Numerous genetic and epigenetic alterations cause functional changes in cell biology underlying cancer. These hallmark functional changes constitute potentially tissue‐independent anticancer therapeutic targets. We hypothesized that RNA‐Seq identifies gene expression changes that underly those hallmarks, and thereby defines relevant therapeutic targets. To test this hypothesis, we analysed the publicly available TCGA‐TARGET‐GTEx gene expression data set from the University of California Santa CruzToil recompute project using WGCNA to delineate co‐correlated ‘modules’ from tumour gene expression profiles and functional enrichment of these modules to hierarchically cluster tumours. This stratified tumours according to T cell activation, NK‐cell activation, complement cascade, ATM, Rb, angiogenic, MAPK, ECM receptor and histone modification signalling. These correspond to the cancer hallmarks of avoiding immune destruction, tumour‐promoting inflammation, evading growth suppressors, inducing angiogenesis, sustained proliferative signalling, activating invasion and metastasis, and genome instability and mutation. This approach did not detect pathways corresponding to the cancer enabling replicative immortality, resisting cell death or deregulating cellular energetics hallmarks. We conclude that RNA‐Seq stratifies tumours along some, but not all, hallmarks of cancer and, therefore, could be used in conjunction with other analyses collectively to inform precision therapy. Hanahan and Weinberg posited cancer unifying changes in cell biology : resisting cell death, sustaining proliferative signalling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality and inducing angiogenesis.Individualized cancer therapy requires identifying contributors to these hallmarks, that is targetable biochemical pathways and genetic interactions. Presently, this includes histopathological, DNA, cytogenic and proteomic analyses.The work of Kaczowski et al led us to hypothesize that cancer‐associated mutations and epimutations alter expression of co‐correlated groups of genes independent of cancer type and are detectable primarily in cancer tissue by RNA‐Seq. We reasoned that assessing for co‐correlated groups of genes is arguably more sensitive to changes in expression of gene networks underlying biological processes than is identifying common processes among individual transcripts. To test this, we analysed gene expression profiles from the University of California, Santa Cruz (UCSC) Toil recompute of the TCGA, Therapeutically Applicable Research to Generate Effective Treatments (TARGET)22.1TcgaTargetGtex_rsem_gene_tpm (TTG) data set)  non‐coding entries leaving 19,672 protein‐coding entries  Table .These co2.2Because non‐cancerous primary site‐specific gene expression might obscure cancer signatures, we used two methods to subtract non‐cancer expression data. We analysed data before and after correction Figure B.Pc, a t×q matrix of cancer primary sites, and Pn, a t×r matrix of normal tissue primary sites. q and r are the number of cancer and normal samples, respectively, and t is the number of primary sites. We used two gene expression matrices:C, a s×q matrix of cancer gene expression from the T‐C‐PS data set  was calculated by first defining the mean normal expression Ltissue was calculated as:The tissue‐corrected gene expression matrix Y by both the total number of primary sites, t and the number of cancers within each primary site, r:The grand normal‐corrected data set  was calculated by partitioning matrix ml was calculated as before. Finally, the grand mean‐corrected gene expression matrix Lgrand as calculated as:2.3WGCNA package in R (version 1.68).For clustering analysis, the genes profiled were restricted firstly to protein‐coding genes because mechanisms of tumorigenesis are currently better understood for the protein‐coding transcriptome Figure A. Second2.4WebGestaltR package  in the 2.5find_k function from the dendextend R package (version 1.12.0); this function estimates k using maximal average silhouette widths.k groups to assign cancers to a cluster.Clusters of similar cancers were defined by hierarchical clustering 3To test the hypotheses that cancer‐inducing gene expression changes are detectable by RNA‐Seq and traverse cancer primary sites, we analysed the TTG data set Table  and rest3.1P ≤ .049). The remaining three modules were enriched for cancer‐relevant processes. The grey60 and purple modules were, respectively, enriched for natural killer (NK) cell signalling and T cell receptor (TCR) signalling (Table P ≤ 2.9*10‐5), axes characteristic of the avoiding immune destruction hallmark. The midnight blue module was enriched for histone modification signalling (Table P ≤ 10‐12), a component of the genome instability and mutation hallmark.Analysis of the uncorrected data set showed that subtle expression differences in both hallmark cancer and cancer‐unrelated, tissue‐specific pathways differentiated clusters. WGCNA categorized 991 genes into 17 modules. Eleven of those modules were enriched for functional pathway annotations: brown, cyan, green, grey60, light yellow, midnight blue, pink, purple, red, tan and turquoise. Eight modules were enriched for tissue‐specific processes: brown, cyan, green, light yellow, pink, red, tan and turquoise (Table P ≤ 10‐16). The brown and green modules did not show differential expression . Post hoc analysis by Dunn's Test for pairwise differences in module expression between clusters showed significantly different expression for four of six pairwise cluster comparisons for the turquoise module, five of six comparisons for the purple, red and tan modules, and six of six comparisons for the cyan, grey60, light yellow, midnight blue and pink modules , prostate (20%), ovary (18%) and kidney (12%) cancers Figure A. ClusteP ≤ 3.2*10‐9). The light yellow module was expressed higher in breast cancers than other sites . The pink module was expressed higher in liver cancers than other sites . The red, tan and turquoise modules were expressed higher in brain cancers than other sites .The cyan module was expressed higher in stomach and prostate cancers than other sites . The green module was expressed higher in skin cancers than in prostate, liver, kidney, brain or breast cancers .The expression of the grey60, midnight blue and purple modules differed between pairs of primary sites but without an appreciable pattern .Post hoc analysis by Tukey's Test determined pairwise differences in module expression between primary sites Table . The bro3.2Because of the potential for cancer‐unrelated primary site‐specific pathways to obfuscate tumorigenic signatures, we repeated the analyses Figure D after cP ≤ .05). Of these 16 modules, 7 were enriched for tissue‐specific processes: black, blue, cyan, dark green, light cyan, light yellow and turquoise (Table P ≤ .004), processes globally dysregulated in cancer,P ≤ .05) and genes corresponding to mitogenic signalling axes (Table P ≤ .05), markers of the tumour‐promoting inflammation and evading immune destruction hallmarks.WGCNA identified 3573 genes distributed into 27 modules in the tissue‐corrected data. Of those modules, 16 were enriched for functional pathway annotations: black, blue, brown, cyan, dark green, green, green yellow, grey60, light cyan, light yellow, magenta, pink, purple, red, turquoise and white (Table se Table . The remOS2Table ); componEL)Table  and in h2E)Table ; the for2E)Table , a charaP ≤ 2.2*10‐16). Post hoc analysis by Dunn's Test to assess pairwise differences in module expression showed differential expression for 38 of 45 cluster comparisons for the blue and purple modules, 39 of 45 comparisons for the turquoise module, 40 of 45 comparisons for the black and dark green modules, 41 of 45 comparisons for the cyan, green yellow, grey60, light cyan and light yellow modules, 42 of 45 comparisons for the brown, green, magenta, red and white modules, and 44 of 45 comparisons for the pink module , testis (24%) and uterine (9%) cancers .Post hoc analysis by Tukey's Test determined pairwise differences in module expression between primary sites Table . The exp3.3Given modules enriched for normal tissue processes in the tissue‐corrected data, we corrected for non‐cancer gene expression using the grand mean expression of each gene in all non‐cancer primary sites. This stratified tumours according to some hallmarks.P ≤ .05). Of those 12 modules, 4 were enriched for tissue‐specific processes: blue, green yellow, magenta and yellow (Table P ≤ .05). The remaining 8 modules were enriched for cancer‐relevant processes. The tan module was enriched for markers of angiogenesis (Table P ≤ .01), that is the inducing angiogenesis hallmark. The turquoise module was enriched for cell cycle progression pathways (Table P ≤ .03), a component of the evading growth suppressors hallmark. The cyan and light cyan modules were, respectively, enriched for markers of the epithelial to mesenchymal transition (EMT) and extracellular matrix (ECM) receptor and adhesion signalling (Table P ≤ .02), components of the activating invasion and metastasis hallmark. The black, green, pink and salmon modules were enriched for NK cell, T cell or inflammatory signalling (Table P ≤ .05), components of the tumour‐promoting inflammation and evading immune destruction hallmarks.WGCNA identified 1084 genes in 17 modules, of which 12 were enriched for functional pathway annotations: black, blue, cyan, green, green yellow, light cyan, magenta, pink, salmon, tan, turquoise and yellow (Table P ≤ 2.2*10‐16).Hierarchical clustering of the 1,084 genes in grand mean‐corrected WGCNA modules defined 10 clusters. These clusters were characterized by distinct expression of modules Figure C. All moPost hoc analysis by Dunn's Test for pairwise differences in module expression showed differential expression for 31 of 45 cluster comparisons for the magenta module, 37 of 45 for the black module, 