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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 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.

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

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.

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

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]

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.

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.

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.

Antiangiogenic agents’ effects are elicited by VN, a process that improves tumor oxygenation and chemotherapy delivery, factors implicated in chemo-sensitivity. A biomarker that allowed monitoring this process non-invasively could help guiding individual therapeutic decisions. As hypoxia can be tracked with FMISO-PET, and hypoxia is influenced by vessel abnormality, we reasoned that a dynamic FMISO-PET monitoring of tumors treated with antiangiogenics at early timepoints could be informative of VN. Three pancreas cancer PDXs, (Panc215, Panc 286 and Panc219) were studied. Animals (athymic nude mice) were randomized to a window-of-opportunity (WOP) short course (4 days) of the multikinase inhibitor antiangiogenic agent dovitinib (DOV, a VEGFR1-3, PDGFRA/B and FGFR inhibitor) or vehicle. The WOP was preceded and followed by FMISO-PET and FDG-PET, as well as tumor sampling in order to assess vessel architecture and density (CD31 and confocal acquisition), 10Kda-dextran extravasation (vessel abnormality), tumor necrosis (H&E), and hypoxia staining (pimomidazole immunohistochemistry, in order to study the correspondence between FMISO-PET and tissue hypoxia evolution). After the WOP, animals were randomized to the addition of GEM or vehicle to their treatment. Tumor growth inhibition (TGI) was the parameter under study, comparing for each PDX the TGI of GEM, DOV or the combination versus vehicle. DOV did not exert significant TGI in any of the three models. FMISO-PET SUV levels evolved during the WOP: the SUV increased 53% and 72% for Panc286 and Panc219 when treated with vehicle, but remained unchanged during WOP for Panc215. The SUV changes were modulated by DOV: administered during the WOP, FMISO-PET SUV in Panc215 remained stable, but abrogated the increase in Panc219, and induced a 10% decrease in Panc285. FDG-PET showed that the areas that turned FMISO-PET-negative with DOV were still viable in the three models, confirmed by histologic examination (no significant change in the percentage of necrotic area surface). DOV did not add TGI effect to GEM in Panc215 but it improved TGI an additional >80% and 75% compared to GEM alone in Panc286 and Panc219 (p = 0.02 and 0.001). Dovitinib reduced vessel tortuosity and dextran extravasation in Panc286 and Panc219, whereas did not change these parameters in Panc215 during the WOP. FMISO-PET mirrored pimomidazole staining evolution during the WOP. Intratumor gemcitabine concentration increased >2fold after DOV vs. vehicle during the WOP in Panc286 and Panc219, whereas did not change in Panc215. In conclusion, FMISO-PET accurately monitors the changes following VN induced by DOV: hypoxia and vessel leakage reversal, and increased interstitial concentration of GEM. FMISO-PET monitoring during the WOP detect in which tumors antiangiogenic treatment exerts positive effects added to chemotherapy. Citation Format: Tamara Mondejar, Elena Hernadez-Agudo, Marisa Soto-Montenegro, Diego Megias, David Cebrian, Jesus Sanchez, Francisca Mulero, Ramon Colomer, Manuel Hidalgo, Manuel Desco, Miguel Quintela-Fandino. 18F-misonidazole PET (FMISO-PET) monitors vascular normalization (VN) and predicts benefit from antiangiogenic treatment plus chemotherapy in pancreas cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1493. doi:10.1158/1538-7445.AM2015-1493

Objective Our aim was to evaluate liver graft integrity and function using scintigraphy and ultrasonography in a porcine model of auxiliary heterotopic liver transplantation with portal vein arterialization (AHLT-PVA). Materials and Methods Using Doppler ultrasonography we evaluated eight AHLT-PVA by parenchymal echogenicity, portal and arterial anatomy, and portal and biliary system flow. Two types of scintigraphy were performed: microaggregated human albumin colloid scintigraphy and diisopropyl iminodiacetic acid (DISIDA) scintigraphy, both labeled with 99mTc. Results The animals were distributed into two groups. The first group consisted of three animals with clinical suspicion of graft dysfunction, in which the ultrasonographic study revealed areas of parenchymal destructuring. In the scintigraphic study, heterogenous uptake was observed; there was no uptake in one animal. Necropsy of these three animals revealed areas of graft necrosis. The second group consisted of five animals with good clinical evolutions, in which the ultrasonographic study showed portal dilation, portal flow with arterial spiculations, and homogenous echogenicity of the hepatic parenchyma. The scintigraphic study revealed homogenous uptake by the graft and an elimination speed of the hepatobiliary agent similar to that of the native liver. Conclusions An heterogenous echostructure of the graft provided a sign of poor prognosis indicating necrosis in the same way as heterogenous uptake or nonuptake of radioisotope upon scintigraphy. Scintigraphy is a good method to evaluate biliary function and bile elimination. In an AHLT-PVA, the main ultrasound findings derived from arterialization were dilation of the portal system and portal flow with arterial spiculations.

Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent for T2D/obesity. Native FGF21 has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF21. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for > 1 year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF21. Furthermore, FGF21 overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF21 gene therapy to treat obesity, insulin resistance, and T2D. This work was supported by grants from Ministerio de Economía y Competi- tividad (MINECO) and FEDER, Plan Nacional I+D+I (SAF2014-54866R), andGeneralitat de Catalunya (2014SGR1669and ICREA Academia Award to F.B.), Spain, from the European Commission (MYOCURE, PHC-14-2015 667751) and the European Foundation for the Study of Diabetes (EFSD/MSD European Research Programme on Novel Therapies for Type 2 Diabetes,2013). V.J. was recipient of a post-doctoral research fellowship from EFSD/ Lilly. E.C., V.S., and C.M. received a predoctoral fellowship from Ministerio de Educación, Cultura y Deporte, and J.R. from Ministerio de Economía y Competitividad, Spain. The authors thank Marta Moya and Maria Molas for technical assistance. Sí