest for foetuses, with an average post-treatment foetal death of approximately 29.8 min. As for the direct euthanasia method assessed, foetal time to death after intraplacental injection of sodium pentobarbital was approximately 14 min. Significant differences among the different mouse strains employed were found. Based on the results obtained in our study, we consider that the administration of a sodium pentobarbital overdose by intraplacental injection to be an effective euthanasia method for murine foetuses.

Coronary heart disease is one of the main causes of death in the developed world, and treatment success remains modest, with high mortality rates within 1 year after myocardial infarction (MI). Thus, new therapeutic targets and effective treatments are necessary. Short telomeres are risk factors for age-associated diseases, including heart disease. Here we address the potential of telomerase (Tert) activation in prevention of heart failure after MI in adult mice. We use adeno-associated viruses for cardiac-specific Tert expression. We find that upon MI, hearts expressing Tert show attenuated cardiac dilation, improved ventricular function and smaller infarct scars concomitant with increased mouse survival by 17% compared with controls. Furthermore, Tert treatment results in elongated telomeres, increased numbers of Ki67 and pH3-positive cardiomyocytes and a gene expression switch towards a regeneration signature of neonatal mice. Our work suggests telomerase activation could be a therapeutic strategy to prevent heart failure after MI.

Resumen —La gammagrafia con 99mTc-MIBI se ha propuesto como metodo de diagnostico por imagen de una gran variedad de tumores malignos. En el momento actual, el mecanismo por el cual las celulas malignas captan y concentran el 99mTc-MIBI no es del todo conocido. Algunas neoplasias mamarias no muestran captacion de 99mTc-MIBI. El objetivo de este estudio es determinar si existe correlacion entre la captacion de 99mTc-MIBI por parte del tumor y distintos parametros anatomopatologicos implicados en la agresividad tumoral. Para ello se ha estudiado 100 pacientes con cancer de mama. A todas ellas se les realizo gammagrafia mamaria con 99mTc-MIBI calculandose mediante un analisis semicuantitativo la ratio tumor/fondo en cada proyeccion. Tras la cirugia se determino el tamano tumoral, invasion ganglionar axilar, grado histologico (Scarff Bloom Richardson), grado nuclear, indice mitotico, presencia de atipia y receptores hormonales (estrogenos y progesterona). Resultados Hemos encontrado una correlacion estadisticamente significativa (p Conclusiones La captacion del 99mTc-MIBI en el cancer de mama esta correlacionado con el grado de diferenciacion tumoral: A menor diferenciacion celular tumoral (mayor agresividad) mayor captacion. Por otro lado no se ha encontrado correlacion entre la captacion de 99mTc-MIBI y los parametros anatomopatologicos clasicos que definen la agresividad tumoral, como son el tamano y la invasion ganglionar axilar.

Resumen —La presencia de ganglios locorregionales afectados debe considerarse como uno de los factores pronosticos mas importantes en el cancer de mama. En la actualidad el clinico esta condicionado por la absoluta falta de una metodologia capaz de proporcionar un juicio preoperatorio atendible sobre los ganglios axilares que, en caso de negatividad, permita evitar su extirpacion quirurgica. En nuestro estudio pretendemos evaluar la utilidad de la gammagrafia mamaria con 99mTc-MIBI en el diagnostico preoperatorio de la invasion ganglionar axilar y analizar la relacion entre la captacion del radiofarmaco y la presencia de mayor numero de ganglios afectados. Material y metodo Estudiamos 84 pacientes con cancer de mama. A todas ellas se les realizo gammagrafia mamaria con 99mTc-MIBI calculandose mediante un analisis semicuantitativo la ratio tumor/fondo en cada proyeccion. Tras la cirugia se determino la invasion ganglionar axila. Los resultados de la gammagrafia mamaria con 99mTc-MIBI en la deteccion de invasion ganglionar axilar fueron sensibilidad 36%, especificidad 100%, valor predictivo positivo 100% y valor predictivo negativo 48%. En nuestro estudio no encontramos correlacion entre la captacion de 99mTc-MIBI en la lesion primaria y el numero de ganglios axilares invadidos. Conclusion La gammagrafia mamaria con 99mTc-MIBI puede aportar datos complementarios al diagnostico pre-operatorio de la invasion ganglionar axilar, basandonos en la elevada especificidad y valor predictivo positivo de la tecnica.