38 of 45 for the salmon module, 40 of 45 for the green yellow, pink and tan modules, 41 of 45 for the turquoise and yellow modules, 42 of 45 for the light cyan module, 43 of 45 for the blue and green modules, and 44 of 45 for the cyan module Table .To investigate whether anatomical cancer primary site corresponded with cluster assignment, we evaluated the primary site composition of each cluster. Clusters 1, 3 and 5 were primary site heterogeneous. Cluster 1 was primarily composed of ovarian, (56%), testis (19%) and kidney (16.8%) cancers . The magenta module was expressed higher in skin cancers than in prostate, pancreas, ovary, lung, liver, kidney, brain, colon and breast cancers . The expression of black, blue, cyan, green, light cyan, pink, salmon, tan, turquoise and yellow modules differed for many pairwise comparisons of primary sites but did not have an appreciable pattern .Post hoc analysis by Tukey's Test determined pairwise differences in module expression between primary sites Table . The gre3.4P ≤ .05, Table To evaluate whether cancer clusters express hallmarks independently of primary site, we assessed the stratification of a primary site across clusters and the primary site diversity within clusters. For the former, we counted the number of clusters in which the null hypothesis of the hypergeometric test was rejected  high expression of genes enriched for TCR signalling and for angiogenic signalling, (b) low expression of both TCR signalling and angiogenic signalling genes, and (c) high expression of TCR signalling and low expression of angiogenic signalling genes Figure C. AlthouMultimodal treatment targets multiple hallmarks concurrently because the strong selective pressure on cancer cell populations 4.2Our analyses did not detect three of Hanahan and Weinberg's hallmarks as gene expression modules stratifying tumours. These hallmarks were resisting cell death, deregulating cellular energetics and enabling replicative immortality.TERT.TERT alone is insufficient as a transcriptomic network to be detected by our analyses.Strong transcriptomic signatures are not expected for the sustained enabling replicative immortality hallmark. This hallmark is predominantly characterized by the expression of The deregulating cellular energetics hallmarks have a strong transcriptomic footprint.Like the cellular energetics hallmark, the resisting cell death hallmark has a strong transcriptomic footprint. It is characterized by the subversion of the regulatory and functional elements of the cellular apoptosis machinery.4.3Analyses of the uncorrected data set showed that brain, oesophageal, ovarian, prostate, stomach and uterine cancers were stratified across clusters Table . Due to Suggesting the dependence of clustering on the anatomical primary site, analyses of the tissue‐corrected data set found that no cancer types were stratified across clusters Table . The norAnalyses of the grand mean‐corrected data set showed that breast, oesophageal and lung cancers were stratified across clusters Table , suggest4.4Previous investigations into transcriptomic subdivisions of cancer observed several of the pathways that we identified. Specifically, pancreatic,4.5The detection of some cancer hallmark signatures is encouraging, given the conservative approach  and the following limitations of our study. First, the data set did not contain isoform‐specific expression, and this prevented the incorporation of alternatively spliced transcripts into our analysis. Alternatively, spliced transcripts play important roles in cancer cell biology 5BRAF‐ERK‐CREB1), the activating invasion and metastasis , and the genome instability and mutation (histone modification) hallmarks. Additionally, cancer clusters differentiated by the above hallmarks stratified breast, oesophageal and lung cancers, highlighting the possibility of targeting transcriptomic features independent of anatomical primary site. Future studies are required to determine the therapeutic and prognostic relevance of these findings and to assess the impact of including transcriptomic features that we did not analyse.RNA‐Seq detects some hallmarks of cancer and those hallmarks stratified some, but not all, cancer types. We consistently identified signatures corresponding to the tumour‐promoting inflammation and avoiding immune destruction hallmarks , the evading growth suppressors , the inducing angiogenesis , the sustained proliferative signalling (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.CB conceived the hypothesis tested in this study. GF and PC developed the methodology to test that hypothesis. All code for data analysis and processing was developed by GF. GF wrote the manuscript with input and revisions provided by CB, PC and SM. SM and CB aided the clinical and biological relevance of conclusions drawn from data analysis. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


One of the main focuses of tissue engineering is to search for tridimensional scaffold materials, complying with nature's properties for tissue regeneration. Determining material biocompatibility is a fundamental step in considering its use. Therefore, the purpose of this study was to analyze osteoblast cell adhesion and viability on different materials to determine which was more compatible for future bone regeneration. Tridimensional structures were fabricated with hydroxyapatite, collagen, and porous silica. The bovine bone was used as material control. Biocompatibility was determined by seeding primary osteoblasts on each tridimensional structure. Cellular morphology was assessed by SEM and viability through confocal microscopy. Osteoblast colonization was observed on all evaluated materials' surface, revealing they did not elicit osteoblast cytotoxicity. Analyses of four different materials studied with diverse compositions and characteristics showed that adhesiveness was best seen for HA and viability for collagen. In general, the results of this investigation suggest these materials can be used in combination, as scaffolds intended for bone regeneration in dental and medical fields. Bone regeneration has increasingly turned into a very promising field, with use of resorbable biomaterials in combination with cells . Tissue Scaffolds act as templates mimicking the functions of the extracellular matrix (ECM), where cells can interact and differentiate into their native phenotype. It is then necessary for the scaffold to meet the following conditions: the scaffold must be biocompatible, nontoxic, nonimmunogenic, easy to elaborate, and biodegradable. In addition, it must allow for proper cell survival and signaling. Moreover, cells must be capable of growth, cellular remodeling, and reorganization , 7. HencOne of the most important characteristics of the scaffold is a high interconnected porosity to enable vascularization for nutrient and gas diffusion, which permits waste disposal –10. AddiThus, a proper porosity can improve mechanical retention between the scaffold and the host's environment, achieved as a result of ECM unspecified protein adsorption through a formed layer. This allows for a response from the cells in regard to the biomaterial they are in contact with, thus enabling new tissue formation . Rustom Another aspect to consider is scaffold mechanical resistance to load and forces. The scaffold must provide properties as close as possible to the host's natural environment, to be degraded only when the new tissue is properly formed . CurrentAlso, porous silicon seems promising for scaffold use in bone repair, since this material has been shown to promote osteoblast adherence and initiate maturation process , 25. FurIn spite of these discoveries, the ideal scaffold for bone regeneration has not been found. Hence, the objective of this work was to analyze osteoblast attachment and viability on four different materials: bovine bone (control), hydroxyapatite (HA), porous silicon, and collagen to determine a possible scaffold fabrication candidate for bone regeneration.3)2·4H2O) was placed with 1,500 mL 0.12 M diammonium hydrogen phosphate ((NH4)2HPO4) at 1.0 mL/min. To adjust the pH to 12, 75 mL of ammonium hydroxide (NH4OH) was added. Subsequently, the solution was heated at 90°C, stirred for 1 hour, and incubated for 10 days at room temperature. The obtained precipitate was washed several times with distilled water to neutralize the pH, dried at 250°C for 1 h, and then calcinated at 1,000°C for 3 h.Hydroxyapatite synthesis was carried out by the wet technique. In a three-necked flask, 900 mL of 0.33 M calcium nitrate tetrahydrate  to prevent microorganism growth. Subsequently, the bone was rinsed with water to remove traces of cleaning and disinfecting solutions. It was then subjected to heat treatment at 1,000°C for 3 h to remove all organic materials .θ), pitch size 0.02° (2θ), time per step 0.4 S, CuKα1 radiation λ = 1.5406 Å, and LynxEye detector. Crystalline phase qualitative identification present in the samples was carried out by comparison between reflections of the profile measured with diffraction profile reflections reported by the International Center for Diffraction Data (ICDD) (Powder Diffraction File) using search-match software [XRD was performed on HA and bovine bone samples. X-ray diffractograms were obtained on a PANalytical X'Pert PRO MPD (Netherlands) device with the following parameters: measuring range between 2° and 90° (2software .2H5OH) (Merck KGaA) concentration [1 : 2] and a cell specially designed for this purpose  and approved by Research Ethics Committee at the Pontificia Universidad Javeriana act 002 of 2009.