Resumen La gammagrafia mamaria con 99m Tc-MIBI ha demostrado ser de utilidad como complemento a la mamografia en el diagnostico del cancer de mama. Existen grupos concretos de pacientes en las que la mamografia encuentra aun mas disminuida su especificidad. Objetivo En este estudio nos planteamos valorar la utilidad de la gammagrafia mamaria con 99m Tc-MIBI, en aquellas situaciones que mamograficamente plantean problemas diagnosticos, como ocurre en las pacientes con: mamas densas, mujeres jovenes y mamas intervenidas y radiadas. Material y metodo Estudiamos un total de 201 pacientes; 109 pacientes con patron mamografico de mamas densas, 8 pacientes menores de 30 anos y 24 pacientes con mamas intervenidas y radiadas. Los resultados fueron en el diagnostico de mamas densas una sensibilidad del 88% para el MIBI frente al 81% para la mamografia y especificidad del 90% frente al 28%. En las mujeres menores de 30 anos la sensibilidad para el MIBI fue del 100% frente a la mamografia con sensibilidad del 50%, la especificidad fue del 100% frente al 20%. En las mujeres con mamas intervenidas, MIBI sensibilidad 80% frente a mamografia 80%, especificidad MIBI 100% y mamografia 42%. Conclusiones La gammagrafia mamaria con 99m Tc-MIBI es una excelente tecnica de diagnostico, por su elevada especificidad, complementando a la mamografia en el diagnostico de cancer de mama en pacientes con mamas densas, mujeres jovenes y mamas intervenidas y radiadas.

The cellular microenvironment plays a relevant role in cancer development. We have reported that mesenchymal stromal/stem cells (MSCs) deficient for p53 alone or together with RB (p53(-/-)RB(-/-)) originate leiomyosarcoma after subcutaneous (s.c.) inoculation. Here, we show that intrabone or periosteal inoculation of p53(-/-) or p53(-/-)RB(-/-) bone marrow- or adipose tissue-derived MSCs originated metastatic osteoblastic osteosarcoma (OS). To assess the contribution of bone environment factors to OS development, we analyzed the effect of the osteoinductive factor bone morphogenetic protein-2 (BMP-2) and calcified substrates on p53(-/-)RB(-/-) MSCs. We show that BMP-2 upregulates the expression of osteogenic markers in a WNT signaling-dependent manner. In addition, the s.c. coinfusion of p53(-/-)RB(-/-) MSCs together with BMP-2 resulted in appearance of tumoral osteoid areas. Likewise, when p53(-/-)RB(-/-) MSCs were inoculated embedded in a calcified ceramic scaffold composed of hydroxyapatite and tricalciumphosphate (HA/TCP), tumoral bone formation was observed in the surroundings of the HA/TCP scaffold. Moreover, the addition of BMP-2 to the ceramic/MSC implants further increased the tumoral osteoid matrix. Together, these data indicate that bone microenvironment signals are essential to drive OS development.

Phosphoinositide-3-kinases (PI3K) are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. PI3K is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the rapid identification of ETP-46992, within 2-aminocarbonyl imidazo [1,2-a] pyrazine series, with suitable pharmacokinetic (PK) properties that allows the establishment of mechanism of action and efficacy in vivo studies. ETP-46992 showed tumor growth inhibition in a GEMM mouse tumor model driven by a K-RasG12V oncogenic mutation and in tumor xenograft models with PI3K pathway deregulated (BT474).