µg/ml streptomycin , and 1.25 µg/ml fungizone  and incubated at 37°C and 5% CO2. The medium was changed twice a week until cells reached confluence. After seven days, cells migrating out of the tissue were visualized. The cells obtained were characterized by RT-PCR for osteocalcin (OCN), osteopontin (OPN), alkaline phosphatase (ALP), and bone sialoprotein (BSP) gene expression.Osteoblasts were isolated, following the method reported by Ducheyne and Qiu . First, 6 osteoblasts were seeded on each material. Cells were maintained in DMEM supplemented with 10% FBS and 200 μg/ml penicillin/streptomycin for 7 days, followed by scanning electron microscopy (SEM) and fluorescent confocal scanning for biocompatibility evaluation.Primary human osteoblasts seeded into 12-well plates at a cell density of 50,000 cells/well on HA, silicon, bovine bone, and collagen matrices were evaluated for biocompatibility. To this end, manufactured porous matrices were sterilized with ethylene oxide and 1 × 10After one week, culture matrices with cells were fixed with 3% buffered glutaraldehyde solution, followed by dehydration with increasing ethanol concentrations , and dried at a critical point ending with gold coating. Morphology was evaluated using SEM on a JEOL Model JSM-6490 LV operated under high vacuum mode, equipped with sensors allowing for scattered electron imaging.n = 4) by fluorescence microscopy using the live/dead kit . This kit discriminates living cells, by the presence of ubiquitous esterase activity, which is determined by the enzymatic conversion of nonfluorescent calcein into intensely fluorescent calcein. The polyanionic dye calcein is retained inside living cells, producing an intense green fluorescence (excitation at 495 nm and emission at 515 nm). Furthermore, if plasma membrane integrity is lost, then ethidium homodimer-1 (EthD-1) enters the cells. An increase in bright red fluorescence is observed in dead cells (excitation at 495 nm with emission at 635 nm) caused by EthD-1 binding to nucleic acids. Staining was performed on the 3D matrix seeded without additional procedures and cut with a microtome.Cell viability was assessed on matrices  similarity to human bone characteristics, as was the control bovine bone; (2) structural component of nonorganic phase such as hydroxyapatite; (3) structural component of organic phase, i.e., collagen; and (4) last, a material with high mechanical strength characteristics such as porous silicon.In this study, scaffolds were prepared with four different materials to evaluate Hydroxyapatite has been one of the most used bioceramics in dental reconstruction and bone tissue regeneration due to its biocompatibility, osteoconductivity, and lack of cytotoxicity , 33. HowRomanelli et al. developed a nanofibrous gel scaffold to which titanium nanoparticles and nanocrystals were added to simulate bone tissue, achieving osteoblast differentiation . In thisμm were obtained, which fall within the size range of bovine bone microporosities used as control (range from 0.1 µm to approximately 5 µm). It was identified that osteoblasts interacted with each other and adhered to the surface, extending their filopodia. This suggests HA could provide a suitable microenvironment for cell development. Furthermore, the technique can be improved allowing pore size control, which supports nutrient transport and toxin disposal ..55].Osteoblast adhesion to substrates is mediated via adhesion molecules. The cell-matrix adhesions mechanically interlink internal actin filaments and matrix, leading to the formation of focal adhesions, focal plaque, or focal contact. Such focal contacts are tenacious adhesion structures, which remain attached to the substratum, even after forceful cell detachment  56]..56].Collectively, all materials assayed provided different properties that need further optimization to achieve a substrate mimicking a natural environment. The best material providing a compatible surface for cell adhesion was bovine bone, followed by HA, porous silicon, and collagen. In contrast, after one week of culture, collagen sustained the most number of viable cells.In this study, osteoblast cell morphology, adhesiveness, and viability were evaluated as a possible biocompatible indicator. We observed a favorable cellular response between the cells seeded onto the prepared scaffold. Depending on the substrate, different responses were observed. Adhesiveness was best seen for HA and viability for collagen. These materials have been widely reported as alternatives for 3D scaffold development –60. ThisBone tissue engineering still holds many challenges. Scaffold material characteristics are key to reach the ultimate goal: cellular recognition, tissue specific commitment, repair, and regeneration. Therefore, it is of special interest to focus on using a combination of materials to provide mutual benefits. This interaction will tackle the weaknesses of one material by complementing with the strengths of the other material, leading to an ideal preparation for future scaffold establishment in tissue regeneration.


During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast‐inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.Astronauts experience osteoporosis‐like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of  The rapid and vigorous bone loss is one of the key challenges that remain to be solved to enable humans to pursue healthy lives in outer space. Bone mass often reflects a balance between bone formation by osteoblasts and bone resorption by osteoclasts. An increasing number of studies have demonstrated the effects of microgravity on bone metabolism and osteoblast activity, and increased bone resorption under microgravity has been widely reported.κB (RANK), which is a receptor for the RANK ligand (RANKL). RANK ligand is expressed in some stromal cells such as osteoblasts and bone marrow cells, and is required for osteoclastogenesis and osteoclast activation.In bone tissue, differentiation and activation of osteoclasts are generally influenced by interactions with osteoblastic lineage cells.Teleost scales are calcified tissues that exhibit multiple similarities with mammalian membrane bone tissues.Antiresorptive drugs, such as bisphosphonates, are expected to prevent bone loss during space flight.Investigating whether melatonin increases an osteoclast‐inhibiting hormone, CALCITONIN, in goldfish scales and whether the increased CALCITONIN regulates osteoclast function via paracrine system could offer insights on the role of melatonin. The present study performed space experiments to analyze bone metabolism in goldfish scale during space flight to determine the melatonin‐dependent regulatory mechanisms of osteoclasts.22.1Carassius auratus) specimens were purchased from Higashikawa Fish Farm  and were artificially fertilized at Tokyo University of Marine Science and Technology. Experiments with growing fish (10‐12 cm) were performed according to the recommendations in the ethical guidelines of Kanazawa University and Tokyo Medical and Dental University.Goldfish  and 96‐well plates  with and without melatonin  as illustrated in Figure Subsequently, the scales in 96‐well plates were frozen at −95°C for tartrate‐resistant acid phosphatase (TRAP) activity analysis.Some scales were incubated with melatonin (1 μM) in F‐μg conditions and processed for gene expression analysis.2.4The scales fixed with 4% PFA were incubated in 0.1% Triton X‐100 for 10 minutes. They were then used for actin ring formation and TRAP activity analysesFor the immunohistochemical analysis, the fixed scales were used as whole‐mount samples or cryosections. They were incubated with a blocking solution and then with primary antibodies: anti‐acetylserotonin O‐methyltransferase (ASMT) , anti‐CALCITONIN (×50 000),Asmt were prepared using a cDNA fragment containing 324‐1244 bases in GU205783. The signals of specific transcripts were detected using a kit (Boehringer Mannheim). The sections were counterstained with methyl green.For in situ hybridization, digoxigenin (DIG)‐labeled sense and antisense single‐stranded RNA probes for goldfish 2.52 observation areas. Mean measurements from the six areas were regarded as representative values for each scale.Regenerating scales had enlarged foci with complex mesh‐like networks of grooves Figure B. In the2 were recorded, and the mean number of nuclei per multinucleated osteoclast and the percentage of osteoclasts with actin rings were calculated. The width of the grooves at the middle of each groove, actin ring size, and the percentage of groove lengths covered with actin rings were measured using NIH Image J (https://imagej.nih.gov/ij/).The numbers of the osteoclasts per mm2.6Calcitonin  was used for the standardization procedure.Total RNA isolation and cDNA synthesis were performed using a kit (Qiagen GmbH).n Figure . Elongat2.7arylalkylamine N‐acetyltransferase (Aanat) and acetylserotonin O‐methyltransferase (Asmt) expression levels between space flight and ground control conditions in medaka brain samples, raw fastq files of RNA‐sequencing (RNA‐seq) data were downloaded from the DNA Data Bank of Japan database (accession no. DRA003542).Aanat and Asmt candidates, the longest transcripts that exhibited the highest homologies  were predicted to be the promising transcripts in this study.To compare 2.8The regenerating scales were incubated in a culture medium containing 10 nM to 1 μM of melatonin for 1, 2, and 4 days. Some scales were incubated with Mel‐R antagonist luzindole , and melatonin (1 μM) for 4 days. In addition, the CALCITONIN concentrations in the media were measured using ELISA for salmon CALCITONIN antibodyTo examine the effects of melatonin in mammalian bones, 10 calvariae excised from 2‐day‐old Wistar rats were cut in half, and two of the calvaria halves were incubated in a well with BGJb medium  containing melatonin (1 µM) or vehicle at 37°C for 1 day. The CALCITONIN concentrations in the culture media (n = 5 for each group) were measured as described above. Experiments with rats were performed according to the recommendations in the ethical guidelines of Okayama University.2.9t test or paired t test was performed to analyze significant differences between the two groups. In addition, one‐way repeated‐measures analysis of variance (ANOVA) or one‐way ANOVA followed by Tukey's post hoc test was used to examine significant differences in the values among three or four groups.Student's 33.1Aanat and Asmt mRNA levels in the regenerating scales were also significantly higher than those in the ontogenic scales  significantly increased in F‐μg condition  significantly increased under F‐μg condition , osterix (Osx), type 1 collagen 1a (Col1a), and osteocalcin (Ocn)—were lower in F‐μg scales than in F‐1g scales , and Ef1a, in regenerating scales cultured in dish for 1 day, 2 days, and 4 days were analyzed by qPCR. We then presented raw Ct values for housekeeping genes analyzed (Table Ef1α was higher than those of β‐Actin and Gapdh (Table Ef1α expression level does not cause misinterpretation of the target genes' expression in our qPCR analyses. In addition, CALCITONIN levels detected in the culture medium of the scales increased significantly by day 4 (Figure CALCITONIN and Mel‐R were immunolocalized in mononuclear cells forming the scale fibrillary plate Figure A‐D. The 2 Figure F‐H. It in Figure I. Validaed Table , as welled Table . In the dh Table . It was 4 Figure J. CALCIT4 Figure K.4In the experiments based both on the ISS and on the ground, to the best of our knowledge, we are the first to demonstrate a novel melatonin action pathway on bone metabolism using goldfish scales Figure , a bone We chose goldfish scales as experimental materials suitable for the analysis of bone metabolism in outer space. Although fish scales and mammalian mineralized skeleton are both derived from dermal skeleton,Calcitonin mRNA expression and CALCITONIN release into media. In addition, Mel‐R was detected in the scale osteoblasts (Figure We detected melatonin and melatonin‐synthesizing enzymes in the scale Figure . We alsos Figure C,D. The s Figure K, indicaAanat and Asmt expression levels decreased in the medaka brain including the pineal gland in space (Figure In the space experiments, we could evaluate resorptive activities using increased groove widths as indicators, and activated osteoclasts in the regenerating scales Figure . Our rese Figure B,C.Rankl:Opg (Figure Rankl:Opg could be due to the activation of osteoclast and bone resorption. Increased expression level of Cox2a could facilitate the upregulation of Rankl, since COX2 is an isozyme of cyclooxygenase, which is required for the synthesis of prostaglandin E2, a stimulator of bone resorption.Microgravity increased the expression ratio of g Figure G, a key Mmp‐9, Ctsk, Rankl, and Cox2a, and the Rankl:Opg ratio at F‐1g (Figure Rank (Figure Rank in osteoclastic cells and Rankl in osteoblastic cells, as above reported in mammalian cells. The suppression of Rankl or RANKL by melatonin has been widely reported.Opg expression in goldfish scales. These effects of melatonin induced the downregulation of the Rankl:Opg ratio, which should contribute to the suppression of Rankl‐mediated osteoclastogenesis. These results were consistent with the previous report that the effects of melatonin were mediated through signaling pathway mediated by Mel‐R, especially its MT2 subtype, on osteoblasts.Cox2a expression (Figure 2, a stimulator of RANKL expression.Osx and Col1a, was maintained by melatonin (Figure Under microgravity conditions, melatonin maintained the gene expression levels detected at F‐1g for g Figure A,B,E,G,Hk Figure D. The ren Figure H, which n Figure K,L, implPth1rIrs1Cebpb,Fos,Twist1,Osr2To comprehensively identify the genes whose expression levels were altered in the scales kept in F‐μg condition, we performed next‐generation sequencing analysis Figure . Our anaCalcitonin expression on day 4 in ground basis experiment (Figure Calcitonin mRNA expression translates to the induced expression of CALCITONIN in fish scales on day 4. Notably, Calcitonin expression in the regenerating scales significantly decreased during space flight (Figure Calcitonin expression in bone tissues under microgravity conditions. Consistent with our findings, the serum CALCITONIN in monkeys exhibited modest decreases during space flight,Calcitonin expression in bone tissues was counteracted by treatment with melatonin (Figure In the present study, we observed a loss of the melatonin effect on t Figure H. One pot Figure J,K. ThisTo further assess the morphological changes taking place in osteoclasts and the mechanism by which melatonin could suppress bone loss during space flight, we cultured the regenerating scales under simulated microgravity conditions created using a 3‐dimensional clinostat (Ground‐µg; Figure Circadian clocks regulate various biochemical, cellular, and physiological processes with a periodicity of approximately 24 hours in order to maximize an organism's physiological efficiency.Calcitonin and the downregulation of Rankl in osteoblasts (Figure Overall, the present study provides evidence that melatonin suppresses the bone‐resorbing activity of osteoclasts in bone tissues under microgravity conditions via the upregulation of s Figure . In humaMI, MJT, AH, KK, YT, TY, TS, IT, YF, YM, AK, RA, MN, YS, TN, MN, HI, SW, HA, KM, MS, HM, HF, MM, HU, SY, RM, TI, AH, and NS performed experiments. AH, SY, TS, HS, KO, and NS planned the space experiment. ME and TT prepared materials. TT, HN, NS, MN, TK, TH, KH, IT, TY, VSC, KI, TS, SE, YMT, AK, AS, YW, AT, AH, and NS analyzed the data. MI, YF, YT, JH, AH, and NS wrote the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Cardiac function, myocardial fibrosis, and the apoptosis of cardiomyocytes were observed at 4 weeks after treatment. In addition, EPC retention and the angiogenesis of ischemic myocardium were evaluated. Labeling with magnetic nanoparticles exhibited minimal influence to the biological functions of EPCs. The transplantation of magnetized EPCs guided by an external magnet significantly improved the cardiac function, decreased infarction size, and reduced myocardial apoptosis in MI rats. Moreover, enhanced aggregations of magnetized EPCs in the infarcted border zone were observed in rats with external magnet‐guided transplantation, accompanied by the significantly increased density of microvessels and upregulated the expression of proangiogenic factors, when compared with non‐external‐magnet‐guided rats. The magnetic field‐guided transplantation of magnetized EPCs was associated with the enhanced aggregation of EPCs in the infarcted border zone, thereby improving the therapeutic efficacy of MI.Low retention of endothelial progenitor cells (EPCs) in the infarct area has been suggested to be responsible for the poor clinical efficacy of EPC therapy for myocardial infarction (MI). This study aimed to evaluate whether magnetized EPCs guided through an external magnetic field could augment the aggregation of EPCs in an ischemia area, thereby enhancing therapeutic efficacy. EPCs from male rats were isolated and labeled with silica‐coated magnetic iron oxide nanoparticles to form magnetized EPCs. Then, the proliferation, migration, vascularization, and cytophenotypic markers of magnetized EPCs were analyzed. Afterward, the magnetized EPCs (1 × 10 AlthougCell therapy is an innovative, exciting, and promising therapeutic option for ischemic disease, which possesses the potential to fundamentally heal necrotic organs through cell regeneration  were isolated from the long bone of male syngeneic adult Sprague‐Dawley (SD) rats  by density gradient centrifugation using lymphocyte separation medium . Then, the MNCs were separated and resuspended with EGM‐2 SingleQuots , containing 2% fetal bovine serum, hydrocortisone, human fibroblast growth factor‐basic (hFGF‐B), VEGF, recombinant human‐epidermal growth factor (rhEGF), recombinant analogue insulin‐like growth factor 1 (R3‐IGF‐1), ascorbic acid, glycated albumin‐1000 (GA‐1000), and heparin. Subsequently, the cell suspension was seeded into human‐fibronectin‐coated  60‐mm dishes and incubated at 37°C in an atmosphere containing 95% air and 5% COThe identification of EPCs were performed by 1,1’‐dioctadecyl‐3,3,3’,3’‐tetramethylindocarbocyanine (DiI)‐labeled acetylated low density lipoprotein  uptake and the fluorescein isothiocyanate (FITC)‐labeled Ulex europaeus agglutinin (UEA)‐1  binding co‐staining experiment. Briefly, after 7 days in culture, the EPCs were washed with phosphate buffer saline (PBS) and incubated with Dil‐ac‐LDL (10 μg/ml) at 37°C for 4 hours. Then, FITC‐UEA‐1 (10 μg/ml) was added and incubated for 1 hour. Finally, the cells were incubated with 4’,6‐diamidino‐2‐phenylindole (DAPI). Differentiating EPCs were recognized as dually positive for Dil‐ac‐LDL uptake and FITC‐UEA‐1 binding when observed by fluorescence microscopy.2.23O4 core at the center, and surrounded by a silica layer in the outer surface. Adherent EPCs were washed with PBS and incubated with nanoparticles that dissolved into serum‐free endothelial basal medium‐2 (EBM‐2) with various concentrations of 0, 10, 25, 50, 100, and 200 μg/ml for 12 hr. To identify the labeling efficiency of nanoparticles and its influence on cell vitality at different doses, Prussian blue staining and flow cytometry assay were performed, respectively. For Prussian blue staining, at the end of the co‐incubation, labeled and unlabeled EPCs on the glass coverslips were fixed with 4% formaldehyde. Then, these cells were stained with a mixture solution that consisted of 2% potassium ferrocyanide and 2% HCl solution in equal proportions for 30 min. Thereafter, cell nuclei were counterstained with nuclear fast red. The vitality of EPCs, incubated with different concentrations of nanoparticles, was measured by Annexin V‐FITC and propidium iodide double staining, as previously described . The nanoparticles were 60 nm in diameter, characterized with a 40‐nm Fe2.3All in vitro experiments for determining whether nanoparticle labeling would affect the normal properties of EPCs were performed at 24 hr after transfection.2.3.1The