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKTSer473 phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-RasG12V oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures. This work was supported by Ministerio de Ciencia e Innovación (PET2008-0168, CSD2009-00088), and Ministerio de Educación y Ciencia y Fondo Social Europeo (PTQ05-02-02498 and Fundación PCM, MAT 2006-08720). These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Escherichia-coli-produced recombinant human BMP-2 (rhBMP-2) was generously supplied by Noricum S.L. (Spain). We thank Alba Cuenca-Moya (Noricum S.L.) and Virginia Gutierrez (Pharmakine) for their technical support. Sí

Ellis-van Creveld syndrome protein homolog (Evc) was previously shown to mediate expression of Indian hedgehog (Ihh) downstream targets in chondrocytes. Consequently disruption of the Ihh/Pthrp axis was demonstrated in Evc(-/-) mice, but the full extent of Evc involvement in endochondral development was not totally characterized. Herein we have examined further the Evc(-/-) growth plate in a homogeneous genetic background and show that Evc promotes chondrocyte proliferation, chondrocyte hypertrophy and the differentiation of osteoblasts in the perichondrium, hence implicating Evc in both Pthrp-dependent and Pthrp-independent Ihh functions. We also demonstrate that Evc, which localizes to osteoblast primary cilia, mediates Hedgehog (Hh) signaling in the osteoblast lineage. In spite of this, bone collar development is mildly affected in Evc(-/-) mutants. The onset of perichondrial osteoblastogenesis is delayed at the initial stages of endochondral ossification in Evc(-/-) mice, and in later stages, the leading edge of expression of osteoblast markers and Wnt/ß-catenin signaling components is located closer to the primary spongiosa in the Evc(-/-) perichondrium owing to impaired osteoblast differentiation. Additionally we have used Ptch1-LacZ reporter mice to learn about the different types of Hh-responsive cells that are present in the perichondrium of normal and Evc(-/-) mice. Evc mediates Hh target gene expression in inner perichondrial cells, but it is dispensable in the external layers of the perichondrium. Finally, we report cranial base defects in Evc(-/-) mice and reveal that Evc is essential for intrasphenoidal synchondrosis development., This work was funded by the Spanish Ministry of Science and Innovation (SAF-62291 and SAF-17901), the European Union (LSHM-CT-2007-03741) and the Ramón Areces Foundation.

RASopathies are a class of developmental syndromes that result from congenital mutations in key elements of the RAS/RAF/MEK signaling pathway. A well-recognized RASopathy is the cardio-facio-cutaneous (CFC) syndrome characterized by a distinctive facial appearance, heart defects, and mental retardation. Clinically diagnosed CFC patients carry germ-line mutations in four different genes, B-RAF, MEK1, MEK2, and K-RAS. B-RAF is by far the most commonly mutated locus, displaying mutations that most often result in constitutive activation of the B-RAF kinase. Here, we describe a mouse model for CFC generated by germ-line expression of a B-RafLSLV600E allele. This targeted allele allows low levels of expression of B-RafV600E, a constitutively active B-Raf kinase first identified in human melanoma. B-Raf+/LSLV600E mice are viable and display several of the characteristic features observed in CFC patients, including reduced life span, small size, facial dysmorphism, cardiomegaly, and epileptic seizures. These mice also show up-regulation of specific catecholamines and cataracts, two features detected in a low percentage of CFC patients. In addition, B-Raf +/LSLV600E mice develop neuroendocrine tumors, a pathology not observed in CFC patients. These mice may provide a means of better understanding the pathophysiology of at least some of the clinical features present in CFC patients. Moreover, they may serve as a tool to evaluate the potential therapeutic efficacy of B-RAF inhibitors and establish the precise window at which they could be effective against this congenital syndrome., Work in the laboratory of M.D. was funded by Consorcios Estratégicos Nacionales en Investigación Técnica Program (CDTEAM) Grant TEC2008-06715-C02-01, Centro de Investigación Biomédica en Red Program Grants CB06/01/0079 and PNSD 2007-2010, Fondo de Investigación Sanitaria (FIS) Grant CP08/00017, and Fundación de la Mutua Madrileña del Automovil (FMMA). E.M.B.W. was the holder of a United Kingdom National Institute for Health Research Academic Clinical Fellowship and was supported by the Manchester Biomedical Research Centre. Work in the laboratory of X.R.B. was funded by National Institutes of Health Grant R01CA073735, Spanish Ministry of Science and Innovation (MICINN) Grants SAF2009-07172 and RD06/0020/0001, Autonomous Government of Castilla y León (GR97), and Asociación Española contra el Cáncer. Work in the laboratory of M.B. was supported by European Union- Framework Programme Grants LSHG-CT-2006-037188 and LSHG-CT-2007-037665 (to M.B.), European Research Council Grant ERC-AG/250297-RAS AHEAD (to M.B.), MICINN Grants SAF2006-11773 and CSD2007-00017 (to M.B.), FMMA (to M.B.), FIS Grant PI042124, and Autonomous Community of Madrid Grant GR/SAL/0349/2004.