migration capacities of labeled and unlabeled EPCs were evaluated by the transwell chamber assay , CD34 , and VEGFR . Then, the percentages of these biomarkers were analyzed by fluorescence‐activated cell sorting . CD133 was regarded as a biomarker of hematopoietic stem cell lineage, whereas VEGFR and CD34 were considered as biomarkers of endothelial cell lineage.2.3.4EPCs could synthesize and release varieties of pro‐angiogenesis cytokines, such as VEGF, IGF‐1, FDGF, SDF‐1α, and β‐FGF. The protein expression of neovascularization‐related cytokines in these two groups was measured by western blot analysis.2.4To determine whether the external magnetic field could manipulate the magnetized EPCs, their distribution with the intervention of an external magnetic field at a static status was first explored. In brief, after 24 hr of co‐incubation, the magnetized EPCs were digested and seeded into 60‐mm dishes with or without a magnet under the bottom center of the dishes. After incubation for 24 hr, the cells were fixed with 4% formaldehyde and stained with DAPI. Cell concentration degrees at different areas were observed by fluorescence microscopy.To better mimic the blood flow status caused by heart constriction in vitro, magnetized EPCs were resuspended with PBS and collected into 15‐ml centrifuge tubes. Then, these were located onto a swaying shaker, with or without a magnet field, and fixed on the outer surface. After 15 min, the difference between the locations of the cells from these two groups were observed.2.55, 5 × 105, 1 × 106, and 5 × 106. At 24 hr after transplantation, blood samples were collected from each group, and the plasma level of cardiac troponin I (cTnI) was measured by the enzyme‐linked immunosorbent assay to determine whether any microcirculation embolism occurred, according to manufacturer's instructions.Previous studies have reported that cell transplanting over a certain amount would lead to the embolization of myocardial microcirculation, and compromise the therapeutic efficacy of the cell therapy. Therefore, before the transplantation experiment, the optimal EPC amount for transplantation was determined, which was associated with the maximal cell retention in the ischemia area without leading to microvascular embolism at the first place. A concentration gradient study was designed, in which 15 non‐infarcted female rats were randomly divided into five groups, and transplanted with the following number of EPCs via the tail vein: 0, 1 × 10To further verify whether the transplantation with the optimal amount of nanoparticle‐labeled EPCs has any influence on the normal physiological function of rats, serum parameters of renal function (BUN and Cr), liver function (GOT and GPT), and iron content (SI and Ferritin) were measured at 1 and 4 weeks after transplantation using commercially available assay kits.2.6Female SD wild‐type rats  were provided by Wuhan University Experiment Animal Center. All experimental procedures were conducted in accordance with the Institutional Guidelines and Guide for the Care and Use of Laboratory Animals . The rat MI model was established, as previously described. In brief, animals were fasted for 12 hr before surgery and anesthetized with intraperitoneally injected with pentobarbital sodium (60 mg/kg). Then, a volume‐controlled small animal respirator was used to support the breathing of rats through a trachea cannula, and an electrocardiograph was used to continuously monitor for standard body electrocardiogram. Next, a left thoracotomy was performed, and a 6‐0 silk suture on a small curved needle was passed through the myocardium located approximately 2–3 mm from the junction between the left atrium and arterial cone, to ligate the left anterior descending artery (LAD). A successful MI model was confirmed by the elevated ST segment in Leads‐II and the regional paleness of the myocardial surface.2.7n = 10), rats were subjected to surgical manipulation without LAD occlusion and injected with 100 μl of PBS via the tail vein; (b) MI control group , rats were subjected to LAD occlusion and injected with 100 μl of PBS via the tail vein; (c) MI + EPCs transplantation group , rats were subjected to LAD occlusion and transplanted with 1 × 106 EPCs suspended in 100 μl of PBS via the tail vein; (d) MI + EPCs transplantation + Magnet group , rats were subjected to LAD occlusion and transplanted with 1 × 106 EPCs via the tail vein, followed by the application of a cylindrical magnet (0.39 T) above the infarct area of the heart for 1 hr; (e) MI + nanoparticle‐labeled EPCs transplantation group , rats were subjected to LAD occlusion and transplanted with 1 × 106 nanoparticle‐labeled EPCs via the tail vein; (f) MI + nanoparticle‐labeled EPCs transplantation + Magnet group , rats were subjected to LAD occlusion and transplanted with 1 × 106 nanoparticle‐labeled EPCs via the tail vein, followed by the application of a magnet above the infarct area of the heart for 1 hr.Cell therapy was performed at 1 week after MI surgery. Then, 10 sham‐operated (SO) and 50 MI female rats were divided into the following six groups: (a) SO group , cells were also transfected with adenovirus that overexpressed enhanced green fluorescent protein (GFP) as a visible marker. At 4 weeks after cell therapy, the animals were killed, and the hearts were harvested and frozen in optimal cutting temperature (OCT) compound. Myocardial tissue samples from the border zone of the infarct area were prepared and cut into 5‐μm sections with a thickness of 100 μm. To evaluate the retention of labeled EPCs and determine whether the external magnetic field could increase their aggregation in the myocardium, Prussian blue staining was performed. In brief, the specimen sections were incubated with a mixture solution of 2% potassium ferrocyanide and 2% hydrochloric acid, and counterstained with nuclear fast red.+ cells in each group. Cardiomyocytes in the representative slides were marked with the primary antibody of mouse anti‐α‐actinin, whereas GFP+ cells were marked with the primary antibody of rabbit anti‐GFP, followed by staining with secondary antibodies, accordingly. Then, the nuclei were stained with DAPI. All images were observed using a fluorescence microscope, and the mean number of GFP+ cells in one high‐power field in each group was calculated.Previous studies have reported that nanoparticles may be stagnated in tissues because of cell death or exocytosis, causing the false‐positive phenomenon for engraftment. On this account, immunocytochemistry was performed to exhibit GFP2.9To quantitatively analyze the retention of transplanted EPCs in the heart, real‐time polymerase chain reaction (PCR) was performed at 4 weeks after cell therapy in four cell transplantation groups. As mentioned above, EPCs isolated from male donor SD rats were transplanted to female recipients, and the cell engraftment numbers could be speculated by determining the retention of the sex determining region Y (SRY) gene located on the Y chromosome in the heart of female rats. Briefly, the whole heart was harvested, weighed, and homogenized. Then, the genomic DNA was isolated from myocardial tissues using commercial kits . The TaqMan assay  was used to quantify the number of transplanted cells with the rat SRY gene as a template . A standard curve for the amplification multiples of rat SRY genes isolated from EPCs and cells numbers was generated. As a result, on the basis of the amplification multiples measured by PCR in each sample, the retained cell numbers could be calculated. The cell retention rate was calculated as the cell number of EPCs detected in the heart over the number of total transplanted EPCs.2.10The apoptosis of cardiomyocytes was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, according to manufacturer's instructions . Anti‐α‐actinin antibody was used to mark the cardiomyocytes, whereas red fluorescent dye and DAPI were used to label the apoptotic nuclei and entire nuclei, respectively. The apoptosis index was calculated as the ratio of TUNEL‐positive cells (red) to total cardiomyocytes (blue). Myocardial remodeling after infarction was evaluated by Masson's staining, as previously demonstrated. Areas stained in blue indicated the deposition of collagen fibrils.2.11At 4 weeks after cell transplantation, the rat hearts collected from each group were excised and transected at the plane of the papillary muscle, followed by freezing in the OCT compound. 10 μm sections with a thickness of 100 μm were obtained, and Masson's trichrome staining was performed, according to manufacturer's instructions. Thereafter, images were acquired using a Pannoramic MIDI Slide scanner. Then, the percentage of infarcted areas in each section was calculated.The left ventricular function at each of the indexed time points was detected by transthoracic echocardiography using the MyLab 30CV ultrasound system . Rats were given anesthesia by inhaling isoflurane, and ultrasound images were obtained from the parasternal short axis at the level of the mid‐papillary muscle from at least three separate cardiac cycles. Then, left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured.2.12To investigate the capillary density of different groups, the endothelial cells of representative sections were stained with the antibody of CD31. Moreover, because previous studies have indicated that transplanted EPCs could differentiate into mature endothelial cells and participate into the process of neovascularization, the antibody of GFP was used to mark endothelial cells that differentiated from the transplanted EPCs. After incubation with the secondary antibodies, images were photographed under a fluorescence microscope. The number of both CD31 and GFP‐positive cells were counted in at least three randomly high‐power fields.2.13Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) was performed, as previously described in studies. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) served as an internal control. The primer sequences for the real‐time RT‐PCR are listed, as follows: Collagen I forward: 5′‐TGACTGGAAGAGCGGAGAGT‐3′, Collagen I reverse: 5′‐GAATCCATCGGTCATGCTCT‐3′; Collagen III forward: 5′‐TTTGTGCAATGTGGGACCTG‐3′, Collagen III reverse: 5′‐AATGGGATCTCTGGGTTGGG‐3′; TGF‐β forward: 5′‐CACTCCCGTGGCTTCTAGTG‐3′, TGF‐β reverse: 5′‐GGACTGGCGAGCCTTAGTTT‐3′; GAPDH forward: 5′‐ACAGCAACAGGGTGGTGGAC‐3′, GAPDH reverse: 5′‐TTTGAGGGTGCAGCGAACTT‐3′.For the western blot assay, the cells or myocardial samples were lysed, and proteins were extracted according to the specification of manufacturers. Total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electrophoretically transferred onto polyvinylidene ﬂuoride membranes . Then, 5% nonfat dried milk were used to block the nonspecific binding sites in the membranes, and the membranes were incubated with the primary antibodies of VEGF , IGF‐1 , FDGF , SDF‐1α , β‐FGF , ANP , BNP , Bax , and Bcl‐2  overnight, followed by incubation with the corresponding secondary antibodies. The protein bands were visualized using an enhanced chemiluminescence system . GAPDH was used as an internal loading control.2.14t test was used for comparisons between two groups. One‐way analysis of variance was used for comparisons among multiple groups, and Student–Newman–Keuls (SNK)‐q tests were used for subsequent analyses between multiple comparisons. p value of <0.05 was considered statistically significant. All statistical analyses were performed using SPSS 19.0 software.Continuous values were presented as mean ± standard deviation. The Student's 33.1As shown in Figure 3.2The incorporation of nanoparticles and EPCs were achieved by endocytosis. To confirm the optimal labeling condition, labeling efficiency was determined by Prussian blue staining, and EPC vitality was further measured at various nanoparticle concentrations by the ﬂow cytometric analysis. As shown in Figure 3.3p > 0.05). Similarly, the proliferation curve of the cells in the labeled and unlabeled groups were not statistically different in the successive 5 days . These results indicated that labeling with nanoparticles has no significant impact on cell proliferation and migration ability.As shown in Figure p > 0.05). In addition, the cell phenotype of labeled and unlabeled cells was determined. As expected, the expression of biomarkers, such as CD133, CD34, and VEGFR, in cells from these two groups were practically unanimous . Cell synthesis and secretion capacity were also explored by analyzing the protein expression of angiogenic and cardiac protective factors. As shown in Figure p > 0.05).Neovascularization is of an important capacity of endothelial cell lineage. To determine whether nanoparticle labeling compromised the angiogenesis ability of EPCs in vitro, a tube formation assay was performed. As shown in Figure 3.4p < 0.05). Moreover, the magnetized EPCs also exhibited satisfying magnetic responsibility in turbulent status. As shown in Figure To investigate the magnetic responsibility of nanoparticle‐labeled EPCs, their distributions with or without the guidance of an external magnetic field was observed. As shown in Figure 3.56 EPCs, indicating that transplanting this amount of EPCs could obstruct the myocardial microcirculation. Accordingly, 1 × 106 was considered as the “safe amount” of EPCs for transplantation. To further evaluate whether this amount of EPCs for transplantation would affect the normal physiological functions of rats, the serum markers of renal function and liver function, and the metabolic state of iron element were measured, accordingly. As shown in Table 6 was chosen as the optimum number of cells for the subsequent in vivo experiment.To determine the optimal number of EPCs for transplantation in vivo, serum cTnI levels were measured at different cell transplantation concentrations. As shown in Figure 3.6+ area in the group with an external magnetic field was much larger than that without an external magnetic field (p < 0.05). To eliminate the error of possible “false‐positive phenomenon”, cells that expressed GFP by adenovirus transfection were used to trace the retention of transplanted EPCs. As shown in Figure + cells. Interestingly, the GFP+ cell number in one high‐power field was not statistically different among the MI + EPCs group, MI + EPCs + M group, and MI + Fe‐EPCs group (p > 0.05).As shown by the results of the histological examination, magnetized EPCs that accumulated in the myocardium were positive for Prussian blue staining. However, the unlabeled EPCs could not be stained by Prussian blue. Figure p < 0.05). Moreover, these retention rates were not statistically different among the MI + EPCs group, MI + EPCs + M group and MI + Fe‐EPCs group (p > 0.05), but were consistent in terms of the number of GFP+ cells in these three groups. These results demonstrate that magnetic field guidance could significantly increase the retention of magnetized EPCs in the ischemic myocardium. These above results also indicate that magnetic iron oxide nanoparticles labeling had no obvious influence on the engraftment of EPCs, and that the external magnetic field has no significant impact on the retention of unlabeled EPCs.These results above were highly consistent with the data of the RT‐PCR. As shown in Figure 3.7+ cells were observed in all EPC transplantation groups, and the number of TUNEL+ cells in the MI + Fe‐EPCs + M group was further reduced, when compared with the MI + Fe‐EPCs group (p < 0.05). In addition, magnetic guidance could further decrease the proapoptotic protein level of Bax, but increase the antiapoptotic protein level of Bcl‐2. The Masson's staining result revealed that magnetic guidance could further reduce the fibrosis area , which again demonstrates that magnetic iron oxide nanoparticles labeling has no significant adverse impact on the normal function of EPCs.To determine whether increased EPC retention could further ameliorate myocardial remodeling and improve cardiac function, heart morphology and echocardiography were measured. At 4 weeks after cell therapy, rats in the MI group exhibited the most serious ventricular dilatation, the most extensive infarct area, and the thinnest left ventricular wall thickness. Conversely, all groups that accepted cell therapy had ameliorated myocardial remodeling and preferable cardiac morphology. Interestingly, rats in the MI + Fe‐EPCs + M group exhibited the best protective efficacy, with a minimal infarct size and the most compact ventricular structure Figure a. At 1 w3.9+/GFP+ cell number, indicating that more endothelial cells were differentiated from the transplanted EPCs in this group. However, CD31+/GFP+ cells in the other three cell therapy groups were rarely observed. Furthermore, the expression of pro‐angiogenesis proteins VEGF, IGF‐1, FDGF, SDF‐1α, and β‐FGF were also observed. As shown in Figure +/GFP+ cell and pro‐angiogenesis proteins expression were found among the MI + EPCs group, MI + EPCs + M group, and MI + Fe‐EPCs group (p > 0.05), which demonstrate that magnetic iron oxide nanoparticles labeling exerted no significant adverse influence on the differentiation and secretion functions of EPCs.The cells, that were both CD31 and GFP‐positive, confirmed that endothelial cells were differentiated from the transplanted EPCs. As shown in Figure 4The pivotal issue for the survival of ischemia organs rest with the formation of new blood vessels, which is especially true when it comes to MI (Losordo & Dimmeler, 3O4 core with a diameter of 60 nm, the nanoparticles exhibited excellent biocompatibility and higher cell endocytosis efficiency without any obvious side effects. In addition, compared with SPION, the silica outer surface endowed the rigid structure of nanoparticles, thereby keeping it insensitive and stable, regardless of the changes in pH and temperature. Moreover, the silica layer acts as a protective shell, which prevents the Fe3O4 core from quick degradation. Finally, the particular structure of the material not only maintains the long‐term reactiveness of the particle to the external magnetic field, but also prevents the release of a large amount of iron element, which may result in cell cytotoxicity (Li et al., An important advantage of the present experimental study was the application of silica‐coated magnetic iron oxide nanoparticles, instead of traditional SPION, for the labeling of EPCs. The material possesses multiple advantages. Silica is classified as “Generally Recognized as Safe” by the FDA (Chen et al., Stem cell‐based cell therapy has become an attractive and potential treatment strategy for MI (Hou, Kim, Woo, & Huang, 6 EPCs were transplanted, which displayed no obvious adverse influence to the normal physiological function of rats. The time point of EPC infusion after MI was another critical issue. Indeed, there is a paradox in cell transfer time selection, and myocardial interstitial edema and severe inflammatory response persists at the acute phase after MI. Therefore, cell transplantation efficiency at this time window might be restricted. However, EPCs exhibited a limited homing efficiency at 7–9 days after MI (Mu et al., The dose and curative effect are often concentration dependent in pharmaceutical studies. However, this is not the situation for cell therapy. In fact, researchers have demonstrated that the transplantation of excessive cells would obstruct the microcirculation. Moreover, the external magnetic field would further increase the aggregation of magnetized cells, thereby exacerbating the risk of coronary embolism (Huang et al., +/CD31+ cells demonstrates that transplanted EPCs could directly differentiate into endothelial cells. However, the number of GFP+/CD31+ cells is rare, indicating that the direct mechanism may be less important. Furthermore, it was found that pro‐angiogenesis cytokines, such as VEGF, IGF‐1, FDGF, SDF‐1α, and