Objective— Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. Methods and Results— We produced biodegradable, injectable polylactic coglycolic acid–based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood–derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin + vascular smooth muscle cells, indicating more efficient vessel stabilization. Conclusion— Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

Genetic overexpression of the protein deacetylase Sir2 increases longevity in a variety of lower organisms, and this has prompted a great interest on the effects of its closest mammalian homologue, Sirt1, on aging and cancer. We have generated transgenic mice moderately overexpressing Sirt1 under its own regulatory elements (Sirt1-tg). Old Sirt1-tg mice present lower levels of DNA damage, decreased expression of the aging-associated gene p16Ink4a, a better general health and fewer spontaneous carcinomas and sarcomas. These effects, however, were not sufficiently potent to affect longevity. To further extend these observations, we developed a metabolic syndrome-associated liver cancer model in which wild-type mice develop multiple carcinomas. Sirt1-tg mice show a reduced susceptibility to liver cancer and exhibited improved hepatic protection from both DNA damage and metabolic damage. Together, these results provide direct proof for the anti-aging activity of Sirt1 in mammals and for its tumour suppression activity in aging- and metabolic syndrome-associated cancer.

SummaryInappropriate drug delivery, secondary toxicities, and persistent chemo- and immunoresistance have traditionally compromised treatment response in melanoma. Using cellular systems and genetically engineered mouse models, we show that melanoma cells retain an innate ability to recognize cytosolic double-stranded RNA (dsRNA) and mount persistent stress response programs able to block tumor growth, even in highly immunosuppressed backgrounds. The dsRNA mimic polyinosine-polycytidylic acid, coadministered with polyethyleneimine as carrier, was identified as an unanticipated inducer of autophagy downstream of an exacerbated endosomal maturation program. A concurrent activity of the dsRNA helicase MDA-5 driving the proapoptotic protein NOXA resulted in an efficient autodigestion of melanoma cells. These results reveal tractable links for therapeutic intervention among dsRNA helicases, endo/lysosomes, and apoptotic factors.

The progressive accumulation of DNA damage is thought to be one of the driving forces that initiates ageing. However, the nature of the damage that arises endogenously is still ill-defined. A known source of endogenous damage is replicative stress (RS), which is intrinsically associated to DNA replication and prevented mainly by the ATR kinase. Here, we have developed a murine model of the human Seckel Syndrome characterized by a severe deficiency in ATR. Seckel mice suffer high levels of RS during embryogenesis when proliferation is widespread, but which decrease to marginal levels in postnatal life. In spite of this decrease, adult Seckel mice present accelerated ageing, which is further aggravated in the absence of p53 due to a further increase of RS. Together, these results support the concept that endogenous RS, particularly in utero, contributes to the onset of ageing in postnatal life and this is counterbalanced by the RS-limiting role of the checkpoint proteins ATR and p53.