β‐FGF, were extremely abundant at the ischemia region. As a result, it was speculated that this indirect mechanism might contribute more therapeutic benefits.Early studies have indicated that the therapeutic effect of EPCs mainly relay on its direct transdifferentiation into endothelial cells to keep the integrity of the endothelial layer, which is called, the “direct mechanism”, whereas subsequent studies have suggested that pro‐angiogenesis cytokines released by engrafted EPCs at the injury site to facilitate the self‐repair and extension of endothelial cells might play a more important role, namely, the “indirect mechanism”. In the current study, it was found that the transplantation of EPCs could ameliorate MI‐induced injury through both of the above mechanisms. The existence of GFPThere were several limitations in the current study. First, the EPCs transplanted in the experiment were early outgrowth EPCs. Different results might be obtained if late outgrowth EPCs would be used. Second, cell transplantation was performed only after MI. Some clinical studies have revealed that cell therapy could improve the cardiac function in the short term. However, no significant improvement in cardiac function could be observed in the long run, such as at 6 months after implantation. As the retention of stem cells in the body tends to decrease over time, repeated transplantation might enhance its long‐term efficacies. Third, mere changes in the cardiac function at 4 weeks after EPC implantation were observed. Fourth, for patients with cardiovascular diseases, the normal function of EPCs would be compromised. As a result, the strategy of EPC auto‐transplantation may probably be not as effective as that in the animal experiment. Fifth, a rat model of MI was used in the current study. Large animal experiments are still needed to determinate the corresponding EPC transplantation number and magnetic field parameter before clinical application. In addition, it should be noted that multiple mechanisms mediate cell loss after transplantation. With this rationale, a combination of magnetic targeting and multiple joint mechanisms, such as gene modification and drugs, to enhance cell engraftment capability, might be more effective.In conclusion, the current study demonstrates that the magnetic field‐guided transplantation of magnetized EPCs is associated with the enhanced aggregation of EPCs in the infarcted border zone, thereby improving their therapeutic efficacy for MI. Moreover, the safety of silica‐coated magnetic iron oxide nanoparticle‐labeled EPCs was confirmed in the current study. These results suggest that the magnetic field‐guided transplantation of magnetized EPCs may become a feasible new therapeutic strategy for MI.B‐F.Z., J.C., and H.J. have made substantial contributions to conception and design; B‐F.Z. and J.C. have done acquisition of data; Q.H. and S.Y. have done the analysis and interpretation of the data; B‐F.Z. and X‐P.L. were involved in drafting the manuscript; H.J. and J.C. revised it critically for important intellectual content.The authors declare that there are no conflict of interests.


Here, we report on those patients receiving retreatment with bortezomib.Studies have shown that bortezomib retreatment is effective in relapsed/refractory multiple myeloma (MM). The observational, prospective electronic VELCADEConsenting adults scheduled to receive bortezomib for MM were enrolled at 162 sites across Europe, Canada, Brazil, Russia, and Turkey between 2006 and 2010. Retrospective data on prior therapies and prospective observational data after bortezomib initiation were captured electronically at baseline, after every bortezomib cycle, and every 12 weeks after discontinuation or progression. Investigator‐assessed responses and adverse events (AEs) were evaluated.Ninety‐six of 873 patients enrolled to eVOBS received bortezomib as first retreatment for progressive disease during the prospective observation period. Median age was 62 years, 53% were male, and median number of prior therapies at retreatment was 4. Overall, 41% of patients initiated bortezomib retreatment in combination with dexamethasone, 16% in combination with lenalidomide, and 21% received monotherapy. Rate of partial response or better (≥PR) was 75% at initial bortezomib therapy, including 44% complete response (CR)/near CR (nCR); at retreatment, ≥PR rate was 46%, including 15% CR/nCR. Median progression‐free survival was 11.4 months  from start of initial bortezomib treatment and 6.4 months (95% CI: 4.4‐7.2) from start of retreatment. Median overall survival from start of retreatment was 17.6 months (95% CI: 14.4‐23.5). Of the 96 patients retreated with bortezomib, 77% reported an AE. Peripheral neuropathy during bortezomib retreatment occurred in 49% of patients, including 10% grade 3/4.These data suggest that retreatment with bortezomib is a feasible option for patients with relapsed/refractory MM. Retreatment with bortezomib is, therefore, a viable option for patients with progressive disease (PD), either as a subsequent or later line of therapy after initial bortezomib treatment. A number of retrospective studies,electronic VELCADE OBservational Study (eVOBS) that was designed to study the efficacy and safety of bortezomib‐based therapies for MM in real‐world medical practice.While the efficacy and safety of bortezomib‐based therapies for retreatment have been shown in the highly controlled clinical trial setting, these findings may not reflect those observed in routine medical practice, where the patient population can differ substantially from that selected by strict clinical trial entry criteria. To date, however, data on the use of bortezomib retreatment in the “real‐world” oncology practice setting are limited. To address this gap, we conducted a sub‐analysis of the prospective, international, non‐interventional, 22.1Electronic VELCADE OBservational Study was an open‐label, non‐interventional, observational study designed to collect prospective data from MM patients undergoing bortezomib‐based therapy within any of the locally approved indications in the real‐world oncology practice setting. Details of study design and conduct have been published recently.Patients were enrolled between June 2006 and December 2010 at clinical practices in Belgium, Brazil, Canada, France, Greece, Russia, Spain, Sweden, and Turkey. The study was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice, and applicable regulatory requirements, and was approved by an Independent Ethics Committee or Institutional Review Board in all participating countries. All patients provided written informed consent in accordance with local legislation.2.2The overall objective of eVOBS was to evaluate the clinical outcomes associated with bortezomib‐based therapies in real‐world medical practice.2.3Patients' MM treatment histories during the year prior to starting bortezomib were recorded retrospectively. Additional information, including patient demographics and disease characteristics, scheduled bortezomib dose, concomitant medications, and laboratory parameters, were obtained at initiation of bortezomib treatment (baseline). Observational data were then collected prospectively over a 3‐year period following initiation of the first cycle of bortezomib. Data were captured electronically at baseline and after every bortezomib cycle, with the exception of serious adverse events (AEs), which were reported within 24 hours of the knowledge of the event. Any bortezomib dose adjustments or cycle delays were documented.Each site used and recorded its own existing methods and criteria for response assessment. MM disease stage was assessed at the time of diagnosis using Durie‐Salmon or International Staging System criteria. Responses were assessed by investigators applying modified European Group for Blood and Marrow Transplant (EBMT),2.4All time‐to‐event endpoints were analyzed using Kaplan‐Meier and Cox proportional hazards regression analyses. Patients lost to follow‐up or who discontinued bortezomib treatment without a reason were censored in all time‐to‐event analyses. Kaplan‐Meier analyses were stratified according to baseline characteristics  and best response to bortezomib. The two‐sided log‐rank test was used to assess the significance of any differences between the stratified data; the conventional significance threshold of 0.05 was used across all analyses. As described in Terpos et al, missing data were not substituted nor imputed.33.1In total, 1573 patients who initiated bortezomib treatment for MM, at any of the 162 surveyed centers, were enrolled into the eVOBS registry. Due to concerns about data quality, 700 patients enrolled in Russia were excluded from the primary study analysis, leaving 873 evaluable patients. Demographics and baseline characteristics for the entire eVOBS population have been reported previously.n = 29), Brazil (n = 17), France (n = 11), Greece (n = 26), Spain (n = 4), Sweden (n = 4), and Turkey (n = 5). Although patients were also enrolled into the eVOBS study at clinics in Canada, none underwent bortezomib retreatment for PD during the prospective observational phase.Of the 873 patients with MM who received bortezomib‐based therapy during the 3‐year prospective observational phase, 96 (11%) underwent retreatment with bortezomib for PD during this period. The number of retreated patients enrolled by country was Belgium , and the median number of prior therapies and time since diagnosis was higher. In the retreatment population, the median age (62 years) and proportion of males (53%) were both slightly lower than in the overall population .3.2The majority of patients undergoing bortezomib retreatment received this therapy as their fourth or fifth line Table . The mosn = 42, 44%), autologous stem cell transplantation , and thalidomide .In total, 91 (95%) patients had received an alternative treatment for MM between initial bortezomib and bortezomib retreatment. Of these, 49 (51%) had one alternative treatment, 25 (26%) had two, 12 (13%) had three, and 5 (5%) had four intermediate treatments. The most common intermediate treatments were lenalidomide plus dexamethasone  patients started bortezomib retreatment at 1.3 mg/m2 and 18 (19%) did so at a reduced dose (≤1.0 mg/m2) (Table Compared with 85 (89%) who started initial bortezomib treatment at 1.3 mg/m3.3n = 35, 36%), EBMT response criteria , other (not specified) criteria , and SWOG response criteria . Overall, 75% of patients achieved a best response of ≥PR following initial bortezomib therapy, compared with 46% of patients who underwent bortezomib retreatment (Kaplan‐Meier estimates). This included 44% and 15% of patients with CR/nCR, respectively. Median time to ≥PR was 1.4 months  for initial bortezomib treatment and 1.7 months (95% CI: 1.3‐2.1) for retreatment.Criteria used for response assessment in the subset of bortezomib‐retreated patients were M‐protein reduction . In patients who had received one or two prior therapies, 70% achieved ≥PR at retreatment, compared with 48% for three prior therapies and 39% for four or more prior therapies . There was no significant difference in the ≥PR rate to bortezomib retreatment in patients stratified by a TFI of <6 versus ≥6 months between end of previous treatment line and bortezomib initiation. Of the 96 patients receiving bortezomib retreatment, 12 received a second retreatment with bortezomib following PD, of whom one patient achieved CR and one achieved PR.We investigated factors that may influence response to retreatment. In total, 68% of patients who had achieved CR/nCR with initial bortezomib achieved ≥PR on retreatment; this figure was 39% in patients who had achieved PR and 20% in patients who had achieved ≤MR with initial bortezomib  and OS (P = 0.0779) from the start of bortezomib retreatment in patients who achieved CR/nCR versus PR versus ≤MR with bortezomib retreatment  from the start of bortezomib retreatment and depth of response after initial bortezomib treatment.In bortezomib‐retreated patients, median follow‐up from the start of initial bortezomib was 35 months (range 9‐60), and from the start of retreatment, 11 months (range 0‐30). All medians for time to event endpoints were generated using Kaplan‐Meier and Cox proportional hazards regression analyses. Median PFS in bortezomib‐retreated patients was 11.4 months (95% CI: 9.1‐12.7) from the start of initial bortezomib treatment and 6.4 months (95% CI: 4.4‐7.2) from the start of retreatment. Median OS was 41.8 months (95% CI: 33.7‐not estimable) from the start of initial bortezomib treatment and 17.6 months (95% CI: 14.4‐23.5) from the start of bortezomib retreatment. There was no statistically significant difference in PFS , or PFS , from the start of retreatment in patients with a TFI of ≥6 versus <6 months between initial bortezomib and subsequent line of therapy  or OS , bone pain (n = 2), disease progression (n = 2), neutropenia (n = 2), and pneumonia (n = 2). Eight (8%) patients died during retreatment.Of the 96 patients who underwent retreatment with bortezomib post‐PD, 74 (77%) reported AEs Table . In tota3.5.1In the 96 bortezomib‐retreated patients, 66 (69%) had PN during initial bortezomib treatment , while 47 (49%) had PN during bortezomib retreatment   of patients included in this analysis were enrolled at clinics in countries within the European Union. Most of these patients received bortezomib‐dexamethasone or bortezomib monotherapy for initial bortezomib treatment, and bortezomib retreatment within eVOBS. These approaches are in line with the current European approval status of bortezomibOf the 96 patients included in this study who were eventually retreated with bortezomib, 75% achieved ≥PR after their initial bortezomib treatment, including 44% CR/nCR. These rates are slightly higher than the 69% ≥PR rate and 37% CR/nCR rate observed in the overall eVOBS study population following initial bortezomib‐based treatment.In our retreated cohort, 46% of patients achieved ≥PR with bortezomib retreatment, including 15% CR/nCR. The ≥PR rate with bortezomib retreatment reported here is comparable with overall response rates reported with bortezomib retreatment in previous prospective clinical trials,The ≥PR rates observed with bortezomib retreatment in this study are encouraging, considering the advanced disease stage and heavily pretreated nature of the retreated population. This is consistent with previous studies demonstrating that bortezomib retreatment is feasible in later lines of therapy and can produce responses in a considerable proportion of patients.Notably, 95% of patients retreated with bortezomib in eVOBS had received at least one alternative treatment for MM between initial bortezomib and bortezomib retreatment, which may have impacted on the observed ≥PR rate. Although infrequent in clinical practice, 12 patients in our population received a second retreatment with bortezomib after PD, with two patients going on to achieve ≥PR.Consistent with previous prospectiveIn addition, there was a non‐statistically significant trend for a higher ≥PR rate in patients who had undergone fewer therapies prior to retreatment. While no significant difference in ≥PR rate was observed between patients who had a TFI ≥6 or <6 months in this study, higher overall response ratesN = 873) showed no substantial impact on survival distributions by best response (EBMT criteria vs other methods).Limitations to the survey approach used for data collection in the eVOBS study include the variable criteria used for response assessment, which may have impacted the efficacy findings. However, this is the reality of clinical care across sites and countries, and although a substantial proportion of responses were assessed by M‐protein or non‐specified criteria, the overall eVOBS study population  was longer than from the start of retreatment (6.4 months), in accordance with the disease course of an increasingly aggressive cancer in later lines of therapy.2) at initiation of retreatment compared with initial bortezomib treatment (70% vs 89%), which may have made a minor contribution to the observed lower response rates following retreatment. Although rates of discontinuation due to AEs were similar between bortezomib retreatment and initial bortezomib (19% vs 20%), a lower percentage of patients completed the planned course of treatment , and discontinuations due to PD were higher . These findings may reflect lower treatment tolerance, together with increased disease refractoriness, in this population of patients who had received multiple lines of therapy for MM prior to bortezomib retreatment.Median number of cycles received at bortezomib retreatment was encouraging when compared with the number received at initial bortezomib treatment . A lower proportion of patients received standard‐dose bortezomib  retreatment with bortezomib is a feasible option for patients with RRMM, even among heavily pretreated patients, and (2) retreatment may produce better responses than originally achieved during the initial course of bortezomib treatment. Our results regarding survival among patients with deeper responses to bortezomib retreatment and/or a longer TFI following initial bortezomib treatment warrant further investigation.If confirmed in future prospective studies, bortezomib retreatment for those patients responding below PR following initial bortezomib exposure would benefit from an additional treatment option. Furthermore, subcutaneous bortezomib administration is now available and has demonstrated improved tolerability compared with IV infusion.This analysis was supported by Janssen Research and Development.None declared.C. Hulin has received honoraria/consulting fees from Janssen, Celgene, Takeda, Amgen, and Novartis; J. de la Rubia has received personal fees from Amgen, Celgene and Janssen; M. Dimopoulos has received honoraria for participation in advisory boards with Janssen, Celgene, Takeda, Amgen, and Novartis; E. Terpos has received research funding from Amgen, Janssen, Takeda, Celgene/Genesis, honoraria from Amgen, BMS, Celgene/Genesis, Janssen, Novartis, Takeda, and GSK, is a steering committee member for Amgen and a data monitoring committee member for Celgene; E. Katodritou has received research support and personal fees from Janssen Cilag; V. Hungria has received personal fees for board membership and consultancy from Janssen‐Cilag, and payment for lectures including service on speakers' bureaus; A.‐M. Stoppa has received personal fees for consultancy and board membership from Janssen, Celgene, and Amgen; D. Sargin has received personal fees from Janssen; A. Belch has received personal fees from Janssen; J. Diels reports employment and shareholdings of Janssen, Johnson & Johnson; R.A. Olie reports employment and shareholdings of Janssen, Johnson & Johnson, and shareholdings of Amgen; D. Robinson Jr reports employment and shareholdings of Janssen, Johnson & Johnson; A. Potamianou reports employment and shareholdings of Janssen, Johnson & Johnson; H. van de Velde reports employment at Takeda Pharmaceuticals and stockholdings of Janssen, Johnson & Johnson; M. Delforge has received research grants from Janssen and Celgene, consulting fees/honoraria from Janssen, and personal fees from Janssen, Celgene and Amgen; H. De Samblanx, J. Aagesen, and A. Sioni have no conflicts to disclose. The financial relationships listed did not impact the conclusions of the study.Conceptualization: ET, JD, RO, APFormal Analysis: MeDi, ET, EK, AS, JD, RO, AP, HvdV, MiDeFunding Acquisition: APInvestigation: MeDi, ET, EK, VH, DS, AS, JD, ROResources: CH, JdlR, MeDi, HDS, ABWriting—Original Draft Preparation: CH, JdlR, MeDi, ET, EK, VH, HDS, AMS, JA, DS, AS, AB, JD, RO, DR, AP, HvdV, MiDeWriting—Review and Editing: CH, JdlR, MeDi, ET, EK, VH, HDS, AMS, JA, DS, AS, AB, JD, RO, DR, AP, HvdV, MiDe